| 1. |
- Björkqvist, Maria, et al.
(author)
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The R6/2 transgenic mouse model of Huntington's disease develops diabetes due to deficient {beta}-cell mass and exocytosis.
- 2005
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In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 14:5, s. 565-574
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Journal article (peer-reviewed)abstract
- Diabetes frequently develops in Huntington's disease (HD) patients and in transgenic mouse models of HD such as the R6/2 mouse. The underlying mechanisms have not been clarified. Elucidating the pathogenesis of diabetes in HD would improve our understanding of the molecular mechanisms involved in HD neuropathology. With this aim, we examined our colony of R6/2 mice with respect to glucose homeostasis and islet function. At week 12, corresponding to end-stage HD, R6/2 mice were hyperglycemic and hypoinsulinemic and failed to release insulin in an intravenous glucose tolerance test. In vitro, basal and glucose-stimulated insulin secretion was markedly reduced. Islet nuclear huntingtin inclusions increased dramatically over time, predominantly in ß-cells. ß-cell mass failed to increase normally with age in R6/2 mice. Hence, at week 12, ß-cell mass and pancreatic insulin content in R6/2 mice were 35±5 and 16±3% of that in wild-type mice, respectively. The normally occurring replicating cells were largely absent in R6/2 islets, while no abnormal cell death could be detected. Single cell patch-clamp experiments revealed unaltered electrical activity in R6/2 ß-cells. However, exocytosis was virtually abolished in ß- but not in {alpha}-cells. The blunting of exocytosis could be attributed to a 96% reduction in the number of insulin-containing secretory vesicles. Thus, diabetes in R6/2 mice is caused by a combination of deficient ß-cell mass and disrupted exocytosis.
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| 2. |
- Cheviet, Severine, et al.
(author)
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Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis
- 2006
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 119:14, s. 2912-2920
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Journal article (peer-reviewed)abstract
- Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237 +/- 13 versus 279 +/- 3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.
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| 3. |
- Granhall, Charlotte, et al.
(author)
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Separately inherited defects in insulin exocytosis and beta-cell glucose metabolism contribute to type 2 diabetes.
- 2006
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In: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 55:12, s. 3494-3500
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Journal article (peer-reviewed)abstract
- The effects of genetic variation on molecular functions predisposing to type 2 diabetes are still largely unknown. Here, in a specifically designed diabetes model, we couple separate gene loci to mechanisms of P-cell pathology. Niddm1i is a major glucose-controlling 16-Mb region in the diabetic GK rat that causes defective insulin secretion and corresponds to loci in humans and mice associated with type 2 diabetes. Generation of a series of congenic rat strains harboring different parts of GK-derived Niddm1i enabled fine mapping of this locus. Congenic strains carrying the GK genotype distally in Niddm1i displayed reduced insulin secretion in response to both glucose and high potassium, as well as decreased single-cell exocytosis. By contrast, a strain carrying the GK genotype proximally in Niddm1i exhibited both intact insulin release in response to high potassium and intact single-cell exocytosis, but insulin secretion was suppressed when stimulated by glucose. Islets from this strain also failed to respond to glucose by increasing the cellular ATP-to-ADP ratio. Changes in P-cell mass did not contribute to the secretory defects. We conclude that the failure of insulin secretion in type 2 diabetes includes distinct functional defects in glucose metabolism and insulin exocytosis of the P-cell and that their genetic fundaments are encoded by different loci within Niddm1i.
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| 4. |
- Holmkvist, Johan, et al.
(author)
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Polymorphisms in the gene encoding the voltage-dependent Ca(2+) channel Ca (V)2.3 (CACNA1E) are associated with type 2 diabetes and impaired insulin secretion
- 2007
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In: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 50:12, s. 2467-2475
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Journal article (peer-reviewed)abstract
- AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion is dependent on the electrical activity of beta cells; hence, genes encoding beta cell ion channels are potential candidate genes for type 2 diabetes. The gene encoding the voltage-dependent Ca(2+) channel Ca(V)2.3 (CACNA1E), telomeric to a region that has shown suggestive linkage to type 2 diabetes (1q21-q25), has been ascribed a role for second-phase insulin secretion. METHODS: Based upon the genotyping of 52 haplotype tagging single nucleotide polymorphisms (SNPs) in a type 2 diabetes case-control sample (n = 1,467), we selected five SNPs that were nominally associated with type 2 diabetes and genotyped them in the following groups (1) a new case-control sample of 6,570 individuals from Sweden; (2) 2,293 individuals from the Botnia prospective cohort; and (3) 935 individuals with insulin secretion data from an IVGTT. RESULTS: The rs679931 TT genotype was associated with (1) an increased risk of type 2 diabetes in the Botnia case-control sample [odds ratio (OR) 1.4, 95% CI 1.0-2.0, p = 0.06] and in the replication sample (OR 1.2, 95% CI 1.0-1.5, p = 0.01 one-tailed), with a combined OR of 1.3 (95% CI 1.1-1.5, p = 0.004 two-tailed); (2) reduced insulin secretion [insulinogenic index at 30 min p = 0.02, disposition index (D (I)) p = 0.03] in control participants during an OGTT; (3) reduced second-phase insulin secretion at 30 min (p = 0.04) and 60 min (p = 0.02) during an IVGTT; and (4) reduced D (I) over time in the Botnia prospective cohort (p = 0.05). CONCLUSIONS/INTERPRETATION: We conclude that genetic variation in the CACNA1E gene contributes to an increased risk of the development of type 2 diabetes by reducing insulin secretion.
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| 5. |
- Ivarsson, Rosita, et al.
(author)
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Myosin 5a controls insulin granule recruitment during late-phase secretion.
- 2005
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In: Traffic: the International Journal of Intracellular Transport. - : Wiley. - 1398-9219. ; 6:11, s. 1027-1035
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Journal article (peer-reviewed)abstract
- We have examined the importance of the actin-based molecular motor myosin 5a for insulin granule transport and insulin secretion. Expression of myosin 5a was downregulated in clonal INS-1E cells using RNAinterference. Stimulated hormone secretion was reduced by 46% and single-cell exocytosis, measured by capacitance recordings, was inhibited by 42% after silencing. Silencing of Slac-2c/MYRIP, which links insulin granules to myosin 5a, resulted in similar inhibition of single-cell exocytosis. Antibody inhibition of the myosin 5a-Slac-2c/MYRIP interaction significantly reduced the recruitment of insulin granules for release. The pool of releasable granules independent of myosin 5a activity was estimated to approximately 550 granules. Total internal reflection microscopy was then applied to directly investigate granule recruitment to the plasma membrane. Silencing of myosin 5a inhibited granule recruitment during late phase of insulin secretion. In conclusion, we propose a model where insulin granules are transported through the actin network via both myosin 5a-mediated transport and via passive diffusion, with the former playing the major role during stimulatory conditions.
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| 6. |
- Ivarsson, Rosita, et al.
(author)
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Redox control of exocytosis - Regulatory role of NADPH, thioredoxin, and glutaredoxin
- 2005
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In: Diabetes. - 1939-327X. ; 54:7, s. 2132-2142
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Journal article (peer-reviewed)abstract
- Cellular redox state is an important metabolic variable, influencing many aspects of cell function like growth, apoptosis, and reductive biosynthesis. In this report, we identify NADPH as a candidate signaling molecule for exocytosis in neuroendocrine cells. In pancreatic beta-cells, glucose acutely raised the NADPH-to-NADP(+) ratio and stimulated insulin release in parallel. Furthermore, intracellular addition of NADPH directly stimulated exocytosis of insulin granules. Effects of NADPH on exocytosis are proposed to be mediated by the redox proteins glutaredoxin (GRX) and thioredoxin (TRX) on the basis of the following evidence: 1) Expression of GRX mRNA is very high in beta-cells compared with other studied tissues, and GRX protein expression is high in islets and in brain; 2) GRX and TRX are localized in distinct microdomains in the cytosol of beta-cells; and 3) microinjection of recombinant GRX potentiated effects of NADPH on exocytosis, whereas TRX antagonized the NADPH effect. We propose that the NADPEVGRX/ TRX redox regulation mediates a novel signaling pathway of nutrient-induced insulin secretion.
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| 7. |
- Jing, Xingjun, et al.
(author)
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CaV2.3 calcium channels control second-phase insulin release.
- 2005
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In: Journal of Clinical Investigation. - 0021-9738. ; 115:1, s. 146-154
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Journal article (peer-reviewed)abstract
- Concerted activation of different voltage-gated Ca2+ channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type CaV2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in CaV2.3-knockout (CaV2.3–/–) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca2+ channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological CaV2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca2+ signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single CaV2.3–/– ß cells. CaV2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the CaV2.3–/– mouse. We propose a specific role for CaV2.3 Ca2+ channels in second-phase insulin release, that of mediating the Ca2+ entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.
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| 8. |
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| 9. |
- Krus, Ulrika, et al.
(author)
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Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in the ATP:ADP ratio in rat pancreatic islets.
- 2006
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In: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 49:7, s. 1578-1586
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Journal article (peer-reviewed)abstract
- AIMS/HYPOTHESIS: The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion. METHODS: Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K(+) channel (K(ATP)-channel) conductance. RESULTS: We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca(2+)-induced exocytosis and K(ATP)-channel conductance in single cells, were unaffected. Insulin secretion induced by alpha-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K(ATP)-independent actions of fuel secretagogues are operational, as well as the first and K(ATP)-dependent phase. CONCLUSIONS/INTERPRETATION: Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.
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| 10. |
- Li, Dai-Qing, et al.
(author)
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Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3.
- 2009
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In: Cell metabolism. - : Elsevier BV. - 1932-7420 .- 1550-4131. ; 10:4, s. 309-15
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Journal article (peer-reviewed)abstract
- Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.
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