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Clinical Trial
. 2013 Feb 8;288(6):3938-51.
doi: 10.1074/jbc.M112.385682. Epub 2012 Dec 24.

Regulation of Glucagon Secretion in Normal and Diabetic Human Islets by γ-Hydroxybutyrate and Glycine

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Free PMC article
Clinical Trial

Regulation of Glucagon Secretion in Normal and Diabetic Human Islets by γ-Hydroxybutyrate and Glycine

Changhong Li et al. J Biol Chem. .
Free PMC article

Abstract

Paracrine signaling between pancreatic islet β-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-(13)C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in β-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels.

Figures

FIGURE 1.
FIGURE 1.
T2D human islets have impaired insulin secretion and oxygen consumption and different glucose-mediated glucagon suppression and GABA shunt. A shows insulin secretion in response to glucose in the presence of 4.0 mm AAM in normal (filled circles with black line; n = 5) and T2D islets (T2D-αGR, open circles with dashed gray line; T2D-αNGR, triangles with dashed black line; n = 3 for each). Versus T2D-αNGR and normal, * indicates p < 0.05; versus T2D-αGR and normal, # indicates p < 0.05; 25 mm glucose versus 0 mm glucose, a indicates p < 0.05; 25 mm glucose versus 5 mm glucose, b indicates p < 0.05. B shows glucagon secretion (inset shows the percent changes). Versus T2D-αNGR and normal, * indicates p < 0.05; versus 0 mm glucose, a indicates p < 0.05. C shows the glucose stimulation of islet oxygen consumption in normal and T2D islets (normal, black-filled bars (n = 5); T2D-αGR, gray-filled bars; T2D-αNGR, open bars (n = 3 for each); also shown in D and E). Versus 0 mm glucose (G, 0), a indicates p < 0.01; versus 25 mm glucose (G, 25), b indicates p < 0.01; versus normal, * indicates p < 0.05. D shows GABA/glutamate ratios. Versus normal, * indicates p < 0.05; versus 0 mm glucose, a indicates p < 0.05. E shows expression data for selected genes detected by quantitative PCR. Versus normal, * indicates p < 0.05. Data are presented as mean ± S.E. (error bars). GK, glucokinase; PC, pyruvate carboxylase; GDH, glutamate dehydrogenase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.
FIGURE 2.
FIGURE 2.
Human islets express GHB loop and produce GHB in response to glucose stimulation. A shows gene expression detected by RT-PCR of the enzymes of the GHB loop including SSA reductase, GHB dehydrogenase, and the putative GHB receptor (TSPAN-17) in four different preparations of normal human islets. B shows GHB production and release in batch-incubated normal human islets. After 60-min preincubation, batches of 500 islets were incubated with 0 (open bars) or 10 mm glucose (filled bars) for another 60 min, and GHB was determined in islet homogenates and the incubation supernatants. Versus 0 mm glucose, * indicates p < 0.05 (n = 3). C and D show GHB release and its [13C]GHB enrichment from experiments with [U-13C]glucose (compare with results in Tables 1 and 2). Normal, filled circles with black line (n = 5); T2D-αGR, open circles with dashed gray line (n = 3); T2D-αNGR, triangles with dashed black line (n = 3). Versus T2D-αGR, * indicates p < 0.05; versus 0 mm glucose, a indicates p < 0.05; versus 5 mm glucose, b indicates p < 0.05. Data are presented as mean ± S.E. (error bars). MPE, mole percent enrichment.
FIGURE 3.
FIGURE 3.
GHB produced via GABA shunt mediates glucose suppression of glucagon secretion. A–C show GABA levels and glucagon and insulin secretion in normal islets in the absence (filled circles with solid line) or in the presence of 1.55 mm vigabatrin (open triangles with dashed line). A, islet intracellular GABA levels; B, glucagon secretion; C, insulin secretion. Versus untreated islets, * indicates p < 0.01; versus 0 mm glucose, a indicates p < 0.05; n = 4. D shows glucagon secretion stimulated by an amino acid mixture in batch-incubated normal human islets and inhibition of amino acid-stimulated glucagon secretion by the GHB agonist 3-CPA. After 60-min preincubation, batches of 50 islets were incubated with different treatments for another 60 min. Versus AAM stimulation, * indicates p < 0.01, and # indicates p < 0.05; n = 4. Data are presented as mean ± S.E. (error bars).
FIGURE 4.
FIGURE 4.
Glycine stimulates cytosolic calcium influx and glucagon secretion via strychnine-sensitive glycine receptor in normal human islets. Cytosolic calcium ([Ca2+]i) levels were measured by dual wavelength fluorescence microscopy using Fura-2 as the calcium indicator. Data are presented as a black line (mean values), and S.E. values (error bars) are given in gray. A shows [Ca2+]i responses to a 4.0 mm amino acid mixture and 10 mm glucose (G 10) (n = 5). B shows insulin secretion from batch-incubated normal human islets. Versus 0 mm glucose and amino acid mixture, # indicates p < 0.05; n = 4. C shows effects of different concentrations of glycine on [Ca2+]i (n = 4). D shows dose-dependent strychnine inhibition of [Ca2+]i stimulated by 0.2 or 0.5 mm glycine (n = 4). E shows gene expression (upper panel) detected by RT-PCR of glycine receptors α1 and β. Results are representative for comparable results from three separate preparations of normal human islets. The lower panel of E shows the presence of glycine receptor protein detected by Western blotting in two batches of normal human islets. Data are presented as mean ± S.E. (error bars).
FIGURE 5.
FIGURE 5.
Glycine stimulates glucagon secretion and [Ca2+]i influx, and this effect is inhibited by glucose and strychnine. A shows glucagon secretion from perifused normal human islets in response to stimulation by a glycine ramp (0–5 mm over a period of 30 min; n = 3). B shows different [Ca2+]i responses to glycine and glucose stimulation and potassium chloride (KCl) in single dispersed human islet cells (data are representative for four separate experiments with similar results). The cell indicated by the black line is sensitive to glycine stimulation and is likely the α-cell; the cell indicated by the gray line is sensitive to glucose stimulation and is likely the β-cell. [Ca2+]i levels were measured by dual wavelength fluorescence microscopy using Fura-2 as the calcium indicator. C and D show glucagon and insulin secretion in response to glycine stimulation and the effects of glucose, strychnine, and the GHB receptor antagonist NCS-382 in batch-incubated normal human islets. After 60-min preincubation, batches of 50 islets were then incubated with different treatments as indicated in the figure for another 60 min. Versus 0.5 mm glycine, * indicates p < 0.05; n = 4. Data are presented as mean ± S.E. (error bars). G, glucose.
FIGURE 6.
FIGURE 6.
GHBDH and receptors of glycine and GHB are α-cell-specific in mouse islets. A shows relative gene expression of insulin, glucagon, GAD, SSA reductase, GHBDH, and receptors of GHB and glycine in intact mouse islets and purified β-cells from GFP transgenic mice with β-cell-specific expression (open bars, whole islets; filled bars, purified β-cells). GAPDH was used as a reference gene. ND, not detectable; n = 3. B shows the calcium response to glycine and glucose stimulation in a single GFP-positive or -negative islet cell from GFP transgenic mice (data are representative for three separate experiments with similar results). [Ca2+]i levels were measured by dual wavelength fluorescence microscopy using Fura-2 as the calcium indicator. C and D show glucagon and insulin secretion in isolated batch-incubated normal mouse islets. Versus glucose-free basal conditions, a indicates p < 0.05; versus 1 mm glycine stimulation, b indicates p < 0.01, and c indicates p < 0.05; n = 5. Data are presented as mean ± S.E. (error bars). GHBR, GHB receptor; G, glucose.
FIGURE 7.
FIGURE 7.
Opposite effects of glycine and GHB on glucagon secretion and the metabolic interaction between β- and α-cells. α-Cells are stimulated by glycine via its receptor and are inhibited by GHB produced by β-cells also via its receptor. During glucose stimulation of insulin secretion, increased generation of α-ketoglutarate (α-KG) from the TCA cycle supports enhanced flux through the GABA shunt, which leads to increased production of GHB. GHB is generated from SSA via SSA reductase. Vigabatrin acts as a GABA transaminase (GABA-T) inhibitor. Strychnine inhibits the glycine receptor. 3-CPA is a GHB agonist, and NCS-382 is a GHB antagonist. ATP and GTP inhibit glutamate dehydrogenase (GDH). SSADH, SSA dehydrogenase; AAs, amino acids.

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