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Research ArticleEndocrinology
Open Access | 
10.1172/jci.insight.178754
1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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1Department of Endocrinology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
2Department of Physiology and
3Department of Nutritional Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.
4Metabolism Research Group, Division of Advanced Diagnostics, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Address correspondence to: Ziyi Zhang, No.3 Qingchun East Rd., Hangzhou, China, 310016. Phone: 86.150.6816.9258; Email: zhangziyi@zju.edu.cn. Or to: Feihan F. Dai or Michael B. Wheeler, Medical Sciences Building, Room 3352, 1 King’s College Cir., Toronto, Ontario, Canada, M5S 1A8. Phone: 416.978.6737; Email: f.dai@utoronto.ca (FFD); michael.wheeler@utoronto.ca (MBW).
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Published April 22, 2025 - More info
Published in Volume 10, Issue 8 on April 22, 2025
AbstractGlycine and β-alanine activate glycine receptors (GlyRs), with glycine known to enhance insulin secretion from pancreatic islet β cells, primarily through GlyR activation. However, the effects of GlyR activation on β cell proliferation have not been examined. Here, we aim to investigate the potential proliferative effects of glycine and β-alanine on islets. In vitro experiments on mouse and human islets revealed that glycine and β-alanine, via GlyR activation, stimulated the proliferation of β cells and α cells, without affecting insulin or glucagon secretion. Further analysis indicated the involvement of the PI3K/mTORC1/p70S6K signaling pathway in this process. Inhibition of GlyRs and PI3K/mTORC1/p70S6K signaling attenuated proliferative effects of glycine and β-alanine. In vivo and ex vivo studies supported these findings, showing increased β and α cell mass after 12 weeks of oral administration of glycine and β-alanine, with no changes in insulin secretion or glucose homeostasis under normal conditions. However, during an acute insulin resistance induced by insulin receptor antagonist S961, glycine and β-alanine enhanced insulin secretion and reduced blood glucose levels by increasing β cell secretory capacity. These findings demonstrate glycine and β-alanine in vivo and in vitro promote islet cell proliferation via GlyR activation and the PI3K/mTORC1/p70S6K pathway, potentially providing a target to enhance islet capacity.
IntroductionType 2 diabetes (T2D) is characterized by impaired glucose homeostasis caused by insufficient insulin secretion from pancreatic β cells in the face of insulin resistance (1). As a result, methods aimed at early diagnosis of T2D and therapeutic strategies to enhance β cell mass and function have been a major focus of recent research efforts. To this end, glycine supplementation has been shown to increase circulating insulin (2–4), making this nonessential amino acid a possible target for enhancing insulin secretion. It has further been demonstrated that glycine enhances insulin secretion directly from human β cells of T2D and non-T2D donors (5) and that circulating levels of glycine are inversely correlated with T2D risk (6–11), insulin resistance (12–14), impaired glucose tolerance (15, 16), and obesity (14, 17). Taken together, these studies suggest that increased levels of glycine augment β cell function, and low levels of circulating glycine are associated with dysmetabolism and T2D.
In the CNS, glycine acts primarily as an inhibitory neurotransmitter, activating ligand-gated chloride channels, also known as glycine receptors (GlyRs), to generate inhibitory postsynaptic potentials that are important for processing sensory and motor cues (18, 19). Recently, it has been shown that GlyRs, notably α1 subunit–containing GlyRs, are expressed in β cells and that glycine, via GlyRs, stimulates insulin secretion. Mechanistically, it was shown that glycine initiated depolarizing Cl– currents in β cells that enhance action potential firing and Ca2+ entry triggering insulin secretion. Thus, in β cells, glycine appears to act as an excitatory neurotransmitter via GlyRs. Besides glycine, GlyRs can also be pharmacologically activated by other amino acids, including β-alanine and taurine (20, 21).
γ-Aminobutyric acid (GABA) type A (GABAA) receptors are also permeable to Cl–, and their general structure mirrors that of GlyRs. Numerous studies have shown that GABAA receptors are expressed in pancreatic islet cells and that GABA can stimulate both α and β cell proliferation. To this end, we have previously demonstrated that GABA administration increases β cell mass to enhance insulin secretion and augment glucose tolerance in vivo (22). GABA treatment also promotes β cell proliferation via the GABAA receptor through the phosphatidylinositol-3 kinase/ mammalian target of rapamycin complex 1/p70S6K (PI3K/ mTORC1/p70S6K) pathway in vitro (22, 23). Given the fact that GABAA receptors and GlyRs are both ionotropic receptors and that their activation leads to increased Cl– ion conductance, we hypothesize that GlyRs activation may also lead to the proliferation of islet cells. Indeed, glycine has been shown to be associated with cellular proliferation in multiple tissues and cells. Jain and colleagues reported that glycine consumption and the mitochondrial glycine biosynthetic pathway were strongly correlated with proliferation across cancer cells, whereas blocking glycine uptake and biosynthesis led to impaired cell proliferation (24). Lin et al. demonstrated that glycine could enhance satellite cell proliferation through activating mTORC1 (25). Glycine also activates the Akt/mTOR signaling pathway in primary mouse hepatocytes and may play a role in liver regeneration (26). Overall, these findings suggest that glycine can promote cell proliferation, but the effects of GlyR activation on islet cell proliferation still need to be clarified.
Therefore, in the present study, we aimed to examine the effects of glycine and β-alanine, which both activate GlyRs (19), on the islet proliferation and function both in vivo and in vitro, and we explore the potential mechanism underlying those effects.
ResultsEffects of glycine and β-alanine on islet cell proliferation in primary mouse islets and human islets in vitro. To investigate whether glycine and β-alanine, which both activate GlyRs, had direct effects on islet cell proliferation, mouse islets were treated with 1 mM glycine or 1 mM β-alanine for 5 days (Figure 1A) in vitro. Since interstitial glycine concentrations are expected to exceed the circulating level (~300 μM), in particular because pancreatic β cells store and release glycine as previously described by Yan-Do et al. (5), we rationalized the use of 1 mM glycine or β-alanine to treat primary mouse and human islets in the present study. Harmine, a DYRK1A inhibitor and mitogenic compound, was used as positive control of islet cell proliferation (27). Ki67 staining was used to evaluate the proliferation of both β cells and α cells in dispersed islets. In mouse islets, glycine treatment significantly increased costaining of insulin+Ki67+ and glucagon+Ki67+ by 80% ± 3.6% (P < 0.001) and 129% ± 25% (P < 0.01), respectively (Figure 1, B and C). Similarly, β-alanine administration significantly increased costaining of insulin+Ki67+ and glucagon+Ki67+ by 123% ± 19% (P < 0.001) and 157% ± 29% (P < 0.01), respectively (Figure 1, B and C). These observations suggest glycine and β-alanine induce proliferation of both β and α cells in mouse islets.
Figure 1Glycine and β-alanine stimulate both β cell and α cell proliferation in mouse islets in vitro. (A) Workflow for in vitro treatment of mouse islets. (B and C) Representative images of Ki67+ cells and islet cell proliferation measurement in mouse islets treated with vehicle, 1 mM glycine, or 1 mM β-alanine for 5 days in vitro. Harmine (10 μM) was used as a positive control for islet cell proliferation (n = 5 mice used for each treatment). We used 30 islets per mouse, with an average of 9,761 cells per sample to derive the data. Cell proliferation rate was calculated by normalizing Ki67+ β cell/α cell numbers to total β cell/α cell numbers on cytospin slides. Data are shown as mean ± SEM. Statistical significance was assessed using an unpaired t test, with Holm-Bonferroni correction applied for multiple comparisons. ***P < 0.001, **P < 0.01, compared with the control group. White arrows indicate Ki67-positive proliferating α cells and β cells.
There are 4 known isoforms of α-subunit (α1–α4) and a single β-subunit of GlyR in most species (18, 19) including humans, where GLRA4, a gene encoding GlyRα4, is thought to be a pseudogene (28). In line with previous studies (29–32), we demonstrated that GlyRs were expressed in mouse and human islets by performing quantitative PCR (qPCR) (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.178754DS1) and immunofluorescence staining (Supplemental Figure 1). Next, strychnine, a specific GlyR inhibitor (19), was used to determine whether glycine/β-alanine–induced islet proliferation was mediated through the activation of GlyR. With strychnine, the proliferation of both β cell and α cell induced by glycine and β-alanine was blocked (P < 0.001) (Figure 2, A and B). In contrast, harmine-induced proliferation remained unaffected by strychnine (Figure 2, A and B). This suggested that, unlike harmine action, glycine and β-alanine induced the islet cell proliferation through GlyR.
Figure 2Glycine and β-alanine stimulate both β cell and α cell proliferation in mouse islets via glycine receptor in vitro. (A and B) Representative images of Ki67+ cells and islet cell proliferation in dispersed mouse islets treated with vehicle, 1 mM glycine, 1 mM β-alanine, or 10 μM Harmine, with or without 1 μM strychnine for 5 days (n = 5 mice used for each treatment). Data are depicted as mean ± SEM. Statistical significance was determined by using unpaired t test, with Holm-Bonferroni correction applied for multiple comparisons. ***P < 0.001, **P < 0.01, compared with the control group. ###P < 0.001, compared with the glycine or β-alanine treatment group. GlyR, glycine receptor; STR, strychnine. Scale bar: 50 μm. White arrows indicate Ki67+ proliferating α cells and β cells.
In cultured human islets, 5 days treatment of glycine or β-alanine enhanced the proliferation of both β and α cells, compared with corresponding controls (Figure 3, A–C). Glycine treatment significantly increased costaining of insulin+Ki67+ or glucagon+Ki67+ by 81% ± 34% (P < 0.05) or 76% ± 44% (P = 0.05), respectively (Figure 3, B and C). Similarly, β-alanine administration also increased costaining of insulin+Ki67+ or glucagon+Ki67+ by 72% ± 20% (P < 0.01) or 89% ± 48% (P = 0.07), respectively (Figure 3, B and C). Like the observations in mouse islets, we found that these positive effects on cell proliferation induced by glycine/β-alanine could be blocked by cotreatment with strychnine (Figure 3D), implicating GlyR activation in primary human islets.
Figure 3Glycine and β-alanine stimulate both β cell and α cell proliferation in human islets via glycine receptor in vitro. (A) Workflow for in vitro treatment of human islets. (B and C) Representative images of Ki67+ cells and islet cell proliferation in human islets treated with vehicle, 1mM glycine, or 1 mM β-alanine for 5 days in vitro. Harmine (10 μM) was used as a positive control for islet cell proliferation. We used 30 islets per donor, with an average of 7,490 cells per sample to derive the data. Cell proliferation was calculated by normalizing Ki67+ β cell/α cell numbers to total β cell/α cell numbers on cytospin slides. Scale bar: 50 μm. White arrows indicate Ki67+ proliferating α cells and β cells. (D) Islet cell proliferative in primary human islets treated with vehicle, 1 mM glycine, or 1 mM β-alanine, with or without 1 μM strychnine for 5 days. Data are depicted as mean ± SEM. Statistical significance was determined by using Mann-Whitney U test or unpaired t test dependent on dataset normality test, with Holm-Bonferroni correction applied for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. #P < 0.05, compared with the glycine or β-alanine treatment group. STR, strychnine. We used islets from 11 donors, 7 of 11 donors have accessible HbA1c values, all falling within the normal range.
Effects of glycine or β-alanine treatment on insulin secretion and content in mouse islets in vitro. After treating isolated mouse islets with glycine or β-alanine for 5 days, we evaluated glucose-stimulated insulin secretion (GSIS). No change was observed in insulin secretion under the treatment of low glucose (2 mM), high glucose (11 mM), or high glucose + KCl, the latter of which depolarizes β cells to maximally stimulate insulin exocytosis (Figure 4A). Moreover, total insulin content was not changed (Figure 4B). After normalization to DNA content/cell number, there was no significant difference in insulin secretion or total insulin content among the control, glycine, and β-alanine group (Figure 4, C and D). In an acute study on human islets where glycine was added during GSIS, it was found that the addition of glycine did not significantly affect insulin secretion, regardless of treatment (low glucose [2 mM] or high glucose [11 mM]; Supplemental Figure 2). Moreover, neither glycine nor β-alanine induced glucagon secretion in vitro in mouse islets, with l-arginine serving as a positive control (Supplemental Figure 3).
Figure 4Glycine and β-alanine treatment had no effect on glucose-stimulated insulin secretion (GSIS) and total islet insulin content in mouse islets in vitro. (A) Mouse islets were treated with vehicle, 1 mM glycine or 1 mM β-alanine for 5 days in vitro. Insulin secretion under the treatment of low glucose (2 mM), high glucose (11 mM), and high glucose + KCl was measured. (B) Total insulin content of 20 islets treated with vehicle, 1 mM glycine, or 1 mM β-alanine for 5 days in vitro. (C and D) Insulin secretion and total insulin content normalized to total DNA content. Data are depicted as mean ± SEM. n = 4–5 mice used for each treatment. Statistical significance was determined by using unpaired t test, with Holm-Bonferroni correction applied for multiple comparisons. There were no significant differences among the groups, with P > 0.05.
Islet proliferation and expansion is enhanced in mice supplemented with glycine and β-alanine, but no changes are observed in glucose tolerance. To explore the effects of glycine and β-alanine administration on islet cell proliferation in vivo, we used wild-type FVB strain (WT FVB) male mice supplemented with 2% glycine or 2% β-alanine in drinking water for 12 weeks and performed IHC to evaluate β cell and α cell proliferation (Figure 5A). In control mice, without glycine or β-alanine supplement, we found that the circulating glycine was 344.6 ± 84.9 μM in the plasma (Figure 5B), which is consistent with previous reports (5). After oral glycine treatment, there was a 5.7-fold increase in circulating glycine concentration (1,981.2 ± 314.2 μM versus 344.6 ± 84.9 μM in the control) (Figure 5B). Targeted metabolomics was used to assess circulating amino acid levels after glycine treatment, revealing that, aside from isoleucine, the levels of 19 other amino acids remained unchanged following glycine administration (Supplemental Figure 4).
Figure 5Glycine and β-alanine stimulate both β cell and α cell proliferation in mouse islets in vivo. (A) Workflow for in vivo oral administration of 2% glycine and 2% β-alanine drinking water to mice and subsequent assessment. (B) Plasma glycine concentration. (C) Isolated islet size. Scale bars: 500 μm. (D) Total insulin content in 20 isolated mouse islets. (E and F) Representative images of Ki67+ cells and islet cell (both β cell and α cell) proliferation in mice treated with vehicle, 2% glycine, or 2% β-alanine for 12 weeks in vivo. We used 30 islets per mouse, with an average of 17,778 cells per sample to derive the data. Data are depicted as mean ± SEM. n = 7–9 in each group. Scale bar: 50 μm. White arrows indicate Ki67+ proliferating α cells and β cells. Statistical significance was determined by using unpaired t test, with Holm-Bonferroni correction applied for multiple comparisons where appropriate. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.
Examining isolated islets, we observed that the mean size of islets treated by glycine and β-alanine was significantly greater than that of the control group (Figure 5C), as was total insulin content (Figure 5D), compared with control mice. Employing Ki67 staining on dispersed islet cells, we found Ki67+ β cells and Ki67+ α cells were significantly increased in glycine and β-alanine treated groups, compared with controls (Figure 5, E and F). In further studies examining pancreatic islets, we observed a significant increase in the β cell mass, β cell number/area, and α cell mass, but no changes in the mean β or α cell size were observed (Figure 6, A–G), ruling out cell hypertrophy as a factor. These data suggest that there was a significant increase in islet cell proliferation after the administration of glycine or β-alanine.
Figure 6Glycine and β-alanine stimulate both β cell and α cell proliferation in mouse islets in vivo. (A) Representative images of insulin-stained pancreatic sections from mice administered glycine or β-alanine for 12 weeks. Scale bar: 200 μm. (B–G) Analysis of these sections were performed by calculating β cell mass (B), β cell number/area (C), average β cell size (D), α cell mass (E), α cell number/area (F), and average α cell size (G). n = 9–12 samples were analyzed in each group, with sections from 2 different levels in each sample stained and analyzed. Statistical significance was determined by using unpaired t test (B, C, E, and G) or Mann-Whitney U test (D and F) dependent on dataset normality test, with Holm-Bonferroni correction applied for multiple comparisons. *P < 0.05, compared with the control group.
Next, we examined the long-term effects of glycine and β-alanine on key metabolic parameters and islet function (Figure 7A). No changes were observed in body weight, fasting plasma glucose, fasting insulin, or fasting glucagon in glycine- or β-alanine–treated groups compared with the control (Figure 7, B–E). Moreover, there was no significant difference in glucose tolerance and insulin sensitivity between glycine- and β-alanine–treated groups and the controls (Figure 7, F–H). After 12 weeks of treatment, no changes in insulin secretion were observed ex vivo in the mice supplemented with glycine/β-alanine or regular water (Figure 7, I and J). These results suggested glycine or β-alanine supplementation has no effect on glucose homeostasis or insulin secretion in vivo under normal/physiological conditions.
Figure 7Glycine and β-alanine administration did not influence glucose homeostasis in vivo upon glucose stimulation. (A) Workflow for treatment and in vivo and in vitro assessment. (B–H) Mice were treated with 2% glycine or 2% β-alanine through drinking water for 12 weeks and were then assessed for glucose homeostasis by evaluating body weight (B), fasting glucose (C), fasting insulin (D), fasting glucagon level (mice were treated with 2% glycine or normal drinking water for 5 weeks before glucagon assessment) (E), glucose changes during OGTT (F), insulin levels during OGTT (G), and glucose levels during ITT (H). n = 6–10 in each group. (I and J) glucose-stimulated insulin secretion in 20 islets isolated from mice treated with vehicle, 2% glycine or 2% β-alanine, with or without normalization by DNA content/cell number. n = 5–7 in each group. Data are depicted as mean ± SEM. Statistical significance was determined by using unpaired t test (B, D, E, and I), 1-way ANOVA (F–H) or Mann-Whitney U test (C and J) dependent on dataset normality test, with Holm-Bonferroni correction applied for multiple comparisons.
Treatment of glycine or β-alanine improves glucose homeostasis in mice with acute severe insulin resistance. Next, we sought to determine whether glycine or β-alanine treatment enhanced islet function under a severe acute metabolic stress condition. The compound S961, a specific insulin receptor antagonist (33), was used to elicit insulin resistance in control and glycine- or β-alanine–treated mice (Figure 8A). Prior long-term administration of glycine or β-alanine for 12 weeks led to lower plasma glucose 2 hours after S961 injection (Figure 8B). This could be attributed to the higher plasma insulin levels observed at the same time point (Figure 8C). These data reveal that both glycine and β-alanine can enhance islet proliferation and functional capacity to secrete insulin in vivo, particularly under metabolic stress conditions such as an acute insulin resistance.
Figure 8Improved glucose tolerance and insulin secretion in S961-induced transient insulin resistance in glycine- and β-alanine–treated mice. (A) Workflow for in vivo oral administration of 2% glycine and 2% β-alanine drinking water to mice. (B and C) Blood glucose levels and plasma insulin levels were measured hourly after S961 injection (30 nmol/kg). Data are depicted as mean ± SEM. n = 9–12 in each group. Statistical significance was determined by using unpaired t test, with Holm-Bonferroni correction applied for multiple comparisons. *P < 0.05, *P < 0.01, compared with the control group.
Glycine and β-alanine induce islet proliferation via the Akt/mTOR pathway in mouse islets. To gain mechanistic insights into how glycine or β-alanine may promote islet cell proliferation, we examined downstream pathways known to be linked to both GlyR activation and cellular proliferation (23, 26, 34). Glycine has previously been shown to activate the Akt/mTOR signaling pathway in primary mouse hepatocytes and has been linked to liver regeneration (26). Since this cytosolic signaling pathway has also been implicated in β cell growth (23), we aimed to determine if Akt/mTOR was required for glycine- and β-alanine–mediated islet cell proliferation. In isolated mouse islets, glycine or β-alanine treatment for 5 days significantly increased costaining of Ki67+/insulin+ or Ki67+/glucagon+, respectively (Figure 9, B and C), compared with the corresponding control groups. Interestingly, the addition of wortmannin (PI3K antagonist), rapamycin (mTORC1/2 inhibitor), or PF-4708671 (p70S6K inhibitor) significantly reduced the Ki67+/insulin+ or Ki67+/glucagon+ cell number, indicating glycine/β-alanine–induced islet cell proliferation effects were blunted in mouse islets (Figure 9, A–C). These data suggest that glycine and β-alanine induced islet proliferation is dependent upon an intact PI3K/mTORC1/p70S6K signaling pathway. The lack of an effect of glycine/β-alanine on insulin secretion, as shown above, would suggest that under the conditions studied, the islet proliferation effect of glycine/β-alanine is not mediated directly by insulin.
Figure 9Glycine and β-alanine stimulate islet cell proliferation through PI3K/mTOR/p70S6K signaling pathway in mouse islets. (A) Outline of the PI3K/mTOR/p70S6K signalling pathway and selective pathway inhibitors. (B and C) Mouse islets were exposed to various treatments for 5 days in vitro (n = 5 for each group). Cell proliferation was assessed by normalizing Ki67+ β cell/α cell numbers to total β cell/α cell numbers on cytospin slides. Glycine: 1 mM; β-alanine: 1 mM; Wortmannin (PI3K antagonist): 100 nM; PF-4708671 (p70S6K inhibitor): 10 μM; Rapamycin (mTORC1/2 inhibitor): 10 nM. Data are depicted as mean ± SEM. Statistical significance was determined by using unpaired t test, with Holm-Bonferroni correction applied for multiple comparisons. **P < 0.01, ***P < 0.001, compared with the control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the glycine or β-alanine treatment group. n = 4–5 in each group.
DiscussionIn our current study, we show that 5 days of treatment of glycine or β-alanine induced proliferation of pancreatic β cells and α cells in the isolated mouse islets in vitro but did not change GSIS from these islets. In vivo, in the mice supplemented with glycine or β-alanine for 12 weeks, we also observed that the proliferation of pancreatic β cells and α cells was significantly enhanced, but glucose tolerance or insulin secretion were not affected when examined under normal physiological conditions. However, when challenged by S961-induced acute insulin resistance, those mice supplemented with glycine or β-alanine had enhanced insulin secretion. Thus, the reduced glucose level may be attributed to an increased capacity to secrete insulin due to the increased β cell mass. These effects appear to require a functional PI3K/mTORC1/p70S6K signaling pathway.
Islet cells and neurons share many similarities in their physiology (35, 36), function (37–40), and gene expression (41, 42). Indeed, the inhibitory neurotransmitter GABA was detected in pancreatic β cells and was shown to play roles in insulin secretion (43) and β cell proliferation (22, 23, 44). Our previous studies demonstrated that GABA administration led to modest increases in β cell mass, insulin secretion, and glucose tolerance in vivo (22). GABA treatment also promoted β cell proliferation via the GABAA receptor through the PI3K/mTORC1/p70S6K pathway in vitro (22, 23). Similar to GABA, the related inhibitory neurotransmitter glycine (45) was also d
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