Weiping Han

Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice

Vertebrates express at least 15 different synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 synaptotagmins, however, have not been established. By analogy to the role of synaptotagmin-1, -2, and -9 in neurotransmission, these other synaptotagmins may serve as Ca2+ transducers regulating other Ca2+-dependent membrane processes, such as insulin secretion in pancreatic β-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic β-cells. To determine whether synaptotagmin-7 regulates Ca2+-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic β cells.

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas.

Hormones such as glucagon are secreted by Ca2+‐induced exocytosis of large dense‐core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic β‐cells secreting insulin revealed that synaptotagmin‐7 alone is not sufficient to mediate Ca2+‐dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin‐1 and ‐7 collaborate as Ca2+ sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca2+ sensors in large dense‐core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin‐7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca2+‐triggered glucagon release from α‐cells than insulin secretion from β‐cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca2+‐triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin‐7, nearly abolished Ca2+‐triggered glucagon secretion. Moreover, single‐cell capacitance measurements confirmed that pancreatic α‐cells lacking synaptotagmin‐7 exhibited little Ca2+‐induced exocytosis, whereas all other physiological and morphological parameters of the α‐cells were normal. Our data thus identify synaptotagmin‐7 as a principal Ca2+ sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca2+ sensors in neurotransmitter, neuropeptide, and hormone secretion.

Wip1-Dependent Regulation of Autophagy, Obesity, and Atherosclerosis

Obesity and atherosclerosis-related diseases account for over one-third of deaths in the western world. Controlling these conditions remains a major challenge due to an incomplete understanding of the molecular pathways involved. Here, we show that Wip1 phosphatase, a known negative regulator of Atm-dependent signaling, plays a major role in controlling fat accumulation and atherosclerosis in mice; specifically, Wip1 deficiency prevents both conditions. In the course of atherosclerosis, deletion of Wip1 results in suppression of macrophage conversion into foam cells, thus preventing the formation of atherosclerotic plaques. This process appears to be independent of p53 but rely on a noncanonical Atm-mTOR signaling pathway and on selective autophagy in regulation of cholesterol efflux. We propose that the Wip1-dependent control of autophagy and cholesterol efflux may provide avenues for treating obesity and atherosclerosis.

Calcium-sensing beyond neurotransmitters: functions of synaptotagmins in neuroendocrine and endocrine secretion.

Neurotransmitters, neuropeptides and hormones are released through the regulated exocytosis of SVs (synaptic vesicles) and LDCVs (large dense-core vesicles), a process that is controlled by calcium. Synaptotagmins are a family of type 1 membrane proteins that share a common domain structure. Most synaptotagmins are located in brain and endocrine cells, and some of these synaptotagmins bind to phospholipids and calcium at levels that trigger regulated exocytosis of SVs and LDCVs. This led to the proposed synaptotagmin-calcium-sensor paradigm, that is, members of the synaptotagmin family function as calcium sensors for the regulated exocytosis of neurotransmitters, neuropeptides and hormones. Here, we provide an overview of the synaptotagmin family, and review the recent mouse genetic studies aimed at understanding the functions of synaptotagmins in neurotransmission and endocrine-hormone secretion. Also, we discuss potential roles of synaptotagmins in non-traditional endocrine systems.

FoxO1 inhibits leptin regulation of pro-opiomelanocortin promoter activity by blocking STAT3 interaction with specificity protein 1.

Leptin controls food intake and energy expenditure by regulating hypothalamic neuron activities. Leptin exerts its actions through complex signaling pathways including STAT3 phosphorylation, nuclear translocation, and binding to target gene promoter/cofactor complexes. Deficient or defective leptin signaling leads to obesity, which may be caused by insufficient leptin levels and/or resistance to leptin signaling. To understand the molecular mechanisms of leptin resistance, we studied the regulation of pro-opiomelanocortin (POMC) gene expression by leptin. We show that phospho-STAT3 activates POMC promoter in response to leptin signaling through a mechanism that requires an SP1-binding site in the POMC promoter. Furthermore, FoxO1 binds to STAT3 and prevents STAT3 from interacting with the SP1·POMC promoter complex, and consequently, inhibits STAT3-mediated leptin action. Our study suggests that leptin action could be inhibited at a step downstream of STAT3 phosphorylation and nuclear translocation, and provides a potential mechanism of leptin resistance in which an increased FoxO1 antagonizes STAT3-mediated leptin signaling.

Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte

Astrocytes release a variety of signaling molecules including glutamate, d-serine, and ATP in a regulated manner. Although the functions of these molecules, from regulating synaptic transmission to controlling specific behavior, are well documented, the identity of their cellular compartment(s) is still unclear. Here we set out to study vesicular exocytosis and glutamate release in mouse hippocampal astrocytes. We found that small vesicles and lysosomes coexisted in the same freshly isolated or cultured astrocytes. Both small vesicles and lysosome fused with the plasma membrane in the same astrocytes in a Ca2+-regulated manner, although small vesicles were exocytosed more efficiently than lysosomes. Blockade of the vesicle glutamate transporter or cleavage of synaptobrevin 2 and cellubrevin (both are vesicle-associated membrane proteins) with a clostridial toxin greatly inhibited glutamate release from astrocytes, while lysosome exocytosis remained intact. Thus, both small vesicles and lysosomes contribute to Ca2+-dependent vesicular exocytosis, and small vesicles support glutamate release from astrocytes.

Identification of Specific Cell-Surface Markers of Adipose-Derived Stem Cells from Subcutaneous and Visceral Fat Depots

Summary Adipose-derived stem/stromal cells (ASCs) from the anatomically distinct subcutaneous and visceral depots of white adipose tissue (WAT) differ in their inherent properties. However, little is known about the molecular identity and definitive markers of ASCs from these depots. In this study, ASCs from subcutaneous fat (SC-ASCs) and visceral fat (VS-ASCs) of omental region were isolated and studied. High-content image screening of over 240 cell-surface markers identified several potential depot-specific markers of ASCs. Subsequent studies revealed consistent predominant expression of CD10 in SC-ASCs and CD200 in VS-ASCs across 12 human subjects and in mice. CD10-high-expressing cells sorted from SC-ASCs differentiated better than their CD10-low-expressing counterparts, whereas CD200-low VS-ASCs differentiated better than CD200-high VS-ASCs. The expression of CD10 and CD200 is thus depot-dependent and associates with adipogenic capacities. These markers will offer a valuable tool for tracking and screening of depot-specific stem cell populations.

In Vivo hyperpolarized carbon‐13 magnetic resonance spectroscopy reveals increased pyruvate carboxylase flux in an insulin‐resistant mouse model

The pathogenesis of type 2 diabetes is characterized by impaired insulin action and increased hepatic glucose production (HGP). Despite the importance of hepatic metabolic aberrations in diabetes development, there is currently no molecular probe that allows measurement of hepatic gluconeogenic pathways in vivo and in a noninvasive manner. In this study, we used hyperpolarized carbon 13 (13C)‐labeled pyruvate magnetic resonance spectroscopy (MRS) to determine changes in hepatic gluconeogenesis in a high‐fat diet (HFD)‐induced mouse model of type 2 diabetes. Compared with mice on chow diet, HFD‐fed mice displayed higher levels of oxaloacetate, aspartate, and malate, along with increased 13C label exchange rates between hyperpolarized [1‐13C]pyruvate and its downstream metabolites, [1‐13C]malate and [1‐13C]aspartate. Biochemical assays using liver extract revealed up‐regulated malate dehydrogenase activity, but not aspartate transaminase activity, in HFD‐fed mice. Moreover, the 13C label exchange rate between [1‐13C]pyruvate and [1‐13C]aspartate (kpyr‐>asp) exhibited apparent correlation with gluconeogenic pyruvate carboxylase (PC) activity in hepatocytes. Finally, up‐regulated HGP by glucagon stimulation was detected by an increase in aspartate signal and kpyr‐>asp, whereas HFD mice treated with metformin for 2 weeks displayed lower production of aspartate and malate, as well as reduced kpyr‐>asp and 13C‐label exchange rate between pyruvate and malate, consistent with down‐regulated gluconeogenesis. Conclusion: Taken together, we demonstrate that increased PC flux is an important pathway responsible for increased HGP in diabetes development, and that pharmacologically induced metabolic changes specific to the liver can be detected in vivo with a hyperpolarized 13C‐biomolecular probe. Hyperpolarized 13C MRS and the determination of metabolite exchange rates may allow longitudinal monitoring of liver function in disease development. (HEPATOLOGY 2013)

Reduced body weight and increased energy expenditure in transgenic mice over-expressing soluble leptin receptor.

Background Soluble leptin receptor (OBRe), one of several leptin receptor isoforms, is the only bona fide leptin binding protein in plasma. Our earlier studies demonstrated that OBRe modulates leptin levels in circulation. Both clinical and in vitro studies have shown that OBRe expression is inversely correlated to body weight and leptin levels. However, it is not clear whether OBRe plays an active role, either in collaboration with leptin or independently, in the maintenance of body weight. Methodology/Principal Findings To investigate the function of OBRe in the regulation of energy homeostasis, we generated transgenic mice that express OBRe under the control of human serum amyloid P (hSAP) component gene promoter. The transgene led to approximately doubling of OBRe in circulation in the transgenic mice than in wild type control mice. Transgenic mice exhibited lower body weight at 4 weeks of age, and slower rate of weight gain when compared with control mice. Furthermore, transgenic mice had lower body fat content. Indirect calorimetry revealed that transgenic mice had reduced food intake, increased basal metabolic rate, and increased lipid oxidation, which could account for the differences in body weight and body fat content. Transgenic mice also showed higher total circulating leptin, with the majority of it being in the bound form, while the amount of free leptin is comparable between transgenic and control mice. Conclusions These results are consistent with the role of OBRe as a leptin binding protein in regulating leptins bioavailability and activity.

Pancreatic regulation of glucose homeostasis

In order to ensure normal body function, the human body is dependent on a tight control of its blood glucose levels. This is accomplished by a highly sophisticated network of various hormones and neuropeptides released mainly from the brain, pancreas, liver, intestine as well as adipose and muscle tissue. Within this network, the pancreas represents a key player by secreting the blood sugar-lowering hormone insulin and its opponent glucagon. However, disturbances in the interplay of the hormones and peptides involved may lead to metabolic disorders such as type 2 diabetes mellitus (T2DM) whose prevalence, comorbidities and medical costs take on a dramatic scale. Therefore, it is of utmost importance to uncover and understand the mechanisms underlying the various interactions to improve existing anti-diabetic therapies and drugs on the one hand and to develop new therapeutic approaches on the other. This review summarizes the interplay of the pancreas with various other organs and tissues that maintain glucose homeostasis. Furthermore, anti-diabetic drugs and their impact on signaling pathways underlying the network will be discussed.