Induction of insulin secretion in engineered liver cells by nitric oxide

BACKGROUND: Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway. RESULTS: Expression of either human insulin or the beta cell specific transcription factors PDX-1, NeuroD1 and MafA in the Hepa1-6 cell line or primary liver cells via adenoviral gene transfer, results in production and secretion of insulin. Although, the secretion of insulin is not significantly increased in response to high glucose, treatment of these engineered liver cells with L-arginine stimulates insulin secretion up to three-fold. This L-arginine-mediated insulin release is dependent on the production of nitric oxide. CONCLUSION: Liver cells can be engineered to produce insulin and insulin secretion can be induced by treatment with L-arginine via the production of nitric oxide

Glimepiride Administered in Chow Reversibly Impairs Glucose Tolerance in Mice

Sulfonylureas are a class of antidiabetes medications prescribed to millions of individuals worldwide. Rodents have been used extensively to study sulfonylureas in the laboratory. Here, we report the results of studies treating mice with a sulfonylurea (glimepiride) in order to understand how the drug affects glucose homeostasis and tolerance. We tested the effect of glimepiride on fasting blood glucose, glucose tolerance, and insulin secretion, using glimepiride sourced from a local pharmacy. We also examined the effect on glucagon, gluconeogenesis, and insulin sensitivity. Unexpectedly, glimepiride exposure in mice was associated with fasting hyperglycemia, glucose intolerance, and decreased insulin. There was no change in circulating glucagon levels or gluconeogenesis. The effect was dose-dependent, took effect by two weeks, and was reversed within three weeks after removal. Glimepiride elicited the same effects in all strains evaluated: four wild-type strains, as well as the transgenic Grn−/− and diabetic db/db mice. Our findings suggest that the use of glimepiride as a hypoglycemic agent in mice should proceed with caution and may have broader implications about mouse models as a proxy to study the human pharmacopeia

\u3cem\u3eSphk2\u3csup\u3e−/−\u3c/sup\u3e\u3c/em\u3e Mice are Protected from Obesity and Insulin Resistance

Sphingosine kinases phosphorylate sphingosine to sphingosine 1‑phosphate (S1P), which functions as a signaling molecule. We have previously shown that sphingosine kinase 2 (Sphk2) is important for insulin secretion. To obtain a better understanding of the role of Sphk2 in glucose and lipid metabolism, we have characterized 20- and 52-week old Sphk2−/− mice using glucose and insulin tolerance tests and by analyzing metabolic gene expression in adipose tissue. A detailed metabolic characterization of these mice revealed that aging Sphk2−/− mice are protected from metabolic decline and obesity compared to WT mice. Specifically, we found that 52-week old male Sphk2−/− mice had decreased weight and fat mass, and increased glucose tolerance and insulin sensitivity compared to control mice. Indirect calorimetry studies demonstrated an increased energy expenditure and food intake in 52-week old male Sphk2−/− versus control mice. Furthermore, expression of adiponectin gene in adipose tissue was increased and the plasma levels of adiponectin elevated in aged Sphk2−/− mice compared to WT. Analysis of lipid metabolic gene expression in adipose tissue showed increased expression of the Atgl gene, which was associated with increased Atgl protein levels. Atgl encodes for the adipocyte triglyceride lipase, which catalyzes the rate-limiting step of lipolysis. In summary, these data suggest that mice lacking the Sphk2 gene are protected from obesity and insulin resistance during aging. The beneficial metabolic effects observed in aged Sphk2−/− mice may be in part due to enhanced lipolysis by Atgl and increased levels of adiponectin, which has lipid- and glucose-lowering effects

Transcriptional Activity of the Islet β Cell Factor Pdx1 is Augmented by Lysine Methylation Catalyzed by the Methyltransferase Set7/9

The transcription factor Pdx1 is crucial to islet β cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using β cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in β cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in β cells (SetΔβ). SetΔβ mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and β cell function

Obesity and Diabetes Cause Cognitive Dysfunction in the Absence of Accelerated β-Amyloid Deposition in a Novel Murine Model of Mixed or Vascular Dementia

Mid-life obesity and type 2 diabetes mellitus (T2DM) confer a modest, increased risk for Alzheimer\u27s disease (AD), though the underlying mechanisms are unknown. We have created a novel mouse model that recapitulates features of T2DM and AD by crossing morbidly obese and diabetic db/db mice with APPΔNL/ΔNLx PS1P264L/P264L knock-in mice. These mice (db/AD) retain many features of the parental lines (e.g. extreme obesity, diabetes, and parenchymal deposition of β-amyloid (Aβ)). The combination of the two diseases led to additional pathologies-perhaps most striking of which was the presence of severe cerebrovascular pathology, including aneurysms and small strokes. Cortical Aβ deposition was not significantly increased in the diabetic mice, though overall expression of presenilin was elevated. Surprisingly, Aβ was not deposited in the vasculature or removed to the plasma, and there was no stimulation of activity or expression of major Aβ-clearing enzymes (neprilysin, insulin degrading enzyme, or endothelin-converting enzyme). The db/AD mice displayed marked cognitive impairment in the Morris Water Maze, compared to either db/db or APPΔNLx PS1P264L mice. We conclude that the diabetes and/or obesity in these mice leads to a destabilization of the vasculature, leading to strokes and that this, in turn, leads to a profound cognitive impairment and that this is unlikely to be directly dependent on Aβ deposition. This model of mixed or vascular dementia provides an exciting new avenue of research into the mechanisms underlying the obesity-related risk for age-related dementia, and will provide a useful tool for the future development of therapeutics

Induction of insulin secretion in engineered liver cells by nitric oxide-6

<p><b>Copyright information:</b></p><p>Taken from "Induction of insulin secretion in engineered liver cells by nitric oxide"</p><p>http://www.biomedcentral.com/1472-6793/7/11</p><p>BMC Physiology 2007;7():11-11.</p><p>Published online 17 Oct 2007</p><p>PMCID:PMC2121102.</p><p></p>sulin or control adenovirus, after treatment with 1 or 25 mM glucose for 1 h. Total insulin secretion is expressed as μU/ml * 10cells

Induction of insulin secretion in engineered liver cells by nitric oxide-4

<p><b>Copyright information:</b></p><p>Taken from "Induction of insulin secretion in engineered liver cells by nitric oxide"</p><p>http://www.biomedcentral.com/1472-6793/7/11</p><p>BMC Physiology 2007;7():11-11.</p><p>Published online 17 Oct 2007</p><p>PMCID:PMC2121102.</p><p></p>ndividually or in combination was determined using a mouse insulin ELISA kit. After incubation with the corresponding adenoviruses, the cells were incubated first over night with 1 mM glucose and then transferred to KRB buffer containing 25 mM glucose. Total insulin secretion is expressed as μU/ml * 10cells. B. Hepa1-6 cells expressing all three transcription factors, PDX-1, NeuroD1 and MafA were incubated for 1 h in KRB buffer containing 1 mM glucose with or without 20 mM L-arginine and 100 μM L-NNA. The amount insulin in the media was quantified by an insulin ELISA assay. The data are averages of five (n = 5) independent experiments

Induction of insulin secretion in engineered liver cells by nitric oxide-5

<p><b>Copyright information:</b></p><p>Taken from "Induction of insulin secretion in engineered liver cells by nitric oxide"</p><p>http://www.biomedcentral.com/1472-6793/7/11</p><p>BMC Physiology 2007;7():11-11.</p><p>Published online 17 Oct 2007</p><p>PMCID:PMC2121102.</p><p></p>el A) or a combination of PDX-1, NeuroD1 and MafA adenoviruses (panel B). After incubation on 1 mM glucose for about 16 h, the cells were transferred to KRB buffer containing 1 mM glucose in the presence or absence of 20 mM L-arginine and 100 μM L-NNA. Insulin secretion in the media was measured in three independent experiments and is expressed as fold difference of insulin secretion of 1 mM glucose incubated cells

Induction of insulin secretion in engineered liver cells by nitric oxide-1

<p><b>Copyright information:</b></p><p>Taken from "Induction of insulin secretion in engineered liver cells by nitric oxide"</p><p>http://www.biomedcentral.com/1472-6793/7/11</p><p>BMC Physiology 2007;7():11-11.</p><p>Published online 17 Oct 2007</p><p>PMCID:PMC2121102.</p><p></p>rred to 1 mM glucose with or without 20 mM L-arginine in the presence or absence of the NOS inhibitor L-NNA (100 μM) for 1 h. The amount of secreted insulin in the medium was determined and is expressed as fold difference, where insulin secretion on 1 mM glucose was set as 1-fold. Values are expressed as means ± SD for n = 5 in each group. B. Effect of NO donor sodium nitroprusside (SNP) on insulin secretion in Hepa1-6 cells. Insulin secretion in Hepa1-6 cells incubated with the human insulin adenovirus was measured after incubation of cells with 1 mM glucose, with or without 20 mM L-arginine or 100 μM SNP for 1 h (n = 3)