UCP2 Regulates the Glucagon Response to Fasting and Starvation

Glucagon is important for maintaining euglycemia during fasting/starvation, and abnormal glucagon secretion is associated with type 1 and type 2 diabetes; however, the mechanisms of hypoglycemia-induced glucagon secretion are poorly understood. We previously demonstrated that global deletion of mitochondrial uncoupling protein 2 (UCP2−/−) in mice impaired glucagon secretion from isolated islets. Therefore, UCP2 may contribute to the regulation of hypoglycemia-induced glucagon secretion, which is supported by our current finding that UCP2 expression is increased in nutrient-deprived murine and human islets. Further to this, we created α-cell–specific UCP2 knockout (UCP2AKO) mice, which we used to demonstrate that blood glucose recovery in response to hypoglycemia is impaired owing to attenuated glucagon secretion. UCP2-deleted α-cells have higher levels of intracellular reactive oxygen species (ROS) due to enhanced mitochondrial coupling, which translated into defective stimulus/secretion coupling. The effects of UCP2 deletion were mimicked by the UCP2 inhibitor genipin on both murine and human islets and also by application of exogenous ROS, confirming that changes in oxidative status and electrical activity directly reduce glucagon secretion. Therefore, α-cell UCP2 deletion perturbs the fasting/hypoglycemic glucagon response and shows that UCP2 is necessary for normal α-cell glucose sensing and the maintenance of euglycemia

Characterization of Zinc Influx Transporters (ZIPs) in pancreatic beta cells: roles in regulating cytosolic zinc homeostasis and insulin secretion

Zinc plays an essential role in the regulation of pancreatic beta cell function, affecting important processes including proinsulin biosynthesis, glucose-stimulated insulin secretion, and cell viability. Mutations in zinc efflux transport protein ZnT8, have been linked with both type 1 and type 2 diabetes, further supporting an important role for zinc in glucose homeostasis. However, very little is known about how cytosolic zinc is controlled by zinc influx proteins (ZIPs). In the current study, we have examined the beta cell and islet ZIP transcriptome and show consistent high expression of ZIP6 (Slc39a6) and ZIP7 (Slc39a7) genes across human, mouse islets and MIN6 beta cells. Modulation of ZIP6 and ZIP7 expression significantly altered cytosolic zinc influx in pancreatic beta cells, indicating an important role for ZIP6 and ZIP7 in regulating cellular zinc homeostasis. Functionally, this deregulated cytosolic zinc homeostasis led to impaired insulin exocytosis and insulin secretion. In parallel studies, we identified both ZIP6 and ZIP7 as potential interacting proteins with GLP-1R by a membrane yeast-two-hybrid (MYTH) assay. Knock-down of ZIP6 but not ZIP7 in MIN6 beta cells impaired the protective effects of GLP-1 on fatty acid-induced cell death possibly via reduced p-ERK pathway. Thus, our data suggests that ZIP6 and ZIP7 function as two important zinc influx transporters to regulate cytosolic zinc concentrations and insulin secretion in beta cells. In particular, ZIP6 is also capable of directly interacting with GLP-1R to facilitate the protective effect of GLP-1 on beta cell survival

Zip4 mediated zinc influx stimulates insulin secretion in pancreatic beta cells.

Zinc has an important role in normal pancreatic beta cell physiology as it regulates gene transcription, insulin crystallization and secretion, and cell survival. Nevertheless, little is known about how zinc is transported through the plasma membrane of beta cells and which of the class of zinc influx transporters (Zip) is involved. Zip4 was previously shown to be expressed in human and mouse beta cells; however, its function there is still unknown. Therefore, the aim of this study was to define the zinc transport role of Zip4 in beta cells. To investigate this, Zip4 was over-expressed in MIN6 beta cells using a pCMV6-Zip4GFP plasmid. Organelle staining combined with confocal microscopy showed that Zip4 exhibits a widespread localization in MIN6 cells. Time-lapse zinc imaging experiments showed that Zip4 increases cytoplasmic zinc levels. This resulted in increased granular zinc content and glucose-stimulated insulin secretion. Interestingly, it is unlikely that the increased glucose stimulated insulin secretion was triggered by a modulation of mitochondrial function, as mitochondrial membrane potential remained unchanged. To define the role of Zip4 in-vivo, we generated a beta cell-specific knockout mouse model (Zip4BKO). Deletion of the Zip4 gene was confirmed in Zip4BKO islets by PCR, RT-PCR, and immuno-histochemistry. Zip4BKO mice showed slightly improved glucose homeostasis but no change in insulin secretion during an oral glucose tolerance test. While Zip4 was not found to be essential for proper glucose homeostasis and insulin secretion in vivo in mice, this study also found that Zip4 mediates increases in cytoplasmic and granular zinc pools and stimulates glucose dependant insulin secretion in-vitro

Sympathetic tone dictates the impact of lipolysis on FABP4 secretion

Levels of circulating fatty acid binding protein 4 (FABP4) protein are strongly associated with obesity and metabolic disease in both mice and humans, and secretion is stimulated by β-adrenergic stimulation both in vivo and in vitro. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of adipose triglyceride lipase (ATGL) and was absent from adipose tissue explants from mice specifically lacking ATGL in their adipocytes (ATGLAdpKO). Here, we find that upon activation of β-adrenergic receptors in vivo, ATGLAdpKO mice unexpectedly exhibited significantly higher levels of circulating FABP4 as compared with ATGLfl/fl controls, despite no corresponding induction of lipolysis. We generated an additional model with adipocyte-specific deletion of both FABP4 and ATGL (ATGL/FABP4AdpKO) to evaluate the cellular source of this circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the source of elevated FABP4 levels in ATGLAdpKO mice was indeed from the adipocytes. ATGLAdpKO mice exhibited significantly elevated corticosterone levels, which positively correlated with plasma FABP4 levels. Pharmacological inhibition of sympathetic signaling during lipolysis using hexamethonium or housing mice at thermoneutrality to chronically reduce sympathetic tone significantly reduced FABP4 secretion in ATGLAdpKO mice compared with controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for in vivo stimulation of FABP4 secretion from adipocytes, which can be induced through sympathetic signaling

Deletion of Zip4 in Zip4BKO mice.

<p>A. PCR results from skin, liver and islet DNA samples showing the wild type (WT), LoxP/LoxP and recombined Zip4 allele. The Zip4 WT allele (187bp) is present in all the different sample of the control RipCre mouse. In the Zip4BKO, LoxP/LoxP Zip4 allele (227bp) is present in all the tissues. Islet DNA of Zip4BKO displays recombined Zip4 gene (400bp) which shows effectiveness and specificity of the Cre lox recombination system. B. qPCR of Zip4 from control RipCre and Zip4BKO isolated islets. C. qPCR was using the ileum as a positive control. D&E immunohistochemistry experiments were performed on dispersed islet cells to co-stain for insulin and Zip4 (D) and glucagon and Zip4 (E). F. Dithizone staining and its quantification in RipCre and Zip4BKO islets.</p

mRNA expression of zinc transporters in islets of Zip4BKO mice.

<p>Zip family and Znt8 transcript expression in Zip4BKO mouse islets normalized to control RipCre mouse islets.</p

Role of Zip4 in insulin secretion in MIN6 cells.

<p>A. Glucose stimulated insulin secretion (GSIS) from MIN6 transfected with control vector pCMV-eGFP and MIN6 cells overexpressing Zip4 (pCMV6-Zip4GFP) in presence (10 mM G) or absence of glucose (0 mM G). B. Total insulin content. C. Representative images (left panels) and average mitochondrial membrane potential traces (right panel) monitored in MIN6 cells with Rhodamine123 in absence of glucose (0 mM G), 10 mM glucose (10 mM G), and 10 mM glucose and 20 mM sodium azide (NaN3). D. mRNA expression of genes indicated in MIN6 cells.*. p˂0.05; ***. p˂0.001.</p

In-vivo characterization of Zip4BKO mice.

<p>A. Average body weights of 8 week old RipCre and Zip4BKO mice. B. Oral glucose tolerance test in RipCre and Zip4BKO mice and the corresponding area under the blood glucose curve (C). D. Plasma insulin secretion measured during oral glucose tolerance test. *. p˂0.05.</p

Localization of Zip4 in the pancreas.

<p>Bright field images of Zip4 immuno-cytochemistry in mouse pancreatic sections.</p