Effects of Huangjiu, Baijiu and Red Wine Combined With High-Fat Diet on Glucose and Lipid Metabolism: Aggravate or Alleviate?

Abstract

Aim

To compare effects on certain health indices in rodents of different doses of alcoholic beverages, huangjiu (Chinese yellow wine), red wine and baijiu (Chinese liquor) combined with high-fat diet (HFD) and the pure HFD.

Methods

A total of 80 rats were randomly divided into eight groups and treated with (a) basal diet (3.5 kcal/g); (b) HFD (19.5% w/w lard, 4.5 kcal/g) and (c) HFD with low or high doses of separate alcoholic beverages (2.5 and 5 g/kg ethanol, respectively) for 28 weeks.

Results

Chronic drinking when combined with HFD was associated with reduced body weight, fat accumulation and serum TNF-α level, serum TG, TC and LDL-C levels, and improved glucose tolerance (OGTT) and insulin sensitivity (ITT), hepatic enzymes; elevated levels or activities of the antioxidant enzymes like superoxide dismutase, catalase and glutathione reductase, reduced the content of lipid peroxidation productions such as malondialdehyde, in comparison with the pure HFD intake. In addition, compared with HFD, drinking plus HFD improved microbiota dysbiosis, down-regulated the ratio of Firmicutes/Bacteroidetes and promoted the growth of some probiotics including Prevotellaceae_UCG-001 and norank_f__Bacteroidales_S24-7_group.

Conclusion

Overall, the three beverages showed different impacts on indicators but red wine showed the most ‘beneficial’ effects. Of course, higher ethanol dosages can be expected to cause overall negative health effects, and harms of high fat intake can be prevented by healthier diet.

INTRODUCTION

High-fat diets (HFDs) are becoming more common in many countries. More than 600 million people have a daily HFD, and there are already 1.9 billion overweight adults worldwide (Sandouk and Lansang, 2017). Excessive intake of fat can lead to disorder of lipid metabolism, glycometabolism and liver function, increase the generation of reactive oxygen species (ROS) and break the oxidative balance, inducing hyperlipidemia, and diseases such as obesity, atherosclerosis, myocardial infarction, diabetes and some types of cancer (Wires et al., 2017, Ribeiro et al., 2018). Intestinal flora plays an important role in digestion, fermentation and metabolism, which are closely related to overall health (Nash et al., 2018). HFD can affect the composition and quantity of intestinal microbiota by changing the redox state and destroying the microenvironment of the intestine, resulting in decreased community diversity with probiotics decreasing and harmful bacteria increasing (Murphy et al., 2015, Zhang et al., 2019).

Some 2.3 billion people worldwide drinking alcohol daily (Global status report on alcohol and health 2018). Alcohol causes hepatitis, cirrhosis, various cancers, pancreatic damage, heart disease and mitochondrial damage directly or indirectly (Rocco et al., 2014, Cai et al., 2014, Yuan et al., 2015). Heavy drinking appears to induce intestinal microbiota disorders, resulting in ecological imbalance and excessive growth of intestinal flora (Voutilainen and Kärkkäinen, 2019; Bishehsari et al., 2017), releasing endotoxins to the blood and finally induced hepatic inflammation (Engen et al., 2015). However, some studies find moderate alcohol consumption can increase the abundance of Prevotella and Bacteroides (Queipo-Ortuño et al., 2012), which may help regulate glucose and lipid metabolism (Moschen et al., 2012). Furthermore, moderate alcohol consumption has been associated with lower risk of coronary heart disease (Camargo et al., 1997), total mortality (Solomon et al., 2000, Diem et al., 2003) and risk of type 2 diabetes and vascular disease (Wannamethee et al., 2003, Artero et al., 2015), although a causal role is debated.

Baijiu is a traditional Chinese beverage distilled from fermented grain and consists of various bioactive components with physiological effects (Sun et al., 2015, Zhao, Sun, et al., 2017a). Chinese yellow wine (huangjiu), an ancient beverage, has been claimed to have health-promoting effects due to its antioxidant activity (Que et al., 2006, Guo et al., 2007). Red wine, now widely consumed around the world, is also known for its functional components and bioactivities (Lingua et al., 2016, Li and Sun, 2019).

So we ask here whether alcohol combined with HFD consumption will aggravate or alleviate the effects of HFD on the health, with or without a dose effect, and we compare those three alcoholic beverages. We study glucose and lipid metabolism in rats for up to 28 weeks, measuring body weight, fat accumulation, blood lipids, blood sugar, liver function, serum cytokines and intestinal microbiota.

MATERIALS AND METHODS

Chemicals and reagents

Niulanshan Erguotou Chinese liquor (56% vol) was purchased from Beijing Shunxin Agriculture Co., Ltd. Niulanshan Winery (Beijing, China). Great Wall dry red wine (13% vol) was purchased from China Great Wall Wine Co., Ltd (Hebei, China). Huadiao Chinese yellow wine (15% vol)—the 3 years of Shaoxing wine was purchased from Shaoxing Nverhong Wine Co., Ltd (Zhejiang, China). Edible alcohol was purchased from Meihekou City Fukang Alcohol Co., Ltd (Jilin, China). Commercial assay kits of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate transferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were obtained from Dirui Medical Technology Co., Ltd. (Jilin, China). Assay kits of insulin, adiponectin, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and free fatty acid (FFA) were supplied by Beijing Huaying Biotechnology Research Institute (Beijing, China). Superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and malondialdehyde (MDA) kits were provided by Biyuntian Biotechnology Research Institute (Shanghai, China). Ethanol, chloroform, hydrochloric acid and other reagents were of analytical grade and were offered by Beijing Reagent Factory (Beijing, China). Glucose was furnished by Henan Zhongtai Food Co., Ltd. (Henan, China). Short-acting insulin injection was bought from Jiangsu Wanbang Biochemical Pharmaceutical Co., Ltd. (Jiangsu, China).

Preparation of alcohol beverages

The ethanol content of huangjiu and red wine was determined and then concentrated by the multistage flasher. After that, the ethanol content of the concentrated huangjiu and red wine was determined again, and the edible alcohol was backfilled to replenish the lost ethanol to 45% vol. As for baijiu, the ethanol content was determined and directly diluted to 45% vol.

Animals and diets

The protocol was approved by the Animal Ethics Committee of the Beijing Key Laboratory of Functional Food from Plant Resources (Permit A330–2018-1) and strictly conducted in accordance with the National Institute of Health guidelines for animal care. Eighty male Sprague–Dawley rats (180–220 g) were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China) [Certificate SCXK (Beijing) 2016–0011] and housed in Laboratory Animal Center, College of Food Science and Nutritional Engineering, China Agricultural University, kept at 23 ± 2°C and relative humidity 45 ± 5% with good ventilation and 12 h light/darkness cycle. After 1 week of adaptive feeding, the rats were randomly divided into eight groups (n = 10 each): control group with basal diet (C), high-fat group with high-fat diet (HF), high-fat diet with low-dose huangjiu (2.5 g/kg ethanol) group (HFLH), high-fat diet with high-dose huangjiu (5 g/kg ethanol) group (HFHH), high-fat diet with low-dose red wine (2.5 g/kg ethanol) group (HFLW), high-fat diet with high-dose red wine (5 g/kg ethanol) group (HFHW), high-fat diet with low-dose baijiu (2.5 g/kg ethanol) group (HFLB), high-fat diet with high-dose baijiu (5 g/kg ethanol) group (HFHB). The amount of ethanol in low-dose and high-dose groups was equivalent to about 2 and 4 standard drinks (1 drink = 12 g ethanol) in humans, respectively. Rats in control group were fed with normal diet; rats in HF and drinking groups were fed with HFD. The composition of the basal diet and high-fat diet is shown in Table 1. Rats in each group were given free access to water or diet. Body weight and food consumption were measured once a week and daily, respectively. The experiment was continued for 28 weeks.

Organ coefficient

At the end of the study, liver, epididymal, perirenal and abdominal fat were removed and weighed immediately after rats being sacrificed. Organ weights were normalized to body weight as organ coefficient. The tissues used for histological analysis were determined as follows and all the tissues were stored at −80°C for further analysis.

Serum biochemical profiles

After fasting for one night, the rats were narcotized and the blood samples were taken from orbital venous. The blood sample was incubated at 4°C for 24 h and then centrifuged for 15 min at 4000 g, 4°C to obtain serum. TG, TC, HDL-C, LDL-C, AST, ALT and ALP were measured by an automatic analyzer followed instructions of the corresponding commercial assay kits. The liver lipids were extracted after homogenizing and hepatic TG, TC and FFA levels were determined in the same way as the serum lipid profiles.

Table 1

Composition of experimental diets

Nutrient	Basal diet	High-fat diet
Protein (g/kg)	200.0	187.3
Fat (g/kg)	45.0	227.7
Carbohydrate (g/kg)	570.0	424.7
Crude fiber (g/kg)	37.0	27.6
Vitamin (g/kg)	2.1	1.6
Mineral (g/kg)	32.2	29.2
Ash (g/kg)	65.3	48.6
Sodium cholate (g/kg)	0.0	2.0
Total energy (kcal/g)	3.5	4.5

Serum cytokines and oxidative stress status

The concentrations of adiponectin, TNF-α, and IL-6 were measured using an ELISA kit after serum centrifugation, and the specific procedures were carried out according to the kits’ instructions. The content of SOD, CAT, GR and MDA in the liver were measured by commercial kits as the specifications, respectively.

Oral glucose, insulin tolerance and insulin resistance tests (OGTT, ITT and HOMA-IR)

For OGTT, blood glucose was determined via the blood from tail vein of fasting rats by a glucometer. Rats were fasted for 12 h, intragastrically administered with glucose solution at a dose of 2.0 g/kg bw, then the tail blood was collected at subsequent time intervals of 0, 30, 60, 90 and 120 min. For ITT, short-acting insulin (0.6 IU/kg bw) was injected intraperitoneally after fasting for 4 h, and blood tests were performed as for the OGTT. Total areas under the curve (AUC) of OGTT and ITT were calculated using a trapezoidal method. Fasting blood glucose was measured by using a glucometer via tail blood (12 h), while fasting insulin was determined by the commercial kit. HOMA-IR index was calculated as follows:

$$\begin{eqnarray*} \mathrm{HOMA}-\mathrm{IR}\;\mathrm{index} &\!\!\!\!\!=\!\!\!\!\!& \mathrm{fasting}\kern0.17em \mathrm{blood}\kern0.17em \mathrm{glucose}\kern0.17em \mathrm{level}\;\left(\mathrm{mmol}/\mathrm{L}\right)\\ &&\times \mathrm{fasting}\kern0.17em \mathrm{insulin}\kern0.17em \mathrm{level}\;\left(\mathrm{mU}/\mathrm{L}\right)/22.5. \end{eqnarray*}$$

Histological analysis

Hepatic tissue and fat tissue were placed in a 10% neutral formalin and embedded in paraffin. All the tissues were sliced and stained with hematoxylin and eosin (HE) for the measurement of cellular necrosis and degeneration, inflammatory cell infiltration, fibrosis and edema.

Intestinal microbiota

Colonic contents in rats were collected in individual sterile cryotube and stored at −80°C. The total DNA was extracted via the EZNA soil DNA extraction kit (Omega Bio-tek, Norcross, GA, USA) according to instructions, the DNA concentration and purity were determined by NanoDrop2000 spectrophotometer (Thermo Fisher, MA, USA) and the DNA extraction quality was detected by 1% agarose gel electrophoresis. The V3-V4 region of the bacterial 16S ribosomal RNA gene was subjected to PCR amplification using primers 338F (5′-ACTCCTACGGGAGGCAGC-AG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′).

The second-generation Illumina MiSeq sequencing platform along with multivariate statistical methods were used to detect the diversity of the V3-V4 region of the bacterial 16S rRNA gene. Related library construction and the Miseq high-throughput sequencing process were performed by Majorbio (Shanghai, China). Operational taxonomic units (OTUs) were clustered at 97% similarity, and analyses of α-diversity, community composition and PCoA were performed.

Statistical analysis

All diagrams were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). The results were expressed as mean ± standard deviation (Mean ± SD). Statistical comparisons were performed with a one-way analysis of analysis of variance test (ANOVA) and post hoc Student–Newman–Keuls analyses by SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Significant difference was considered at P < 0.05.

RESULTS

Effects of three kinds of alcoholic beverages combined with HFD on body weight, intake of food and energy

Compared with the control group, HF increased body weights, particularly after 7 weeks (Fig. 1A). Alcohol moderated weight gain induced by HFD: high-dose drinking more than low-dose drinking groups. From the result of final weight (Table 2), HFHB group significantly decreased by 29.9% when compared with the HF, while the weight of HFHW group decreased by 27.6% (P < 0.05), and there was no significant difference compared with the control group. Followed by the HFLW, HFHH, HFLH and HFLB, all drinking combined with HFD groups showed a decrease in body weight compared with HF group.

Fig. 1.

Effect of three kinds of alcoholic beverages combined with high-fat diet on body weight in rats during experimental period and blood lipids.

(A) Body weight; (B) serum total triglyceride level; (C) serum total cholesterol level; (D) serum high-density lipoprotein cholesterol level; (E) serum low-density lipoprotein cholesterol level. Values are expressed as the mean ± SD (n ≥ 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

Table 2

Effect of three kinds of alcoholic beverages on food intake, energy intake, initial weight and final weight in rats combined with HFD

Groups	Intake of food (g/d)	Intake of energy (kcal/d)	Initial weight (g)	Final weight (g)
C	25.12 ± 1.69e	87.54 ± 5.90a	238.19 ± 8.91a	618.47 ± 42.29a
HF	24.05 ± 1.97d	108.18 ± 8.85d	237.72 ± 8.39a	837.10 ± 73.68c
HFLH	19.10 ± 2.27b	95.33 ± 9.79b	238.22 ± 11.49a	658.42 ± 56.17ab
HFHH	17.26 ± 3.42a	95.87 ± 15.12b	238.94 ± 9.33a	644.52 ± 45.63ab
HFLW	20.25 ± 2.46c	100.63 ± 11.02c	238.51 ± 7.84a	611.77 ± 58.56a
HFHW	17.82 ± 3.65a	97.09 ± 16.63b	238.03 ± 8.24a	606.44 ± 34.88a
HFLB	20.25 ± 2.54c	100.52 ± 11.87c	243.10 ± 9.25a	702.27 ± 60.04b
HFHB	17.42 ± 3.46a	94.48 ± 17.12b	237.98 ± 8.24a	586.71 ± 53.80a

Values are expressed as the mean ± SD (n ≥ 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

The intake of food and energy in each group was also recorded in this study (Table 2). Rats eating HFD significantly decreased food intake but significantly increased the intake of energy. All drinking groups significantly reduced the intake of food and energy compared with HF groups, whereas high-dose drinking groups showed lower food intake compared with low-dose drinking groups. The results indicated that drinking combined with HFD could reduce the weight gain caused by the pure high-fat diet, which may be related to reduced energy intake. But energy intake seemed not the only factor, as the energy intake of the high-dose drinking groups (HFHW and HFHB) was higher than that of the control group, whereas the body weight was lower.

Effects of three kinds of alcoholic beverages combined with HFD on coefficient of liver and fat

Compared with the control group, the liver coefficient in HF was significantly increased (P < 0.05, Table 3). However, all the drinking combined with HFD groups showed a decrease trend in liver coefficient compared with HF group, and no significant difference was found between drinking groups and the control group. In addition, high-dose drinking could to some extend increase the liver coefficient compared with low-dose drinking. The coefficient of perirenal fat, epididymal fat and abdominal fat in HF was enhanced compared to the control group (P < 0.05). The fat coefficient of each drinking group showed a downward trend compared with HF, but it showed an upward trend compared with the control group. Among them, the visceral fat accumulation in the low-dose drinking group was higher than that in the corresponding high-dose drinking group. In addition, HFLH showed the highest total visceral fat coefficient, whereas HFHW had the lowest total visceral fat coefficient.

Table 3

Effect of three kinds of alcoholic beverages combined with HFD on coefficient of liver and fat

Groups	Liver coefficient (g/100 g bw)	Perirenal fat coefficient (g/100 g bw)	Epididymal fat coefficient (g/100 g bw)	Abdominal fat coefficient (g/100 g bw)	Total visceral fat coefficient (g/100 g bw)
C	2.21 ± 0.14a	0.56 ± 0.11a	1.92 ± 0.24a	1.77 ± 0.33a	4.68 ± 0.59a
HF	2.45 ± 0.22b	0.99 ± 0.17d	3.20 ± 0.57d	5.72 ± 1.05e	10.15 ± 0.96e
HFLH	2.21 ± 0.12a	0.78 ± 0.07c	2.87 ± 0.12cd	3.59 ± 0.52d	7.33 ± 0.69d
HFHH	2.31 ± 0.14ab	0.74 ± 0.14bc	2.33 ± 0.45ab	3.27 ± 0.51cd	6.64 ± 0.98cd
HFLW	2.20 ± 0.15a	0.83 ± 0.12cd	2.47 ± 0.36bc	3.10 ± 0.64bcd	5.94 ± 0.78bc
HFHW	2.29 ± 0.10ab	0.56 ± 0.08a	2.45 ± 0.20bc	2.39 ± 0.26ab	5.33 ± 0.50ab
HFLB	2.23 ± 0.09a	0.86 ± 0.11cd	2.77 ± 0.24bcd	2.78 ± 0.39bc	6.64 ± 0.10cd
HFHB	2.25 ± 0.08a	0.61 ± 0.05ab	2.62 ± 0.24bc	2.79 ± 0.50bc	5.89 ± 0.42bc

Values are expressed as the mean ± SD (n ≥ 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

Effects of three kinds of alcoholic beverages combined with HFD on blood lipids

As shown in Fig. 1B–E, after 28 weeks of HFD, serum TG, TC and LDL-C were significantly elevated compared with the control group, which indicated that long-term intake of HFD could induce the occurrence of hyperlipidemia. Interestingly, drinking combined with HFD could to some extent induce a decreasing trend in serum TG, TC and LDL-C, an increasing trend in HDL-C compared with HF, which meant drinking combined with HFD may alleviate the dyslipidemia caused by HFD. In addition, high-dose drinking groups showed higher HDL-C and LDL-C content and lower level of serum TG. Among the three kinds of alcoholic beverages, red wine showed more effective on improving lipid disorders.

Effects of three kinds of alcoholic beverages combined with HFD on blood glucose

After 28-week treatment for HFD, glucose metabolism disorders occurred in these rats (Fig. 2). In detail, HFD significantly increased the level of fasting blood sugar, fasting insulin, AUC of OGTT, AUC of ITT and HOMA-IR compared with the control, which indicated that continuous intake of HFD induced insulin resistance, resulting in decreased ability of self-regulation of insulin and peripheral insulin sensitivity. Drinking combined with HFD significantly decreased the fasting blood glucose level and the AUC of OGTT also induced a downward trend of fasting insulin, AUC of ITT and HOMA-IR. The results above indicated that drinking combined with HFD could alleviate the glucometabolic disorder caused by the pure HFD. In addition, high dose of drinking especially high dose of baijiu intake could stimulate the release of insulin, and even induced higher HOMA-IR, but the results of OGTT and ITT showed that no insulin resistance was found among the high-dose drinking groups.

Fig. 2.

Effect of three kinds of alcoholic beverages combined with high-fat diet on blood glucose.

(A) Concentration of fasting blood glucose; (B) area under curve (AUC) value of oral glucose tolerance test; (C) area under curve (AUC) value of insulin tolerance test; (D) content of fasting insulin; (E) homeostasis model assessment–insulin resistance index. Values are expressed as the mean ± SD (n ≥ 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

Effects of three kinds of alcoholic beverages combined with HFD on liver function

HFD significantly increased the activity of ALT, AST and ALP, which indicated that long-term intake of HFD might induce hepatic damage (Fig. 3A–C). Among drinking groups, huangjiu, red wine and baijiu combined with HFD showed a trend of decreasing the activity of AST compared with HFD. Low-dose red wine and baijiu combined with HFD also reduced activities of ALT and ALP compared with HFD, whereas high-dose drinking did not, indicating that low-dose drinking of red wine and baijiu could to some extent alleviate hepatic injury compared with the pure HFD’s effect.

Fig. 3.

Effect of three kinds of alcoholic beverages combined with high-fat diet on liver function and the flake of hematoxylin and eosin (HE) staining of liver tissue in rats.

(A) Serum alanine transferase activity; (B) serum aspartate transferase activity; (C) serum alkaline phosphatase activity; (D) HE staining of liver. Values are expressed as the mean ± SD (n ≥ 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

Effects of three kinds of alcoholic beverages combined with HFD on hepatic lipids and antioxidant capacity

In comparison with the control, HFD intake significantly reduced the activities of SOD, CAT and GR in the liver, and increased the hepatic content of MDA, indicating that HFD decreased the hepatic antioxidant capacity and induced lipid peroxidation in the liver (Fig. 4A–D). Alcohol was associated with a trend towards increasing the activity of SOD, with high-dose drinking groups showing greater trend than the low-dose drinking groups, meanwhile HFHW and HFHB significantly elevated the activity of SOD compared with HF. Drinking combined with HFD increased CAT activity compared with HF except for HFHB, and low-dose drinking was more effective. Drinking significantly increased GR activity compared with HF, and HFLW and HFHB had the better effect. All three kinds of alcoholic beverages combined with HFD significantly reduce the MDA content when compared with HF, and red wine was associated with the greatest reduction. When compared with the control group, no difference of liver MDA content was found in huangjiu or red wine groups, but in the baijiu groups, it was increased.

Fig. 4.

Effect of three kinds of alcoholic beverages combined with high-fat diet on hepatic lipids and antioxidant capacity.

(A) Hepatic superoxide dismutase activity; (B) hepatic catalase activity; (C) hepatic glutathione reductase activity; (D) hepatic malondialdehyde content; (E) hepatic total triglyceride content; (F) hepatic total cholesterol content; (G) hepatic free fatty acid content. Values are expressed as the mean ± SD (n ≥ 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

After 28 weeks of HFD, hepatic levels of TG, TC and FFA were significantly increased compared with the control (Fig. 4E–G). In comparison with HF, hepatic TG and TC content in HFHW were significantly decreased, and all drinking groups showed down-trend in reducing the content of hepatic FFA. High dose of red wine intake exhibited better effect on inhibiting the hepatic accumulation of TG and TC, while high dose of baijiu showed greatest effect in reducing the hepatic level of FFA.

Effects of three kinds of alcoholic beverages combined with HFD on serum cytokines

Serum levels of IL-6, TNF-α and adiponectin were measured in this study and results are shown in Fig. 5A–C. After 28-week treatment of HFD, serum content of IL-6 and adiponectin was significantly decreased and content of TNF-α was significantly increased compared with the control. Drinking combined with HFD was associated with raised IL-6 level, while reducing the release of TNF-α and adiponectin compared with HF. However, low-dose of red wine showed the greatest effect on increasing the content of adiponectin.

Fig. 5.

Effect of three kinds of alcoholic beverages combined with high-fat diet on serum cytokines and the flake of hematoxylin and eosin (HE) staining of epididymal adipose tissue in rats.

(A) Serum interleukin-6 content; (B) serum tumor necrosis-α content; (C) serum adiponectin content; (D) HE staining of epididymal adipose tissue. Values are expressed as the mean ± SD (n ≥ 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

Effects of three kinds of alcoholic beverages combined with HFD on pathological morphology of liver and fat cell

Histopathological changes of the liver in rats were observed in HE staining and the results are shown in Fig. 3D. There was no steatosis or edema in liver of the control group, and the structure of the hepatic lobule was normal; no fibrosis was found in the portal vein and no necrosis in liver tissues. In HF, there was diffuse steatosis and swelling degeneration of hepatocytes, dilatation and perivascular fibrosis of portal vein along with fat vacuoles. The degree of hepatic steatosis was reduced compared with HF with all varieties of alcoholic beverages. In comparison with HF, HFHW and HFHB reduced the degree of hepatocyte edema, whereas huangjiu groups and HFLW increased it. The intake of red wine and baijiu inhibited the fragmentation and necrosis of hepatocytes compared with HF, and the effect in high-dose groups was more than in low-dose groups. However, in huangjiu groups, fragmentation and necrosis of hepatocytes was greater.

The area of adipocytes in HF was increased compared with control (Fig. 5D). All alcoholic beverages were associated with limiting the increase of rat adipocytes, with red wine showing the greatest effect. In addition, long-term intake of HFD caused inflammatory cell infiltration in most of the rats’ adipose tissue. Drinking increased inflammatory infiltration together with HFD, but there were no differences in the effects of different varieties of beverages.

Effects of three kinds of alcoholic beverages combined with HFD on gut microbiota

As shown in Fig. 6A, compared with the control, the amount of OTUs was significantly decreased after 28 weeks of HFD. However, drinking combined with HFD increased the amount of OTUs in comparison with HF, and high-dose drinking showed higher OTUs amount. In alpha diversity analysis, HF showed a decreasing tendency in richness (Chao1) and variety (Shannon and Simpson) of microbiota community (Fig. 6B–D). However, drinking combined with HFD was associated with some elevation of microbial richness and diversity in comparison with HF. Furthermore, as the dose of alcohol increased, the richness and diversity of intestinal microbial increased. Among the three beverages, high dose of huangjiu and baijiu showed more effective on microbial diversity (Simpson). Unweighted UniFrac-based PCoA revealed beta diversity of microbiota community (Fig. 6E), demonstrating that the intestinal flora in control, HF and drinking groups were different.

Fig. 6.

Effect of three kinds of alcoholic beverages combined with high-fat diet on alpha and beta diversity of gut microbiota.

(A–D) Alpha diversity was evaluated by (A) observed OTUs; (B) Chao1 index; (C) Shannon index and (D) Simpson index. (E) Plots of unweighted UniFrac-based PCoA. Values are expressed as the mean ± SD (n = 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

At the phylum level, HFD significantly elevated Firmicutes/ Bacteroidetes (F/B) ratio compared with control (Fig. 7); however, alcohol was associated with lower F/B ratio compared with HF. Among the three beverages, low-dose groups were associated more effect on the ratio of F/B than high-dose groups. At the family level, HF exhibited a reduction in levels of Bacteroidales_S24_7_group, Rikenellaceae, Prevotellaceae, Ruminococcaceae and Peptostreptococcaceae compared with control, along with remarkable augmentation in levels of Lachnospiraceae and Desulfovibrionaceae (Fig. 8). Drinking combined with HFD changed the abundance of all the families above with different extent compared with HF. A heat map was made to manifest the composition of column in different groups at genus level. The top 50 different bacterial abundances were shown with hierarchical clustering, and the shade of color indicated relative abundance (Fig. 9A). HF showed up-regulation in relative abundance of Lactobacillus, unclassified_f_Lachnospiraceae, Roseburia and Lachnospiraceae_NK4A136_group, together with a decrease in norank_f_bacteroidales_S24_7_group, prevotella-1 and prevotellaceae_UCG-001. However, the abundance of these bacteria was up- or down-regulated in the alcohol/HFD groups and was similar to the control (i.e. now high fat) group (Fig. 9B–F). The same result was obtained according to the heat map. As for Lactobacillus, Roseburia and Lachnospiraceae_NK4A136_group, HF has the darkest color, while as for norank_f_bacteroidales_S24_7_group and prevotellaceae_UCG-001, HF has the lightest color.

Fig. 7.

Effect of three kinds of alcoholic beverages combined with high-fat diet on the composition of gut microbiota at phylum level.

(A) Bacterial profile at phylum level; (B) ratio of Firmicutes/Bacteroidetes. Values are expressed as the mean ± SD (n = 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

Fig. 8.

Effect of three kinds of alcoholic beverages combined with high-fat diet on the composition of gut microbiota at family level.

(A) Bacterial profile at family level; (B) relative abundance of Bacteroidales_S24-7_group; (C) relative abundance of Desulfovibrionaceae; (D) relative abundance of Lachnospiraceae; (E) relative abundance of Prevotellaceae. Values are expressed as the mean ± SD (n = 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

Fig. 9.

Effect of three kinds of alcoholic beverages combined with HFD on the composition of gut microbiota at genus level.

(A) Heatmap analysis on the OTU level; (B) relative abundance of Lactobacillus; (C) relative abundance of Roseburia; (D) relative abundance of Lachnospiraceae_NK4A136_group; (E) relative abundance of Prevotellaceae_UCG-001; (F) relative abundance of norank_f__Bacteroidales_S24-7_group. Values are expressed as the mean ± SD (n = 6). Mean values without a common letter are significantly different (P < 0.05). C, control; HF, high fat; HFLH, high fat and low dose of huangjiu; HFHH, high fat and high dose of huangjiu; HFLW, high fat and low dose of wine; HFHW, high fat and high dose of wine; HFLB, high fat and low dose of baijiu; HFHB, high fat and high dose of baijiu.

DISCUSSION

We assessed many variables in this study and have not made any statistical correction for the large number of tests conducted. However, the sum of evidence we believe shows important findings about long-term high-fat diet (HFD) induced obesity in rats in whom energy intake exceeded expense, excess energy getting stored in adipocytes in the form of TG, which increased in volume and eventually caused fat accumulation and obesity (Kim et al., 2015). Adipose tissue sectioning also showed that HFD caused inflammatory cell infiltration. Fat cells not only store extra energy but also secrete cytokines such as adiponectin, IL-6 and TNF-α, which are related to the regulation of energy metabolism, inflammation and insulin resistance (Popko et al., 2010, Farias et al., 2019). We found that fat accumulation in rats in HF was increased compared with control, and serum adiponectin content was decreased, in agreement with Kubota et al.(2007) who observed that adiponectin was available to enhance food intake and reduce energy consumption, but the secretion of adiponectin generally decreased with the increase of fat capacity. In addition, long-term intake of HFD increased the release of TNF-α, which could further inhibit the secretion of adiponectin and resulting in insulin resistance (Whitehead et al., 2006). Results of OGTT and ITT showed that HFD impaired the ability of insulin self-regulation and reduced peripheral insulin sensitivity, causing disorder of glucose metabolism consistent with previous studies (Riant et al., 2009).

We found that alcoholic beverages combined with HFD reduced body weight, intake of energy and visceral fat coefficient compared with pure HFD intake. In addition, it was found that high-dose drinking was associated more with controlling body weight and abdominal fat deposition than low-dose drinking. The results of pathological section revealed that the area of adipocytes in drinking groups was significantly lower than that in HF, and rats in high-dose drinking groups had smaller fat cells compared with low-dose drinking. These results above were consistent with the trend of body weight and fat coefficient and in agreement with the report of Nelson’s animal experiment and Addolorato’s human test (Addolorato et al., 1998, Nelson et al., 2016). It is concluded that the reasons why drinking controlled weight gain and fat accumulation in this experiment were as follows: firstly, the rats that had excessive alcohol intake in drinking group, especially in high-dose drinking groups, were in a state of drowsiness for a long time, decreasing the intake of food and energy. Secondly, long-term drinking may inhibit the appetite of rats. Thirdly, absorptive and digestive function of rats may be damaged because of much alcohol consumption (Addolorato et al., 1997). Fourthly, chronic alcohol exposure may stimulate adipose tissue lipolysis (Zhong et al., 2012). Huangjiu, red wine and baijiu impacted differently on body weight and fat accumulation combined with HFD, and red wine was associated more strongly with reduced weight gain and fat accumulation, which may be related to the polyphenols contained in the red wine. Studies have found that resveratrol could reduce weight gain and lipid accumulation induced by HFD and can interfere with fat absorption by activating the AMP-activated protein kinase (AMPK) pathway (Dasgupta and Milbrandt, 2007, Cantó et al., 2009). This may explain the two phenomena that appear in this experiment: firstly, there was no significant difference in energy intake between HFLB and HFLW, but the weight of rats in HFLB was significantly higher than HFLW; secondly, the energy intake of rats with huangjiu was significantly lower than that with red wine, oppositely, the visceral fat coefficient of rats in huangjiu groups was significantly higher.

The impact of drinking on blood lipid is controversial. Results displayed that alcoholic beverages combined with HFD decreased the levels of serum TC, TG and LDL-C, whereas increased the level of HDL-C compared with HFD intake alone, indicating that drinking could improve the lipid metabolism disorder induced by HFD. The reason may be that the intake of food and energy in drinking combined with HFD groups are lower than those in HF. In addition, it may be because drinking promoted the transport of blood lipids to the liver and stimulated hepatic lipid accumulation (higher hepatic TG and TC levels in drinking combined with HFD than HF, Fig. 4) (Dasgupta and Milbrandt, 2007). Drinking combined with HFD decreased the level of LDL-C compared with pure HFD, which might lower the risk of cardiovascular diseases caused by HFD. This result was consistent with Yuan’s study, which claimed that moderate ethanol consumption alleviated high-fat diet–induced cardiac contractile, intracellular Ca²⁺ anomalies and mitochondrial injury (Yuan et al., 2015). The effects of huangjiu, red wine and baijiu combined with HFD on lipid metabolism in rats were discrepant. In general, red wine was associated with greater reduction of serum TG, TC, LDL-C level and hepatic TG, TC and FFA content than huangjiu and baijiu. It was noteworthy that although huangjiu was rich in bioactive ingredients, it still caused a decrease in serum HDL-C level in rats and an increased trend in hepatic TG and TC content. The explanation may be that huangjiu production introduces some harmful substances such as urethane, formaldehyde, biogenic amine and aflatoxin (Hugenholtz, 2016). Besides, huangjiu used in this study was semi-dry, which indicated that sugar in huangjiu could to some extent affected the lipid metabolism in rats.

Drinking combined with HFD reduced fasting blood glucose, enhancing glucose tolerance and peripheral insulin sensitivity compared with pure HFD group (Fig. 2). Note that HFHB significantly promoted the secretion of insulin and elevated HOMA-IR; however, no insulin resistance was obtained in HFHB from the results of OGTT and ITT. Interestingly, the effect of alcohol consumption on glycometabolism remains controversial. Some studies indicated that heavy alcohol drinking enhanced the risk for type 2 diabetes (T2D), thereby damaging glucose homeostasis and inducing insulin resistance (Kim et al., 2013). In addition, chronic ethanol exposure could be reducing insulin sensitivity and glucose tolerance through down-regulating glucose transporter 4 expression via an AMPK-dependent pathway in adipocytes (Feng et al., 2010). However, some research showed that long-term intake of alcohol seemed to be associated with improved glycemic control in T2D, and the reason may be due to improved insulin sensitivity (Pietraszek et al., 2010). Previous research also suggested that the effect of drinking on the increase of fasting blood glucose may be through the influence of signaling pathway in adipose tissue and the inhibitory effect on gluconeogenesis (Mokuda et al., 2004). According to the results of this study, drinking increased insulin sensitivity and glucose tolerance; the underlying mechanism should be further clarified.

Chronic consumption of alcohol is known to elevate activities of ALT and AST in serum, along with hepatic oxidative stress such as lipid peroxidation in liver (higher levels of MDA) and decreased contents or activities of antioxidant enzyme (SOD, CAT and GR), even worse after combined with HFD (Giriwono et al., 2010, Zhao, Wang, et al., 2017b, Guo et al., 2015). Nevertheless, the present study found that the effect of drinking on liver function was related to the amount and variety of alcoholic beverages consumed. ALT and AST are the most important indicators for evaluating liver damage, ALT is mainly distributed in the cytoplasm (Glinghammar, 2009) and AST is present in cytoplasm and mitochondria (Otto-Ślusarczyk et al., 2016). Increased activity of ALT in the serum indicates injury in the hepatocyte with cellular constituents leaking into serum, whereas elevated AST activity suggested necrosis in hepatocyte and dysfunction in mitochondria (Zhao et al., 2016). All three alcoholic beverages combined with HFD decreased the activity of AST compared with HFD, suggesting that drinking alleviated the damage of mitochondria in hepatocytes caused by long-time consumption of HFD. Drinking aggravated the release of ALT induced by HFD, although the extent of hepatic damage did not increase according to the results of histopathological section in liver. SOD, CAT and GR are key enzymes in the antioxidant system, whereas MDA is one of the lipid peroxidation products (Tsikas, 2017). The present study showed that drinking combined with HFD could alleviate hepatic oxidative stress caused by the pure intake of HFD, and red wine exhibited greater effect than huangjiu and baijiu, which may be associated with higher content of bioactive substances. Although high-dose of baijiu increased levels or activities of SOD and GR, MDA content was significantly higher than that with red wine and huangjiu, which should be further studied.

It was worth noting that HFD led to a decrease, while supplement of alcoholic beverages in the diet led to an increase, in microbial diversity, which conflicted with Peterson et al. (2017) who observed that chronic ethanol exposure caused reductions in bacterial alpha diversity. At the present study, we found that drinking modulated the gut microbiota not at the phylum level but at the family and genus level. It was found that the predominant phyla in gut microbiota were Bacteroidetes, Firmicutes, Proteobacteria and Verrucomicrobia, which coincided with the finding of Chang et al. (2015). The ratio of Firmicutes/Bacteroidetes was proved to have a connection with the degree of obesity, and we found that supplement of alcohol reversed the increase in F/B ratio caused by HFD, consistent with preceding results of weight loss in drinking groups (Koliada et al., 2017). At the family level, alcohol ingestion significantly reduced the relative abundance of the Lachnospiraceae and obviously increased the relative abundance of the Bacteroides, which was in keeping with preceding results that drinking inhibited weight gain and fat accumulation in rats (Turnbaugh et al., 2006; Zhao et al., 2016). At the genus level, red wine and huangjiu restrained the increase of relative abundance of Lactobacillus in rats compared with pure HFD, while Lactobacillus abundance in HFHB was close to HF. Thus, red wine and huangjiu were more effective on improving inflammatory response and insulin resistance; however, HFHS may induce higher risk of insulin resistance as found by Rocha-Ramírez et al. (2017).

In conclusion, long-term intake of alcoholic beverages like huangjiu, red wine and baijiu combined with HFD can reduce weight gain, fat accumulation and inflammatory cytokines release, improve glucose and lipid metabolism, alleviate hepatic function damage and oxidant stress, and regulate the composition of intestinal flora compared with pure consumption of HFD (based on metabolism parameters, oxidative parameters, hepatic function and intestinal flora). Although all conclusions derived from animal experiments, there are implications for human health albeit with advice to limit dosage of drinking to avoid alcohol’s extensive negative effects. Furthermore, avoiding a high-fat diet is, of course, also a very important health message.

Availability of data

The data underlying this article will be shared on reasonable request to the corresponding author.

Authors’ contributions

L.Z., F.Z. and B.J. designed research; L.Z., H.O. and N.Z. analyzed data; L.Z. and N.Z. performed research; L.Z. and H.O. wrote the paper; F.Z., C.W. and B.J. contributed new reagents or analytic tools and developed software necessary to perform and record experiments.

ACKNOWLEDGEMENTS

This work was funded by the National Key Research and Development Program of China (grant number 2018YFD0400403) and the China Postdoctoral Science Foundation (grant number 2019TQ0011).

Conflicts of interest

The authors declare no conflict of interest.

Author notes

Liang Zhao and Hanying Ouyang contributed equally to this work.