Palmitate inhibits insulin gene expression by altering PDX-1 nuclear localization and reducing MafA expression in isolated rat islets of Langerhans.

Abnormalities in lipid metabolism have been proposed as contributing factors to both defective insulin secretion from the pancreatic beta cell and peripheral insulin resistance in type 2 diabetes. Previously, we have shown that prolonged exposure of isolated rat islets of Langerhans to excessive fatty acid levels impairs insulin gene transcription. This study was designed to assess whether palmitate alters the expression and binding activity of the key regulatory factors pancreas-duodenum homeobox-1 (PDX-1), MafA, and Beta2, which respectively bind to the A3, C1, and E1 elements in the proximal region of the insulin promoter. Nuclear extracts of isolated rat islets cultured with 0.5 mm palmitate exhibited reduced binding activity to the A3 and C1 elements but not the E1 element. Palmitate did not affect the overall expression of PDX-1 but reduced its nuclear localization. In contrast, palmitate blocked the stimulation of MafA mRNA and protein expression by glucose. Combined adenovirus-mediated overexpression of PDX-1 and MafA in islets completely prevented the inhibition of insulin gene expression by palmitate. These results demonstrate that prolonged exposure of islets to palmitate inhibits insulin gene transcription by impairing nuclear localization of PDX-1 and cellular expression of MafA.

Syncollin inhibits regulated corticotropin secretion from AtT-20 cells through a reduction in the secretory vesicle population.

Syncollin is a 13 kDa protein that is highly expressed in the exocrine pancreas. Syncollin normally exists as a doughnut-shaped homo-oligomer (quite probably a hexamer) in close association with the luminal surface of the zymogen granule membrane. In the present study, we examine the effect of expression of syncollin in AtT-20 neuroendocrine cells, which do not normally express this protein. Efficient expression was achieved by infection of the cells with adenoviral constructs encoding either untagged or GFP (green fluorescent protein)-tagged syncollin. Both forms of the protein were sorted into corticotropin (ACTH)-positive secretory vesicles present mainly at the tips of cell processes. Neither form affected basal corticotropin secretion or the constitutive secretion of exogenously expressed secreted alkaline phosphatase. In contrast, regulated secretion of corticotropin was inhibited (by 49%) by untagged but not by GFP-tagged syncollin. In parallel, untagged syncollin caused a 46% reduction in the number of secretory vesicles present at the tips of the cell processes. Syncollin-GFP was without effect. We could also show that native syncollin purified from rat pancreas was capable of permeabilizing erythrocytes. We suggest that syncollin may induce uncontrolled permeabilization of corticotropin-containing vesicles and subsequently destabilize them. Both forms of syncollin were tightly membrane-associated and appeared to exist as homooligomers. Hence, the lack of effect of syncollin-GFP on regulated exocytosis suggests that the GFP tag interferes in a subtler manner with the properties of the assembled protein.

Insulin secretory deficiency and glucose intolerance in Rab3A null mice.

Insulin secretory dysfunction of the pancreatic beta-cell in type-2 diabetes is thought to be due to defective nutrient sensing and/or deficiencies in the mechanism of insulin exocytosis. Previous studies have indicated that the GTP-binding protein, Rab3A, plays a mechanistic role in insulin exocytosis. Here, we report that Rab3A(-/-) mice develop fasting hyperglycemia and upon a glucose challenge show significant glucose intolerance coupled to ablated first-phase insulin release and consequential insufficient insulin secretion in vivo, without insulin resistance. The in vivo insulin secretory response to arginine was similar in Rab3A(-/-) mice as Rab3A(+/+) control animals, indicating a phenotype reminiscent of insulin secretory dysfunction found in type-2 diabetes. However, when a second arginine dose was given 10 min after, there was a negligible insulin secretory response in Rab3A(-/-) mice, compared with that in Rab3A(+/+) animals, that was markedly increased above that to the first arginine stimulus. There was no difference in beta-cell mass or insulin production between Rab3A(-/-) and Rab3A(+/+) mice. However, in isolated islets, secretagogue-induced insulin release (by glucose, GLP-1, glyburide, or fatty acid) was approximately 60-70% lower in Rab3A(-/-) islets compared with Rab3A(+/+) controls. Nonetheless, there was a similar rate of glucose oxidation and glucose-induced rise in cytosolic [Ca(2+)](i) flux between Rab3A(-/-) and Rab3A(+/+) islet beta-cells, indicating the mechanistic role of Rab3A lies downstream of generating secondary signals that trigger insulin release, at the level of secretory granule transport and/or exocytosis. Thus, Rab3A plays an important in vivo role facilitating the efficiency of insulin exocytosis, most likely at the level of replenishing the ready releasable pool of beta-granules. Also, this study indicates, for the first time, that the in vivo insulin secretory dysfunction found in type-2 diabetes can lie solely at the level of defective insulin exocytosis.