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Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects.

Swisa A, Granot Z, Tamarina N, Sayers S, Bardeesy N, Philipson L, Hodson DJ, Wikstrom JD, Rutter GA, Leibowitz G, Glaser B, Dor Y - J. Biol. Chem. (2015)

Bottom Line: However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood.Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway.This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

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Insulin secretion in LKB1-deficient β cells is KATP-dependent.A, insulin levels during an islet perifusion assay. Switching medium from 2.8 to 16.7 mm glucose (G16.7) causes higher secretion in LKB1-deficient islets. Diazoxide (Dia) (100 μm) abolishes secretion in both control and mutant islets, and further addition of 30 mm KCl triggers a second peak of secretion. Data represent the mean of 2 groups of islets taken from different mice at age of 2.5 months. B, insulin secretion during islet perifusion with glyburide. Glyburide (1 μm) triggers dramatically more insulin secretion from LKB1-deficient islets in either low or high glucose. Data represent mean of data from three (control) and five (LKB1-deficient) mice. C, serum insulin and glucose levels after administration of nifedipine (Nif) to lox/lox and LKB1-deficient mice after an overnight fast. Nifedipine (10 mg/kg in 5% DMSO) was injected at time 0. Glucose was injected at 45 min. Glucose was measured at 0, 45, and 60 min, and insulin was measured at 60 min. Mice were 3–12 months old. n = 6–9 mice per group. D, glucose-stimulated insulin secretion from perifused islets treated with nifedipine. Dashed lines, nifedipine was added before high glucose. Solid lines, nifedipine was added 15 min after the addition of high glucose. In both cases no significant difference was observed between lox/lox and βLKB islets in the presence of nifedipine. Statistical significance is shown for the experiment where nifedipine was added after high glucose. E, left, representative plots of calcium influx after glucose stimulation of wild type and βLKB islets. Islets were perifused with KRB buffer containing 2.8 or 16.7 mm glucose or 30 mm KCl as indicated. Intracellular calcium is calculated by the ratio of emission at 340- and 380-nm wavelengths using Fura-2 dye. Each plot represents the average ratio of 8–20 islets taken from one mouse. Right, calculation of 6 parameters of calcium response in lox/lox and βLKB islets, based on the calcium plots. Mice were 6 months old, n = 3 per genotype. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ns, p > 0.05.
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Figure 2: Insulin secretion in LKB1-deficient β cells is KATP-dependent.A, insulin levels during an islet perifusion assay. Switching medium from 2.8 to 16.7 mm glucose (G16.7) causes higher secretion in LKB1-deficient islets. Diazoxide (Dia) (100 μm) abolishes secretion in both control and mutant islets, and further addition of 30 mm KCl triggers a second peak of secretion. Data represent the mean of 2 groups of islets taken from different mice at age of 2.5 months. B, insulin secretion during islet perifusion with glyburide. Glyburide (1 μm) triggers dramatically more insulin secretion from LKB1-deficient islets in either low or high glucose. Data represent mean of data from three (control) and five (LKB1-deficient) mice. C, serum insulin and glucose levels after administration of nifedipine (Nif) to lox/lox and LKB1-deficient mice after an overnight fast. Nifedipine (10 mg/kg in 5% DMSO) was injected at time 0. Glucose was injected at 45 min. Glucose was measured at 0, 45, and 60 min, and insulin was measured at 60 min. Mice were 3–12 months old. n = 6–9 mice per group. D, glucose-stimulated insulin secretion from perifused islets treated with nifedipine. Dashed lines, nifedipine was added before high glucose. Solid lines, nifedipine was added 15 min after the addition of high glucose. In both cases no significant difference was observed between lox/lox and βLKB islets in the presence of nifedipine. Statistical significance is shown for the experiment where nifedipine was added after high glucose. E, left, representative plots of calcium influx after glucose stimulation of wild type and βLKB islets. Islets were perifused with KRB buffer containing 2.8 or 16.7 mm glucose or 30 mm KCl as indicated. Intracellular calcium is calculated by the ratio of emission at 340- and 380-nm wavelengths using Fura-2 dye. Each plot represents the average ratio of 8–20 islets taken from one mouse. Right, calculation of 6 parameters of calcium response in lox/lox and βLKB islets, based on the calcium plots. Mice were 6 months old, n = 3 per genotype. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ns, p > 0.05.

Mentions: To clarify how βLKB islets secrete more insulin in response to glucose, we perturbed steps in the triggering pathway for insulin secretion. Treatment with diazoxide, a KATP channel opener, completely abolished glucose-stimulated secretion in perifused βLKB islets (Fig. 2A). This suggested that closure of KATP channels and membrane depolarization are essential for enhanced secretion in the mutants. However, when channels were forced to open with diazoxide and KCl was added, βLKB islets secreted more insulin than controls, consistent with the findings in Fig. 1E. Thus in the face of a similar degree of membrane depolarization, βLKB islets secrete more insulin, indicating enhancement of secretion at a distal step of the pathway (Fig. 2A). Furthermore, treatment with glyburide (forcing the closure of KATP channels) led to higher insulin secretion from cultured βLKB islets both at basal (2.8 mm) and stimulating (16.7 mm) glucose concentrations (Fig. 2B). In addition, in vivo administration of a high dose of glyburide boosted plasma insulin levels to a higher degree in βLKB mice compared with controls (data not shown). This further indicates a component boosting GSIS downstream to membrane depolarization.


Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects.

Swisa A, Granot Z, Tamarina N, Sayers S, Bardeesy N, Philipson L, Hodson DJ, Wikstrom JD, Rutter GA, Leibowitz G, Glaser B, Dor Y - J. Biol. Chem. (2015)

Insulin secretion in LKB1-deficient β cells is KATP-dependent.A, insulin levels during an islet perifusion assay. Switching medium from 2.8 to 16.7 mm glucose (G16.7) causes higher secretion in LKB1-deficient islets. Diazoxide (Dia) (100 μm) abolishes secretion in both control and mutant islets, and further addition of 30 mm KCl triggers a second peak of secretion. Data represent the mean of 2 groups of islets taken from different mice at age of 2.5 months. B, insulin secretion during islet perifusion with glyburide. Glyburide (1 μm) triggers dramatically more insulin secretion from LKB1-deficient islets in either low or high glucose. Data represent mean of data from three (control) and five (LKB1-deficient) mice. C, serum insulin and glucose levels after administration of nifedipine (Nif) to lox/lox and LKB1-deficient mice after an overnight fast. Nifedipine (10 mg/kg in 5% DMSO) was injected at time 0. Glucose was injected at 45 min. Glucose was measured at 0, 45, and 60 min, and insulin was measured at 60 min. Mice were 3–12 months old. n = 6–9 mice per group. D, glucose-stimulated insulin secretion from perifused islets treated with nifedipine. Dashed lines, nifedipine was added before high glucose. Solid lines, nifedipine was added 15 min after the addition of high glucose. In both cases no significant difference was observed between lox/lox and βLKB islets in the presence of nifedipine. Statistical significance is shown for the experiment where nifedipine was added after high glucose. E, left, representative plots of calcium influx after glucose stimulation of wild type and βLKB islets. Islets were perifused with KRB buffer containing 2.8 or 16.7 mm glucose or 30 mm KCl as indicated. Intracellular calcium is calculated by the ratio of emission at 340- and 380-nm wavelengths using Fura-2 dye. Each plot represents the average ratio of 8–20 islets taken from one mouse. Right, calculation of 6 parameters of calcium response in lox/lox and βLKB islets, based on the calcium plots. Mice were 6 months old, n = 3 per genotype. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ns, p > 0.05.
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Figure 2: Insulin secretion in LKB1-deficient β cells is KATP-dependent.A, insulin levels during an islet perifusion assay. Switching medium from 2.8 to 16.7 mm glucose (G16.7) causes higher secretion in LKB1-deficient islets. Diazoxide (Dia) (100 μm) abolishes secretion in both control and mutant islets, and further addition of 30 mm KCl triggers a second peak of secretion. Data represent the mean of 2 groups of islets taken from different mice at age of 2.5 months. B, insulin secretion during islet perifusion with glyburide. Glyburide (1 μm) triggers dramatically more insulin secretion from LKB1-deficient islets in either low or high glucose. Data represent mean of data from three (control) and five (LKB1-deficient) mice. C, serum insulin and glucose levels after administration of nifedipine (Nif) to lox/lox and LKB1-deficient mice after an overnight fast. Nifedipine (10 mg/kg in 5% DMSO) was injected at time 0. Glucose was injected at 45 min. Glucose was measured at 0, 45, and 60 min, and insulin was measured at 60 min. Mice were 3–12 months old. n = 6–9 mice per group. D, glucose-stimulated insulin secretion from perifused islets treated with nifedipine. Dashed lines, nifedipine was added before high glucose. Solid lines, nifedipine was added 15 min after the addition of high glucose. In both cases no significant difference was observed between lox/lox and βLKB islets in the presence of nifedipine. Statistical significance is shown for the experiment where nifedipine was added after high glucose. E, left, representative plots of calcium influx after glucose stimulation of wild type and βLKB islets. Islets were perifused with KRB buffer containing 2.8 or 16.7 mm glucose or 30 mm KCl as indicated. Intracellular calcium is calculated by the ratio of emission at 340- and 380-nm wavelengths using Fura-2 dye. Each plot represents the average ratio of 8–20 islets taken from one mouse. Right, calculation of 6 parameters of calcium response in lox/lox and βLKB islets, based on the calcium plots. Mice were 6 months old, n = 3 per genotype. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ns, p > 0.05.
Mentions: To clarify how βLKB islets secrete more insulin in response to glucose, we perturbed steps in the triggering pathway for insulin secretion. Treatment with diazoxide, a KATP channel opener, completely abolished glucose-stimulated secretion in perifused βLKB islets (Fig. 2A). This suggested that closure of KATP channels and membrane depolarization are essential for enhanced secretion in the mutants. However, when channels were forced to open with diazoxide and KCl was added, βLKB islets secreted more insulin than controls, consistent with the findings in Fig. 1E. Thus in the face of a similar degree of membrane depolarization, βLKB islets secrete more insulin, indicating enhancement of secretion at a distal step of the pathway (Fig. 2A). Furthermore, treatment with glyburide (forcing the closure of KATP channels) led to higher insulin secretion from cultured βLKB islets both at basal (2.8 mm) and stimulating (16.7 mm) glucose concentrations (Fig. 2B). In addition, in vivo administration of a high dose of glyburide boosted plasma insulin levels to a higher degree in βLKB mice compared with controls (data not shown). This further indicates a component boosting GSIS downstream to membrane depolarization.

Bottom Line: However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood.Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway.This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

Show MeSH
Related in: MedlinePlus