Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and ...human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity.
Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily ...evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreER(Tm) and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1(PB)-CreER(Tm);R26R(lacZ) mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mice carrying a targeted disruption of the prostaglandin E2 (PGE2) E-prostanoid receptor 3 (EP3) gene, Ptger3, were fed a high-fat diet (HFD), or a micronutrient matched control diet, to investigate ...the effects of disrupted PGE2-EP3 signaling on diabetes in a setting of diet-induced obesity. Although no differences in body weight were seen in mice fed the control diet, when fed a HFD, EP3−/− mice gained more weight relative to EP3+/+ mice. Overall, EP3−/− mice had increased epididymal fat mass and adipocyte size; paradoxically, a relative decrease in both epididymal fat pad mass and adipocyte size was observed in the heaviest EP3−/− mice. The EP3−/− mice had increased macrophage infiltration, TNF-α, monocyte chemoattractant protein-1, IL-6 expression, and necrosis in their epididymal fat pads as compared with EP3+/+ animals. Adipocytes isolated from EP3+/+ or EP3−/− mice were assayed for the effect of PGE2-evoked inhibition of lipolysis. Adipocytes isolated from EP3−/− mice lacked PGE2-evoked inhibition of isoproterenol stimulated lipolysis compared with EP3+/+. EP3−/− mice fed HFD had exaggerated ectopic lipid accumulation in skeletal muscle and liver, with evidence of hepatic steatosis. Both blood glucose and plasma insulin levels were similar between genotypes on a control diet, but when fed HFD, EP3−/− mice became hyperglycemic and hyperinsulinemic when compared with EP3+/+ fed HFD, demonstrating a more severe insulin resistance phenotype in EP3−/−. These results demonstrate that when fed a HFD, EP3−/− mice have abnormal lipid distribution, developing excessive ectopic lipid accumulation and associated insulin resistance.
Glucose Metabolism In Vivo in Four Commonly Used Inbred Mouse Strains
Eric D. Berglund 1 ,
Candice Y. Li 1 2 ,
Greg Poffenberger 3 ,
Julio E. Ayala 1 2 ,
Patrick T. Fueger 4 ,
Shannon E. Willis 3 ,
...Marybeth M. Jewell 3 ,
Alvin C. Powers 1 3 5 and
David H. Wasserman 1 2
1 Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee
2 Vanderbilt University–NIH Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, Nashville, Tennessee
3 Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University School of Medicine, Nashville,
Tennessee
4 Departments of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina
5 VA Tennessee Valley Healthcare System, Nashville, Tennessee
Corresponding author: Eric Berglund, eric.d.berglund{at}vanderbilt.edu
Abstract
OBJECTIVE —To characterize differences in whole-body glucose metabolism between commonly used inbred mouse strains.
RESEARCH DESIGN AND METHODS —Hyperinsulinemic-euglycemic (∼8.5 mmol/l) and -hypoglycemic (∼3.0 mmol/l) clamps were done in catheterized, 5-h-fasted mice
to assess insulin action and hypoglycemic counter-regulatory responsiveness. Hyperglycemic clamps (∼15 mmol/l) were done to
assess insulin secretion and compared with results in perifused islets.
RESULTS —Insulin action and hypoglycemic counter-regulatory and insulin secretory phenotypes varied considerably in four inbred mouse
strains. In vivo insulin secretion was greatest in 129X1/Sv mice, but the counter-regulatory response to hypoglycemia was
blunted. FVB/N mice in vivo showed no increase in glucose-stimulated insulin secretion, relative hepatic insulin resistance,
and the highest counter-regulatory response to hypoglycemia. In DBA/2 mice, insulin action was lowest among the strains, and
islets isolated had the greatest glucose-stimulated insulin secretion in vitro. In C57BL/6 mice, in vivo physiological responses
to hyperinsulinemia at euglycemia and hypoglycemia were intermediate relative to other strains. Insulin secretion by C57BL/6
mice was similar to that in other strains in contrast to the blunted glucose-stimulated insulin secretion from isolated islets.
CONCLUSIONS —Strain-dependent differences exist in four inbred mouse strains frequently used for genetic manipulation and study of glucose
metabolism. These results are important for selecting inbred mice to study glucose metabolism and for interpreting and designing
experiments.
Footnotes
Published ahead of print at http://diabetes.diabetesjournals.org on 8 April 2008.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit,and the work
is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted April 3, 2008.
Received November 15, 2007.
DIABETES
Pancreatic Islet Production of Vascular Endothelial Growth Factor-A Is Essential for Islet Vascularization, Revascularization,
and Function
Marcela Brissova 1 ,
Alena Shostak 1 ,
Masakazu Shiota 2 ,
...Peter O. Wiebe 2 ,
Greg Poffenberger 1 ,
Jeannelle Kantz 2 ,
Zhongyi Chen 1 ,
Chad Carr 1 ,
W. Gray Jerome 3 4 ,
Jin Chen 4 5 ,
H. Scott Baldwin 6 ,
Wendell Nicholson 1 ,
David M. Bader 7 ,
Thomas Jetton 8 ,
Maureen Gannon 1 2 and
Alvin C. Powers 1 2 9
1 Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville,
Tennessee
2 Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee
3 Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee
4 Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee
5 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee
6 Division of Pediatric Cardiology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee
7 Stahlman Laboratory, Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville,
Tennessee
8 Division of Endocrinology and Metabolism, Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont
9 Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee
Address correspondence and reprint requests to Alvin C. Powers, Division of Diabetes, Endocrinology, and Metabolism, 715 PRB,
Vanderbilt University, Nashville, TN 37232. E-mail: al.powers{at}vanderbilt.edu
Abstract
To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined
pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult
islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of
islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth
factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal
pancreatic insulin content and β-cell mass, mice with β-cell–reduced VEGF-A expression had impaired glucose-stimulated insulin
secretion. By vascular or diffusion delivery of β-cell secretagogues to islets, we showed that reduced insulin output is not
a result of β-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that
the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity
and, consequently, the amount of insulin delivered into the systemic circulation.
Ang, angiopoietin
VEGF, vascular endothelial growth factor
Footnotes
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org .
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted July 19, 2006.
Received May 18, 2006.
DIABETES
Abstract
Selective inhibitors of sodium glucose cotransporter-2 (SGLT2) are widely used for the treatment of type 2 diabetes and act primarily to lower blood glucose by preventing glucose ...reabsorption in the kidney. However, it is controversial whether these agents also act on the pancreatic islet, specifically the α cell, to increase glucagon secretion. To determine the effects of SGLT2 on human islets, we analyzed SGLT2 expression and hormone secretion by human islets treated with the SGLT2 inhibitor dapagliflozin (DAPA) in vitro and in vivo. Compared to the human kidney, SLC5A2 transcript expression was 1600-fold lower in human islets and SGLT2 protein was not detected. In vitro, DAPA treatment had no effect on glucagon or insulin secretion by human islets at either high or low glucose concentrations. In mice bearing transplanted human islets, 1 and 4 weeks of DAPA treatment did not alter fasting blood glucose, human insulin, and total glucagon levels. Upon glucose stimulation, DAPA treatment led to lower blood glucose levels and proportionally lower human insulin levels, irrespective of treatment duration. In contrast, after glucose stimulation, total glucagon was increased after 1 week of DAPA treatment but normalized after 4 weeks of treatment. Furthermore, the human islet grafts showed no effects of DAPA treatment on hormone content, endocrine cell proliferation or apoptosis, or amyloid deposition. These data indicate that DAPA does not directly affect the human pancreatic islet, but rather suggest an indirect effect where lower blood glucose leads to reduced insulin secretion and a transient increase in glucagon secretion.
The G6PC1, G6PC2 and G6PC3 genes encode distinct glucose-6-phosphatase catalytic subunit (G6PC) isoforms. In mice, germline deletion of G6pc2 lowers fasting blood glucose (FBG) without affecting ...fasting plasma insulin (FPI) while, in isolated islets, glucose-6-phosphatase activity and glucose cycling are abolished and glucose-stimulated insulin secretion (GSIS) is enhanced at submaximal but not high glucose. These observations are all consistent with a model in which G6PC2 regulates the sensitivity of GSIS to glucose by opposing the action of glucokinase. G6PC2 is highly expressed in human and mouse islet beta cells however, various studies have shown trace G6PC2 expression in multiple tissues raising the possibility that G6PC2 also affects FBG through non-islet cell actions. Using real-time PCR we show here that expression of G6pc1 and/or G6pc3 are much greater than G6pc2 in peripheral tissues, whereas G6pc2 expression is much higher than G6pc3 in both pancreas and islets with G6pc1 expression not detected. In adult mice, beta cell-specific deletion of G6pc2 was sufficient to reduce FBG without changing FPI. In addition, electronic health record-derived phenotype analyses showed no association between G6PC2 expression and phenotypes clearly unrelated to islet function in humans. Finally, we show that germline G6pc2 deletion enhances glycolysis in mouse islets and that glucose cycling can also be detected in human islets. These observations are all consistent with a mechanism by which G6PC2 action in islets is sufficient to regulate the sensitivity of GSIS to glucose and hence influence FBG without affecting FPI.
Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells, and changes in their expression are associated with developmental state and ...diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled more than 40,000 cells from normal human islets by single-cell RNA-Seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-Seq, MAFA/MAFB-coexpressing β cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.
We generated a mouse model (MIP-Luc-VU-NOD) that enables non-invasive bioluminescence imaging (BLI) of beta cell loss during the progression of autoimmune diabetes and determined the relationship ...between BLI and disease progression. MIP-Luc-VU-NOD mice displayed insulitis and a decline in bioluminescence with age which correlated with beta cell mass, plasma insulin, and pancreatic insulin content. Bioluminescence declined gradually in female MIP-Luc-VU-NOD mice, reaching less than 50% of the initial BLI at 10 weeks of age, whereas hyperglycemia did not ensue until mice were at least 16 weeks old. Mice that did not become diabetic maintained insulin secretion and had less of a decline in bioluminescence than mice that became diabetic. Bioluminescence measurements predicted a decline in beta cell mass prior to the onset of hyperglycemia and tracked beta cell loss. This model should be useful for investigating the fundamental processes underlying autoimmune diabetes and developing new therapies targeting beta cell protection and regeneration.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Endogenous β cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that β cell recovery following ...hypervascularization-induced β cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during β cell loss, and recruited MΦs are essential for β cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during β cell recovery and leads to increased β cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of β cells, ECs, and MΦs reveals that β cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in β cells. Collectively, these findings suggest a new β cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate β cell self-renewal.
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