Diabetes and Nonalcoholic Fatty Liver Disease: A Pathogenic Duo

Table 1.

Summary of relative strength of associations of NAFLD and its subtypes with prediabetes and types of diabetes based on human studies

NAFLD pathological subcategory	Type 1 diabetes^c	Prediabetes^c (IFG, IGT)	Type 2 diabetes	Refs.
Simple steatosis	+	++	++	1, 70, 76, 78
NASH with high NAS^a	+/−	++	++	77
NASH with substantial fibrosis (F2-F3)	+/−	++	+++	2, 63, 71, 73, 77–79
Cirrhosis due to NAFLD (includes F4)^b	+/−	+++	++++	21, 71, 118

NAFLD pathological subcategory	Type 1 diabetes^c	Prediabetes^c (IFG, IGT)	Type 2 diabetes	Refs.
Simple steatosis	+	++	++	1, 70, 76, 78
NASH with high NAS^a	+/−	++	++	77
NASH with substantial fibrosis (F2-F3)	+/−	++	+++	2, 63, 71, 73, 77–79
Cirrhosis due to NAFLD (includes F4)^b	+/−	+++	++++	21, 71, 118

^a

NAS is the sum of standardized scores of steatosis, inflammation, and hepatocyte ballooning at pathology (see Section IV).

^b

Overlaps clinically with hepatogenous diabetes.

^c

Based on cross-sectional studies only.

Diabetes has not only been associated with fibrosis in NAFLD in cross-sectional studies but also has been shown to be a marker of progression for fibrosis in longitudinal series. In a study of histologically proven NAFLD in 432 patients, type 2 diabetes was found to be an independent risk factor for fibrosis progression to moderate and severe fibrosis (2). In another clinical cohort of 103 patients with average age of 45 ± 11 yr who underwent serial liver biopsy at an average interval of 3.2 ± 3.0 yr (range, 0.3–21.3 yr), and in whom 42% had diabetes, preexisting diabetes was found to be a predictor of fibrosis progression on multivariate analysis with a regression coefficient of 0.35 (P = 0.007) (63). As previously discussed, the increased rates of NASH and fibrosis and also apparent increases in fibrosis progression observed in populations with diabetes are of great importance because NASH, particularly with increasing fibrosis, appears to be far more significant than simple steatosis in leading to adverse morbidity and mortality outcomes, both liver related and otherwise (1, 3, 4, 74).

The condition of prediabetes, which includes impaired fasting glucose (IFG) and IGT, is present when hyperglycemia occurs, but by definition, it is not into the blood glucose range of diabetes mellitus (75). Prediabetes reflects relative insulin deficiency, and it is along a continuum from normal plasma glucose to diabetes range levels. As reported in Table 1, in the limited number of series where it has been examined, prediabetes was found to be associated with an increased risk of NAFLD. In a large Korean cohort of 99,969 individuals without clinical evidence of diabetes, the risk of NAFLD on ultrasound increased with an increasing level of glycosylated hemoglobin (HbA1c) (OR, 1.44 at HbA1c 5–5.4%, 2.62 at HbA1c 5.5–5.9%, and 7.18 at HbA1c 6.0–6.4%) when compared with the lowest group by HbA1c category (HbA1c ≤ 4.9%; P < 0.001) (76). In that study, prediabetes range blood glucose levels will have cosegregated mainly with the highest HbA1c readings of 6.0–6.4%, where the odds of NAFLD were greatest. A study undertaking liver biopsies on recruited patients (n = 73) found that those with any degree of NAFLD were more likely to have IGT (29%), compared with the normal glucose tolerant controls (14%) (P < 0.0001) (77). In an Australian study of 70 patients not known to have diabetes, with fasting glucose levels below 7.0 mmol/liter, but with NAFLD as determined by ultrasound, 24% had IGT and 10% had diabetes on standard oral glucose tolerance test (OGTT) (78). Over half of the NAFLD patients with a normal fasting glucose had abnormal glucose tolerance as detected by OGTT. In addition, irrespective of the status of glucose tolerance, 2-h hyperinsulinemia during the OGTT occurred in all subjects with NAFLD, and fasting insulin resistance (HOMA-IR) was found in 73%.

Prediabetes in small cross-sectional series examining NAFLD has been associated more so with increased NASH rather than simple steatosis. In one publication (n = 73), prediabetes (IFG or IGT) occurred in 20% of patients with simple steatosis and in 55% of NASH patients (P = 0.036), and in that study, a higher 2-h plasma glucose reading on the OGTT correlated, albeit weakly, with higher fibrosis stage (r = 0.25; P = 0.046); stage 3–4 liver fibrosis occurred in none of the controls with normal glucose regulation, in 4% with prediabetes, and in 17% of those with newly diagnosed type 2 diabetes (77). By univariate analysis, prediabetes presence (IFG or IGT) as well as hyperinsulinemia and insulin resistance each correlated with NAFLD, although relative relationships with NASH were not reported (77). Interestingly, in a larger series (n = 173) in patients without a history of diabetes, hyperinsulinemia 2 h after oral glucose loading, rather than hyperglycemia, was independently associated with the severity of NASH and fibrosis (P = 0.0001; OR, 3.56; 95% CI, 1.61–7.86), suggesting that circulating insulin, rather than glucose, is a better marker of histological severity in NAFLD (79).

In contrast to findings in type 2 diabetes, prediabetes has not yet been studied in longitudinal series to determine whether it is associated with fibrosis progression in NASH and also the strength of such association compared with that present in type 2 diabetes and NASH with fibrosis. Larger, prospective studies are needed to better define the histological findings of NASH, including fibrosis progression with the continuous variables of hyperinsulinemia, insulin resistance, and hyperglycemia caused by relative insulin deficiency.

B. NAFLD predicts diabetes development

In approaching the relationship between NAFLD and diabetes from another perspective, multiple studies supported by a recent meta-analysis have shown that NAFLD is associated with insulin resistance and diabetes and that NAFLD presence predicts the development of diabetes (1, 78–80). In a Korean study of 11,091 individuals without diabetes at baseline, where fasting insulin levels were also measured and divided into quartiles, the 5-yr (crude) OR for developing type 2 diabetes in the presence of ultrasound evidence of fatty liver at baseline was 5.05 (95% CI, 2.08–12.29) for the lowest insulin quartile and 6.34 (95% CI, 3.58–11.21) for the highest insulin quartile. After multivariate adjustment, including for baseline glucose level, the OR for the highest insulin quartile remained significant at 2.42 (95% CI, 1.23–4.75) (81). Bae et al. (82), also from Korea, using data from 2049 individuals without diabetes, showed that the multivariate adjusted hazard ratio (HR) for developing diabetes in subjects who had both NAFLD on ultrasound and IFG at baseline was 8.95 (95% CI, 6.49–12.35) at 4 yr. Musso et al. (1) reported a 3.51 multiple-adjusted OR for incident diabetes in patients with evidence of ultrasonographic/histological NAFLD in their recent meta-analysis of three large community-based cohorts with a range of follow-up from 4 to 10 yr.

VII. Diabetes Complications Are Linked to NAFLD

A. Macrovascular disease

NAFLD has been associated with an increased prevalence of both micro- and macrovascular complications in patients with type 1 or 2 diabetes (4). The most studied association is the increased risk of macrovascular, and in particular cardiovascular, disease in patients with NAFLD, with or without diabetes. Although large, long-term, prospective studies are needed to address current limitations of available data, there is a growing body of evidence to suggest that cardiovascular disease is the leading cause of death in patients with advanced NAFLD and that NAFLD is associated with an increased risk of incident cardiovascular disease that is independent of the risk conferred by traditional risk factors and components of the metabolic syndrome (4, 83). In addition, NASH presence appears to afford a greater risk than simple steatosis (84, 85). From studies of NAFLD with histopathological grading, cardiovascular disease was the most frequent cause of death in 173 patients with biopsy-proven NAFLD who were followed for 13 yr (57). Ekstedt et al. (60) found that the 14-yr risk of death from cardiovascular disease was higher by a factor of two among 129 patients with NASH than in the general population. Furthermore, Söderberg et al. (85) recently confirmed that NASH, not simple steatosis, was associated with increased mortality from all causes, and from cardiovascular disease and liver-related causes among patients with NAFLD who were followed for a mean period of 21 yr (4, 85). Subclinical detection methods using surrogate markers such as carotid intima-media thickness and pulse wave velocity support these clinical findings (84, 86).

Data exploring any association between NAFLD and cardiovascular disease in patients with diabetes is more limited. In a large study involving approximately 3000 unselected patients with type 2 diabetes (87) and in a smaller series of type 1 diabetes (70), the prevalence of coronary, cerebrovascular, and peripheral vascular disease was remarkably higher among patients with NAFLD. This finding was independent of traditional macrovascular risk factors; duration of diabetes; extent of glycemic control; use of lipid-lowering, antidiabetic, antihypertensive, or antiplatelet medications; and components of the metabolic syndrome. Moreover, NAFLD, as assessed by magnetic resonance (MR) spectroscopy (MRS), was associated with reduced myocardial perfusion in patients with type 2 diabetes who were known to have coronary artery disease. This was found to be independent of traditional risk factors, visceral fat mass, and insulin sensitivity (assessed by euglycemic hyperinsulinemic clamp) (4, 88). In addition, in a cohort of 247 Child-Pugh class A patients with biopsy-confirmed NAFLD with advanced fibrosis or cirrhosis, a history of diabetes and hypercholesterolemia was associated with vascular events (i.e., nonfatal myocardial infarction, nonfatal stroke, and vascular death) and vascular-related death. The adjusted OR for vascular event and death combined in the presence of diabetes in this cohort was 10.43 (95% CI, 1.26–82.53; P = 0.03) (89). This increase in risk is higher than the risk that diabetes has been estimated to carry for vascular disease in the general population by a factor of approximately 5 (90).

Some counterbalance to the argument that NAFLD is an independent risk factor for cardiovascular disease is demonstrated by two negative studies in type 2 diabetes that found no association with NAFLD and increased carotid intima-media thickness or coronary, aortic, or carotid calcium (91, 92) and studies that have failed to show an association for NAFLD and markers of cardiovascular disease such as carotid intima-media thickness after statistical adjustments were made for traditional risk factors (83, 93).

B. NAFLD and the microvascular complications of diabetes

Although data are limited, being sourced from observational studies and largely from one group, an association between NAFLD and CKD has been suggested, including in populations with diabetes. While this association must be confirmed in larger, controlled studies and across broader ethnic groups, in patients with either type 1 or 2 diabetes, the presence of NAFLD as determined by ultrasound has been associated with an increased prevalence of microalbuminuria and chronic kidney disease (CKD) (3, 94, 95). Prospective studies have also demonstrated an increased incidence of CKD in patients with type 2 diabetes and NAFLD. As with cardiovascular disease risk, the degree of liver inflammation also appears to be important. A cross-sectional study looking at NASH severity by histopathology demonstrated an association between the NASH/fibrosis stage and decreasing mean values of estimated glomerular filtration rate (eGFR), independently of traditional macrovascular risk factors, insulin resistance, and metabolic syndrome components (3, 96).

Clarifying the association of NAFLD with CKD is particularly important in those with significant liver disease requiring liver transplant because CKD is a well-recognized determinant of long-term morbidity and mortality in liver transplant recipients. In a recent retrospective case review on patients undergoing liver transplantation at a single center in the United Kingdom between January 2000 and December 2008, where NASH patients and patients with cryptogenic cirrhosis and three components of the metabolic syndrome were compared with a group matched for age, sex, model end-stage liver disease score, and eGFR by the modified diet in renal disease equation (68.7 vs. 69.3 ml/min/1.73 m²) at baseline, the eGFR was 8.46 ml/min/m² lower (P < 0.001) in the NASH/cryptogenic cirrhosis group 3 months after transplant after adjusting for effects of BMI, tacrolimus levels, diabetes mellitus, hypertension, and HCC. In addition, 31.2% of patients in the NASH/cryptogenic cirrhosis group progressed to stage IIIb CKD by 2 yr, compared with 8.3% in the control group (P = 0.009). This finding supports the concept that less renal toxic immunosuppressive regimens should be considered in NASH patients after liver transplant (97).

In addition to the association with CKD, Targher et al. (98) have also observed a higher prevalence of diabetic retinopathy in patients with diabetes and NAFLD diagnosed by ultrasound in a cohort of 202 patients with type 1 diabetes. The age- and sex-adjusted prevalence of retinopathy was 53.2 vs. 19.8% (P < 0.0001) in this cohort, and NAFLD was associated with prevalent retinopathy with an OR of 3.3 (95% CI, 1.4–7.6; P < 0.005) on multivariate analysis. This finding was independent of age, sex, diabetes duration, medication use, HbA1c, and the presence of the metabolic syndrome (98).

Interestingly, de Lédinghen et al. (99), when looking at liver fibrosis assessed noninvasively by Fibrotest and FibroScan (see Section IX.B pertaining to assessment of liver fibrosis) in 277 hospitalized patients with type 1 or 2 diabetes (52% with type 1 diabetes), found that the absence of retinopathy (OR, 5.4; 95% CI, 1.44–20.57; P = 0.01) but a past history of foot ulcer (OR, 5.2; CI, 1.50–18.03; P = 0.009) was associated with a higher prevalence of liver fibrosis by multivariate analysis in type 2 diabetes. In type 1 diabetes, no factor was associated with liver fibrosis (99). Clearly, more studies are needed to make conclusions about the impact of NAFLD on the micro- and macrovascular complications of diabetes.

C. Cancer risk in NAFLD and diabetes

Apart from the traditional vascular complications associated with diabetes, more recent evidence has suggested links between diabetes and various cancers, particularly HCC (100), breast cancer, and colorectal cancer (101). Several systemic reviews and one retrospective cohort study demonstrated an increased adjusted HR for HCC in patients with diabetes from 1.73- to 3.64-fold. This risk appears to be independent of alcohol and viral hepatitis but to vary with diabetes duration and other comorbidities (102–104). In particular, a large meta-analysis of participant data from 97 prospective studies, including 123,205 deaths in 820,900 people, found that the HR for HCC in people with diabetes was 2.16 (95% CI, 1.62–2.88) when compared with people without diabetes after adjustment for age, smoking status, and BMI (105). Because NAFLD is clearly linked to an increased risk of HCC, by assumption, the presence of diabetes in NAFLD may confer additional risk. Indeed, diabetes was present in 59% of a recent cohort of 87 patients with NASH who developed HCC (40).

Several studies have also linked NAFLD to adenomatous polyps of the bowel, and in particular high-grade right-sided lesions (74, 106). Whether the effect of diabetes and NAFLD on cancer risk is synergistic is not yet clear, but the pathogenic mechanisms behind these shared associations with neoplasm are likely to have a common basis.

D. Other associations with NAFLD relevant to diabetes

NAFLD and diabetes are each associated with cognitive impairment (107–109) and bacterial sepsis (110, 111). In a small population of obese individuals who underwent liver biopsy during bariatric surgery, mild cognitive impairment, as defined by extensive psychometric testing, was found in five of 11 patients with NASH in the absence of cirrhosis. This finding was associated both with markers of inflammation and with hyperammonemia. In this same study, 29 patients with simple steatosis did not have mild cognitive impairment or markers of inflammation or hyperammonemia (109). In addition, in a study of 247 hospitalized patients with evidence of NAFLD on ultrasound compared with 100 gender- and age-matched controls, the adjusted OR of recurrent (at least two) bacterial infections per year for a 3-yr period was 3.0 (95% CI, 2.6–4.2; P < 0.001) (111).

Mild cardiac dysfunction has also been noted in populations with NAFLD and type 2 diabetes. In a small population (n = 50) of patients with well-controlled type 2 diabetes without a history of ischemic heart disease, patients with NAFLD detected by ultrasound (n = 32) had early features of left ventricular (LV) diastolic dysfunction, independent of hypertension and several other potential confounders (OR, 30.9; 95% CI, 2.45–79.2; P = 0.008 on multivariate analysis) (112). Previously, NAFLD presence in a nondiabetic, normotensive population has been linked to increased risk of early LV diastolic dysfunction and impaired LV metabolism (113). Further studies are needed to clarify any association of NAFLD with LV diastolic dysfunction, including in diabetes.

VIII. Pathogenic Mechanisms in the Relationship of NAFLD and Diabetes

A. Evidence derived from animal models

As described earlier in this review, diabetes does appear to be an independent risk factor for NAFLD progression in humans, especially the fibrosis in NASH. Animal models of diabetes and NAFLD help to further characterize the pathogenic mechanisms potentially involved in progression of NAFLD in humans, but in a more controlled manner than in human studies.

NAFLD as a model in animals can be achieved by various means, and the challenge is to achieve the pathology of human NAFLD, especially NASH, in the correct metabolic context (114). The literature contains many different mouse models that exhibit histological evidence of hepatic steatosis and more variably, steatohepatitis, and although none to date fully replicate the human phenotype (115, 116), NASH animal models are receiving increased attention, and their relevance to human disease is increasingly being realized (117). NASH animal models include diet-induced, methionine-choline-deficient approaches, which induce only transient insulin resistance and without weight gain (118, 119). Genetic models of leptin gene or receptor knockout in diabetic ob/ob and db/db mice, respectively (118, 120), with the added metabolic insult of a methacholine deficient diet, will create all the histological features of NASH (121), and exacerbate hepatic fatty acid oxidation through functional leptin deficiency (122).

Models of NAFLD that include diabetes mellitus are summarized in Table 2, including their relative strengths and weaknesses. One particularly attractive genetic model of the metabolic syndrome and NASH involves a homozygous truncating mutation in the Alms1 gene (foz/foz). This leads to overfeeding, metabolic syndrome, type 2 diabetes, and on exposure to a high-fat and/or cholesterol diet, NASH with fibrosis (123, 124).

Table 2.

Animal models of NAFLD that include diabetes

Model basis	Model subtype	Advantages of the model	Disadvantages of the model	Role implicated for diabetes in the model	Refs.
Genetically manipulated ± nutritional modification	ob/ob mice-leptin deficient	Reliable development of diabetes, extreme obesity, and metabolic syndrome	Role of diabetes in NASH development is difficult to examine. Model needs further hits to progress to NASH	Not systematically studied using diabetes therapy	120, 121
	db/db mice-leptin resistant	Reliable development of diabetes, metabolic syndrome	Role of diabetes in NASH development is difficult to examine. Model needs further hits to progress to NASH	Not systematically studied using diabetes therapy	118, 121
	Goto-Kakizaki inbred rat	Reliable 2 diabetes onset after 18 wk of age	Model develops NASH, but without significant fibrosis	After 4 months high-fat feeding, 1 month metformin or insulin, + atorvastatin, decreased oxidative stress and inflammation	125
	Alms1 homozygous (foz/foz) mutant mice; some fed high fat and/or cholesterol	Reliable development of diabetes, metabolic syndrome, and all features of NASH including progressive fibrosis	Role of diabetes added to metabolic syndrome in NAFLD development is difficult to examine	PPAR-α agonist therapy improves blood glucose and steatosis but not liver inflammation	123, 124, 129
Wild-type with chemical or nutritional modification	ICR neonatal mice injected up to 5 consecutive days with monosodium glutamate	Reliable induction of severe obesity at 24 wk, then diabetes, progressive NAFLD with all NASH features by 54 wk	Mechanism of effect of MSG on diabetes development has not been fully determined. Not physiological in the pathological insult used	Not systematically studied	132
	Outbred Sprague-Dawley rats (male), fed 2% (wt/wt) cholesterol	All features of metabolic syndrome and NASH develop including progressive fibrosis, consistently after 24 wk	Diabetes development occurs late, after wk 36–48 and after NASH is well-developed. Diet very high in cholesterol	Role of preexisting diabetes in NASH pathogenesis cannot be studied	226
	Wild-type c57BL/6J mice with high-fat feeding then timed induced type 2 diabetes	Reflects human disease process of NAFLD progression to NASH with fibrosis. Role of diabetes can be interrogated	Needs prolonged 15 wk of high-fat feeding to induce insulin resistance before diabetes induced by low-dose streptozotocin	Diabetes added to high-fat feeding accelerates NASH fibrosis progression, which is prevented by insulin	6

Model basis	Model subtype	Advantages of the model	Disadvantages of the model	Role implicated for diabetes in the model	Refs.
Genetically manipulated ± nutritional modification	ob/ob mice-leptin deficient	Reliable development of diabetes, extreme obesity, and metabolic syndrome	Role of diabetes in NASH development is difficult to examine. Model needs further hits to progress to NASH	Not systematically studied using diabetes therapy	120, 121
	db/db mice-leptin resistant	Reliable development of diabetes, metabolic syndrome	Role of diabetes in NASH development is difficult to examine. Model needs further hits to progress to NASH	Not systematically studied using diabetes therapy	118, 121
	Goto-Kakizaki inbred rat	Reliable 2 diabetes onset after 18 wk of age	Model develops NASH, but without significant fibrosis	After 4 months high-fat feeding, 1 month metformin or insulin, + atorvastatin, decreased oxidative stress and inflammation	125
	Alms1 homozygous (foz/foz) mutant mice; some fed high fat and/or cholesterol	Reliable development of diabetes, metabolic syndrome, and all features of NASH including progressive fibrosis	Role of diabetes added to metabolic syndrome in NAFLD development is difficult to examine	PPAR-α agonist therapy improves blood glucose and steatosis but not liver inflammation	123, 124, 129
Wild-type with chemical or nutritional modification	ICR neonatal mice injected up to 5 consecutive days with monosodium glutamate	Reliable induction of severe obesity at 24 wk, then diabetes, progressive NAFLD with all NASH features by 54 wk	Mechanism of effect of MSG on diabetes development has not been fully determined. Not physiological in the pathological insult used	Not systematically studied	132
	Outbred Sprague-Dawley rats (male), fed 2% (wt/wt) cholesterol	All features of metabolic syndrome and NASH develop including progressive fibrosis, consistently after 24 wk	Diabetes development occurs late, after wk 36–48 and after NASH is well-developed. Diet very high in cholesterol	Role of preexisting diabetes in NASH pathogenesis cannot be studied	226
	Wild-type c57BL/6J mice with high-fat feeding then timed induced type 2 diabetes	Reflects human disease process of NAFLD progression to NASH with fibrosis. Role of diabetes can be interrogated	Needs prolonged 15 wk of high-fat feeding to induce insulin resistance before diabetes induced by low-dose streptozotocin	Diabetes added to high-fat feeding accelerates NASH fibrosis progression, which is prevented by insulin	6

Reliable development of diabetes refers to the onset of diabetes that occurs in the majority of animals.

ICR, Imprinting control region.

Table 2.

Animal models of NAFLD that include diabetes

Model basis	Model subtype	Advantages of the model	Disadvantages of the model	Role implicated for diabetes in the model	Refs.
Genetically manipulated ± nutritional modification	ob/ob mice-leptin deficient	Reliable development of diabetes, extreme obesity, and metabolic syndrome	Role of diabetes in NASH development is difficult to examine. Model needs further hits to progress to NASH	Not systematically studied using diabetes therapy	120, 121
	db/db mice-leptin resistant	Reliable development of diabetes, metabolic syndrome	Role of diabetes in NASH development is difficult to examine. Model needs further hits to progress to NASH	Not systematically studied using diabetes therapy	118, 121
	Goto-Kakizaki inbred rat	Reliable 2 diabetes onset after 18 wk of age	Model develops NASH, but without significant fibrosis	After 4 months high-fat feeding, 1 month metformin or insulin, + atorvastatin, decreased oxidative stress and inflammation	125
	Alms1 homozygous (foz/foz) mutant mice; some fed high fat and/or cholesterol	Reliable development of diabetes, metabolic syndrome, and all features of NASH including progressive fibrosis	Role of diabetes added to metabolic syndrome in NAFLD development is difficult to examine	PPAR-α agonist therapy improves blood glucose and steatosis but not liver inflammation	123, 124, 129
Wild-type with chemical or nutritional modification	ICR neonatal mice injected up to 5 consecutive days with monosodium glutamate	Reliable induction of severe obesity at 24 wk, then diabetes, progressive NAFLD with all NASH features by 54 wk	Mechanism of effect of MSG on diabetes development has not been fully determined. Not physiological in the pathological insult used	Not systematically studied	132
	Outbred Sprague-Dawley rats (male), fed 2% (wt/wt) cholesterol	All features of metabolic syndrome and NASH develop including progressive fibrosis, consistently after 24 wk	Diabetes development occurs late, after wk 36–48 and after NASH is well-developed. Diet very high in cholesterol	Role of preexisting diabetes in NASH pathogenesis cannot be studied	226
	Wild-type c57BL/6J mice with high-fat feeding then timed induced type 2 diabetes	Reflects human disease process of NAFLD progression to NASH with fibrosis. Role of diabetes can be interrogated	Needs prolonged 15 wk of high-fat feeding to induce insulin resistance before diabetes induced by low-dose streptozotocin	Diabetes added to high-fat feeding accelerates NASH fibrosis progression, which is prevented by insulin	6

Model basis	Model subtype	Advantages of the model	Disadvantages of the model	Role implicated for diabetes in the model	Refs.
Genetically manipulated ± nutritional modification	ob/ob mice-leptin deficient	Reliable development of diabetes, extreme obesity, and metabolic syndrome	Role of diabetes in NASH development is difficult to examine. Model needs further hits to progress to NASH	Not systematically studied using diabetes therapy	120, 121
	db/db mice-leptin resistant	Reliable development of diabetes, metabolic syndrome	Role of diabetes in NASH development is difficult to examine. Model needs further hits to progress to NASH	Not systematically studied using diabetes therapy	118, 121
	Goto-Kakizaki inbred rat	Reliable 2 diabetes onset after 18 wk of age	Model develops NASH, but without significant fibrosis	After 4 months high-fat feeding, 1 month metformin or insulin, + atorvastatin, decreased oxidative stress and inflammation	125
	Alms1 homozygous (foz/foz) mutant mice; some fed high fat and/or cholesterol	Reliable development of diabetes, metabolic syndrome, and all features of NASH including progressive fibrosis	Role of diabetes added to metabolic syndrome in NAFLD development is difficult to examine	PPAR-α agonist therapy improves blood glucose and steatosis but not liver inflammation	123, 124, 129
Wild-type with chemical or nutritional modification	ICR neonatal mice injected up to 5 consecutive days with monosodium glutamate	Reliable induction of severe obesity at 24 wk, then diabetes, progressive NAFLD with all NASH features by 54 wk	Mechanism of effect of MSG on diabetes development has not been fully determined. Not physiological in the pathological insult used	Not systematically studied	132
	Outbred Sprague-Dawley rats (male), fed 2% (wt/wt) cholesterol	All features of metabolic syndrome and NASH develop including progressive fibrosis, consistently after 24 wk	Diabetes development occurs late, after wk 36–48 and after NASH is well-developed. Diet very high in cholesterol	Role of preexisting diabetes in NASH pathogenesis cannot be studied	226
	Wild-type c57BL/6J mice with high-fat feeding then timed induced type 2 diabetes	Reflects human disease process of NAFLD progression to NASH with fibrosis. Role of diabetes can be interrogated	Needs prolonged 15 wk of high-fat feeding to induce insulin resistance before diabetes induced by low-dose streptozotocin	Diabetes added to high-fat feeding accelerates NASH fibrosis progression, which is prevented by insulin	6

Reliable development of diabetes refers to the onset of diabetes that occurs in the majority of animals.

ICR, Imprinting control region.

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Although there are multiple genetic models of diabetes in animals, few have systematically developed NAFLD models and then studied whether diabetes exacerbates the NAFLD or its progression. This is partly because in the genetic models of NAFLD, diabetes occurs usually as part of the overall syndrome and gene defect (121, 123, 124), or it develops late in the process after NAFLD or NASH is well established (125). Therefore, these models are unable to facilitate systematic examination of the effect of timed onset of diabetes added to a high-fat diet or to a metabolic syndrome phenotype; however, interventions targeting hyperglycemia in diabetes, for example, can be trialled in them. To date, these interventions are relatively few and include hepatic antisteatotic and systemic antiinflammatory effects of a selective dipeptidyl peptidase-IV (DPP-IV) inhibitor in a murine diabetes model (126), statin therapy with either metformin (127) or insulin (128) in db/db fat-fed mice, and peroxisome proliferator-activated receptor (PPAR)-α agonist treatment in the foz/foz cholesterol-fed model (129). This limited series of studies is collated in Table 2 and collectively implicates dyslipidemia, insulin resistance, and possibly hyperglycemia in effects of type 2 diabetes on NAFLD and its progression.

In contrast to the genetic models of obesity with diabetes (118, 120, 121, 123) or the effects of modified diet alone without diabetes (130, 131) or chemically induced obesity resulting later in diabetes (132), some recent animal models of NAFLD have focused on studying effects of timed induction of diabetes on NAFLD progression, in settings arguably more relevant to human diabetes (6). As an example of this, in the authors' published mouse model of NASH, the C57BL/6 mouse is rendered insulin resistant by high-fat feeding with a subsequent increase in total body fat mass, and then partial failure of the pancreatic β-cell is induced chemically by low-dose streptozotocin (6), thus simulating the clinical scenario of development of type 2 diabetes (133). In this model, the induction of diabetes was found to cause more severe NASH, and in particular, progression of fibrosis in NASH (Fig. 1) (6). This effect was attenuated by insulin therapy, indicating that poorly controlled diabetes is required to cause the NASH worsening. This animal model thus reflects what is apparent clinically, where insulin resistance and hyperinsulinemia present from high-fat feeding causes NASH, whereas the addition of type 2 diabetes with relative insulin deficiency causes an additional accelerator for fibrosis progression in NASH.

Figure 1.

Representative liver images of a wild-type mouse model where a high-fat diet fed ad libitum for 15 wk is then combined with induced type 2 diabetes for subsequent 5 wk in C57BL/6 animals (6). Hematoxylin and eosin-stained sections (A–D) show hepatocyte ballooning and lobular inflammatory change in HFD+DM (D, arrows). E–L, Picrosirius red staining of liver sections indicates fibrosis, including in the perisinusoid site, which is most marked in the HFD+DM mice (H and L). Images are magnified at ×400 (A–D; I–L) and ×100 (E–H), respectively. Images in I–L are of the portal tract. The published model shows all histological features of NASH in the HFD+DM group, particularly fibrosis progression (6). D, Open and closed arrows indicate hepatocyte ballooning and inflammatory change, respectively. In each image, scale bar = 200 μm. CHOW alone, Normal diet (A, E, I); DM, diabetes mellitus (B, F, J); HFD, high-fat diet.

In the diabetes and high-fat-fed rodent in combination with increased liver fibrosis in NASH, there was up-regulation of the profibrotic connective tissue growth factor (CTGF), the fibroblast-activating protein mRNA expression as a marker of the activation of the hepatic stellate cell (HSC) responsible for deposition of extracellular matrix in the liver, and also increased TIMP-1 mRNA expression as an inhibitor of the matrix metalloproteinase enzymes that degrade extracellular matrix. Indeed, TIMP-1 mRNA in the high-fat-fed rodents with diabetes was increased 6-fold when compared with chow-fed mice and 3-fold when compared with high-fat-fed mice without diabetes (6), and CTGF mRNA correlated with collagen-I, collagen-III, and TIMP-1 mRNA. Intrahepatic CTGF (61), and TIMP-1 (134) are increased in human NASH, and both molecules are potential hepatic molecular targets to prevent progressive NASH fibrosis in this model. Extension of the mouse model (6) to 37 wk of diabetes results in development of cirrhosis in approximately half of the mice on the ad libitum high-fat diet (7). In summary, this mouse model indicates that on a background of high-fat feeding and insulin resistance, type 2 diabetes causes progression of liver fibrosis in NASH. Recently, chemical diabetes induction preceding high-fat feeding resulting in NASH and HCC has been reported in a C57BL/6 model by other groups in abstract form (135), and it is expected that to help dissect the contribution of diabetes to NASH, further models of timed induction of diabetes will be developed along similar lines.

B. Preclinical and clinical studies examining pathogenic relationships of diabetes and NAFLD

Describing the pathogenesis of NAFLD and NASH in the human with diabetes is complex, and it has been challenging to separate the relative contributions from obesity and other components of the metabolic syndrome. Factors causing NAFLD progression in diabetes are, as yet, not well defined, but they are likely to be an interplay of dysfunctional lipid metabolism and disordered glucose regulation related to insulin resistance with hyperinsulinemia and a relative insulin deficiency, increased oxidative stress, and local and systemic inflammation. Both genetic and environmental conditions are likely to interact to cause NAFLD and NASH and also influence the strong relationship that exists with type 2 diabetes (136, 137).

1. Genetic factors—“common soil”

Genetic susceptibility appears to explain to a large degree familial clustering and ethnic differences in the prevalence of NAFLD/NASH and also why these conditions associate with diabetes and the metabolic syndrome (138). Like diabetes, NAFLD appears to be a heritable disease (139). In addition, familial aggregation of insulin resistance and diabetes in first-degree relatives of patients with NAFLD has been demonstrated (140, 141). A large number of gene variations have been associated with NAFLD (142, 143), but, as is the case with type 2 diabetes, their predictive value is likely to be quite small. In type 2 diabetes, the statistically more important genetic determinants of disease found on genome-wide scanning, such as variation at the TCF7L2 gene, relate mainly to β-cell function rather than to insulin resistance (142, 144). These data reflect that there is a low likelihood of finding a single clear genetic link between type 2 diabetes and NAFLD.

Susceptibility to NAFLD/NASH has been associated with genes influencing insulin sensitivity, in addition to those regulating fatty acid metabolism, oxidative stress, immune regulation, and fibrosis development. Through several genome-wide association studies, the most consistent predictive “NAFLD gene” described involves variations in the adiponutrin/patatin-like phospholipase domain-containing 3 (PNPLA3) gene, with a gene product that is likely to be involved in energy mobilization and storage of lipid droplets (143, 145, 146). Polymorphisms in this gene, the most well-described being rs738409, are thought to alter liver fat content and also to sensitize the liver to environmental stressors, such as obesity or diabetes. The frequency of the rs738409 mutation in Europeans and American Europeans is estimated at 0.23–0.26 (147). Variations in this gene are likely to contribute to racial differences seen in NAFLD prevalence, with people of Hispanic origin demonstrating the highest rates of fatty liver and also polymorphic variation in PNPLA3, followed by individuals of European descent and then African-Americans (143, 148). Specific variations in this domain have also been associated with severe steatosis and progressive fibrosis in adult and pediatric populations. For example, in a small population of 234 patients with type 2 diabetes, the PNPLA3 rs738409 polymorphism was associated with liver fibrosis, assessed noninvasively by FibroTest (a panel of serum markers including bilirubin, γ-glutamyl peptidase, α2 macroglobulin, apolipoprotein A1, and haptoglobulin). This finding was independent of BMI or liver fat content as measured by MRS (149). This functional polymorphism has also been associated with an increased risk of HCC in the setting of cirrhosis (150). The mutation does not appear to be associated with insulin resistance (147) and has not been shown to be increased in type 2 diabetes independent of NAFLD presence (151).

Other single-nucleotide polymorphisms of interest for NAFLD that have been identified in smaller studies include mutations associated with microsomal triglyceride transfer protein (MTP), phosphatidylethanolamine methyltransferase, apolipoprotein C3 (ApoC3), the pregnane X receptor, the superoxide dismutase 2 (SOD2) mitochondrial targeting sequence, and variations in TNF-α, IL-6, and the angiotensin II receptor (143, 152–160). The genetic links between diabetes and NAFLD can be explored by looking at some of these mutations of interest more closely. Further studies examining ApoC3 polymorphisms (T-455C and C-482T) have failed to demonstrate associations with NAFLD in populations other than the original Indian cohort (161, 162), and no relationship of ApoC3 polymorphisms with liver enzymes or insulin resistance was demonstrable by another group (163). In contrast, ApoC3 polymorphisms have been associated with the risk of type 1 and 2 diabetes, with the −482T allele conferring higher risk in lean individuals and the −455C allele being protective for type 2 diabetes in obese individuals (164). Polymorphisms in the SOD2 gene (particularly C47T: rs 4880), thought to play an important role in protection from superoxide radicals, are of ongoing interest in both NAFLD and diabetes. The C allele, when compared with the T allele, results in more efficient transport of SOD2 into the mitochondrial matrix, which can increase the ability of the cell to neutralize superoxide radicals. The T allele has been associated with fibrosis progression in NAFLD by both case-control and intrafamilial association methodologies (165). The T allele has also been associated with nephropathy in type 1 diabetes (166) and retinopathy in type 2 diabetes (167). In addition, meta-analysis demonstrated gene protective effects on the risk of diabetes, diabetic nephropathy, and diabetic retinopathy from the C allele of the C47T polymorphism in the SOD2 gene (168). Functional MTP polymorphisms associated with NAFLD have been associated with incident type 2 diabetes (169). More recently, the MTP −493G/T polymorphism has been shown to be associated with pancreatic β-cell dysfunction in NASH patients without diabetes and also in healthy controls (170). Recent meta-analyses have suggested a link between TNF-α mutation −238 but not −308 and an increased risk for NAFLD (65), but data for −238 and −308 are generally negative for an increased risk of type 2 diabetes (171, 172). TNF-α −308 polymorphism has been associated with type 1 diabetes (173).

Intrauterine growth retardation has also been shown to be associated with NAFLD in pediatric populations and may be related to similar epigenetic changes that have been linked to increased type 2 diabetes risk and development of the metabolic syndrome (174).

2. A seeming paradox: insulin resistance with hyperinsulinemia and a simultaneous relative insulin deficiency in diabetes may cause the progression of NAFLD

Insulin resistance and NAFLD can be seen to exist in a self-perpetuating pathogenic cycle, and insulin resistance is recognized as a fundamental orchestrator of NAFLD/NASH (138). The progression of NAFLD in the setting of insulin resistance is likely to be driven by the pathogenic effects of the subsequent hyperinsulinemia and also by varying degrees of relative insulin deficiency, particularly in diabetes. Insulin resistance is almost invariable in type 2 diabetes, and it is also increasingly recognized as an ongoing issue in many patients with type 1 diabetes, especially those of increasing age (175, 176). By definition, prediabetes and diabetes are states of relative insulin deficiency that, when combined with excess caloric intake and obesity, will commonly contribute to increased portal fatty acids and ectopic fat deposition, including in the liver. Conversely, the subsequent pathogenic changes in fatty liver, especially in the setting of NASH, can lead to exacerbation of insulin resistance and, by assumption, diabetes. The specific factors behind the pathogenic association between NAFLD and diabetes will be addressed below (see Sections VIII.B.2.a and b).

a. Disordered lipid metabolism in NAFLD pathogenesis: insulin resistance, diabetes, and hepatic steatosis.

The steatosis seen in NAFLD is the result of disordered lipid metabolism, where the balance between lipid accumulation and lipid export is disrupted. This is largely the result of the effects of insulin resistance and the subsequent states of hyperinsulinemia and varying degrees of relative insulin deficiency. Because it is difficult to examine turnover of fatty acids in the liver, including their origin and fate in animals, gene expression (steady-state mRNA) and protein levels of key enzymes involved in fatty acid uptake, de novo lipogenesis, transport from the liver, and lipid oxidation are often measured as surrogate markers of lipid metabolism (177). These methods show that in insulin-resistant states, both uptake of exogenously derived fatty acid and de novo hepatic synthesis of fatty acid, exacerbated by decreased lipid export, lead to an increase in lipid synthesis and hepatic lipid content in NAFLD (177).

The mechanisms by which type 2 diabetes mellitus may exacerbate and cause NAFLD progression remain to be fully defined. However, there is evidence to implicate a number of metabolic and cellular mechanisms. First, diabetes presence indicates some degree of relative insulin deficiency. Any degree of insulin lack, especially in the presence of increased body weight or obesity, will cause increased lipase activity and consequent lipolysis in adipose tissues. This will lead to elevated circulating and portal free fatty acids (FFAs) and subsequently to their increased hepatic delivery and uptake (178). Human data demonstrate that insulin resistance at the level of adipose tissue is the critical location for the development of lipotoxic diseases, including NASH (179), and that liver fibrosis worsens as quartiles of adipose insulin tissue resistance worsen (180). Indeed, insulin resistance at the level of the adipocyte may be the primary defect in NAFLD, leading to increased lipolysis and FFA delivery to skeletal muscle and the liver and subsequent secondary increases in insulin resistance in these tissues (83, 180). A recent study including 118 patients with NAFLD and 20 controls without NAFLD found that muscle and liver insulin sensitivity were impaired in patients with NAFLD to a similar degree, whether they had prediabetes or type 2 diabetes, but that adipose tissue insulin resistance worsened in type 2 diabetes (80). The relative insulin deficiency present in type 2 diabetes will thus fail to properly suppress fatty acid flux to the liver.

The second main mechanism by which diabetes may contribute to NAFLD is via the hyperinsulinemia commonly present in type 2 diabetes, especially in the early years after type 2 diabetes diagnosis. Hyperinsulinemia may exacerbate the liver insult caused by the increased portal delivery of fatty acids due to the relative insulin deficiency. Selective resistance to insulin action can occur in insulin-resistant states, leading to potential adverse effects of endogenous hyperinsulinemia present (181, 182). Specifically, as demonstrated in rodent models, insulin resistance may develop at the level of pathways that regulate glucose, including those in the liver, and yet insulin signaling regulating de novo lipogenesis can remain sensitively intact. To illustrate this point, differential sensitivity in insulin-mediated lipogenesis compared with gluconeogenesis pathways was recently reported in rat liver in the intracellular signaling system mammalian target of rapamycin complex 1 and its downstream factor S6 kinase (182). Particularly important to NAFLD, this differential signaling is likely to be involved in the up-regulation of gene expression and activated cleavage of the key enzyme regulating lipid synthesis, sterol regulatory element-binding protein (SREBP)-1, which is induced by both insulin and high-fat diets (136, 183). Up-regulation of SREBP-1 will lead to subsequent increases in de novo lipogenesis compounded by increased delivery of carbohydrate substrate to the liver due to peripheral insulin resistance and reduced capacity to store carbohydrate in muscle as glycogen (184).

Multiple lines of evidence suggest that the stimulatory effect of insulin on fatty acid synthesis is mediated by an increase in SREBP-1c expression, activation, and nuclear localization (185, 186), including through chromatin remodeling (187). Studies in Goto-Kakizaki rats as a model of type 2 diabetes showed that SREBP1c induction by insulin is dependent more on the intracellular insulin receptor docking protein IRS-2 in postreceptor signaling, involving atypical protein kinase C (PKC)-mediated pathways, rather than through IRS-1 (188). In this model, nuclear factor κB (NF-κB) was also induced through this insulin-sensitive IRS-2 dependent pathway. In contrast to intact insulin-mediated IRS-2 signaling, impaired hepatic protein kinase B (PKB/Akt) activation is well documented in diabetes and has been linked mainly to reduced IRS-1 activation by insulin (188). Furthermore, mouse models overexpressing SREBP1c showed that NAFLD develops (189), whereas animal models of type 2 diabetes show increased SREBP1c expression (186). Finally, there are data in obese rodents exhibiting hepatic steatosis reporting that endoplasmic reticulum (ER) stress may activate SREBP1c protein in insulin-resistant states (190).

Increased lipid delivery to the liver in NAFLD is exacerbated by decreased lipid disposal. This is likely to be the result of many factors. SREBP-1 has been shown to reduce the disposal of FFAs by inhibiting their uptake and oxidation by mitochondria (191). Reduced disposal of FFA is also the result of inadequate extrahepatic transport of triglyceride due to an insulin-dependent reduction in production of apolipoprotein B, leading to a relative reduction in very low-density lipoprotein (LDL) production (137, 191, 192).

The increased storage and decreased disposal of intrahepatic lipids are collectively thought to lead to hepatic FFA storage and oxidative capacity being exceeded and accumulation of lipids that can exert toxic effects on the liver. These toxicities are thought to occur via inefficient-mitochondrial (due to mitochondrial uncoupling) and peroxisomal β-oxidation. This process can contribute to the pathogenic cycle of NAFLD and insulin resistance by resulting in further increases in insulin resistance directly, and also through the induction of inflammation, oxidative stress signaling, and ER stress (137, 138, 179, 193, 194). Inflammatory pathways may be another mechanism by which lipids exert toxic effects. Indeed, inflammatory pathways have been reported to be activated by lipids and are also likely to be induced by ER stress (195). Liver X receptors, part of the superfamily of metabolic nuclear receptors involved in many processes correlating with lipid metabolism and also capable of regulating inflammation, are an important link between lipids and inflammation (196).

Notably, in cases where simple steatosis progresses to NASH, evidence suggests that de novo lipogenesis appears to become less prominent, and lobular inflammatory change becomes predominant (49, 197). Although this may reflect that a switch from progressive liver lipid synthesis and accumulation to a more severe phenotype occurs in NASH, it is recognized that the source of most fatty acids for triglyceride synthesis in the liver comes from adipose tissue rather than de novo lipogenesis (198), and that toxic lipid metabolites cause inflammation with parallel continued accumulation of lipid, including triglycerides (199). Thus, collectively, current data suggest that some lipid accumulation continues to occur as hepatic inflammation emerges in NASH.

b. Lipid toxicity may progress simple steatosis to NASH.

The lipids that develop in hepatic steatosis may themselves be toxic to the liver and contribute to NAFLD progression. Specifically, there is evidence that cholesterol, diacylglycerol (DAG), and glycosphingolipids may mediate this toxicity (179, 194). Analysis of circulating lipids utilizing lipidomic screening has shown that increased DAG and triglyceride, impaired peroxisomal polyunsaturated fatty acid metabolism, and also nonenzymatic oxidation of lipid are associated with progression to NASH (200), as are lower levels of phosphatidyl choline and a higher ratio of free cholesterol to phosphatidyl choline (201).

Cholesterol has been implicated as a pathogenic factor for NAFLD progression in the foz/foz mouse with type 2 diabetes fed a high-cholesterol diet. In this model, hyperinsulinemia was identified as the factor leading to hepatic cholesterol accumulation, which then resulted in NASH with fibrosis (202). In addition, insulin applied in vitro to hepatocyte primary cultures in concentrations present in type 2 diabetes in vivo was shown to recapitulate the accumulation of cholesterol, up-regulation of LDL receptor via activation of SREBP-2, and reduced cholesterol biotransformation to bile acids for cholesterol and bile acid excretion (202). A high-fat diet with a high cholesterol level has also been implicated in hepatic immune Kupffer cell activation (50).

Hepatic lipid present in a fatty liver may cause NAFLD progression from simple steatosis to NASH. Although data have shown that hepatic triglyceride (triacylglycerol) is thought to be protective of NASH progression, DAG is a lipid product implicated through downstream atypical PKCε signaling, in exacerbating both systemic insulin resistance (203) and hepatocyte toxicity (204). Lysophosphatidyl choline, derived from phosphatidyl choline, a component of the lipid membrane and also of very LDL, may also contribute to lipotoxicity with increased levels of this phospholipid found to be proportional to disease severity in a small liver biopsy series. Direct lipotoxic effects of lysophosphatidyl choline have also been observed in human liver cell lines, mouse hepatocytes, and in mice (179).

As an additional potential method by which lipid may be toxic in NAFLD, saturated fatty acid may lead to increased hepatic glycosphingolipids, including ceramide (205), which may then contribute to apoptosis of native liver cells as well as inflammatory change (206). Although ceramides have recently also been proposed to cause NAFLD progression through the effects of TNF-α (207), lipidomic screening has not implicated circulating ceramides in NAFLD and NASH pathogenesis, and further studies are required to determine whether they have a role.

c. Hepatic inflammation and oxidative stress.

The main inflammatory factors and cellular pathways implicated in causing NASH, compared with profiles in simple steatosis, have recently been extensively reviewed (208, 209). These are: NF-κB and its downstream pathway; macrophage chemoattractant protein-1 and its receptor, C-C chemokine receptor 2; TNF-α and its signaling; and the interleukins IL-1β, IL-18, and IL-33 (208, 210, 211). c-Jun N-terminal kinase is an important second messenger system activated in inflammation in NASH (212). Hepatocytes are thought to have major proinflammatory cell roles in NASH, as are other cells such as Kupffer cells and, potentially, natural killer cells, natural killer T cells, T cells, sinusoidal endothelial cells, and HSCs (213). The inflammation seen in NAFLD may contribute to defects in insulin signaling. This is supported by data showing that inflammatory signals such as TNF-α and IL-6 affect cellular pathways that intersect with insulin action and appear to activate serine and threonine kinases that have been implicated in increased serine phosphorylation of IRS-1 and subsequent decreased insulin signaling (184). Although it has not been clarified to date whether increasing severity or a certain cytokine profile of hepatic inflammation will cause NASH progression, some data support the hypothesis that the presence of inflammation predicts fibrosis in NASH (214, 215).

Oxidative stress generated from ROS may augment inflammation and up-regulate key factors and pathways of NF-κB activation via inhibitor of κ B kinase, c-Jun N-terminal kinase activation, and toll-like receptor (TLR) signaling (178, 208, 216). Many sources of oxidative stress have been identified in NASH and may be derived from mitochondria, possibly ER, cytochromes P450 2E1 and 4A, and also cytosolic peroxisomes and NADPH oxidase as well as infiltrating and resident inflammatory cells. In a study using both case-control and intrafamilial association methodologies, a consistent association between a functional single-nucleotide polymorphism in the mitochondrial targeting sequence of SOD2, a gene thought to be important in protecting against superoxide radicals, and fibrosis severity in NAFLD provides persuasive genetic evidence that mitochondria-derived oxidative stress is important in the pathogenesis of advanced NAFLD (165). Innate immunity, in particular in the resident macrophage, the Kupffer cell, has also been implicated in inflammation in NASH, possibly secondary to responses after hepatocyte apoptosis (217, 218). The “inflammasome,” which is a larger multimeric structure that regulates caspase 1 activation, is linked to mitochondrial production of ROS and innate immunity (219). Although oxidative stress and related cytokines may not be the primary initiators of liver inflammation in NASH, their roles in insulin resistance, perpetuation of liver necroinflammatory change, and progressive fibrogenesis are thought to be likely (208, 220). Oxidative stress has been linked with altered mitochondrial function, as evidenced by diminished respiratory chain activity and reduced ATP production in steatohepatitis (221).

To date, diabetes has not been reported to magnify inflammatory changes present in NASH either at the level of the hepatic cytokine profile, in the degree of hepatic oxidate stress induced, or in liver histopathological studies of inflammation in NASH (6). In contrast, the circulating level of the adipocyte-derived cytokine, adiponectin, does fall in prediabetes and type 2 diabetes (222), and its reduced levels will in an endocrine manner remove protection against proapoptotic effects of TNF-α on hepatocytes (223). Serum adiponectin levels can distinguish simple steatosis from established NASH (224). Thus, reduced adiponectin could be one link between adipocytes and NASH in type 2 diabetes, including through loss of its direct antiinflammatory effects on the liver.

d. Disordered insulin signaling and hepatic fibrosis.

It can be speculated, but remains to be substantiated, that hyperinsulinemia in type 2 diabetes, possibly in combination with the elevated blood glucose in diabetes, contributes to the fibrosis seen in progressive NASH (77, 79). In the animal model referred to earlier, where diabetes is induced in a high-fat-fed murine model, the diabetic high-fat-fed rodents that showed less hyperglycemia, and yet hyperinsulinemia, demonstrated the most liver fibrosis on histology. This finding suggests that some insulin action in diabetes is necessary for progression of fibrosis and also that hyperglycemia may be a permissive factor for liver fibrosis rather than the most important mediator causing fibrosis in this model of NASH (6). Furthermore, selective hepatic insulin resistance may also play a role in the development of liver fibrosis in NASH. Although insulin resistance in phosphatidylinositol-3 kinase signaling occurs in some tissues, in parallel, selective retention of insulin-mediated signaling via MAPK (ERK1/2) pathways occurs (225). There are limited, recent data in Sprague-Dawley rats to indicate that similar selective postreceptor insulin signaling may occur in the liver in insulin-resistant states, mediated in part by C-reactive protein (226). This may be important because phosphatidylinositol-3 kinase mainly mediates glucose cellular metabolism, and MAPK regulates mainly other cellular effects including mitogenesis and laying down of extracellular matrix. The ERK MAPK pathway has been described in vitro to induce certain profibrotic factors, including CTGF and TGF-β in activated HSCs (227, 228) and to cause proliferation of rat HSCs in primary culture (229). Decreased adiponectin levels seen in diabetes may also contribute to fibrosis progression; adiponectin has antifibrogenic properties, associated with decreased expression of markers of activation of HSCs and increased apoptosis of activated HSCs. Inhibition of leptin signaling, a profibrotic adipokine, via inhibition of JAK2 and specific up-regulation of suppressor of cytokine signaling 3 expression by adiponectin are likely to contribute (230).

3. NAFLD can contribute to development of diabetes by disordered lipid metabolism

One of the primary mechanisms by which NAFLD contributes to type 2 diabetes is by increasing insulin resistance. Patients with NAFLD have significantly increased levels of hepatic and peripheral insulin resistance (skeletal muscle and adipose tissue), and diabetes presence is associated with an apparent further increase in insulin resistance at the level of the adipocyte (80). In NAFLD, insulin resistance is thought to be promoted by the accumulation of fatty-acid metabolites (primarily DAG) within insulin-sensitive tissues, and particularly in the liver (184). Although whole body insulin resistance typically cosegregates with generalized obesity (184), it has been reported that insulin-resistant compared with insulin-sensitive individuals have increased lipid accumulation in their skeletal muscle and liver, but no demonstrable difference in sc or visceral adiposity (231), thus implicating liver more than visceral fat as a marker and possible mediator of insulin resistance. In addition, in people with lipodystrophies, which are an extreme example of insulin resistance but with an overall lack of adiposity, there is an increased prevalence of type 2 diabetes.

Lipodystrophies, as a discrete collection of genetic and acquired disorders involving a lack of adipocytes and hypoleptinemia, are associated with profound hepatic and peripheral insulin resistance. People with lipodystrophy typically have hypertriglyceridemia and ectopic fat deposition, including hepatic steatosis. In a study of a genetic form of this condition, severe congenital generalized lipodystrophy, patients were found to have increased basal rates of glucose production, inability to suppress hepatic glucose production, and inability to stimulate peripheral glucose uptake during hyperinsulinemic-normoglycemic conditions. When given leptin, these patients had a 90% reduction in liver triglyceride content and improved hepatic insulin sensitivity, in addition to a 30% reduction in muscle triglyceride content and an approximate 2-fold increase in insulin-stimulated whole body glucose disposal (184, 232).

The effect of the accumulation of fatty acid metabolites (mainly DAG) within insulin-responsive tissues has been most extensively studied in skeletal muscle via experiments in first-degree, lean, relatives of people with type 2 diabetes, by lipid infusions in normal controls, and by genetic murine models (83). Hepatic steatosis also develops in murine models of peripheral insulin resistance [e.g., models involving muscle-specific deletions of the glucose transporter (GLUT) 4] and is also associated with hepatic insulin resistance in its own right (184). Wild-type mice and rats develop hepatic steatosis after a few days of high-fat feeding that is associated with hepatic insulin resistance, without much change in muscle lipid content or peripheral insulin action. The effect of hepatic steatosis on insulin resistance, as demonstrated in this rodent model, seems linked to decreased signaling of IRS-1 and -2 by the insulin receptor, leading to an impaired ability of insulin to activate hepatic glycogen synthesis and suppress hepatic glucose production (184, 233). A similar defect has been shown in patients with type 2 diabetes. As in skeletal muscle, an isoform of PKC is the likely link. PKCε is expressed in increased concentration and is activated in fatty liver. The activation of PKCε is prevented in mouse models if hepatic steatosis is prevented from developing (184).

Apart from insulin resistance, there is some evidence that insulin secretion may also be defective in people with NAFLD. It is proposed that a sustained elevation in FFAs, as can occur in NAFLD, can lead to pancreatic β-cell lipotoxicity (83). In humans, a sustained elevation of plasma FFA levels has been shown to impair insulin secretion in lean, nondiabetic subjects genetically predisposed to develop type 2 diabetes (234), and it was shown that FFA-induced pancreatic β-cell dysfunction can be rapidly reversed in these subjects by decreasing the release of FFAs from adipose tissue by experimentally inhibiting hormone-sensitive lipase with the nicotinic acid derivative acipimox (83, 235).

4. Chronic hyperglycemia in diabetes may contribute to NAFLD progression

Advanced glycosylation end-products (AGE), the result of nonenzymatic reactions between sugars and proteins, lipids or nucleic acid, and up-regulation of their receptors, are a recognized metabolic outcome of chronic hyperglycemia, as occurs in diabetes. Many of the micro- and macrovascular complications described in diabetes have been attributed to AGEs, due to direct effects of AGEs on the structure and function of proteins and also their indirect effects via intracellular signaling. One of the main intracellular signaling pathways involves activation of NF-κB signaling with subsequent up-regulation of the transcription of intracellular adhesion molecule-1, E-selectin, endothelin 1, tissue factor, vascular epithelial growth factor, and proinflammatory cytokines including TNF-α and IL-6. This pathway also leads to increased expression of the receptor for AGEs (236). Increased levels of AGEs have been observed in NAFLD. It has also been shown in vitro that AGEs promote triglyceride deposition in hepatocytes and fibrogenesis through induction of ROS in HSCs (237, 238).

Hyperglycemia per se can also directly contribute to hepatic steatosis and lipotoxicity by increasing hepatic fat synthesis and reducing fat oxidation by activation of the hepatic transcription factor carbohydrate response element-binding protein (136, 239). Subsequent FFA and DAG accumulation in the liver will reduce the inhibitory effect of insulin on gluconeogenesis, contributing further to hyperglycemia (191, 240).

Preclinical in vitro studies show that hyperglycemia in the presence of hyperinsulinemia can also promote fibrogenesis by causing up-regulation of CTGF and inducing oxidative stress in HSCs, thus promoting fibrogenesis (137, 241). In vitro experiments using rodent HSCs have shown that hyperglycemia can lead to proliferation and activation of these cells in a dose-dependent manner, associated with a biphasic increase in intracellular glucose (242). These increases in intracellular glucose in the HSC are caused by increased cellular synthesis of GLUT2 and by up-regulation of plasma membrane translocation of the glucose transporter GLUT2 (242). Activated rodent HSCs in vitro in the setting of hyperglycemia were shown to have increased abundance of α1 procollagen and α-smooth muscle actin (markers of activation), increased promitogenic factors including platelet-derived growth factor-β receptor, cyclin D1, and antiapoptotic Bcl-2, whereas having reduced abundance of the pro-apoptotic Bax. They also had increased expression of the profibrogenic proteins type-I and type-II TGF-β receptors and CTGF. There were increased levels of intracellular ROS and lipid peroxides, with a decreased level of the antioxidant glutathione, presumed secondary to reduced activity of the rate-limiting enzyme for de novo synthesis of glutathione, glutamate cysteine ligase (242).

5. Endotoxemia may contribute to NAFLD progression

The gut/liver axis is increasingly being recognized as a significant contributor to NAFLD pathogenesis. It is thought that the increased absorption of lipopolysaccharides (LPSs) resulting from a “leaky” small intestinal mucosa may cause activation of the innate immune system, leading to inflammation and increased expression of inflammatory cytokines. Thus, LPSs can indirectly increase insulin resistance via these mechanisms but may also have a direct effect on insulin sensitivity itself (243, 244) and may also impair pancreatic β-cell function (245). Conversely, the accumulation of FFAs in the liver that is exacerbated by insulin resistance can in turn facilitate activation of the innate immune system by direct stimulation of TLR-signaling (243, 246–249), including hepatic TLR-4 (208).

Patients with type 2 diabetes have mean plasma values of LPS 76% higher than controls, and levels in these patients can be significantly reduced with the use of the PPAR-γ agonist, rosiglitazone (250). The increased LPS levels in patients with diabetes are likely to be due in part to their decreased clearance as a result of the detrimental effects of chronic hyperinsulinemia on the hepatic resident phagocytic cell, the Kupffer cell (248, 251). Hyperinsulinemia, in combination with hyperglycemia, may also decrease jejunal motility and increase gastrointestinal transit time (248, 252). These changes to gut motility can lead to small intestinal bacterial overgrowth and a subsequent increased leakiness of the intestinal mucosa (253). The absorption of LPS can also be enhanced by a diet high in saturated fat (248, 254).

6. Environmental common soil of NAFLD and type 2 diabetes

Visceral adiposity is a common phenotype of, and an established risk factor for, type 2 diabetes and also NAFLD (255, 256), and it may contribute to the unfavorable cytokine/adipokine balance and fatty acid metabolism that exists in both of these conditions. In an Australian study of 38 individuals who underwent liver biopsy and MRS to assess for visceral fat content, every 1% increase in visceral fat had an OR of 2.4 for increasing inflammation and an OR of 3.5 for fibrosis on liver histopathology (255). The literature in this area is conflicting, however, because in a study of 2589 individuals from the Framingham Heart Study cohort, where the presence of NAFLD was assessed by abdominal computed tomography (CT), the association between NAFLD and diabetes and IFG was found to be independent of visceral adipose tissue and other obesity measures (257). Similarly, an Italian group used retrospective data on 431 patients with NAFLD confirmed on liver biopsy, split into three groups by waist circumference as a widely accepted indirect measure of visceral adiposity, and found that whereas the prevalence of metabolic derangement and severe steatosis increased with a higher waist circumference, the prevalence of NASH or severe fibrosis (F ≥ 2) was not significantly associated on multivariate analysis. The authors proposed that once NAFLD is present, visceral fat may no longer be a major determinant of the severity of liver damage (258, 259).

Associated with increased adiposity is the tendency for patients with both NAFLD and/or type 2 diabetes to consume a diet higher in saturated fat and fructose, to perform less exercise (138, 260–262), and to smoke (5, 263). Conversely, higher caffeine intake and intense exercise for more than 150 min/wk were associated with less fibrosis on liver biopsy (264, 265). To support the contribution of diet to NAFLD, in a mouse model involving the feeding of a “fast-food” diet (AIN-76 Western Diet, Test Diet; relatively rich in saturated fats, cholesterol and fructose) to genetically unaltered C57BL/6 mice for 25 wk, researchers found that these animals developed similar levels of obesity and insulin resistance to mice fed a high-fat diet, but that they had significantly more hepatomegaly, higher serum cholesterol, higher AST, and significantly more hepatic inflammation, hepatocellular ballooning, and fibrosis (266).

The link between NASH, smoking, and diabetes appears complex. A recent analysis of data collected on 1091 adult subjects from the U.S. multicenter NASH Clinical Research Network found in patients without diabetes the OR of advanced fibrosis was 2.48-fold in patients that had at least a 10-yr smoking history compared with those that had never smoked on multivariate analysis. A dose-response relationship was observed between packet years and fibrosis stage. However, whereas patients with diabetes were at a similar increased risk for advanced fibrosis as those who did not have diabetes who had a significant smoking history, the addition of smoking did not further increase their risk. The authors proposed that smoking may cause hepatic injury by increasing insulin resistance. Therefore, the effect of smoking in diabetes may be obscured by confounding related to the intermediary variable effect of insulin resistance (5).

Biochemical and hormonal links between diabetes and NAFLD include associations with low vitamin D (267–269) and elevated ferritin levels (270, 271), in addition to abnormalities in GH and IGF-I (272–276). Abnormalities in sympathetic outflow and the renin-angiotensin system (RAS) are also shared between these two conditions (191, 277–279). Obstructive sleep apnea may also be important in both NAFLD and diabetes (280–282).

C. Pathogenesis of NAFLD progression summarized

Although the concept of a “two-hit hypothesis” was popular some years ago, where NASH was thought to develop as the result of a pathogenic “second hit” occurring after the initial insult of steatosis (283), it is now recognized that an integrated response to parallel and interacting hepatic insults may be a more correct model to consider in NAFLD progression (50, 138). Current preclinical and clinical data suggest that when added to a high-fat diet with obesity, diabetes can cause increased NASH. As described, the common soil of both genetic predisposition and environmental factors is likely to interact with the milieu of metabolic derangements present in type 2 diabetes to cause NAFLD progression. Diabetes, acting through some degree of relative insulin deficiency causing increased lipolysis and thus increased fatty acid delivery to the liver, combined with hyperinsulinemia and selective hepatic sensitivity to insulin leading to preferential stimulation of certain hepatic lipid synthesis and cholesterol pathways, is likely to be key to the progression of NAFLD in this setting. Selective hepatic response to hyperinsulinemia, combined with ongoing liver inflammation, may then induce profibrotic factors in the liver. In addition, as described above, the hyperglycemia and advanced glycation of diabetes may contribute to increased cellular oxidant stress and induction of proinflammatory and especially profibrotic factors, activating HSCs to cause accumulation of liver extracellular matrix (241). Metabolic and cellular mechanisms by which diabetes may exacerbate NASH, including evidence from animal models, are summarized in the schematic diagrams in Fig. 2 as an overview and Fig. 3 in the context of the liver.

Figure 2.

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Schematic diagram indicating proposed pathogenic process by which type 2 diabetes exacerbates NAFLD and how NAFLD may lead to type 2 diabetes and suboptimal glycemic control. Sequentially, 1) in obese individuals, diabetes onset causes increased lipolysis in adipose tissue and increased fatty acid delivery to the liver; 2) hyperinsulinemia persisting in diabetes and selective hepatic insulin resistance induces de novo lipogenesis early, and later, hepatic fibrosis; 3) hyperglycemia and AGE contribute to hepatocyte death, activation of HSCs, and induction of profibrotic factors, such as CTGF. In turn, hepatic insulin resistance secondary to NAFLD may lead to compensatory hyperinsulinemia and secondary pancreatic β-cell failure with progressive hyperglycemia and type 2 diabetes onset. Asterisk indicates where genetic susceptibility to type 2 diabetes development is thought to play a particularly important role for most diabetes susceptibility genes, for example TCF7L2.

Figure 3.

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Pathogenesis of progressive NAFLD and the proposed mechanism(s) of type 2 diabetes (shown in central purple boxes), in increased lipolysis, hyperinsulinemia, selective insulin resistance, and hyperglycaemia. NAFLD processes of proinflammatory transcription factor up-regulation and Kupffer cell activation are not shown in the schematic; to date, diabetes has not been shown to impact on these pathogenic factors.

D. Type 1 diabetes and liver pathology

Type 1 diabetes is characterized by autoimmune destruction of pancreatic β-cells and absolute insulin deficiency, requiring insulin therapy to sustain life. Classically, a person with type 1 diabetes will not have excess body weight or major features of the metabolic syndrome. In contrast to type 2 diabetes series, clinical and preclinical studies in type 1 diabetes and potential effects in the liver are relatively few. Although in a long-term 10-yr diabetes duration nonhuman primate (baboon) model of type 1 diabetes overt NASH did not occur, at the electron microscopic level, liver sinusoidal epithelium thickness was increased and fenestrations were markedly reduced, potentially affecting clearance of lipoproteins by the liver and producing dyslipidemia (284). Studies of type 1 diabetes in rodents (285) and in humans (286) have previously led to similar findings, with the term “diabetic hepatosclerosis” being used to describe these microangiopathic liver changes that reflect microangiopathy in small vessels in other organs affected by diabetes (287). Therefore, in long-term type 1 diabetes without the metabolic syndrome, it is possible that ultrastructural pathological changes occur in the hepatic sinusoidal spaces causing hepatosclerosis, which is distinct from any subtype of NAFLD. Some people who have type 1 diabetes also display metabolic syndrome features seen in type 2 diabetes (288), and it is possible, but not yet reported, that NAFLD and diabetic hepatosclerosis coexist in people with type 1 diabetes who have the metabolic syndrome phenotype.

IX. Assessing NAFLD in Patients with Diabetes

Current data suggest that adverse outcomes related to NAFLD appear closely linked to the degree of necroinflammatory activity and/or fibrosis in the liver, rather than the presence of steatosis (1). This makes the identification and grading of NASH an essential component to a comprehensive assessment of NAFLD. Indeed, defining the degree of NASH will become even more important as effective treatment options become available. Unfortunately, whereas many reasonable tests exist for detecting fatty liver per se, few established techniques exist for grading the degree of NASH, apart from the invasive method of liver biopsy (68).

A. Screening for NAFLD

1. Imaging

NAFLD may be detected by a variety of imaging techniques including ultrasound, CT scan, and nuclear MR imaging (MRI) and MRS (136, 289–291). Ultrasound uses a combination of hepatorenal echo contrast, liver brightness, deep attenuation, and vascular blurring to make a diagnosis. Due to its low cost, safety profile, and availability, ultrasound is currently the study of choice (68, 69). Ultrasound has a sensitivity of 60–94% and specificity of 84–95% to detect fatty liver (78, 136). Unfortunately, it becomes less sensitive with a decrease in the liver fat content below 33% (68) or with an increase in abdominal obesity (69, 136). Therefore, ultrasound may be missing significant burden of disease, such as in the case of a patient with a highly fibrosed but lipid-poor liver (292), or the fatty liver of an obese patient at higher risk of NASH and its complications (136). Various grading systems have been developed to assess for severity of steatosis in NAFLD with ultrasound, in addition to complementary measures of visceral fat; however, none of them are externally validated and they do not appear to provide any further useful prognostic information about inflammation or fibrosis (293, 294).

2. Liver enzymes

Liver enzymes are not a sensitive marker of NAFLD. In patients with type 2 diabetes and histologically proven NASH, abnormal liver enzymes may be seen in less than 20% of patients, depending on the reference ranges applied (295). Indeed, the most severe forms of liver disease can occur in patients with completely normal liver enzymes (136, 296, 297). One factor contributing to the low rate of liver enzyme abnormality in NAFLD/NASH is likely to be the falsely high levels of traditional reference ranges. These ranges were developed from populations that included individuals with undiagnosed HCV and NAFLD. For this reason, various groups have suggested lowering the upper limit of normal of liver enzyme reference ranges (298). For example, new normal levels for ALT specify serum levels of 19 IU/liter or less for women and 30 IU/liter or less for men (299). In a study of 233 women with class II or III obesity using these reference ranges, only 36% of patients had normal liver enzymes compared with 72.5% using their traditional reference range for ALT of 30 IU/liter or less for women. When comparing these results to liver biopsy in this cohort, the researchers found that the new, lower reference ranges were able to increase the detection of patients with fatty liver and portal fibrosis, but were unhelpful in identifying NASH of any severity (300).

B. Determining the degree of inflammation and fibrosis in the liver

1. Liver biopsy

Liver biopsy remains the “gold standard” method to diagnose NASH, enabling a confirmation of the diagnosis and also a grading of fibrosis and other histological features such as the degree of steatosis, iron deposition, necroinflammatory activity, and associated architectural distortion (66). When reporting studies looking at liver histopathology in type 2 diabetes, some investigators suggest that liver biopsy should be considered in all patients diagnosed with diabetes and hepatic steatosis on ultrasound because of the high prevalence of NASH and advanced fibrosis in these cohorts (71). However, liver biopsy is an invasive procedure with attendant risk of pain, bleeding, or perforation (1/1,000) and, rarely, death (1/10,000). Liver biopsy is also costly, an important factor when considering a condition as common as NAFLD, and is inconvenient to both the patient and physician (66).

Liver biopsy is an imperfect gold standard due to inherent problems that limit its accuracy. Sampling error may occur because the biopsy itself represents only 1/50,000th of the total liver. Paired biopsy studies of NAFLD have demonstrated a rate of discordance for fibrosis stage as high as 22–37% (66, 301, 302). In fact, substantial agreement on paired biopsy samples in NAFLD has only been shown for steatosis (301). This problem is accentuated if the biopsy sample does not contain enough liver tissue (303). Consistency of histological staging is also hampered by relatively high rates of intra- and interobserver variability among histopathologists. When grading fibrosis in NAFLD, intraobserver variability has been estimated at 0.65–0.85 and interobserver variability at 0.84. Like the findings on paired biopsies, intra- and interobserver variabilities are also significant for necroinflammation and ballooning, which in consequence has the potential to affect the diagnosis of NASH or “not NASH” (66, 302).

2. Imaging

As previously mentioned, few imaging studies are available for assessing NAFLD severity. Liver stiffness, as measured by ultrasonographic techniques such as FibroScan or acoustic radiation force impulse (ARFI), or with the less available MR elastography (MRE), have been used to estimate the presence and severity of hepatic fibrosis. These and other methods are summarized in Table 3.

Table 3.

Summary of noninvasive imaging investigations for NAFLD and its progression, with a focus on studies including diabetes

Test	Dx NAFLD	Dx NASH	Assess fibrosis	External validation	Experimental vs. clinical use	Cost	Availability	Factors affecting result	Refs.
Liver ultrasound (ⁱ)	√	x	x	√	C/E	+	++++	Liver fat < 30%, obesity, operator experience, and machine quality	68, 69, 136, 290
Unenhanced liver CT (ⁱⁱ)	√	x	x	√	C/E	++	+++	Liver fat < 30%, protocol used, operator experience, other liver pathology (glycogen/siderosis)	136, 289
Liver MRI/MRS (ⁱⁱⁱ, ^iv)	√	x	x	√	C/E	++++	++	Operator experience	136, 291
Transient elastography (FibroScan) (ⁱⁱⁱ)	x	x	√	√	E	++	++	Higher BMI/obesity, ascites, operator experience, probe used (M vs. XL), ?diabetes	1, 51, 304–306, 308–311
Acoustic force radiation-pulse imaging (ⁱⁱⁱ, ^vi)	x	x	√	√	E	++	++	Higher BMI, motion artifact, operator experience	312, 313
MRE (ⁱⁱⁱ, ^v, ^vi)	x	√	√	x	E	++++	+		314

Test	Dx NAFLD	Dx NASH	Assess fibrosis	External validation	Experimental vs. clinical use	Cost	Availability	Factors affecting result	Refs.
Liver ultrasound (ⁱ)	√	x	x	√	C/E	+	++++	Liver fat < 30%, obesity, operator experience, and machine quality	68, 69, 136, 290
Unenhanced liver CT (ⁱⁱ)	√	x	x	√	C/E	++	+++	Liver fat < 30%, protocol used, operator experience, other liver pathology (glycogen/siderosis)	136, 289
Liver MRI/MRS (ⁱⁱⁱ, ^iv)	√	x	x	√	C/E	++++	++	Operator experience	136, 291
Transient elastography (FibroScan) (ⁱⁱⁱ)	x	x	√	√	E	++	++	Higher BMI/obesity, ascites, operator experience, probe used (M vs. XL), ?diabetes	1, 51, 304–306, 308–311
Acoustic force radiation-pulse imaging (ⁱⁱⁱ, ^vi)	x	x	√	√	E	++	++	Higher BMI, motion artifact, operator experience	312, 313
MRE (ⁱⁱⁱ, ^v, ^vi)	x	√	√	x	E	++++	+		314

Dx, Diagnosis; E, experimental; C, clinical; ?, possibly; √, yes; x, no.

ⁱ⁾

Qualitative;

ⁱⁱ⁾

semiquantitative;

ⁱⁱⁱ⁾

quantitative;

^iv)

MRS is noninvasive reference standard for steatosis assessment;

^v)

limited data suggest that MRE can detect steatohepatitis before fibrosis development with sensitivity 94% and specificity 73% for NASH compared with simple steatosis;

^vi)

limited validation data available.

Table 3.

Summary of noninvasive imaging investigations for NAFLD and its progression, with a focus on studies including diabetes

Test	Dx NAFLD	Dx NASH	Assess fibrosis	External validation	Experimental vs. clinical use	Cost	Availability	Factors affecting result	Refs.
Liver ultrasound (ⁱ)	√	x	x	√	C/E	+	++++	Liver fat < 30%, obesity, operator experience, and machine quality	68, 69, 136, 290
Unenhanced liver CT (ⁱⁱ)	√	x	x	√	C/E	++	+++	Liver fat < 30%, protocol used, operator experience, other liver pathology (glycogen/siderosis)	136, 289
Liver MRI/MRS (ⁱⁱⁱ, ^iv)	√	x	x	√	C/E	++++	++	Operator experience	136, 291
Transient elastography (FibroScan) (ⁱⁱⁱ)	x	x	√	√	E	++	++	Higher BMI/obesity, ascites, operator experience, probe used (M vs. XL), ?diabetes	1, 51, 304–306, 308–311
Acoustic force radiation-pulse imaging (ⁱⁱⁱ, ^vi)	x	x	√	√	E	++	++	Higher BMI, motion artifact, operator experience	312, 313
MRE (ⁱⁱⁱ, ^v, ^vi)	x	√	√	x	E	++++	+		314

Test	Dx NAFLD	Dx NASH	Assess fibrosis	External validation	Experimental vs. clinical use	Cost	Availability	Factors affecting result	Refs.
Liver ultrasound (ⁱ)	√	x	x	√	C/E	+	++++	Liver fat < 30%, obesity, operator experience, and machine quality	68, 69, 136, 290
Unenhanced liver CT (ⁱⁱ)	√	x	x	√	C/E	++	+++	Liver fat < 30%, protocol used, operator experience, other liver pathology (glycogen/siderosis)	136, 289
Liver MRI/MRS (ⁱⁱⁱ, ^iv)	√	x	x	√	C/E	++++	++	Operator experience	136, 291
Transient elastography (FibroScan) (ⁱⁱⁱ)	x	x	√	√	E	++	++	Higher BMI/obesity, ascites, operator experience, probe used (M vs. XL), ?diabetes	1, 51, 304–306, 308–311
Acoustic force radiation-pulse imaging (ⁱⁱⁱ, ^vi)	x	x	√	√	E	++	++	Higher BMI, motion artifact, operator experience	312, 313
MRE (ⁱⁱⁱ, ^v, ^vi)	x	√	√	x	E	++++	+		314

Dx, Diagnosis; E, experimental; C, clinical; ?, possibly; √, yes; x, no.

ⁱ⁾

Qualitative;

ⁱⁱ⁾

semiquantitative;

ⁱⁱⁱ⁾

quantitative;

^iv)

MRS is noninvasive reference standard for steatosis assessment;

^v)

limited data suggest that MRE can detect steatohepatitis before fibrosis development with sensitivity 94% and specificity 73% for NASH compared with simple steatosis;

^vi)

limited validation data available.

a. Transient elastography using FibroScan (EchoSens, Paris, France).

The FibroScan device is being used increasingly as a noninvasive tool to assess for liver fibrosis. Vibrations of mild amplitude (1 mm) and low frequency (50 Hz) are transmitted by a transducer, inducing an elastic shear wave that propagates through the underlying tissues. Pulse-echo ultrasound acquisitions are used to follow propagation of the shear wave and to measure its velocity, which is directly related to tissue stiffness (the elastic modulus): the stiffer the tissue, the faster the shear wave propagates. The device measures a volume that approximates a cylinder 1 cm wide and 4 cm long, 25–65 mm below the skin surface. This volume is at least 100 times larger than that of a needle biopsy sample.

The diagnostic accuracy for measurement of significant (stage ≥ F2) or advanced (stage ≥ F3) fibrosis by FibroScan has been assessed in multiple studies in NAFLD. The diagnostic accuracy and reproducibility of FibroScan for advanced liver fibrosis was good and was unaffected by the severity of steatosis or inflammation in most studies (1). In one study, Wong et al. (304) found the area under the receiver operator characteristic curve (AUROC) for significant fibrosis (perisinusoidal and portal/periportal, F2), advanced fibrosis (septal or bridging fibrosis, F3) and cirrhosis (F4) to be 0.84, 0.93, and 0.95 respectively, in a series of 246 individuals from France and Hong Kong who underwent liver biopsy and FibroScan.

Although FibroScan is of value at the extreme ends of the spectrum to rule out significant pathology and also to identify patients who are likely to have advanced fibrosis, it does result in a large number of individuals having an “intermediate” value requiring further assessment and counseling (51). In addition, in all studies to date using the standard probe, the presence of obesity has been an independent predictor of failure to measure liver fibrosis (1, 305). In the study by Wong et al. (304), over 25% of patients with a BMI of 30 kg/m² or greater had an unsuccessful measurement due to attenuation of shear waves into the liver by sc fat. Moreover, even when FibroScan succeeds in producing a measurement, sc fat tends to lead to an overestimation of liver stiffness (306). These factors are of particular relevance in individuals with diabetes who collectively comprise a population recognized to have a higher BMI than the general population (305), and they have been the driving force behind the development of an extra large (XL) probe for obese patients.

The XL FibroScan probe is useful in obesity because it transmits at a lower frequency (3.5 vs. 2.5 Hz) and has a more sensitive ultrasonic transducer, a deeper focal length, a larger vibration amplitude (3 vs. 2 mm), and a greater depth of measurement below the skin surface (35–75 vs. 25–65 mm), allowing stiffness measurement at a greater depth. In an “in-house” clinical trial, the maximum BMI of a patient with a successful measurement was 61 kg/m² (307). The XL probe has now been validated in at least three studies in NAFLD (308–310). In a study of 276 patients with chronic liver disease and a BMI greater than 28 kg/m², 46% of whom had NAFLD, the failure rate of measurement with the XL probe was significantly lower than with the standard medium (M) probe at 1.1% compared with 16%, respectively. The measurements obtained were also more reliable. The XL probe readings correlated well with the M probe values, with similar AUROCs for significant fibrosis (0.83 vs. 0.86; P = 0.19) and cirrhosis (0.94 vs. 0.91; P = 0.28). Median stiffness readings for fibrosis levels were lower for the XL probe than the M probe (310), perhaps demanding a reassessment of the recommended cutoff values used for defining degrees of fibrosis in individual patients (310). Similar to the M probe in obesity, the XL probe may be less accurate at levels of severe obesity (BMI > 40 kg/m²) due to the attenuation of signal from sc fat (308).

FibroScan appears particularly suitable for screening at-risk populations, such as a large diabetes cohort, given its convenient size, acceptability to patients, and ability to produce an immediate estimate of liver stiffness. In a study using FibroScan as a screening tool for significant liver disease involving 1190 patients over the age of 45 and presenting for a general medical check-up, elevated liver stiffness measurements (>8 kPa) were found in 7.5% of the population (311). Although liver enzymes were found to be normal in 43% of the patients with elevated liver stiffness measurements, a cause for liver disease was determined in all cases by clinical assessment, with or without liver biopsy. NAFLD was detected in 58% (52 from a total of 89) of these patients, being the most common diagnosis. Liver biopsy was obtained in 27 patients, including nine patients with liver stiffness measurement greater than 13 kPa. All nine patients with liver stiffness measurement greater than 13 kPa had cirrhosis on liver biopsy (five secondary to alcoholic liver disease, three due to HCV, and one due to chronic hepatitis B virus). All except one (simple steatosis) of the remaining 17 liver biopsies demonstrated fibrosis, and eight of these 17 liver biopsies were consistent with NAFLD (311).

b. Acoustic radiation force-based shear stiffness (ARFI).

ARFI involves the generation of shear waves in the liver by inducing transient tissue deformations of several microns by acoustic radiation force (312). Advantages of ARFI over FibroScan include the ability to obtain measurement in the presence of ascites and the capacity to use some commercial ultrasound scanners for measurement. The success of this technique also appears to be affected by higher BMI, although reconstructions have been achieved up to a BMI of 66 kg/m² (312). It is also probable that a higher level of ultrasound technical expertise may be required in comparison to the FibroScan, although accuracy of both modalities is likely to be affected by operator experience (305, 312). From limited studies, this technique does appear to produce comparable results to FibroScan (312, 313). More multicenter prospective studies are needed to rigorously evaluate the clinical utility of ARFI in comparison to equivalent techniques and also to establish reliable cutoff values for diagnosis, as a function of controlled patient population variables including gender, age, BMI, and liver disease etiology (312).

c. Magnetic resonance elastography.

MRE involves a drum-like acoustic passive driver positioned against the body wall and secured with an elastic belt. This passive driver is then connected to an active acoustic driver system located outside of the MR scanner room via a polyvinylchloride tube. The active driver generates acoustic vibrations in the body, producing shear wave motion within the liver. This technique has minimal validation data but does appear to be more accurate than ultrasound techniques, particularly at lower levels of fibrosis. In particular, from limited data, MRE appeared to have the capacity to identify NASH before the onset of fibrosis. Therefore, a potential clinical use for this tool may be to differentiate patients with NASH from those with simple steatosis. Financial cost and a lack of access to this technology currently limit its use outside of the research setting (314).

3. Biomarkers and clinical algorithms

Recently, as summarized in Table 4, much attention has been paid to developing and validating biomarkers and clinical algorithms in an attempt to overcome the problems associated with liver biopsy (1). In particular, these techniques could lead to a simple, noninvasive method of screening for and assessing response to treatment in patients with NAFLD/NASH. Unfortunately, this process has been hampered by small and biased sample populations. In addition, because validation must be done by liver biopsy with its inherent inaccuracy, even the perfect noninvasive diagnostic test would not replicate liver biopsy results. It has been estimated that even if biopsy was 90% sensitive and specific, a perfect biomarker could only obtain a maximum AUROC of 0.90 (66).

Table 4.

Summary of clinical and blood test investigations for NAFLD and its progression, with a focus on studies including diabetes

Test	Dx NAFLD	Dx NASH	Assess fibrosis	External validation	Validation in diabetes	Constituents/algorithm	Refs.
Liver enzymes (ⁱ)	√/x	√/x	x	√	√	ALT/AST/GGT and ALP	4, 60, 71, 290, 296–299
NFS (ⁱⁱ)	x	x	√	√	√	−1.675 + 0.037 × age (yr) + 0.094 × BMI (kg/m²) + 1.13 × IFG/diabetes (yes = 1, no = 0) + 0.99 × AST/ALT ratio − 0.013 × platelet ( × 10⁹/liter) − 0.66 × albumin (g/dl)	1
						Rule in disease: > 0.676; rule out disease: < −1.455 (see www.nafldscore.com)
ELF combined clinical algorithm (ⁱⁱⁱ, ^iv, ^v, ^viii)	x	x	√	x	√	ELF discriminant score (DS) = −7.412 + (ln(HA) 0.681) + (ln(PIIINP) 0.775) + (ln(TIMP1) *0.494)	1, 315
						Panel for detecting severe fibrosis = −20.870 + 5.506 ELF (DS) + 4.513 diabetes/IFG (yes = 1, no = 0) − 3.144 AST/ALT ratio − 0.058 BMI> (kg/m²) − 0.026 platelets (×10⁹ /liter) + 0.639 *albumin (g/liter)
Cytokeratin-18 (^iv, ^vi)	x	√	x	√	√	Cytokeratin-18	1, 317, 318
FibroTest (^iv, ^vii, ^viii)	x	x	√	x	√	Formula comprised to weighted sum of macroglobulin, haptoglobin, GGT, age, bilirubin, ApoA1, gender. (see http://www.biopredictive.com)	1, 99, 301

Test	Dx NAFLD	Dx NASH	Assess fibrosis	External validation	Validation in diabetes	Constituents/algorithm	Refs.
Liver enzymes (ⁱ)	√/x	√/x	x	√	√	ALT/AST/GGT and ALP	4, 60, 71, 290, 296–299
NFS (ⁱⁱ)	x	x	√	√	√	−1.675 + 0.037 × age (yr) + 0.094 × BMI (kg/m²) + 1.13 × IFG/diabetes (yes = 1, no = 0) + 0.99 × AST/ALT ratio − 0.013 × platelet ( × 10⁹/liter) − 0.66 × albumin (g/dl)	1
						Rule in disease: > 0.676; rule out disease: < −1.455 (see www.nafldscore.com)
ELF combined clinical algorithm (ⁱⁱⁱ, ^iv, ^v, ^viii)	x	x	√	x	√	ELF discriminant score (DS) = −7.412 + (ln(HA) 0.681) + (ln(PIIINP) 0.775) + (ln(TIMP1) *0.494)	1, 315
						Panel for detecting severe fibrosis = −20.870 + 5.506 ELF (DS) + 4.513 diabetes/IFG (yes = 1, no = 0) − 3.144 AST/ALT ratio − 0.058 BMI> (kg/m²) − 0.026 platelets (×10⁹ /liter) + 0.639 *albumin (g/liter)
Cytokeratin-18 (^iv, ^vi)	x	√	x	√	√	Cytokeratin-18	1, 317, 318
FibroTest (^iv, ^vii, ^viii)	x	x	√	x	√	Formula comprised to weighted sum of macroglobulin, haptoglobin, GGT, age, bilirubin, ApoA1, gender. (see http://www.biopredictive.com)	1, 99, 301

Dx, Diagnosis; ApoA1, apolipoprotein A1; GGT, γ-glutamyl peptidase; HA, hyaluronic acid; √, yes; x, no.

ⁱ⁾

Liver enzyme abnormalities are associated with liver and cardiovascular complications.

ⁱⁱ⁾

On meta-analysis, for F ≥ 3: sensitivity, 64%; specificity, 97%. For not F ≥ 3: sensitivity, 90%; specificity, 60%. AUROC for detection of F ≥ 3 was 0.85 (0.81–0.90).

ⁱⁱⁱ⁾

In a study of 91 patients for fibrosis ≥ F3 (cutoff value, −0.2826): AUROC, 0.98 (95% CI, 0.96–1); sensitivity, 91%; and specificity, 96%.

^iv)

Includes markers that are used in research capacity only with poor standardization across some assays.

^v)

ELF has separate panels for “no,” “moderate,” and “severe” fibrosis.

^vi)

Cytokeratin-18 is a marker of hepatocyte apoptosis with AUROC for detecting NASH 0.82 (0.78–0.88); sensitivity, 78%; specificity, 87% on meta-analysis.

^vii)

For Fibrotest, by meta-analysis, to rule out disease for F≥2: cutoff, <0.3; sensitivity, 76%; specificity, 74%; rule in disease for F≥2: cutoff > 0.7; sensitivity, 32%; specificity, 87%. Bilirubin can affect result.

^viii)

Lack of high-quality external validation data.

Table 4.

Summary of clinical and blood test investigations for NAFLD and its progression, with a focus on studies including diabetes

Test	Dx NAFLD	Dx NASH	Assess fibrosis	External validation	Validation in diabetes	Constituents/algorithm	Refs.
Liver enzymes (ⁱ)	√/x	√/x	x	√	√	ALT/AST/GGT and ALP	4, 60, 71, 290, 296–299
NFS (ⁱⁱ)	x	x	√	√	√	−1.675 + 0.037 × age (yr) + 0.094 × BMI (kg/m²) + 1.13 × IFG/diabetes (yes = 1, no = 0) + 0.99 × AST/ALT ratio − 0.013 × platelet ( × 10⁹/liter) − 0.66 × albumin (g/dl)	1
						Rule in disease: > 0.676; rule out disease: < −1.455 (see www.nafldscore.com)
ELF combined clinical algorithm (ⁱⁱⁱ, ^iv, ^v, ^viii)	x	x	√	x	√	ELF discriminant score (DS) = −7.412 + (ln(HA) 0.681) + (ln(PIIINP) 0.775) + (ln(TIMP1) *0.494)	1, 315
						Panel for detecting severe fibrosis = −20.870 + 5.506 ELF (DS) + 4.513 diabetes/IFG (yes = 1, no = 0) − 3.144 AST/ALT ratio − 0.058 BMI> (kg/m²) − 0.026 platelets (×10⁹ /liter) + 0.639 *albumin (g/liter)
Cytokeratin-18 (^iv, ^vi)	x	√	x	√	√	Cytokeratin-18	1, 317, 318
FibroTest (^iv, ^vii, ^viii)	x	x	√	x	√	Formula comprised to weighted sum of macroglobulin, haptoglobin, GGT, age, bilirubin, ApoA1, gender. (see http://www.biopredictive.com)	1, 99, 301

Test	Dx NAFLD	Dx NASH	Assess fibrosis	External validation	Validation in diabetes	Constituents/algorithm	Refs.
Liver enzymes (ⁱ)	√/x	√/x	x	√	√	ALT/AST/GGT and ALP	4, 60, 71, 290, 296–299
NFS (ⁱⁱ)	x	x	√	√	√	−1.675 + 0.037 × age (yr) + 0.094 × BMI (kg/m²) + 1.13 × IFG/diabetes (yes = 1, no = 0) + 0.99 × AST/ALT ratio − 0.013 × platelet ( × 10⁹/liter) − 0.66 × albumin (g/dl)	1
						Rule in disease: > 0.676; rule out disease: < −1.455 (see www.nafldscore.com)
ELF combined clinical algorithm (ⁱⁱⁱ, ^iv, ^v, ^viii)	x	x	√	x	√	ELF discriminant score (DS) = −7.412 + (ln(HA) 0.681) + (ln(PIIINP) 0.775) + (ln(TIMP1) *0.494)	1, 315
						Panel for detecting severe fibrosis = −20.870 + 5.506 ELF (DS) + 4.513 diabetes/IFG (yes = 1, no = 0) − 3.144 AST/ALT ratio − 0.058 BMI> (kg/m²) − 0.026 platelets (×10⁹ /liter) + 0.639 *albumin (g/liter)
Cytokeratin-18 (^iv, ^vi)	x	√	x	√	√	Cytokeratin-18	1, 317, 318
FibroTest (^iv, ^vii, ^viii)	x	x	√	x	√	Formula comprised to weighted sum of macroglobulin, haptoglobin, GGT, age, bilirubin, ApoA1, gender. (see http://www.biopredictive.com)	1, 99, 301

Dx, Diagnosis; ApoA1, apolipoprotein A1; GGT, γ-glutamyl peptidase; HA, hyaluronic acid; √, yes; x, no.

ⁱ⁾

Liver enzyme abnormalities are associated with liver and cardiovascular complications.

ⁱⁱ⁾

On meta-analysis, for F ≥ 3: sensitivity, 64%; specificity, 97%. For not F ≥ 3: sensitivity, 90%; specificity, 60%. AUROC for detection of F ≥ 3 was 0.85 (0.81–0.90).

ⁱⁱⁱ⁾

In a study of 91 patients for fibrosis ≥ F3 (cutoff value, −0.2826): AUROC, 0.98 (95% CI, 0.96–1); sensitivity, 91%; and specificity, 96%.

^iv)

Includes markers that are used in research capacity only with poor standardization across some assays.

^v)

ELF has separate panels for “no,” “moderate,” and “severe” fibrosis.

^vi)

Cytokeratin-18 is a marker of hepatocyte apoptosis with AUROC for detecting NASH 0.82 (0.78–0.88); sensitivity, 78%; specificity, 87% on meta-analysis.

^vii)

For Fibrotest, by meta-analysis, to rule out disease for F≥2: cutoff, <0.3; sensitivity, 76%; specificity, 74%; rule in disease for F≥2: cutoff > 0.7; sensitivity, 32%; specificity, 87%. Bilirubin can affect result.

^viii)

Lack of high-quality external validation data.

Despite these limitations, several biomarkers and clinical algorithms are currently being used in the research setting and may well in the future offer benefit to the clinician for the diagnosis and monitoring of NAFLD. Biomarker panels that have been used in a research setting and have been internally validated in a NAFLD population with encouraging results include: the NAFLD fibrosis score (NFS) [−1.675 + 0.037 × age in years + 0.094 × BMI (kg/m²) + 1.13 × (yes = 1/no = 0) IFG or diabetes + 0.99 × AST/ALT ratio − 0.013 × platelets (×10⁹ liters) − 0.66 × albumin (g/dl)] (62); the European liver fibrosis panel (ELF) combined clinical algorithm [combination of simple markers panel + ELF; ELF: −7.412 + (ln(hyaluronic acid) × 0.0681) × (ln(PIIINP) × 0.775) + (ln(TIMP-1) × 0.494)] (315); and cytokeratin 18 (316, 317). Cytokeratin 18 and the NFS have also been externally validated, including in populations with diabetes (318). However, among studies there is no consensus regarding a cutoff value for cytokeratin 18. In a recent review by Musso et al. (1), various biomarkers were examined, and the AUROC for the NFS was found to be 0.85 (0.81–0.90) for the detection of a fibrosis score of 3 or greater from 13 studies in 3064 patients, whereas the AUROC for the ELF combined clinical algorithm for fibrosis score of 3 or greater was 0.98 (0.96–1) based on a small study using 91 patients, and the AUROC for cytokeratin 18 to detect NASH was found to be 0.82 (0.78–0.88), based on data from nine studies with a total of 812 patients.

The use of several algorithms combining biomarker panels, such as the NFS and cytokeratin 18, with the FibroScan has been proposed in high-risk individuals, such as those with diabetes, to help identify the patients that may need liver biopsy (1). Although this approach appears useful, further data are needed to validate these biomarker panels and algorithms for use in serial follow-up and also in assessing prognosis.

X. Assessing for Diabetes in the Patient with NAFLD

Given the high prevalence of diabetes in patients with NAFLD, it is mandatory to screen patients with NAFLD for hyperglycemia. An early diagnosis of diabetes in patients with NAFLD would provide an opportunity to direct therapy toward insulin resistance and optimal glycemic control to create a favorable “metabolic memory” that would reduce the subsequent risk of diabetes complications specifically and also perhaps liver complications (319). Traditionally, screening has been done by formal laboratory-based fasting blood glucose levels. However, whereas the cost and resource demands involved in screening the large number of patients with NAFLD needs to be considered, the OGTT has been proposed as a better screening tool for diabetes in NAFLD. The OGTT improves the sensitivity of diabetes detection and also detects patients with IGT, a prediabetic state with established links to cardiovascular risk and early retinopathy (320).

Completely separate from the diagnosis of diabetes, the OGTT may also be useful for assessment of liver fibrosis risk by quantifying insulin and glucose levels after a fixed carbohydrate load (78, 79). Thus, whereas insulin levels are not necessary nor are they used in the diagnosis of diabetes, as described in earlier sections, detecting hyperinsulinemia and insulin resistance parameters after a glucose load may help to assess risk of fibrosis and its progression in NAFLD in those with, and without, glucose dysregulation (78, 79).

By identifying patients with IGT, the opportunity exists for early intervention to prevent or delay type 2 diabetes. Lifestyle intervention projects such as the Diabetes Prevention Program have shown that type 2 diabetes can be delayed or prevented in the majority (58%) of cases. The benefits demonstrated in this program were sustained beyond the initial intervention period; compared with controls, there was a 34% reduction in the relative risk of developing diabetes at follow-up, 10 yr after randomization (321).

Data supporting the use of the OGTT compared with fasting glucose in patients with NAFLD are increasing (77–79, 320). A recent alternative to identifying diabetes using HbA1c estimation (HbA1c ≥ 6.5%), as has recently been endorsed by the World Health Organization and the American Diabetes Association (322, 323), does not appear to offer benefit over fasting glucose in NAFLD (78). NAFLD has been associated with postprandial hyperglycemia more than fasting glucose abnormalities (324), and whereas there are no specific publications addressing the sensitivity of HbA1c measures in diabetes screening in people with NAFLD, it is recognized that HbA1c may be less effective at detecting isolated postprandial hyperglycemia that is into the range defined as diabetes, compared with people who also have fasting hyperglycemia (325). Of further note, HbA1c levels are lower in the setting of cirrhosis (326), largely due to decreased red cell life span. Given that people with NAFLD are at particularly high risk of glucose abnormalities, while awaiting published series to address the topic, there are arguments to caution against routine use of HbA1c to screen for diabetes in patients with NAFLD, and such screening should not be used in the setting of cirrhosis.

XI. Targeting Improved Liver Outcomes in the Patient with Diabetes and NASH

Despite the increasing need for an effective treatment for NASH, there is no specific single intervention that has been shown to be convincingly effective. The approaches are summarized in Table 5. The search for treatments has been hampered by small clinical trials, a lack of longitudinal data, and the need for serial liver biopsy to define improvement. Given the importance of preventing future complications, the main goal of therapy should be to prevent or improve liver fibrosis (1, 59). However, largely due to the limitations already described, no treatment to date has been proven to prevent fibrosis in NASH in a large-scale randomized controlled trial (RCT). To examine this issue, in a systematic literature search of published reports, Hojo and Watanabe (327) selected 38 articles with 47 intervention arms, including 11 high-quality, double-blinded RCTs. Histological features improved in greater than 75% of the intervention arms; however, fibrosis improved in less than 30%. In addition, treatment is likely to be long-term because cessation of any intervention to date has resulted in a reversal of any histological improvement seen (327).

Table 5.

Summary of studies targeting NAFLD and its subgroups, with a focus on those including subjects with diabetes

Intervention	No. of studies	Level of evidence	Participants per study range (n)	% with diabetes, range in studies	Improved	Outcomes possibly improved	Unchanged or worsened	Refs.
Lifestyle intervention and diabetes medicines
Weight loss by lifestyle ± pharmacological intervention	4	I, II, III	31–373	8–100	Liver enzymes steatosis^c,^d, NAS^d, inflammation^d		Fibrosis^d	191, 328, 332
Weight loss by bariatric surgery	3	III, IV	7–381	25–42^a	Liver enzymes, NAS^d, steatosis^d, inflammation^d, hepatocyte ballooning^d	?Fibrosis^d	?Fibrosis^d,^e	337–339
Exercise	6	I, II, III	19–813	25–100^a	Liver enzymes, steatosis^c	NASH^d, fibrosis^d		1, 19, 265, 347–349
Metformin	4	I, II, III	51–671	0–100	?Hepatocyte-ballooning^d,^f	Liver enzymes, ?steatosis^d, ?hepatocyte-ballooning^d	Steatosis^c, ?steatosis^d, inflammation^d, fibrosis^d, NAS^d	1, 358, 361, 387
Thiazolidinediones	4	I, II	55–862	0–100^k	Liver enzymes, steatosis^c,^d, inflammation^d, hepatocellular ballooning^d	?Fibrosis^d	?Fibrosis^d	1, 366–368
Incretins^b	2	I, IV	8 and 3900	100 and NR		Liver enzymes fibrosis^d, NAS^d		379, 380
Nonhypoglycemic pharmacotherapies
Statins^b	2	II	16 and 455	44 and 7	Steatosis^c,^g		Liver enzymes, steatosis^d,^h, inflammation^d,^g, hepatocyte-ballooning^d,^g, fibrosis^d,^g	391, 394
Ezetimibe^b	2	IV	10 and 45	50 and 22	Liver enzymes, steatosis^d, NAS^d, hepatocellular ballooning^d, inflammation^d	?Fibrosis^d, serological markers of fibrosis	?Fibrosis^d	400, 401
PPAR-α agonists^b	2	II, IV	16 and 46	38 and NR	Liver enzymes		Steatosis^d, inflammation^d, hepatocellular ballooning^d, fibrosis^d	403, 405
Angiotensin receptor blockers^b	2	II	54 and 137	NR and 17	Liver enzymes, steatosis^d, hepatocellular ballooning^d,ⁱ, inflammation^d, ⁱ, fibrosis^d,ⁱ			358, 407
Vitamin E	2	II	247 and 173	0	Liver enzymes^j, steatosis^d,^j, inflammation^d,^j, NASH diagnosis^d		Fibrosis^d	361, 367

Intervention	No. of studies	Level of evidence	Participants per study range (n)	% with diabetes, range in studies	Improved	Outcomes possibly improved	Unchanged or worsened	Refs.
Lifestyle intervention and diabetes medicines
Weight loss by lifestyle ± pharmacological intervention	4	I, II, III	31–373	8–100	Liver enzymes steatosis^c,^d, NAS^d, inflammation^d		Fibrosis^d	191, 328, 332
Weight loss by bariatric surgery	3	III, IV	7–381	25–42^a	Liver enzymes, NAS^d, steatosis^d, inflammation^d, hepatocyte ballooning^d	?Fibrosis^d	?Fibrosis^d,^e	337–339
Exercise	6	I, II, III	19–813	25–100^a	Liver enzymes, steatosis^c	NASH^d, fibrosis^d		1, 19, 265, 347–349
Metformin	4	I, II, III	51–671	0–100	?Hepatocyte-ballooning^d,^f	Liver enzymes, ?steatosis^d, ?hepatocyte-ballooning^d	Steatosis^c, ?steatosis^d, inflammation^d, fibrosis^d, NAS^d	1, 358, 361, 387
Thiazolidinediones	4	I, II	55–862	0–100^k	Liver enzymes, steatosis^c,^d, inflammation^d, hepatocellular ballooning^d	?Fibrosis^d	?Fibrosis^d	1, 366–368
Incretins^b	2	I, IV	8 and 3900	100 and NR		Liver enzymes fibrosis^d, NAS^d		379, 380
Nonhypoglycemic pharmacotherapies
Statins^b	2	II	16 and 455	44 and 7	Steatosis^c,^g		Liver enzymes, steatosis^d,^h, inflammation^d,^g, hepatocyte-ballooning^d,^g, fibrosis^d,^g	391, 394
Ezetimibe^b	2	IV	10 and 45	50 and 22	Liver enzymes, steatosis^d, NAS^d, hepatocellular ballooning^d, inflammation^d	?Fibrosis^d, serological markers of fibrosis	?Fibrosis^d	400, 401
PPAR-α agonists^b	2	II, IV	16 and 46	38 and NR	Liver enzymes		Steatosis^d, inflammation^d, hepatocellular ballooning^d, fibrosis^d	403, 405
Angiotensin receptor blockers^b	2	II	54 and 137	NR and 17	Liver enzymes, steatosis^d, hepatocellular ballooning^d,ⁱ, inflammation^d, ⁱ, fibrosis^d,ⁱ			358, 407
Vitamin E	2	II	247 and 173	0	Liver enzymes^j, steatosis^d,^j, inflammation^d,^j, NASH diagnosis^d		Fibrosis^d	361, 367

NR, Not reported; ?, possibly.

^a

Data not available for all studies.

^b

Histological evidence is very limited, and findings should be interpreted with caution.

^c

Measured by noninvasive assessment such as by ultrasound, CT, MRI, or MRS.

^d

On histology.

^e

Mild worsening of fibrosis was seen in the first 12 months of the study by Mathurin et al. (340), with stable disease at 5 yr (although > 95% of study participants had a fibrosis score of <1 at 5 yr).

^f

Pediatric study.

^g

When atorvastatin 20 mg was combined with vitamin E 1000 IU and vitamin C 1 g/d for an average of 3.6 yr.

^h

More patients with diabetes were allocated to the treatment group in this study with the potential to bias results to the negative.

ⁱ

Findings with telmisartan only.

^j

Findings in adults only.

^k

Belfort et al. (368) report that all patients in the study had either diabetes or IGT, but exact numbers of each group were not specified by authors.

Table 5.

Summary of studies targeting NAFLD and its subgroups, with a focus on those including subjects with diabetes

Intervention	No. of studies	Level of evidence	Participants per study range (n)	% with diabetes, range in studies	Improved	Outcomes possibly improved	Unchanged or worsened	Refs.
Lifestyle intervention and diabetes medicines
Weight loss by lifestyle ± pharmacological intervention	4	I, II, III	31–373	8–100	Liver enzymes steatosis^c,^d, NAS^d, inflammation^d		Fibrosis^d	191, 328, 332
Weight loss by bariatric surgery	3	III, IV	7–381	25–42^a	Liver enzymes, NAS^d, steatosis^d, inflammation^d, hepatocyte ballooning^d	?Fibrosis^d	?Fibrosis^d,^e	337–339
Exercise	6	I, II, III	19–813	25–100^a	Liver enzymes, steatosis^c	NASH^d, fibrosis^d		1, 19, 265, 347–349
Metformin	4	I, II, III	51–671	0–100	?Hepatocyte-ballooning^d,^f	Liver enzymes, ?steatosis^d, ?hepatocyte-ballooning^d	Steatosis^c, ?steatosis^d, inflammation^d, fibrosis^d, NAS^d	1, 358, 361, 387
Thiazolidinediones	4	I, II	55–862	0–100^k	Liver enzymes, steatosis^c,^d, inflammation^d, hepatocellular ballooning^d	?Fibrosis^d	?Fibrosis^d	1, 366–368
Incretins^b	2	I, IV	8 and 3900	100 and NR		Liver enzymes fibrosis^d, NAS^d		379, 380
Nonhypoglycemic pharmacotherapies
Statins^b	2	II	16 and 455	44 and 7	Steatosis^c,^g		Liver enzymes, steatosis^d,^h, inflammation^d,^g, hepatocyte-ballooning^d,^g, fibrosis^d,^g	391, 394
Ezetimibe^b	2	IV	10 and 45	50 and 22	Liver enzymes, steatosis^d, NAS^d, hepatocellular ballooning^d, inflammation^d	?Fibrosis^d, serological markers of fibrosis	?Fibrosis^d	400, 401
PPAR-α agonists^b	2	II, IV	16 and 46	38 and NR	Liver enzymes		Steatosis^d, inflammation^d, hepatocellular ballooning^d, fibrosis^d	403, 405
Angiotensin receptor blockers^b	2	II	54 and 137	NR and 17	Liver enzymes, steatosis^d, hepatocellular ballooning^d,ⁱ, inflammation^d, ⁱ, fibrosis^d,ⁱ			358, 407
Vitamin E	2	II	247 and 173	0	Liver enzymes^j, steatosis^d,^j, inflammation^d,^j, NASH diagnosis^d		Fibrosis^d	361, 367

Intervention	No. of studies	Level of evidence	Participants per study range (n)	% with diabetes, range in studies	Improved	Outcomes possibly improved	Unchanged or worsened	Refs.
Lifestyle intervention and diabetes medicines
Weight loss by lifestyle ± pharmacological intervention	4	I, II, III	31–373	8–100	Liver enzymes steatosis^c,^d, NAS^d, inflammation^d		Fibrosis^d	191, 328, 332
Weight loss by bariatric surgery	3	III, IV	7–381	25–42^a	Liver enzymes, NAS^d, steatosis^d, inflammation^d, hepatocyte ballooning^d	?Fibrosis^d	?Fibrosis^d,^e	337–339
Exercise	6	I, II, III	19–813	25–100^a	Liver enzymes, steatosis^c	NASH^d, fibrosis^d		1, 19, 265, 347–349
Metformin	4	I, II, III	51–671	0–100	?Hepatocyte-ballooning^d,^f	Liver enzymes, ?steatosis^d, ?hepatocyte-ballooning^d	Steatosis^c, ?steatosis^d, inflammation^d, fibrosis^d, NAS^d	1, 358, 361, 387
Thiazolidinediones	4	I, II	55–862	0–100^k	Liver enzymes, steatosis^c,^d, inflammation^d, hepatocellular ballooning^d	?Fibrosis^d	?Fibrosis^d	1, 366–368
Incretins^b	2	I, IV	8 and 3900	100 and NR		Liver enzymes fibrosis^d, NAS^d		379, 380
Nonhypoglycemic pharmacotherapies
Statins^b	2	II	16 and 455	44 and 7	Steatosis^c,^g		Liver enzymes, steatosis^d,^h, inflammation^d,^g, hepatocyte-ballooning^d,^g, fibrosis^d,^g	391, 394
Ezetimibe^b	2	IV	10 and 45	50 and 22	Liver enzymes, steatosis^d, NAS^d, hepatocellular ballooning^d, inflammation^d	?Fibrosis^d, serological markers of fibrosis	?Fibrosis^d	400, 401
PPAR-α agonists^b	2	II, IV	16 and 46	38 and NR	Liver enzymes		Steatosis^d, inflammation^d, hepatocellular ballooning^d, fibrosis^d	403, 405
Angiotensin receptor blockers^b	2	II	54 and 137	NR and 17	Liver enzymes, steatosis^d, hepatocellular ballooning^d,ⁱ, inflammation^d, ⁱ, fibrosis^d,ⁱ			358, 407
Vitamin E	2	II	247 and 173	0	Liver enzymes^j, steatosis^d,^j, inflammation^d,^j, NASH diagnosis^d		Fibrosis^d	361, 367

NR, Not reported; ?, possibly.

^a

Data not available for all studies.

^b

Histological evidence is very limited, and findings should be interpreted with caution.

^c

Measured by noninvasive assessment such as by ultrasound, CT, MRI, or MRS.

^d

On histology.

^e

Mild worsening of fibrosis was seen in the first 12 months of the study by Mathurin et al. (340), with stable disease at 5 yr (although > 95% of study participants had a fibrosis score of <1 at 5 yr).

^f

Pediatric study.

^g

When atorvastatin 20 mg was combined with vitamin E 1000 IU and vitamin C 1 g/d for an average of 3.6 yr.

^h

More patients with diabetes were allocated to the treatment group in this study with the potential to bias results to the negative.

ⁱ

Findings with telmisartan only.

^j

Findings in adults only.

^k

Belfort et al. (368) report that all patients in the study had either diabetes or IGT, but exact numbers of each group were not specified by authors.

A. Tailoring traditional diabetes management to the patient with NASH

1. Lifestyle intervention: weight loss

As with type 2 diabetes, the most effective treatment, and therefore the mainstay of management in patients with NASH who are overweight, is directed at weight loss and exercise. Effective and sustained weight loss has been associated with marked improvement in liver enzymes and stable or improved liver histology over time (328–330). A recent systematic review and meta-analysis of randomized trials by Musso et al. (331) reported improvement of hepatic steatosis at 5% or greater weight loss by diet and/or weight-reducing pharmacological therapy, but improvement in NAS only when weight loss was at least 7%. There was no improvement in fibrosis found. In the setting of type 2 diabetes, intensive lifestyle intervention over 12 months with the combination of diet and exercise resulted in a significant reduction in hepatic steatosis, as measured by MRS, in 96 individuals (332). In addition, a recent study of 11 patients (diabetes duration < 4 yr) who were placed on a very low-calorie diet (600 kcal/d) for 8 wk had a 70 ± 5% reduction of hepatic triacylglycerol content as assessed by MR. Associated with this change were significant improvements in glycemic control, hepatic insulin sensitivity, and β-cell function. Pancreatic fat content was also noted to decrease (333).

2. Bariatric surgery

Despite good evidence that sustained weight loss is associated with histological improvement in NAFLD populations, a target of at least 7% is achieved by less than 50% of individuals in clinical trials alone (331). Bariatric surgery is a more definitive technique to achieve significant and sustained weight loss and is currently recommended in patients with diabetes or other obesity-related complications at a BMI above 35 kg/m², who have not achieved recommended weight-loss targets with medical therapies. Data are increasing that bariatric surgery can achieve significant levels of remission of diabetes of up to 95% in populations with obesity, at least in the medium term. This is associated with an apparent immediate reduction in hepatic insulin resistance, followed by a reduction in skeletal muscle intramyocellular fat and insulin resistance (72, 334). A recent position statement by the International Diabetes Federation has recommended that bariatric surgery should also be considered as a treatment option in patients with type 2 diabetes and a BMI of 30–35 kg/m², especially in the presence of other major cardiovascular disease risk factors (335), and there is increasing evidence for benefit in this setting (336). NAFLD could therefore be considered as an indication for bariatric surgery (330) and certainly should influence the decision-making process in a patient with type 2 diabetes, particularly at lower cutoff levels of BMI between 30 and 35 kg/m². There are no RCTs examining bariatric surgery as a treatment for NASH, and therefore no formal recommendations for treatment have been made to date (337). Many small studies of bariatric surgery in patients with NAFLD and obesity have shown an improvement in histology, with a regression in fibrosis and inflammation in some over time (338–340). In addition, data demonstrating a decrease in the hepatic factors involved in regulating liver inflammation and fibrosis support the concept of an expected regression of NASH over a longer period of time (341–343). Interestingly, a significant decrease in fibrosis (and by definition reversal of cirrhosis) and inflammation was seen on serial liver biopsy in a small series of 11 patients with cryptogenic cirrhosis presumed secondary to NASH who underwent biliopancreatic diversion (344). Because cirrhosis may be a relative contraindication to surgery, a liver biopsy and, if appropriate, assessment for complications of cirrhosis such as portal hypertension, should be considered before performing any bariatric procedure (345). NASH per se was not associated with an increased rate of complications after bariatric surgery in a prospective cohort of 437 patients from the United States (346).

3. Lifestyle intervention: exercise

Exercise is a recommended treatment in NAFLD and in type 2 diabetes (347, 348). However, the exact nature, intensity, and duration of exercise required to be effective to ameliorate NAFLD is not yet clear. From recent studies, it appears that intense exercise and resistance training may provide the most benefit, especially when distributed over the week. In a retrospective analysis of data from 813 individuals with biopsy-proven NAFLD who enrolled in the NASH Clinical Research Network, using a self-reported time spent in physical activity, it was found that meeting vigorous [≥75 min/wk at metabolic equivalent (MET) of ≥6] but not moderate (≥150 min/wk of MET 3–5.9) intensity exercise, as per recommendations by the U.S. Department of Health and Human Services and the U.S. Department of Agriculture, was associated with a decreased adjusted odds of having NASH (OR, 0.65; 95% CI, 0.43–0.98) (265). Doubling the recommended time spent in vigorous exercise (i.e., ≥150 min/wk at MET of ≥6) was associated with a decreased adjusted odds of advanced fibrosis (OR, 0.53; 95% CI, 0.29–0.97). Total duration of exercise per week was not associated with NASH stage or fibrosis (265).

In an attempt to find more palatable options for exercise that will be beneficial in NAFLD, a small prospective study of 21 sedentary adults who had clinically defined NAFLD were assigned to 8 wk of resistance training exercise (n = 11) or standard care and demonstrated a 13% relative reduction in liver lipid as measured by MRS in the resistance training group compared with controls (349). Despite a sizeable relative difference in liver fat reduction between the groups, the absolute change of intrahepatic lipid was only 2%. The authors conceded that this reduction in liver fat was quite modest when compared with studies that have demonstrated an approximate 10% reduction after 8-kg weight loss from caloric restriction (349). A further prospective study involving 35 patients with NAFLD (as defined on ultrasound and by elevated serum ALT levels), who were resistant to standard lifestyle intervention, reported favorable results from a 12-wk intervention involving hybrid training, or co-contraction of agonist and antagonist muscles, for 19 min, twice per week. They found significant changes in steatosis grade on ultrasound examination and serum ALT levels in the intervention group when compared with controls; however, this finding was likely to have been affected by a significant reduction in body weight in the intervention group (−0.56 ± 0.60 compared with 2.39 ± 0.56 kg). There were also significant changes in abdominal circumference and body fat in the hybrid training group (350).

To examine the mechanism by which exercise may exert its effects on NAFLD, a group from Yale University studied a small (n = 12) group of healthy, young, lean, insulin-resistant individuals in a crossover trial. They looked at the response to a carbohydrate-rich meal, before and 45 min after exercise on a cross trainer, and found that exercise resulted in a greater than 3-fold increase in muscle glycogen synthesis and an approximate 40% reduction in net hepatic triglyceride synthesis. These changes were also associated with an approximate 30% decrease in hepatic de novo lipogenesis. The authors proposed that exercise may exert its effects on NAFLD by affecting the distribution and fate of ingested carbohydrate and, specifically, by diverting ingested carbohydrate away from the liver and into the muscle by reducing skeletal muscle insulin resistance. Presumably, by increasing carbohydrate uptake into muscle, there would be a reduction in hepatic de novo lipogenesis and hepatic triglyceride synthesis (351).

4. Targeting hyperglycemia with antidiabetic medications

a. Insulin.

Although there is little direct evidence that improving glycemic control in patients with diabetes will improve liver histology, some preclinical data are supportive of this concept (6). In addition, a small clinical study from Japan in which 39 patients with NAFLD and diabetes underwent paired liver biopsy after a median follow-up time of 2.4 yr found that a decrease in HbA1c and the use of insulin were associated with improvement of liver fibrosis independent of age, sex, and BMI. HbA1c was more strongly associated with the improvement in liver fibrosis than the use of insulin after adjustment for each other (χ²; 7.97 vs. 4.58, respectively) (352), suggesting that improved blood glucose may be at least a marker, if not a mechanism, by which the fibrosis in NASH is lessened.

Although some data have supported an association between insulin use in type 2 diabetes and increased risk of HCC and other cancers (353, 354), the studies have significant methodological limitations such that the many confounders, including treatment duration, medication history, and indication for therapy, are inadequately addressed (355, 356).

b. Insulin sensitizers.

Diabetes-specific treatments that have the most data for efficacy in NASH are the insulin sensitizers, including metformin and the PPAR-γ agonists, the thiazolidinediones (TZDs).

Metformin.

Available data indicate improvement in insulin resistance and perhaps liver enzymes with metformin use, but improvement in histology is inconsistent (345, 357–360). The TONIC trial, a randomized, double-blind, double-dummy, placebo-controlled clinical trial with paired liver biopsy over 96 wk that was designed to evaluate whether metformin (1 g/d) or vitamin E (800 IU/d) is an effective treatment in biopsy-proven NAFLD in 173 children (age, 8–17 yr) without diabetes, demonstrated significant improvement in hepatocellular ballooning only in the metformin group compared with placebo (−0.3 vs. 0.1; P = 0.04). There were no significant differences between metformin and placebo when examining other histological features or the primary outcome of sustained improvement in ALT over time. A possible factor affecting this outcome was the relatively low dose of metformin (361).

Despite disappointing results on NAFLD itself, metformin use may be beneficial in minimizing the increased risk of HCC. Metformin has been shown to inhibit cancer cell growth in vitro and in vivo (362). Furthermore, it has been associated with a reduced risk of HCC in patients with type 2 diabetes. Case-control studies have shown that the OR of developing HCC in patients with diabetes treated with metformin reduces to 0.3 when compared with those without this therapy (363, 364). In a retrospective cohort study previously mentioned, using population-based representative claims data from the Taiwan National Health Insurance Database, including 19,349 patients with newly diagnosed type 2 diabetes and 77,396 control patients without diabetes enrolled between 2000 and 2005, Lai et al. (102) found that the use of metformin for a median duration of 2.1 yr was associated with a 51% risk reduction for the development of HCC. A longer duration of metformin therapy appeared to provide more risk reduction in this study (102).

Thiazolidinediones.

Pioglitazone is the most extensively studied and most clinically relevant PPAR-γ agonist in RCT in NASH (365). From available data, histological improvement is significant, with clear reductions in steatosis and inflammation, but only minor effects on fibrosis apparent on paired biopsy. The current findings in regard to fibrosis are hampered by inadequately sized cohorts, including individuals with relatively mild fibrosis, studied for inadequate periods of time, such that the effects of pioglitazone still need further study in NAFLD.

In patients without diabetes, whereas a small study by Aithal et al. (366) did show an improvement in fibrosis when compared with placebo (P = 0.05) after 12 months of pioglitazone 30 mg/d, the largest RCT using pioglitazone, the PIVENS study, failed to show a statistically significant difference in fibrosis grading when compared with placebo (367). A meta-analysis by Musso et al. (331) found no effect of TZDs on fibrosis stage; however, the authors did find that there was a reduced risk of fibrosis progression in those with worsening fibrosis stage. In the setting of diabetes, Belfort et al. (368) recruited 55 patients with IGT or diabetes and performed a small randomized, placebo-controlled trial looking at the effects of pioglitazone 45 mg/d and a hypocaloric diet vs. hypocaloric diet alone over 6 months. They found significant decreases in liver enzymes and liver steatosis and inflammation when compared with placebo. The difference in fibrosis stage was not statistically significant compared with placebo in this study (P = 0.08); however, there was a significant reduction in fibrosis stage in the pioglitazone group when compared with baseline (P = 0.002) (368).

It is apparent from current literature that liver histology has tended to return to pretreatment levels after cessation of TZDs (369, 370), mandating long-term commitment to therapy. As with metformin, the use of pioglitazone in patients with diabetes and NAFLD may have uses outside of its effects on liver histology. For example, pioglitazone use was associated with lower risk of death, myocardial infarction, and stroke in a meta-analysis of studies involving patients with type 2 diabetes (371). Weight gain is a common problem in TZD use, with a mean gain of 2% occurring in up to 75% of patients, and a minority will experience much greater gain (331). However, notably, whereas overall fat increases with TZD use, visceral fat does not change or it decreases, leading to a redistribution of fat stores (372) and an associated improvement in the metabolic profile. Peripheral edema and cardiac failure are well recognized adverse effects of TZD therapy in some patients (371, 373), and long-term therapy with TZDs may also have other significant negative effects on health because increased cardiovascular disease risk has been associated with rosiglitazone (374, 375) and increased bladder cancer risk has been associated with pioglitazone use (376). Problems with osteoporosis and bone fragility and exacerbation of any macular edema present are also of concern (370, 377).

c. Incretin-based therapy.

Newer antidiabetic agents, the incretin mimetics and DPP-IV inhibitors, by acting to increase gut-derived proteins including glucagon-like peptide-1 (GLP-1), have been shown to improve diabetes control by potentiating meal-induced insulin production and by slowing gastric emptying and inhibiting glucagon secretion. Of particular note, these agents have been able to maintain weight neutrality in the case of DPP-IV inhibitors or weight loss in the case of incretin mimetics (378). There is increasing evidence that GLP-1 action is also likely to have direct metabolically desirable effects on the liver (126, 330, 379–382), including suppression of hepatic lipogenesis (383) and stimulation of lipid oxidation (384) and, albeit limited, evidence that it may improve hepatic insulin sensitivity (385, 386). At a cellular level, a G protein-coupled receptor for GLP-1 has been discovered on human hepatocytes. In addition, in vitro treatment with exendin-4, a GLP-1 agonist, directly reduced liver steatosis in human hepatocytes (384, 387). Improvements in hepatocyte insulin sensitivity have also been observed with GLP-1 agonist therapy in this setting (384). In an open-label, uncontrolled trial to assess exenatide (a synthetic analog of exendin-4) safety in subjects with diabetes over 3.5 yr, the treatment was associated with improved liver enzymes, independent of body weight changes and insulin sensitivity (330, 381). A recent meta-analysis of GLP-1 agonists in populations with and without diabetes, including data on liver enzyme tests from 12 of the 25 trials included, found that ALT concentrations decreased after treatment with liraglutide (−2.2 U/liter; −3.6 to −0.9) but not with exenatide (0.7 U/liter; −1.1 to 2.4) on subgroup analysis (379). Evidence that DPP-IV inhibitors confer a liver protective effect is emerging in preclinical studies, and further liver studies with incretin mimetics are reporting similar findings (388). Human trials with DPP-IV inhibitors are under way (330). Although data are promising, enthusiasm for these therapies should be tempered by a lack of long-term data and, in particular, details of long-term side effects (390).

5. Lipid-lowering therapy

a. Statins.

Given the increased risk of macrovascular disease in patients with NASH or diabetes, it is paramount to address any modifiable traditional macrovascular risk factors such as smoking, hyperlipidemia, and hypertension in a cohort of patients with coexisting diabetes and NASH. Lipid lowering of LDL-cholesterol by statin (hydroxymethylglutaryl coenzyme A reductase inhibitor) therapy has obvious therapeutic benefits in addressing cardiovascular risk but also has theoretical benefits in NASH through antiinflammatory, immunomodulatory, and antioxidant properties. Small studies of hyperlipidemic patients with NAFLD demonstrate improvement in liver enzymes and a decrease in liver steatosis on histology (391–393). However, in a small double-blind RCT (n = 16) looking at effects of simvastatin on a small group of biopsy-proven NASH over 12 months, there was no statistically significant change in liver enzymes, hepatic steatosis, necroinflammatory activity, or stage of fibrosis despite a 26% reduction in LDL in the intervention group (394). Thus, current data support the use of statins for risk factor modification rather than primary treatment of NASH per se. Adverse effects of statins on the liver are exceedingly rare and should not be a reason to avoid this therapy in a statin-naive patient with NAFLD (395, 396).

b. Ezetimibe.

Ezetimibe is another lipid-lowering therapy that works by inhibiting Niemann-Pick C1 like 1-dependent cholesterol transport at the brush border of the small intestine. It has been shown to improve liver steatosis and insulin resistance in an obese rat model of metabolic syndrome (397, 398). In humans, Niemann-Pick C1 like 1 mRNA expression is present in both the small intestine and the liver. Ezetimibe may therefore be an effective treatment in NASH by traditional mechanisms and also via specific pathways, such as by the inhibition of biliary cholesterol absorption (399). In two small clinical trials, ezetimibe at a dose of 10 mg/d has been shown to reduce liver enzymes, high-sensitivity C-reactive protein, and type IV collagen (as a surrogate marker of fibrosis), and to improve the NAS on liver biopsy (largely attributable to a decrease in steatosis). In the study by Yoneda et al. (400), there was a nonsignificant decrease in fibrosis stage, but no participants in the study had advanced fibrosis (stage 3–4) at baseline (400, 401).

c. PPAR-α agonists.

Lipid modulation through the use of PPAR-α agonists also has theoretical benefit in the treatment of NAFLD/NASH through their influence on the mitochondrial and peroxisomal fatty acid β-oxidation pathways. PPAR-α has also been implicated in the negative regulation of inflammatory responses (137). Supplementation of η-3 polyunsaturated fatty acid, a PPAR-α ligand, to rodent models of NAFLD has led to decreases in liver steatosis, transaminases, and liver inflammation and fibrosis (402). In humans, reduced transaminases and steatosis have been observed with this treatment (137, 403). Limited small studies in humans with NAFLD using fenofibrate, gemfibrozil, or benzofibrate have shown improvements in biochemistry and imaging (404). However, a study with clofibrate failed to show histological improvement after 12 months of therapy (404, 405).

6. Addressing hypertension

As with lipid-lowering therapy, tailoring medications to treat hypertension has theoretical advantages in patients with NASH and diabetes. In particular, modulation of the RAS has received particular attention in recent NAFLD literature. It has previously been shown that chronic liver injury up-regulates the local tissue RAS, contributing to the recruitment of inflammatory cells and the development of fibrosis (406, 407). Angiotensin II affects insulin signaling and promotes inflammation, endothelial dysfunction, and fibrosis, with direct activation of the HSC (327, 389, 407). In studies of NASH in rats, use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, and in particular telmisartan with the theoretical benefit of concomitant PPAR-γ activity, has led to improved liver histology and markers of fibrosis (406, 408). Human studies of angiotensin receptor blockers and NASH are few and are limited by small numbers. In a small pilot study involving 54 patients with biopsy-proven NASH and systemic hypertension, telmisartan was compared with valsartan. Improvements were greater in plasma lipids, insulin sensitivity and liver steatosis, necroinflammation, and fibrosis in the telmisartan group at 20 months (327, 330, 409). A recent randomized, open-labeled, prospective clinical trial involving 137 patients with biopsy-proven NASH found no benefit of combination therapy with rosiglitazone (4 mg twice daily) plus losartan (50 mg once daily) or rosiglitazone (4 mg twice daily) plus metformin (500 mg twice daily) over rosiglitazone (4 mg twice daily) alone, although doses for metformin and losartan may have been inadequate, as may have been the duration of observation (358).

Modulation of the renin angiotensin aldosterone system may also be achieved with mineralocorticoid receptor antagonists, such as spironolactone. The beneficial effects of an 8-wk treatment of spironolactone in a mouse model with diet-induced diabetes and NAFLD have been reported (410). Early data from a small single-center RCT, including 10 patients treated with vitamin E (400 IU) vs. 10 patients treated with spironolactone (25 mg) plus vitamin E (400 IU) in biopsy-proven NAFLD, have shown a favorable effect on HOMA-IR with the addition of spironolactone in patients with NAFLD. Larger clinical trials are needed to provide definitive data (279).

B. Other agents with potential benefit in the patient with NASH and diabetes

1. Antioxidants

Antioxidants, including vitamins E and C, silymarin (milk thistle), and betaine, have been studied in NAFLD with evidence of limited benefit. Vitamin E has the most significant data supporting its use and is currently the only accepted treatment in pediatric NAFLD. The PIVENS and TONIC trials have been the most recent RCTs to show benefit with vitamin E. The PIVENS study involved 247 individuals with NASH, but without diabetes or cirrhosis, and treatment with pioglitazone 30 mg/d, vitamin E 800 IU/d, or placebo for 96 wk. The primary outcome was histological improvement in the features of NASH, with a P value < 0.025 considered statistically significant given the two planned primary comparisons. Vitamin E therapy, as compared with placebo, was associated with a significantly higher rate of improvement in the composite NAS score (43% compared with 19%; P = 0.001), whereas on the basis of the statistical significance preestablished, pioglitazone was not (34% compared with 19%, respectively; P = 0.04). As compared with placebo, both vitamin E and pioglitazone treatment improved the scores of steatosis, inflammation, ballooning, and serum ALT to a similar extent, but fibrosis did not significantly improve in either group (367). Combination of these two agents may be useful, with a small pilot study suggesting that pioglitazone and vitamin E may be better than pioglitazone alone (345). The TONIC study demonstrated significant improvement in resolution of NASH, hepatocellular ballooning, and NAS in the vitamin E-treated children when compared with placebo but, unlike PIVENS, was unable to demonstrate improvements in liver steatosis or lobular inflammation (361).

Despite the positive findings associated with the use of vitamin E, because treatment is likely to be needed long term, some caution must be applied given the results of several meta-analyses suggesting a small but significant increase in risk of all-cause mortality with supplementation, particularly at doses greater than 400 IU/d (343, 411–413). Given current evidence, vitamin E (α-tocopherol) has been recommended at a dose of 800 IU/d in patients with active NASH (NAS ≥ 4), without diabetes. There is a need for validation of vitamin E as an effective treatment of NASH in patients with diabetes, with close attention paid to long-term safety data (331, 345).

2. Ursodeoxycholic acid (UDCA)

UDCA has theoretical benefits in NASH through its antiapoptotic properties, by decreasing TNF-α levels and perhaps by reducing ER stress and increasing insulin sensitivity. Disappointingly, three placebo-controlled RCTs with UDCA have demonstrated mixed but largely negative results (345, 414–416). A subsequent review has suggested the possible benefit of UDCA plus vitamin E and also the examination of non-UDCA bile acids that may regulate metabolism via the farnesoid X receptor (FXR), a nuclear receptor involved in sensing bile acids. FXR is thought to be a key regulator of lipid and carbohydrate metabolism (137, 417, 418).

3. Novel agents

There have been limited trials of other potential agents in NAFLD. Data in the rat and also small studies in the human suggest that modification of the intestinal flora with probiotics may be a useful adjunct in patients with NAFLD (330). Modulation of the endocannabinoid receptor has been found to affect de novo lipogenesis in the liver and promote steatosis (138). Rimonabant, a cannabinoid 1 receptor antagonist, received attention after it was shown to induce weight loss and improve insulin resistance in clinical trials, but it has recently been withdrawn from the market due to psychiatric complications, primarily depression (138). Studies in animal models and early human studies are proceeding with agents that can modify insulin sensitivity and lipid metabolism via PKC isoform modulation, including via FXR regulation (137, 345). Attention is also being given to agents that can modify cytokines, adipokines, or growth factors such as pentoxifylline, a TNF-α antagonist, and antifibrotic agents that target the hedgehog pathway, thought to mediate hepatocyte apoptosis and regeneration (330, 331, 345, 419). In addition, PPAR-δ agonists, perhaps by increased fatty acid metabolism or by decreased inflammation (mediated via alternative activation of tissue macrophages), have been shown to be protective against diet-induced steatohepatitis in mice fed a methacholine-deficient diet and have limited data in humans suggesting that they are safe (420).

Recent in vitro work examining the benefits of curcumin (the yellow pigment from turmeric) on HSCs in the setting of hyperglycemia is notable. In vitro, curcumin appears to reduce the biphasic intracellular increase in glucose that occurs in HSCs as a result of hyperglycemia, first by blocking membrane translocation of GLUT2 by interrupting p38 MAPK signaling, and then by suppressing gene expression of GLUT2 by stimulating PPAR-γ activation and by attenuating oxidative stress. These changes have led to a reduction in HSC activation and proliferation in vitro that is otherwise seen with the induction of hyperglycemia. The challenge for in vivo studies will be achieving adequate concentrations of curcumin because it has very poor bioavailability (242).

Other agents of interest that have not yet been studied in NAFLD trials but may be of interest due to their effects in diabetes and/or on lipid metabolism include sodium-glucose-linked transport inhibitors as glycosurics, advanced glycation end-product inhibitors, cholesterol ester transfer protein inhibitors, and 11β-hydroxysteroid dehydrogenase type 1 inhibitors (421–424).

XII. Monitoring for Complications of Liver Disease in Patients with NASH and Type 2 Diabetes

NAFLD appears to have lower rates of liver-related complications but similar overall mortality compared with HCV, even after adjustment for potential confounders (89). In particular, HCC rates in cirrhosis related to NASH appear to be less than in cirrhosis related to other causes. From transplant data in the United States, the frequency of HCC among liver transplant recipients with NASH as a primary or secondary indication was 12%, compared with 19% for other indications (P < 0.001). Despite this, patients with cirrhosis secondary to NASH are clearly at risk for development of HCC (56), and diabetes may exacerbate this risk (102). HCC is twice as likely to develop in a patient with type 2 diabetes as in a person with no diabetes (425).

Liver disease is the third leading cause of death in NASH, and HCC represents the main cause of death in this group. In addition, HCC can occur in NASH without cirrhosis. In a cross-sectional Japanese study of 87 patients with HCC secondary to NASH, 59% of whom had diabetes, 11% had stage 1 fibrosis, 17% had stage 2 fibrosis, and 21% had stage 3 fibrosis. Only 51% of the patients with HCC had cirrhosis, or stage 4 fibrosis (40). These findings were replicated in a European study of HCC and chronic liver disease, with 41.7% of HCC secondary to NASH occurring in patients without cirrhosis. This was in contrast to alcoholic liver disease, where 95% of HCC occurred in the setting of cirrhosis in this study (426). Without specific recommendations for NASH, patients found to have advanced fibrosis or cirrhosis should have an ultrasound and an α-fetoprotein measurement every 6–12 months (425). Follow-up for patients with lesser degrees of NASH, with or without fibrosis is less clear, although it may involve regular noninvasive assessment for fibrosis progression.

XIII. Final Comments

End-stage NAFLD, including NASH-related cirrhosis and HCC, is currently the third leading indication for liver transplant worldwide and will likely become the leading indication in the next one to two decades (56, 330). In addition, the sheer and increasing magnitude of the combined problem of NAFLD and diabetes mandates that it should be better recognized by clinicians and also given a high priority for study. Multiple contemporary studies have been able to shed light on possible risk factors for NAFLD/NASH and particularly why diabetes may lead to increased rates and progression of this condition. Despite this, the exact pathogenic mechanisms that link the metabolic milieu that exists in diabetes with NAFLD are far from clear, but substantial improvements in our research tools and in animal models for both NAFLD/NASH and diabetes are helping to advance understanding.

Given the high level of clinical success of bariatric surgery in achieving sustained weight loss and in inducing remission in obese people with type 2 diabetes, RCTs are required to substantiate the degree of change in NASH after particular types of bariatric surgery and to aid in the setting of guidelines for bariatric surgery in people with diabetes and NASH. From a lifestyle perspective, increased focus on exercise type, duration, and intensity is also required to define regimens that will best improve NAFLD and, specifically, NASH.

From available data, it is also clear that the coexistence of NAFLD and diabetes is likely to require a customized pharmacological management, above that used in either condition alone. The identification of effective strategies for both of these conditions is expected to occur as new diabetes-targeted therapies such as the glucosuric agents, AGE inhibitors, and incretin mimetics and DPP-IV inhibitors, in addition to other potentially effective agents, are studied in animal trials with both NASH and diabetes. This knowledge will help pave the way toward much needed RCTs in humans. Clinical trials must be conducted with ample power and continued for adequate duration to allow the demonstration of significant differences in treatment and placebo arms, particularly in relation to fibrosis. This will be increasingly facilitated by the development of accurate noninvasive assessment for initial staging of disease burden and also for monitoring disease progression. It is likely that noninvasive assessment will consist of well-validated algorithms that involve blood-specific markers and also measurements of liver stiffness by imaging technologies. It is also envisioned that, over the course of time, such noninvasive assessments will form part of routine clinical practice, helping to exclude those who require liver-specific investigation and to identify those requiring diagnostic liver biopsy and intensified therapy.

Acknowledgments

We thank Professor James Kench, Head of Tissue Pathology and Diagnostic Oncology Department, Royal Prince Alfred Hospital, for his review of some of the pathology images (shown in Fig. 1).

This work was supported by National Health and Medical Research Council (NHMRC) of Australia Project Grant 632822 on diabetes and NAFLD and a NHMRC Postgraduate Research Scholarship (to K.H.W.).

Disclosure Summary: All authors have nothing to declare.

Abbreviations

AGE
Advanced glycosylation end-products

ALT
alanine aminotransferase

ApoC3
apolipoprotein C3

ARFI
acoustic radiation force impulse

AST
aspartate aminotransferase

AUROC
area under the receiver operating characteristic curve

BMI
body mass index

CI
confidence interval

CKD
chronic kidney disease

CT
computed tomography

CTGF
connective tissue growth factor

DAG
diacylglycerol

DPP-IV
dipeptidyl peptidase-IV

eGFR
estimated glomerular filtration rate

ELF
European liver fibrosis (panel)

ER
endoplasmic reticulum

F
fibrosis stage

FFA
free fatty acid

FXR
farnesoid X receptor

GLP-1
glucagon-like peptide-1

GLUT
glucose transporter

HbA1c
glycosylated hemoglobin

HCC
hepatocellular carcinoma

HCV
hepatitis C virus

HOMA-IR
homeostasis model of assessment for insulin resistance

HR
hazard ratio

HSC
hepatic stellate cell

IFG
impaired fasting glucose

IGT
impaired glucose tolerance

IRS
insulin receptor substrate

LDL
low-density lipoprotein

LPS
lipopolysaccharide

LV
left ventricular

M
medium (probe for FibroScan)

MET
metabolic equivalent

MR
magnetic resonance

MRE
MR elastography

MRI
MR imaging

MRS
MR spectroscopy

MTP
microsomal triglyceride transfer protein

NAFLD
nonalcoholic fatty liver disease

NAS
NAFLD activity score

NASH
nonalcoholic steatohepatitis

NF-κB
nuclear factor κB

NFS
NAFLD fibrosis score

OGTT
oral glucose tolerance test

OR
odds ratio

PIIINP
procollagen-3 N-terminal peptide

PKC
protein kinase C

PNPLA3
adiponutrin/patatin-like phospholipase domain-containing 3 (gene)

PPAR
peroxisome proliferator-activated receptor

RAS
renin-angiotensin system

RCT
randomized controlled trial

ROS
reactive oxygen species

SOD2
superoxide dismutase 2

SREBP
sterol regulatory element-binding protein

TIMP-1
tissue inhibitor of metalloproteinase-1

TLR
toll-like receptor

TZD
thiazolidinedione

UDCA
ursodeoxycholic acid

XL
extra large (probe for FibroScan).

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