Coimbatore B. Srikant

Overexpression of Reg3α increases cell growth and the levels of cyclin D1 and CDK4 in insulinoma cells

Regenerating gene (Reg) family protein Reg3α is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet β-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3α cDNA. Determined using real-time PCR and Western blots, the levels of Reg3α mRNA and protein in Reg3α-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3α-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3α-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that β-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced β-cell mass and diabetes. Our results suggest that Reg3α stimulates β-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.

Reg2 protects mouse insulinoma cells from streptozotocin-induced mitochondrial disruption and apoptosis

We reported previously that pancreas-specific ablation of IGF-I in mice induced an increased expression of regenerating family proteins Reg2 and Reg3β in the pancreas and protected them from streptozotocin (Stz)-induced β-cell damage. We, therefore, assessed the effect of ectopically introduced Reg2 on Stz-induced apoptosis in MIN6 mouse insulinoma cells and report here that Reg2 protects MIN6 cells from Stz-induced apoptosis by attenuating its ability to disrupt mitochondrial membrane integrity, activate caspase-3 and promote poly-ADP ribose polymerase cleavage, and induce apoptosis. These changes correlated with suppression of c-jun N-terminal kinase (JNK) phosphorylation by Stz. Reg2 inhibited Stz-induced proapoptotic events as well as the inactivation of JNK. Inclusion of chemical inhibitor of JNK to Reg2 expressing cells rendered them sensitive to Stz. These data demonstrate that Reg2 protects insulin-producing cells against Stz-induced apoptosis by interfering with its cytotoxic signaling upstream of the intrinsic proapoptotic events by preventing its ability to inactivate JNK.

IGF-I stimulates CCN5/WISP2 gene expression in pancreatic β-cells, which promotes cell proliferation and survival against streptozotocin.

IGF-I is normally produced from hepatocytes and other sources, stimulates protein synthesis, cell survival, and proliferation through receptor-mediated activation of phosphatidylinositol 3-kinase and MAPK, and targets specific molecules within the pancreatic islet cells. The current study was designed to identify novel targets that may mediate its pro-islet actions. Whole-genome cDNA microarray analysis in IGF-I-overexpressing islets identified 82 genes specifically up- or down-regulated. Prominent among them was CCN5/WISP2 whose expression was increased 3- and 2-fold at the mRNA and protein levels. Dual-labeled immunofluorescence revealed that CCN5 expression was low in the β-cells of wild-type islets but was significantly induced in response to IGF-I overexpression. In vitro treatment of mouse islets with IGF-I increased both CCN5 mRNA and protein levels significantly. To define the role of CCN5 in islet cell biology, we stably overexpressed its cDNA in insulinoma MIN6 cells and detected a 2-fold increase in the proliferation of MIN6-CCN5 compared with that in control cells, which correlated with significant elevations in the levels of cyclin D1 and the phosphorylation of Akt and Erk2. Moreover, MIN6-CCN5 cells were found to be resistant to streptozotocin-induced cell death. Using confocal microscopy and subcellular fractionation, we found that overexpressed CCN5 exhibited cytoplasmic accumulation upon stimulation by high glucose. Our results indicate that CCN5, which is minimally expressed in islet β-cells, is strongly and directly induced by IGF-I. CCN5 overexpression stimulates the proliferation of insulinoma cells, activates Akt kinase, and inhibits streptozotocin-induced apoptosis, suggesting that increased CCN5 expression contributes to IGF-I-stimulated islet cell growth and/or survival.

Intestinal adaptation and Reg gene expression induced by antidiabetic duodenal-jejunal bypass surgery in Zucker fatty rats

The antidiabetic mechanism of bariatric surgery includes specific changes in the secretion of incretins. To identify additional players originating from the gut, we evaluated the effects of duodenal-jejunal bypass (DJB) in morbidly obese Zucker fatty rats. A fast relief of hyperglycemia and hyperinsulinemia was achieved even before a significant weight loss occurred. Fourteen days after DJB, we characterized the changes in intestinal histochemistry in the bypassed duodenum and shortcut jejunum that was reanastomosed directly to the starting point of the duodenum and compared with the corresponding regions of sham-operated rats. The bypassed duodenum exhibited mucosal atrophy and apoptosis and decreased proliferative renewal. In shortcut jejunum, DJB resulted in 40% significantly enlarged intestinal circumference and increased epithelial proliferation, especially in putative transit-amplifying (TA) cells and the crypt. Because Reg family proteins promote cell growth and survival, we explored their expression in the intestine. With the use of immunohistochemistry, Reg1, -3α, and -3β were normally expressed in intestinal mucosa. After DJB, the level of Reg1 protein was reduced, whereas Reg3α and -3β were not changed in bypassed duodenum. Downstream in shortcut jejunum, the levels of Reg1 and -3β were greatly induced and especially concentrated in the putative TA cells. Our results revealed significant changes in the integrity and proliferation of the intestinal mucosa as a consequence of DJB, and in cell- and isoform-specific expression of Reg proteins within the replicating mucosal epithelium, and provide evidence indicating that the activation of Reg proteins may contribute to intestinal compensation against increased load and/or to improving insulin sensitivity.

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin‐induced apoptosis, we now demonstrate that mReg2 induces glucose‐regulated peptide 78 (GRP78) expression via the Akt–mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2‐induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt–mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.

Comment on: Turban et al. Optimal Elevation of β-Cell 11β-Hydroxysteroid Dehydrogenase Type 1 Is a Compensatory Mechanism That Prevents High-Fat Diet–Induced β-Cell Failure. Diabetes 2012;61:642–652

Turban et al. (1) recently reported a surprise finding that moderately elevated 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression in the pancreatic β-cells promoted a compensation against high-fat (HF) diet–induced β-cell failure because glucocorticoids are well established to impair insulin secretion and cause β-cell death and insulin resistance in key insulin targets. Rather than accepting the …

Decreased 11β-Hydroxysteroid Dehydrogenase 1 Level and Activity in Murine Pancreatic Islets Caused by Insulin-Like Growth Factor I Overexpression

We have reported a high expression of IGF-I in pancreatic islet β-cells of transgenic mice under the metallothionein promoter. cDNA microarray analysis of the islets revealed that the expression of 82 genes was significantly altered compared to wild-type mice. Of these, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which is responsible for the conversion of inert cortisone (11-dehydrocorticosterone, DHC in rodents) to active cortisol (corticosterone) in the liver and adipose tissues, has not been identified previously as an IGF-I target in pancreatic islets. We characterized the changes in its protein level, enzyme activity and glucose-stimulated insulin secretion. In freshly isolated islets, the level of 11β-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence, 11β-HSD1 was observed exclusively in glucagon-producing, islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced enzymatic activities. Dexamethasone (DEX) and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice, 48-h pre-incubation of DEX caused a significant decrease in insulin release, while the effect of DHC was largely blunted consistent with diminished 11β-HSD1 activity. In order to establish the function of intracrine glucocorticoids, we overexpressed 11β-HSD1 cDNA in MIN6 insulinoma cells, which together with DHC caused apoptosis and a significant decrease in proliferation. Both effects were abolished with the treatment of an 11β-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11β-HSD1 expression and activity within the pancreatic islets, which may mediate part of the IGF-I effects on cell proliferation, survival and insulin secretion.

Somatostatin Receptor Subtype Selectivity for Cytotoxic and Cytostatic Signaling

Somatostatin (SST), originally identified as a hypothalamic peptide inhibitor of growth hormone secretion, was subsequently shown to be widely distributed, to exist in two biologically active forms (SST-14 and SST-28) and to function as an inhibitor of secretion of almost every hormone and growth factor. Initially all the cellular effects of SST were viewed as the consequence of its ability to inhibit hormonal secretion. This notion became untenable with the realization that SST could directly inhibit tumor cell proliferation as initially demonstrated by Liebow et al., [1] and confirmed by numerous subsequent studies that have been reviewed earlier [2-7]. Today SST is best regarded as a unique, pleuripotent, hormone which modulates the functions of central and peripheral nervous systems, exocrine and endocrine organs as well as immune and vascular systems to influence such diverse functions as secretion, motility, cell growth and proliferation. A growing body of evidence has now clearly established that the direct antiproliferative action of SST in tumor cells can induce cytotoxic signals to promote apoptotic cell death or activate signals promoting cytostatic growth arrest. Functional characterization of the five cloned SST receptors (SSTR) have revealed that SST-induced apoptosis and growth arrest occur in a SSTR subtype-selective manner that utilizes tyrosine phosphatase-dependent activation of cytotoxic or cytostatic signaling. This review presents a historical perspective of somatostatin and its receptors, our current understanding of their antiproliferative actions and their clinical relevance.

Somatostatin and its Receptors: Past, Present and the Future

Since its discovery somatostatin (SST) has served as an important hormonal model in understanding the complexities governing the hormone synthesis, processing, secretion and function at a cellular level and its physiology and pathophysiology in health and disease. Initially the distribution, gene expression, processing and physiological actions were defined. Then the heterogeneity and pharmacology of its receptors, as well as the structurefunction characteristics of synthetic agonists of somatostatin receptors (SSTRs) were elucidated. Third, cloning of SSTR subtypes and the attendant explosion of activity in characterizing the molecular biological and pharmacological properties of the SSTR subtypes contributed to a further refinement of our understanding of the biology of SST and SSTRs. Fourth, animal models lacking SST or individual SSTR subtypes generated through gene knockout strategies have enabled the assessment of their physiological importance. The preceding chapters in this book have discussed these issues in detail. The landmark developments in this field (Table 1) have helped change the initial perspective of SST as an endocrine “inhibitory hormone” into one that is an important regulator of embryonic development, neuronal patterning, cell differentiation, proliferation, apoptosis, secretion, motility, and carcinogenic and neoangiogenic processes. Here I reflect on the progress made to date in this field and consider the challenges and promises that lie ahead.