Impaired “Glycine”-mia in Type 2 Diabetes and Potential Mechanisms Contributing to Glucose Homeostasis

Abstract

The onset and/or progression of type 2 diabetes (T2D) can be prevented if intervention is early enough. As such, much effort has been placed on the search for indicators predictive of prediabetes and disease onset or progression. An increasing body of evidence suggests that changes in plasma glycine may be one such biomarker. Circulating glycine levels are consistently low in patients with T2D. Levels of this nonessential amino acid correlate negatively with obesity and insulin resistance. Plasma glycine correlates positively with glucose disposal, and rises with interventions such as exercise and bariatric surgery that improve glucose homeostasis. A role for glycine in the regulation of glucose, beyond being a potential biomarker, is less clear, however. Dietary glycine supplementation increases insulin, reduces systemic inflammation, and improves glucose tolerance. Emerging evidence suggests that glycine, a neurotransmitter, also acts directly on target tissues that include the endocrine pancreas and the brain via glycine receptors and as a coligand for N-methyl-d-aspartate glutamate receptors to control insulin secretion and liver glucose output, respectively. Here, we review the current evidence supporting a role for glycine in glucose homeostasis via its central and peripheral actions and changes that occur in T2D.

Several reports (1–3) support the idea that type 2 diabetes (T2D) progression can be delayed or prevented if action is initiated early. Although recent meta-analyses conclude that not enough information exists to determine whether insulin secretagogue therapy alone can prevent or delay T2D onset (4, 5), lifestyle intervention (1–3), and early intervention with metformin (6) appear effective, at least over the short term (4, 5). Consequently, much research has focused on T2D prevention and early biomarker detection of prediabetes (7, 8). Some putative circulating biomarker metabolites of present interest include 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (9, 10), which may directly impair insulin secretion (9) and can predict T2D up to 5 years before diagnosis (11); elevation of branched chain amino acids (BCAAs) (12, 13), which may play a causative role in insulin resistance (14); and circulating glycine (Fig. 1), a drop in which may be an early indicator of T2D (15–19).

Figure 1.

Putative actions of glycine in glucose homeostasis. Many metabolic effects of circulating glycine, and glycine supplementation, may impact glucose tolerance. The strongest evidence currently available suggests effects in the brain via dorsal vagal complex (DVC) NMDA receptors, systemically by reducing oxidative stress and dampening inflammatory responses, and in the islet to increase insulin secretion via GlyRs.

A number of recent studies have applied high throughput metabolomics technologies to compare metabolite profiles found in healthy participants against those diagnosed with various diseases including cancer, T2D, cardiovascular disease, and obesity (20–22). Cross-sectional studies have identified BCAAs and aromatic amino acids, sugar metabolites (glucose and fructose), and lipids (phospholipids and triglycerides) to be positively associated with insulin resistance and T2D in humans (15, 23). Of all the metabolites examined, relatively few appear negatively associated with T2D risk (23). Glycine, a “nonessential” amino acid that can be synthesized in the body from serine, is consistently and negatively associated with T2D (15–19, 23–26). Participants identified as nondiabetic insulin resistant or having impaired glucose tolerance are found to have reduced circulating glycine (15, 16), as are the nondiabetic children of parents with T2D (27). A similar trend in plasma glycine concentrations is found in obesity. Overweight and obese individuals have lower circulating levels of glycine compared with lean controls (13, 24, 28–30). Finally, in prospective studies ranging between 1 and 19 years, lower glycine concentrations are negatively associated with insulin resistance and are predictive of T2D (17, 18, 23, 31–33). Individuals in the top (fourth) quartile of glycine concentrations have a 1.5-fold lower risk of T2D compared with those in the first quartile (independent of several other risk factors) (18).

Whether dropping glycine levels actively participates in T2D pathogenesis is unknown, but it is relevant to note that (1) interventions that reverse or delay T2D onset are associated with increased plasma glycine concentrations (34–38), and (2) glycine supplementation enhances insulin responses and glucose tolerance (39, 40). Bariatric surgery, a treatment option for obesity shown to reverse T2D in many cases (41–43), is associated with increased circulating glycine levels (34–37). The underlying mechanism for increased glycine is not entirely clear, although the reduction in body weight and improvement in insulin sensitivity may contribute (34). Lifestyle modification, mentioned previously as an early intervention that improves insulin sensitivity and outcomes in T2D (1), remarkably also increases circulating glycine concentrations. In overweight human participants, 6 months of aerobic exercise training increases insulin sensitivity and circulating glycine concentrations (38) and benefits were maintained even up to 15 days after exercise cessation (44). Finally, glycine supplementation is reported to increase insulin secretion, but not action, in first-degree relatives of individuals with T2D (40), improve glucose tolerance in healthy subjects (39), and possibly lower HbA1c in patients with T2D (45).

An exact mechanism for reduced circulating glycine in T2D is not entirely clear, although the precursor serine may also be decreased (19). The strongest evidence however suggests that lowered glycine may be a secondary consequence of excessive free fatty acid (FFA) and BCAA metabolism. FFA metabolism can lead to an accumulation of β-oxidation intermediates (such as acyl-CoA esters). These are conjugated with glycine via the activity of acyl-CoA:glycine-N-acyltransferase, which is responsible for the transesterification of acyl-CoA esters with glycine to produce acyl-glycines (46, 47). Acyl-glycine is membrane permeable and is readily excreted in the urine, thus serving as a mechanism for the elimination of excess β-oxidation intermediates. Similarly, BCAA catabolism byproducts also conjugate to glycine in the liver for excretion (14, 48). Therefore, circulating glycine could be consumed to facilitate excretion of waste products associated with elevated FFAs and BCAAs. Indeed, in a rat model of obesity a BCAA-restricted diet causes an increase in circulating glycine concentrations demonstrating an inverse relationship between BCAA availability and circulating glycine levels (14). The BCAA restricted diet restores acyl-glycines in the urine and the urinary acyl-glycine levels are linearly related to skeletal muscle glycine concentrations (14). These pathways have recently been reviewed (22). Regardless of the mechanism(s), it is important to note that the low circulating glycine levels in prediabetes/T2D are reflected in peripheral tissues (14), and to a lesser extent in the brain (49), where key actions may be exerted on blood glucose control.

Receptors for Glycine

Glycine is a direct agonist of glycine receptors (GlyRs) and a coagonist of N-methyl-d-aspartate (NMDA) receptors. GlyRs are famously known to mediate the action of glycine as an inhibitory neurotransmitter in the central nervous system. These are from a family of pentameric ligand-gated chloride channels (50) that consist of four α subunits (α1 to α4) and one β subunit and can exist as either homomers or heteromers. Activation of the GlyR opens the channel pore and allows negatively charged chloride to enter neurons and inhibit activity (51). Although classically known for their function in neurons and glial cells, GlyRs are expressed elsewhere in the body including the retina (52, 53), the pancreas (54, 55), and immune cells (56–58). Recent single-cell RNA sequencing data has demonstrated that not only do human islets express the GlyR, but among islet cells expression of the GlyRα1 subunit gene (GLRA1) appears highly and specifically expressed in the β-cell (59–63). Many of the studies do not report on the other GlyR subunits, but of the ones that did, GLRA3 was found in the β-cell (62), whereas GLRB is expressed either in β-cells (62) or ductal cells (63).

Aside from activating GlyRs, glycine is a coagonist for the excitatory NMDA receptor, a glutamate receptor which is named due to its selective activation by NMDA. These receptors are tetrameric and typically consist of 2 NR1 and 2 NR2 subunits (64). The NR1 subunit is a mandatory subunit for functional receptors and is responsible for binding glycine, whereas the NR2 subunit binds glutamate. NMDA receptors require two different ligands (glutamate and glycine) and removal of magnesium/zinc block before current can be produced (65). When activated, these receptors are nonselective cation channels that promote membrane depolarization and Ca²⁺ entry, and may be linked to the activation of repolarizing K⁺ channels such as the small-conductance Ca²⁺-activated K⁺ (SK) channels.

Glycine in the Brain: Evidence for NMDA Receptor Predominance in Glucose Homeostasis

Circulating glycine crosses the blood–brain barrier to only a limited extent. Glycine in the cerebrospinal fluid (CSF) is usually much lower than in circulation [a CSF/blood glycine ratio of >0.08 is diagnostic of glycine encephalopathy (66)], and extracellular glycine in the brain is kept low by the action of glycine transporters (GlyTs) that mediate glycine clearance/uptake into glycinergic neurons and glial cells (67). The degree to which reduced circulating glycine in T2D is reflected in the brain, or in central glycine signaling, is therefore unclear, although it is known that glycine supplementation is sufficient to increase brain/CSF glycine in rodents and humans (49, 68, 69).

Evidence suggesting a role for brain GlyRs as regulators of glucose homeostasis is very limited. Orexigenic neurons in the hypothalamus express GlyRs, the activation of which results in hyperpolarization and inhibition of their activity (70, 71). This can be expected to prevent release of the neuropeptide orexin, which would blunt food intake (72–74), insulin-induced glucose uptake (75, 76), and brown adipose tissue–dependent thermogenesis (77). For the most part, although the reduced food intake could contribute to reduced glycemia, most of these actions are not consistent with the known glucose-lowering effect of glycine supplementation and a direct link between brain glycine and glucose homeostasis through these mechanisms has not been demonstrated. Rather, a stronger link exists between brain glycine and activation of NMDA receptors as a mechanism for glycine’s glucose-lowering effect (69, 78).

The dorsal vagal complex (DVC) is an important region regulating hepatic glucose production and hepatic triglyceride-rich very-low-density lipoprotein (VLDL-TG) production (79). The DVC expresses the NMDA receptor and glycine has been demonstrated to potentiate the activation of these receptors to suppress hepatic glucose production (78), hepatic VLDL-TG secretion (69, 80), and food intake (81). Furthermore, the glycine-mediated lowering of glucose production and hepatic VLDL-TG is dependent on hepatic vagal innervation as hepatic vagotomy ablated the effect of central glycine. Pharmacologic inhibition of the NMDA receptor, or adeno-associated virus short hairpin RNA–mediated loss of function, clearly demonstrates that the activation of the NMDA receptor is required for the inhibition of glucose production and inhibition of very-low-density lipoprotein production by centrally administered glycine. These antidiabetic effects are independent of the activation of GlyRs as coinfusion of the GlyR-antagonist strychnine was no different from glycine infusion alone (78).

Similar to direct glycine infusion into the DVC, GlyT inhibition in the DVC by pharmacological inhibition or lentiviral/adenoviral loss of function has been shown to improve plasma glucose levels without affecting plasma insulin (81). By preventing GlyT-mediated clearance of glycine from the DVC, more glycine will be present to activate the DVC NMDA receptors. In this study, long-term GlyT inhibition prevented weight gain in a high-fat-fed rat model with effects seen as early as 4 days of treatment. Here, systemic administration of the GlyT inhibitor had a similar effect on glucose homeostasis. However, like the GlyRs and NMDA receptors, the GlyTs are widely expressed throughout the body, including in the pancreatic islets of Langerhans (55) and macrophages (82).

Glycine, GlyRs, and Inflammation

Inflammation plays an important role in the pathophysiology of T2D. Although chronic inflammation likely has a negative impact on islet function and survival, substantial controversy regarding the specific role of islet inflammation persists and work from us (83) and others (84) suggest important positive effects of inflammatory mediators on glucose tolerance in the short-term, inflammation likely makes key contributions to insulin resistance and impaired central metabolic sensing (85, 86). Furthermore, despite what might be considered modest clinical results at present, anti-inflammatory approaches remain of potential interest in the treatment of T2D (87, 88). A common feature of T2D is chronically elevated oxidative stress marked by proinflammatory factors and reactive oxygen species (ROS) (89), and a deficiency of antioxidants and ROS scavengers (90, 91). Glutathione, an endogenous antioxidant, is synthesized from glycine, cysteine, and glutamate and is consumed when scavenging ROS. A small clinical trial supplemented the substrates for glutathione synthesis (glycine and cysteine) in the diet of participants with T2D and observed an increased glutathione synthesis and plasma reduced glutathione (GSH) concentrations (92). The increased reduced-to-oxidized glutathione ratio suggests that GSH is not being trapped in the oxidized state, but rather participants with T2D have lower total GSH. Markers of plasma oxidative stress and peroxides were decreased following cysteine and glycine supplementation and effects persisted 2 weeks after the termination of the study (45, 92), suggesting an indirect effect of glycine on oxidative stress in the inflammatory state.

Independent of GSH, glycine may also exert a protective effect by directly inhibiting inflammation. Macrophages, T lymphocytes, and neutrophils all express GlyRs, the activation of which suppresses proinflammatory cytokine signaling (93). Briefly, signals such as LPS can bind TLR on macrophages causing depolarization and activation voltage-gated Ca²⁺ channels. The Ca²⁺ influx from the voltage-gated Ca²⁺ channel opening stimulates cytokine production. This may be counteracted by activation of the hyperpolarizing GlyRs. Glycine reduces tumor necrosis factor–α secretion from rat Kupffer cells (94, 95), superoxide production by rat neutrophils (57), and interleukin-1 secretion from human monocytes (96). Furthermore, there is further evidence for glycine’s repression of inflammation in adipose tissue by downregulating tumor necrosis factor–α, uncoupling protein–1, and interleukin-6 while upregulating adiponectin and PPARγ (97–99). The specific details about glycine’s anti-inflammatory actions have been extensively reviewed (93, 100–102).

Glycine and the Islet: GlyR Actions

The role for GlyRs in human islets has been somewhat controversial, although a clearer picture is emerging from recent transcriptomic and functional studies. Some have reported that glycine stimulates glucagon secretion by acting on GlyRs on α-cells (103), and indeed, glycine supplementation appears to increase plasma glucagon levels (39). Our own work shows that glycine stimulates insulin secretion by acting on GlyRs on β-cells (55). In this work, we observed robust GlyR-mediated chloride current in human β-cells that were positively identified by insulin immunostaining. Also consistent with a role for GlyRs in β-cells, as noted previously, single-cell transcriptome studies demonstrate the specificity of GlyRα1 expression in β-cells (59–63). This expression of the GlyR in β-cells may be specific to humans as we and others report difficultly detecting it on mouse and rat islets (55, 104). This may explain why knock down of GlyRs in the rat-derived INS-1 832/13 β-cell line by small-interfering RNA is ineffective at influencing insulin secretion (105). But, as mentioned previously, glycine supplementation increases insulin secretion in vivo and our recent report suggests that glycine directly depolarizes β-cells via GlyRs to increase Ca²⁺ and promote insulin secretion. How does this happen if glycine is an inhibitory neurotransmitter that activates a Cl^– current to suppress electrical activity? Shouldn’t this mechanism decrease insulin secretion?

Unlike GlyR in the nervous system, those in β-cells do not always act in an inhibitory manner (Fig. 2). This is because of an odd quirk of β-cell electrolyte balance. As in some other cell types (106), the Cl^– gradient is slightly different from neurons (107–109). Neurons have a very low intracellular chloride concentration (∼7 mM) (110), whereas the chloride concentration of β-cells is substantially higher (∼32 mM). What this means is that the driving force for Cl^– movement in neurons and islets is different. In electrophysiological terms, the reversal potential for chloride is –80 and –33 mV in neurons and β-cells, respectively. The opening of the GlyRs will therefore hyperpolarize most neurons (inhibitory) but depolarize β-cells (excitatory). Indeed, we recently observed that glycine induces a depolarization and increased Ca²⁺ response in the majority of human β-cells (55). Notably, this response is not uniform across the β-cell population, suggesting heterogeneity in ionic balance among human β-cells (55).

Figure 2.

Differential effects of glycine on membrane potential in β-cells and neurons. (a) In most neurons, the intracellular concentration of chloride ([Cl^–]_i) is very low (∼7 mM) in comparison with that in the surrounding extracellular space. The result is a relatively strong electrochemical gradient favoring the movement of negatively charged chloride into the cell when Cl^– channels (such as GlyR) are open. The result is that activation of GlyR pushes the membrane potential of the neuron toward the hyperpolarized chloride equilibrium potential of these cells (E_Cl = –80 mV). (b) Within β-cells, the [Cl^–]_i is higher (∼32 mM) resulting in a chloride equilibrium potential that is depolarized compared with the resting membrane potential of most cells. Thus, activating GlyR will produce a hyperpolarizing inward Cl^– current (I_Cl) only if the cell membrane potential is already above –33 mV, whereas in most cases, GlyR activation will cause a depolarizing outward I_Cl that tends to bring the β-cell near the threshold for action potential firing.

It would seem that glycine from the blood can activate GlyRs in islets because raising the glycine concentration by oral glycine supplements is able to stimulate insulin secretion (39, 98, 111–113) [and the central action of glycine on glucose levels was shown to occur independent of changes in insulin (78)]. However, glycine concentrations in healthy human plasma varies around ∼200 to 300 µM, which far exceeds the EC₅₀ of the GlyRs (∼100 µM) and is expected to desensitize the receptor (51). Like in the brain, β-cells express glycine transporters (GlyT1 and GlyT2) (55, 59, 61). These clear glycine from the extracellular space into β-cells, where it is stored in vesicles through the action of vesicular amino acid transporters (114). The GlyTs are exceptionally effective at clearing glycine and the local extracellular concentration of glycine is likely to be low, although this is difficult to measure directly. As a result of this glycine uptake and storage by β-cells, a second source of glycine is the β-cells themselves because these secrete glycine in a regulated manner (55).

Interestingly, whereas glycine released upon β-cell stimulation may serve as a feed-forward signal to promote insulin secretion, insulin itself is capable of increasing GlyR activity to further propagate the GlyR feed-forward loop (115). Indeed, activation of the insulin receptor on human β-cells also potentiates GlyR currents, and this can be prevented by inhibition of insulin receptor downstream signaling (55, 115). This raises interesting possibility that impaired insulin signaling in β-cells, such as in “β-cell insulin resistance” proposed to contribute to secretory dysfunction in T2D, results in downregulation of β-cell GlyRs. Indeed, at least one single-cell RNA sequencing study demonstrates that GLRA1 (the gene encoding the GlyRα1 subunit) expression is significantly decreased in β-cells from donors with T2D compared with those of a nondiabetic donor (63) [although another study reports no difference (61)]. In intact islets, an elegant study by Taneera et al. (105) measured gene expression and observed GLRA1 to be inversely proportional to donor HbA1c and body mass index. In vitro, GLRA1 expression is downregulated when islets are incubated in high (18.9 mM) glucose (105). Functionally, and consistent with the aforementioned study, our work demonstrates a downregulation of GlyRα1 immunostaining in β-cells of T2D donors, a reduced GlyR activity, and a near complete inability of insulin to facilitate glycine-induced Cl^– current (55).

Glycine in the Islet: NMDA Receptor Actions

NMDA glutamate receptors have been a focus of recent studies in islets (116), where their activation appears to contribute to the regulation of insulin secretion β-cell excitotoxicity (117). Although these ionotropic receptors are nonselective cation channels (which should depolarize β-cells), their inhibition rather than activation promotes insulin secretion (117). This is suggested to be due to the direct coupling of the relatively small Ca²⁺ influx mediated by these channels with small conductance Ca²⁺-sensitive K+ (SK) channels (118, 119) or the activation of K_ATP channels via the production of NO as a second messenger (120). Either of these effects would tend to hyperpolarize β-cells and dampen electrical activity, although a role for these mechanisms in NMDA receptor regulation of insulin secretion remain to be conclusively demonstrated. NMDA-antagonism has been proposed as a therapeutic approach for T2D (121), although an approach that avoids inhibition of central NMDA receptors would be ideal given their positive role in glucose homeostasis noted previously.

It seems counterintuitive that activation of NMDA receptors causes repolarization of β-cells and inhibition of the NMDA receptors stimulates insulin secretion (117). And likewise, counterintuitive that NMDA receptor inhibition promotes β-cell survival (117)—if we accept that elevated glycine is associated with protection from T2D. But it is important to remember that glycine is only one of several coagonists of the NMDA receptor and other coligands, such as serine (in addition to glutamate of course), may contribute to NMDA receptor activity in islets. Indeed, recent work suggests that knockout of serine racemase, a key enzyme in d-serine synthesis, enhances insulin secretion and islet survival (122)—similar to the effects of NMDA receptor inhibition. Indeed, if we assume that glycine may affect insulin secretion in both the positive (via GlyR) and negative (via NMDA receptor) directions, the ability of NMDA receptor inhibition to promote insulin secretion could in part be due to relief of the negative effects of glycine via the NMDA receptor, allowing glycine-induced GlyR activity to enhance insulin secretion. As such, it would be interesting to determine whether block of GlyR (with strychnine) is sufficient to prevent the insulinotropic effect of NMDA receptor antagonism.

Conclusion

Decreasing plasma glycine with increasing insulin resistance, glucose intolerance, and T2D remains correlative at present. Indeed, decreased glycine levels may be secondary to rather than causative of metabolic dysfunction. However, an overall picture is emerging that suggests a reduction of glycine early in disease progression could exacerbate impaired glucose homeostasis. It is clear that glycine plays important roles in cellular signaling to control glucose homeostasis at both central and peripheral sites, and that glycine supplementation increases insulin secretion and improves glucose tolerance. Circulating glycine in the physiological range may act via GlyRs to limit chronic systemic inflammation and associated insulin resistance, and may enhance insulin secretion from pancreatic β-cells—but the latter may be limited by the suppressive and excitotoxic action of β-cell NMDA receptors. In the brain, glycine signaling via NMDA receptors reduces hepatic glucose output via vagal parasympathetic outflows. At supraphysiological glycine concentrations, for example upon dietary supplementation, inflammation is reduced, insulin secretion is increased and hepatic glucose output is reduced as brain glycine levels increase. This review highlights the potential contributions of glycine to glucose homeostasis and disruption in the glycine signaling that occurs in T2D. In addition to glycine’s role as a major inhibitory neurotransmitter in the central nervous system, glycine is both an excitatory and inhibitory neurotransmitter in the periphery where it may regulate glucose homeostasis through distinct mechanisms targeting inflammation and insulin secretion. The small size of the simplest amino acid may belie its importance and underestimate its role in glucose control.

Abbreviations:

 
• BCAA

  branched chain amino acid

 
• CSF

  cerebrospinal fluid

 
• DVC

  dorsal vagal complex

 
• FFA

  free fatty acid

 
• GLRA1

  GlyRα1 subunit gene

 
• GlyRs

  glycine receptors

 
• GlyT

  glycine transporter

 
• GSH

  reduced glutathione

 
• NMDA

  N-methyl-D-aspartate

 
• ROS

  reactive oxygen species

 
• T2D

  type 2 diabetes

 
• VLDL-TG

  triglyceride-rich very-low-density lipoprotein.

Acknowledgments

We thank Drs. Jessica Yue (University of Alberta) and Christopher Newgard (Duke University) for input and critical reading of the manuscript. Our interest in glycine and its role in regulating pancreatic islet function was initiated by our late colleague and mentor, Dr. Matthias Braun. Work on human islets in the authors’ laboratory is supported by the Alberta Diabetes Foundation and the University of Alberta. P.E.M. holds a 2016–2017 Killam Professorship.

Disclosure Summary: The authors have nothing to disclose.

Author notes

Address all correspondence and requests for reprints to: Patrick E. MacDonald, PhD Alberta Diabetes Institute, University of Alberta, LKS Centre, Room 6-126, Edmonton, Alberta T6G 2R3, Canada. E-mail: [email protected].