Preserving Insulin Secretion in Diabetes by Inhibiting VDAC1 Overexpression and Surface Translocation in ß Cells.

Type 2 diabetes (T2D) develops after years of prediabetes during which high glucose (glucotoxicity) impairs insulin secretion. We report that the ATP-conducting mitochondrial outer membrane voltage-dependent anion channel-1 (VDAC1) is upregulated in islets from T2D and non-diabetic organ donors under glucotoxic conditions. This is caused by a glucotoxicity-induced transcriptional program, triggered during years of prediabetes with suboptimal blood glucose control. Metformin counteracts VDAC1 induction. VDAC1 overexpression causes its mistargeting to the plasma membrane of the insulin-secreting ß cells with loss of the crucial metabolic coupling factor ATP. VDAC1 antibodies and inhibitors prevent ATP loss. Through direct inhibition of VDAC1 conductance, metformin, like specific VDAC1 inhibitors and antibodies, restores the impaired generation of ATP and glucose-stimulated insulin secretion in T2D islets. Treatment of db/db mice with VDAC1 inhibitor prevents hyperglycemia, and maintains normal glucose tolerance and physiological regulation of insulin secretion. Thus, ß cell function is preserved by targeting the novel diabetes executer protein VDAC1.

Mitochondrial proteome analysis reveals altered expression of voltage dependent anion channels in pancreatic ß-cells exposed to high glucose.

Chronic hyperglycemia leads to deterioration of insulin release from pancreatic ß-cells as well as insulin action on peripheral tissues. However, the mechanism underlying ß-cell dysfunction resulting from glucose toxicity has not been fully elucidated. The aim of the present study was to define a set of alterations in mitochondrial protein profiles of pancreatic ß-cell line in response to glucotoxic condition using 2-DE and tandem mass spectrometry. INS1E cells were incubated in the presence of 5.5 and 20 mM glucose for 72 hrs and mitochondria were isolated. Approximately 75 protein spots displayed significant changes (p < 0.05) in relative abundance in the presence of 20 mM glucose compared to controls. Mitochondrial proteins down regulated under glucotoxic conditions includes ATP synthase α chain and δ chain, malate dehydrogenase, aconitase, trifunctional enzyme ß subunit, NADH cytochrome b5 reductase and voltage-dependent anion-selective channel protein (VDAC) 2. VDAC1, 75 kDa glucose-regulated protein, heat shock protein (HSP) 60 and HSP10 were found to be upregulated. The orchestrated changes in expression of VDACs and multiple other proteins involved in nutrient metabolism, ATP synthesis, cellular defense, glycoprotein folding and mitochondrial DNA stability may explain cellular dysfunction in glucotoxicity resulting in altered insulin secretion.

Proteomics and islet research.

The complementary disciplines of genomics and proteomics offer better insights into the molecular mechanisms of diseases. While genomics hunts for defining our static genetic substrate, proteomics explores the structure and function of proteins expressed by a cell or tissue type under specified conditions. In the past decade, proteomics has been revolutionized by the application of techniques such as two-dimensional gel electrophoresis (2DGE), mass spectrometry (MS), and protein arrays. These techniques have tremendous potential for biomarker development, target validation, diagnosis, prognosis, and optimization of treatment in medical care, especially in the field of islet and diabetes research. This chapter will highlight the contributions of proteomic technologies toward the dissection of complex network of signaling molecules regulating islet function, the identification of potential biomarkers, and the understanding of mechanisms involved in the pathogenesis of diabetes.

Proteomic analysis of human adipose tissue after rosiglitazone treatment shows coordinated changes to promote glucose uptake.

The aim of this study was to identify potential protein targets for insulin sensitization in human adipose tissue using unbiased proteomic approaches. Ten moderately obese, but otherwise healthy, subjects were treated with rosiglitazone 4 mg b.i.d. for 14 days and global protein and gene expression changes were monitored. Proteomic analysis revealed distinct up- or downregulation (greater than twofold) in 187 protein spots on the two-dimensional (2-D) gel images between day 0 and day 1 adipose tissue samples. When comparing the protein spots on the gels from day 0 with that of 14-day-treated samples, 122 spots showed differential expression. There was a striking increase in the expression of proteins involved in glucose transporter-4 (GLUT4) granule transport and fusion (actin, myosin-9, tubulin, vimentin, annexins, moesin, LIM, and SH3 domain protein-1), signaling (calmodulin, guanine nucleotide-binding proteins), redox regulation (superoxide dismutase, catalase, ferritin, transferrin, heat shock proteins), and adipogenesis (collagens, galectin-1, nidogen-1, laminin, lamin A/C). However, there was an intriguing absence of correlated changes in mRNA expression, suggesting adaptation at a post-transcriptional level in response to rosiglitazone. Thus, the major changes observed were among proteins involved in cytoskeletal rearrangement, insulin and calcium signaling, and inflammatory and redox signals that decisively upregulate GLUT4 granule trafficking in human adipose tissue. Such orchestrated changes in expression of multiple proteins provide insights into the mechanism underlying the increased efficiency in glucose uptake and improvement of insulin sensitivity in response to rosiglitazone treatment.

Modulation of glucose induced oscillatory [Ca2+]i signals by nitric oxide in ob/ob mouse pancreatic beta-cells.

Nitric oxide (NO) has been embroiled in the regulation of insulin secretion from the pancreatic beta cells and hence for the pathophysiology of diabetes mellitus. The present study was undertaken to assess the effects of hydroxylamine, a nitric oxide donor, on Ca(2+) handling in individual mouse pancreatic beta cells. Cytoplasmic Ca(2+) concentration ([Ca(2+)]i) was measured using dual wavelength microfluorometry and the indicator fura 2. In the presence of 3 mM glucose hydroxylamine raised [Ca(2+)]i in 90% of cells and the response was insensitive to methoxyverapamil and also to the intracellular Ca(2+) ATPase inhibitor, thapsigargin. At 11 mM glucose, the [Ca(2+)]i oscillations were abolished by hydroxylamine in a dose dependent manner. The addition of high concentrations of hydroxylamine (100 microM and 1 mM) resulted in a rapid disappearance of the oscillations with suppression of [Ca(2+)]i to near baseline level in a reversible manner. However, 90% of the beta-cells preserved the oscillatory [Ca(2+)]i activity in the presence of 10 microM hydroxylamine. At sustained elevated [Ca(2+)]i, obtained by depolarization with non metabolizable agonist, tolbutamide (1 mM), there was no effect of hydroxylamine; moreover, the inhibitory effects of hydroxylamine was counteracted by tolbutamide, suggesting that the effects of hydroxylamine is mediated by inhibition of metabolism leading to opening of K(+)(ATP) channel and decrease in Ca(2+) influx. When [Ca(2+)]i was maintained at sustained elevated state by at 11 mM glucose in the presence of glucagon, hydroxylamine at lower concentrations (

Mitochondrial protein patterns correlating with impaired insulin secretion from INS-1E cells exposed to elevated glucose concentrations.

Extended hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and eventually beta-cell apoptosis in individuals with type 2 diabetes mellitus. In an attempt to dissect mechanisms behind the detrimental effects of glucose, we focused on measuring changes in expression patterns of mitochondrial proteins. Impaired GSIS was observed from INS-1E cells cultured for 5 days at 20 or 27 mM glucose compared to cells cultured at 5.5 or 11 mM glucose. After culture, mitochondria were isolated from the INS-1E cells by differential centrifugation. Proteins of the mitochondrial fraction were bound to a strong anionic surface (SAX2) protein array and mass spectra generated by SELDI-TOF-MS. Analysis of the spectra revealed proteins with expression levels that correlated with the glucose concentration of the culture medium. Indeed, such differentially expressed proteins created patterns of protein changes, which correlated with impairment of GSIS. In conclusion, the study reveals the first glucose-induced differentially expressed patterns of beta-cell mitochondrial proteins obtained by SELDI-TOF-MS.

Peroxisomal proteomics, a new tool for risk assessment of peroxisome proliferating pollutants in the marine environment.

In an attempt to improve the detection of peroxisome proliferation as a biomarker in environmental pollution assessment, we have applied a novel approach based on peroxisomal proteomics. Peroxisomal proteins from digestive glands of mussels Mytilus galloprovincialis were analyzed using 2-DE and MS. We have generated a reference 2-DE map from samples obtained in a well-studied reference area and compared this with peroxisomal proteomes from other sequenced genomes. In addition, by comparing 2-DE maps from control samples with samples obtained in a polluted area, we have characterized the peroxisome proliferation expression pattern associated with exposure to a polluted environment. Over 100 spots were reproducibly resolved per 2-DE map; 55 differentially expressed spots were quantitatively detected and analyzed, and 14 of these showed an increase in protein expression of more than fourfold. Epoxide hydrolase, peroxisomal antioxidant enzyme, and sarcosine oxidase (SOX) have been identified by ESI MS/MS, and acyl-CoA oxidase, multifunctional protein, and Cu,Zn-superoxide dismutase were immunolocalized by Western blotting. Our results indicate that a peroxisomal protein pattern associated to marine pollutant exposure can be generated, and this approach may have a greater potential as biomarker than traditional, single-protein markers.

Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry.

Completion of the human genome sequence has provided scientists with powerful resources with which to explore the molecular events associated with disease states such as diabetes. Understanding the relative levels of expression of gene products, especially of proteins, and their post-translational modifications will be critical. However, though the pancreatic islets play a key role in glucose homeostasis, global protein expression data in human are decidedly lacking. We here report the two-dimensional protein map and database of human pancreatic islets. A high level of reproducibility was obtained among the gels and a total of 744 protein spots were detected. We have successfully identified 130 spots corresponding to 66 different protein entries and generated a reference map of human islets. The functionally characterized proteins include enzymes, chaperones, cellular structural proteins, cellular defense proteins, signaling molecules, and transport proteins. A number of proteins identified in this study (e.g., annexin A2, elongation factor 1-alpha 2, histone H2B.a/g/k, heat shock protein 90 beta, heat shock 27 kDa protein, cyclophilin B, peroxiredoxin 4, cytokeratins 7, 18, and 19) have not been previously described in the database of mouse pancreatic islets. In addition, altered expression of several proteins, like GRP78, GRP94, PDI, calreticulin, annexin, cytokeratins, profilin, heat shock proteins, and ORP150 have been associated with the development of diabetes. The data presented in this study provides a first-draft reference map of the human islet proteome, that will pave the way for further proteome analysis of pancreatic islets in both healthy and diabetic individuals, generating insights into the pathophysiology of this condition.

Ca2+ handling of rat pancreatic beta-cells exposed to ryanodine, caffeine, and glucagon.

Reported species differences in the stimulus-secretion coupling of insulin release made it important to compare the Ca2+ handling of rat beta-cells with that previously observed in mice. Single beta-cells and small aggregates were prepared from pancreatic islets of Wistar rats, attached to cover slips and then used for measuring the cytoplasmic Ca2+ concentration ([Ca2+]i) with the ratiometric fura-2 technique. Glucose (11 mM) induced slow oscillations of [Ca2+]i similar to those seen in other species, including humans. Comparison of the oscillations in rat beta-cells with those previously described in mouse revealed that there was a slightly lower frequency and an increased tendency to transformation into sustained [Ca2+]i in response to glucagon or caffeine. Ryanodine (5-20 microM) did not affect existing oscillations but sometimes restored rhythmic activity in the presence of caffeine. Stimulation with glucose resulted not only in oscillations but also in transients of [Ca2+]i sometimes appearing in synchrony in adjacent beta-cells and disappearing after the addition of 200 nM thapsigargin or 20 mM caffeine. The frequency of transients recorded in a medium containing glucagon and methoxyverapamil was higher than seen under similar conditions in mouse beta-cells. Although exhibiting some differences compared with mouse beta-cells, rat beta-cells also have an intrinsic ability to oscillate and to generate the transients of [Ca2+] that are supposed to synchronize the rhythmicity of the islets in the pancreas.