Target For Diabetes Therapy Research Articles

Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve.

Calcific aortic valve disease (CAVD), a degenerative disease characterized by inflammation, fibrosis and calcification, is accelerated in diabetes. Hyperglycemia contributes to this process by mechanisms that still need to be uncovered. We have recently developed a 3D model of the human aortic valve based on gelatin methacrylate and revealed that high glucose (HG) induced osteogenic molecules and increased calcium deposits in a pro-osteogenic environment. To further understand the events leading to calcification in diabetic conditions in CAVD, we analyzed here the inflammatory and remodeling mechanisms induced by HG in our 3D model. We exposed valvular endothelial cells (VEC) and interstitial cells (VIC) to normal glucose (NG) or HG for 7 and 14 days, then we isolated and separated the cells by anti-CD31 immunomagnetic beads. The changes induced by HG in the 3D model were investigated by real-time polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Our results showed that HG induced expression of different cytokines, cell adhesion molecules and matrix metalloproteinases in VEC and VIC. In addition, protein kinase C was increased in VEC and VIC, indicating molecular mechanisms associated with HG induced inflammation and remodeling in both valvular cells. These findings may indicate new biomarkers and targets for therapy in diabetes associated with CAVD.

GLP-1 is an independent predictor of long-term mortality in patients with myocardial infarction complicated by cardiogenic shock – a substudy of the IABP-SHOCK II trial

Abstract Background The incretin hormone Glucagon-like-peptide 1 (GLP-1) is a major stimulus for glucose dependent insulin secretion and holds cardioprotective efficacy. This has made the GLP-1 system a preferred target for diabetes therapy. Secretion of GLP-1 happens in response to nutritional but also inflammatory stimuli. Consequently, marked elevation of circulating GLP-1 levels were found in critically ill patients featuring marked association to markers of inflammation. Purpose Our study sought to investigate GLP-1 levels in patients with cardiogenic shock (CS) complicating myocardial infarction and a possible prognostic correlation to short- and long-term outcome. Methods We serially assessed circulating GLP-1 levels in a prospectively planned biomarker substudy in the IABP-SHOCK II trial. Blood samples were drawn during index PCI and at day 2. The blood was centrifuged immediately, and serum was frozen at −87°C. GLP-1 was measured with a standard ELISA-kit. All-cause mortality at short- (30 days), intermediate- (1 year) and long-term (6 years) follow-up was used for outcome assessment. Results In this study we found circulating GLP-1 to be markedly elevated in patients with myocardial infarction complicated by CS (n=172) at time of index PCI. Patients with fatal short-term outcome (n=70) exhibited higher GLP-1 levels (86 [45–130] pM) at ICU admission in comparison to patients with 30-day survival (48 [33–78] pM; p<0.001) (n=102). In repeated measures ANOVA the course of GLP-1 levels between baseline and day 2 showed a significant interaction between survivors and non-survivors (p=0.04). By univariate Cox-regression analysis GLP-1 levels >median were predictive of short- (hazard ratio [HR] 2.43; 95% confidence interval [CI] 1.50–3.94; p<0.001), intermediate- (HR 2.46; 95% CI 1.62–3.76; p<0.001) and long-term (HR 2.12; 95% CI 1.44–3.11; p<0.001) outcome. This association remained after multivariable correction (HR 2.01; 95% CI 1.37–3.07; p<0.001). In a landmark analysis we found a significant higher mortality in patients with GLP-1 levels >median from day 30 to 1 year (HR 2.56; 95% CI 1.08–6.09; p=0.03). In contrast, beyond 1 year up to 6 years no difference has been observed anymore (HR 1.02; 95% CI 0.41–2.58; p=0.96). Conclusions Elevated plasma levels of GLP-1 are an independent predictor for impaired prognosis in patients with myocardial infarction complicated by CS at short-, intermediate and long-term follow-up. In a landmark analysis this prognostic effect is sustained up to 1 year. The functional relevance of GLP-1 in this context is currently unknown and needs further investigations. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG), German Heart Research Foundation

335-LB: Towards Generating Spatiotemporal Multiscale Models of Pancreatic ß Cells

To characterize biology, we must consider how molecular machines and organelles within a cell interact and traffic, similar to the importance of understanding traffic patterns in major cities. We use an imaging across scales approach to understand architectural changes that occur in pancreatic β-cells during insulin secretion. β-cells produce and release insulin in response to glucose and are therefore a target for diabetes therapies. Diabetes is a worldwide problem affecting hundreds of millions of people with increasing patient numbers every year. To acquire cellular architectural information, we are integrating data from cryo-electron tomography, soft x-ray tomography (SXT), and fluorescence microscopy. We used multiple glucose and GPCR agonist concentrations to investigate the cellular organization changes induced during insulin vesicle biogenesis and trafficking. The SXT method allows for investigating the 3D cellular architecture and is an ideal method for investigating cell-to-cell variabilities. Additionally, we are using machine learning methods for image analysis and autosegmentation which increased the number of conditions and samples we can analyze rapidly. Furthermore, we can use the information from cryo-ET and SXT along with with live cell imaging to assemble a data-driven, dynamic model of insulin granules within pancreatic β-cells. Furthermore, the SXT method provides a unique opportunity for comparing cell-to-cell heterogeneity and will be a useful technique for comparing differences between rodent and human primary cell architectures. The integration of data from different time and spatial scales represents a sophisticated convergence of our understanding of cellular structure and function, which will revolutionize biological discovery, open new dimensions of research, and accelerate advancements in healthcare. Disclosure K.L. White: None. J. Singla: None. R.C. Stevens: Board Member; Self; BirdRock Bio, Danaher Corp. Funding University of Southern California

Mesoscale Architecture of Beta Cells Upon Glucose and Ex-4 Stimulation

To understand biology we must consider how molecular machines and organelles within a cell interact and traffic, similar to the importance of understanding traffic patterns in major cities. We use an imaging across scales approach to understand architectural changes that occur in pancreatic β-cells during insulin secretion. β-cells produce and release insulin in response to glucose and are therefore a target for diabetes therapies. Diabetes is a worldwide problem affecting hundreds of millions of people with increasing patient numbers every year. To acquire cellular architectural information, we are integrating data from cryo-electron tomography, soft x-ray tomography (SXT), and fluorescence microscopy. We imaged INS-1E cells (rat pancreatic β-cell line) under multiple glucose and GPCR agonist concentrations to investigate the cellular organization changes induced during insulin vesicle biogenesis and trafficking. The SXT method allows for investigating the 3D cellular architecture and is an ideal method for investigating cell-to-cell variabilities. Additionally, we are using machine learning methods for image analysis and autosegmentation which increased the number of conditions and samples we can analyze rapidly. Furthermore, we can use the information from cryo-ET and SXT along with with live cell imaging to assemble a data-driven, dynamic model of insulin granules within pancreatic β-cells. The integration of data from different time and spatial scales represents a sophisticated convergence of our understanding of cellular structure and function, which will revolutionize biological discovery, open new dimensions of research, and accelerate advancements in healthcare.

Canavanine increases glucose uptake in C2 C12 cells through the activation of imidazoline I-2B receptors.

Canavanine is a guanidinium derivative that contains the basic structure of the ligand(s) of imidazoline receptor (I-R). Canavanine has been reported to activate the imidazoline I-3 receptor (I-3R) both in vivo and in vitro. Additionally, the activation of the imidazoline I-2B receptor (I-2BR) by guanidinium derivatives may increase glucose uptake. Therefore, the effect of canavanine on the I-2BR was investigated in the present study. Glucose uptake into cultured C2 C12 cells was determined using the radio-ligated tracer 2-[(14) C]-deoxy-glucose. The changes in 5' AMP-activated protein kinase (AMPK) expression were also identified using Western blotting analysis. The canavanine-induced glucose uptake was inhibited in a dose-dependent manner by BU224 (0.01-1 μmol/L), which is a specific I-2BR antagonist, in the C2 C12 cells. Additionally, the canavanine-stimulated AMPK phosphorylation and glucose transporter (GLUT4) expression were also sensitive to BU224 inhibition in the C2 C12 cells. Moreover, both canavanine-stimulated glucose uptake and AMPK phosphorylation were attenuated by high concentrations of amiloride (1-2 μmol/L), which is another established I-2BR inhibitor, in a dose-dependent manner in C2 C12 cells. Additionally, compound C abolished the canavanine-induced glucose uptake and AMPK phosphorylation at a concentration (0.1 μmol/L) sufficient to inhibit AMPK. In conclusion, these data demonstrated that canavanine has an ability to activate I-2BR through the AMPK pathway to increase glucose uptake, which indicates I-2BR as a new target for diabetic therapy.

Editorial: Molecular Endocrinology Articles in the Spotlight for November 2013

Welcome to the November issue of Molecular Endocrinology. I am pleased to report that beginning with this issue, the editors will identify a single article each month to be made freely available upon publication. In addition, all minireviews will be freely available immediately upon publication effective with this issue. Our first freely available article is by Mao et al., “Circadian Gating of Epithelial-to-Mesenchymal Transition in Breast Cancer Cells via Melatonin-Regulation of GSK3 .” In this study, the authors investigated the connection between circadian/melatonin disruption and breast cancer risk in shift workers. Their studies used a unique xenograft model with human breast cancer cells maintained in nude rats. Disruption of nighttime melatonin production by exposure of the host to light at night has far-reaching biological consequences, including disruption and activation of key proliferative/survival pathways in the xenograft human breast cancer cells including MAPK/ERK and PI3K/AKT, which repress glycogen synthase kinase 3 (GSK3 ) activity and drive epithelial-tomesenchymal transition. Thus, this study identifies a molecular mechanism that could contribute to the increased risk of breast cancer incidence in industrialized nations characterized by excessive use of artificial light during the night. “20-HETE Induces Hyperglycemia through the cAMP/PKA-PhK-GP Pathway” by Lai et al. demonstrated that hyperglycemia and hypertension in CYP4F2 transgenic mice were ameliorated with HET0016 [a selective 20-hydroxyeicosatetraenoic acid (20-HETE) inhibitor] treatment. Furthermore, 20-HETE was shown to activate glycogen phosphorylase, which is a target for diabetes therapy. Therefore, through induction of glycogenolysis through the cAMP/protein kinase A-phosphorylase kinase-glycogen phosphorylase pathway, 20HETE could participate in the common pathogenesis of hypertension and hyperglycemia. This study implicates 20-HETE as a potential new target for hypertension and hyperglycemia therapy. Williams et al.’s “The microRNA (miR)-199a/214 Cluster Mediates Opposing Effects of Progesterone and Estrogen on Uterine Contractility during Pregnancy and Labor” investigates the role of the clustered miRNAs, miR-199a-3p and miR-214, as important mediators of the opposing action of progesterone (P4) vs. estradiol-17 (E2) on myometrial quiescence and contractility during pregnancy and labor. Specifically, P4 and E2 exert opposite effects on these clustered miRs with P4 triggering induction and E2 repression via differential effects on the zinc finger, E-box binding homeobox protein (ZEB1). Furthermore, miR199a-3p and miR-214 act to block TNF-induced myometrial contractility via an inhibition of cyclooxygenase-2, an enzyme that catalyzes the formation of proinflammatory cytokines. Their findings in mice and humans provide new insight into strategies to prevent preterm birth as specific targets are identified that respond differentially to endogenous steroid hormones during normal labor. These studies and others in this issue highlight the impact of endocrinology research in both prevention and treatment of many conditions. Congratulations to these researchers, as well as those who produced all of the excellent science in this issue, on broadening our knowledge in such diverse areas. We also hope you enjoy the new freely available articles that will hereafter be a monthly feature in Molecular Endocrinology. Donald B. DeFranco, Ph.D. Editor-in-Chief, Molecular Endocrinology

New Horizons in Cellular Regulation by Inositol Polyphosphates: Insights from the Pancreaticβ-Cell

Studies of inositol polyphosphates in the pancreatic β-cell have led to an exciting synergism between new discoveries regarding their cellular roles and new insights into β-cell function. Because the loss or malfunction of the β-cell is central to diabetes, these studies open the possibility of new pharmacological interventions in a disease that has reached epidemic proportions worldwide. Using the β-cell as our prime but not exclusive example, we examine the inositol polyphosphates in three main groups: 1) inositol 1,4,5-trisphosphate and its influence on Ca(2+) signaling, specifically in a cell in which cytoplasmic-free Ca(2+) concentration is principally increased by plasma membrane standing voltage-gated Ca(2+) channels; 2) higher inositol polyphosphates including a novel second messenger inositol 3,4,5,6-tetrakisphosphate and a regulatory role for inositol hexakisphosphate in β-cell Ca(2+) homeostasis and exo- and endocytosis; and 3) inositol pyrophosphates and their role in β-cell exocytosis, together with the exciting possibility of being novel targets for therapy in diabetes. We conclude with some of the new perspectives that are likely to become apparent in the next few years.

Mediation of AMP Kinase in the Increase of Glucose Uptake in L6 Cells Induced by Activation of Imidazoline I-2 Receptors

Recent work using radioactive tracer indicates that activation of imidazoline I2 receptor (I2R) by guanidinium derivatives may increase the glucose uptake in the skeletal muscle. However, the effect of I2R activation on nonradioactive glucose uptake is still unknown. The ability of glucose uptake in cultured L6 cells is then determined using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) as a fluorescence indicator. The changes in 5'-AMP-activated protein kinase (AMPK) expression were also identified by Western blot analysis. In the present study, 2-(2-benzofuranyl)-2-imidazoline (2-BFI) is used to stimulate I2R while 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is applied to activate AMPK directly. Both compounds can increase 2-NBDG in L6 cells in a concentration-dependent manner. Meanwhile, compound C at concentrations sufficient to inhibit AMPK blocked this increase of glucose uptake by 2-BFI or AICAR. However, only 2-BFI-induced glucose uptake action was dose-dependently blocked by BU224, a specific I2R antagonist, in L6 cells. Moreover, AMPK phosphorylation was markedly increased by 2-BFI or AICAR in L6 cells. Similarly, only the effect of 2-BFI was attenuated by BU224 in L6 cells. Thus, we suggest that AMPK is mediated in I2R activation for increase of glucose uptake in the skeletal muscle cell and I2R will be a new target for diabetic therapy.

Editorial: Molecular Endocrinology Articles in the Spotlight for November 2012

Welcome to the November issue of Molecular Endocrinology. I am pleased to report that beginning with this issue, the editors will identify a single article each month to be made freely available upon publication. In addition, all minireviews will be freely available immediately upon publication effective with this issue. Our first freely available article is by Mao et al., “Circadian Gating of Epithelial-to-Mesenchymal Transition in Breast Cancer Cells via Melatonin-Regulation of GSK3 .” In this study, the authors investigated the connection between circadian/melatonin disruption and breast cancer risk in shift workers. Their studies used a unique xenograft model with human breast cancer cells maintained in nude rats. Disruption of nighttime melatonin production by exposure of the host to light at night has far-reaching biological consequences, including disruption and activation of key proliferative/survival pathways in the xenograft human breast cancer cells including MAPK/ERK and PI3K/AKT, which repress glycogen synthase kinase 3 (GSK3 ) activity and drive epithelial-tomesenchymal transition. Thus, this study identifies a molecular mechanism that could contribute to the increased risk of breast cancer incidence in industrialized nations characterized by excessive use of artificial light during the night. “20-HETE Induces Hyperglycemia through the cAMP/PKA-PhK-GP Pathway” by Lai et al. demonstrated that hyperglycemia and hypertension in CYP4F2 transgenic mice were ameliorated with HET0016 [a selective 20-hydroxyeicosatetraenoic acid (20-HETE) inhibitor] treatment. Furthermore, 20-HETE was shown to activate glycogen phosphorylase, which is a target for diabetes therapy. Therefore, through induction of glycogenolysis through the cAMP/protein kinase A-phosphorylase kinase-glycogen phosphorylase pathway, 20HETE could participate in the common pathogenesis of hypertension and hyperglycemia. This study implicates 20-HETE as a potential new target for hypertension and hyperglycemia therapy. Williams et al.’s “The microRNA (miR)-199a/214 Cluster Mediates Opposing Effects of Progesterone and Estrogen on Uterine Contractility during Pregnancy and Labor” investigates the role of the clustered miRNAs, miR-199a-3p and miR-214, as important mediators of the opposing action of progesterone (P4) vs. estradiol-17 (E2) on myometrial quiescence and contractility during pregnancy and labor. Specifically, P4 and E2 exert opposite effects on these clustered miRs with P4 triggering induction and E2 repression via differential effects on the zinc finger, E-box binding homeobox protein (ZEB1). Furthermore, miR199a-3p and miR-214 act to block TNF-induced myometrial contractility via an inhibition of cyclooxygenase-2, an enzyme that catalyzes the formation of proinflammatory cytokines. Their findings in mice and humans provide new insight into strategies to prevent preterm birth as specific targets are identified that respond differentially to endogenous steroid hormones during normal labor. These studies and others in this issue highlight the impact of endocrinology research in both prevention and treatment of many conditions. Congratulations to these researchers, as well as those who produced all of the excellent science in this issue, on broadening our knowledge in such diverse areas. We also hope you enjoy the new freely available articles that will hereafter be a monthly feature in Molecular Endocrinology. Donald B. DeFranco, Ph.D. Editor-in-Chief, Molecular Endocrinology

Glucose-Induced Isoform of ChREBP

A glucose-induced alternative isoform of ChREBP, a transcription factor that promotes lipogenesis, may be a target for diabetes therapy.

Activation of Imidazoline I-2B Receptors by Allantoin to Increase Glucose Uptake into C2C12 Cells

Allantoin, an active principle of the yam, belongs to the group of guanidinium derivatives and has been reported to lower plasma glucose in diabetic animals. Recent evidence indicates that activation of the imidazoline I(2B) receptor (I(2B)R) by guanidinium derivatives also increases glucose uptake; however, the effect of allantoin on I(2B)R is still unknown. Glucose uptake into cultured C₂C₁₂ cells was determined using 2-[¹⁴C]-deoxy-D-glucose as a tracer. The changes in 5'-AMP-activated protein kinase (AMPK) expression were also identified by Western blotting analysis. The allantoin-induced glucose uptake action was dose-dependently blocked by BU224, a specific I₂R antagonist, in C₂C₁₂ cells. Moreover, AMPK phosphorylation by allantoin was found to be dose-dependently increased in C₂C₁₂ cells using AICAR treatment as a reference. In addition, both actions of allantoin, the increases in glucose uptake and AMPK phosphorylation, were dose-dependently attenuated by amiloride in C₂C₁₂ cells. Moreover, compound C at concentrations sufficient to inhibit AMPK blocked the allantoin-induced glucose uptake and AMPK phosphorylation. Thus, we suggest that allantoin can activate I(2B)R to increase glucose uptake into cells, and propose I(2B)R as a new target for diabetic therapy.

Activation of Imidazoline I-2B Receptor by Metformin to Increase Glucose Uptake in Skeletal Muscle

Metformin (dimethylbiguanide) belongs to guanidinium-derivative and is widely used for treatment of diabetic disorders in clinic. Metformin lowers blood glucose in diabetic animals through increase of glucose uptake into skeletal muscle. Recent evidence indicates that activation of imidazoline I2B receptor (I2BR) by guanidinium-derivatives also increased glucose uptake; however, the effect of metformin on I2BR is still unknown. The blood glucose levels were determined by a glucose kit. The ability of glucose uptake into isolated skeletal muscle or cultured C2C12 cells was determined using 2-[14C]-deoxyglucose as tracer. The expressions of 5' AMP-activated protein kinase (AMPK) and glucose transporter 4 (GLUT-4) were identified by Western blotting analysis. The metformin-induced blood glucose-lowering action was dose-dependently blocked by BU224, a specific I2R antagonist, in Wistar rats. Also, similar reversion by BU224 was observed in isolated skeletal muscle regarding the metformin-induced glucose uptake. Moreover, AMPK phosphorylation by metformin was concentration-dependently reduced by BU224 in isolated skeletal muscle. In addition, signals for metformin increased glucose uptake were identified via I2R/PI3K/PKC/AMPK dependent pathway in C2C12 cells. Thus, we suggest that metformin can activate I2BR to increase glucose uptake and I2BR will be a new target for diabetic therapy.

Lack of TRPM2 Impaired Insulin Secretion and Glucose Metabolisms in Mice

OBJECTIVETRPM2 is a Ca2+-permeable nonselective cation channel activated by adenosine dinucleotides. We previously demonstrated that TRPM2 is activated by coapplication of heat and intracellular cyclic adenosine 5′-diphosphoribose, which has been suggested to be involved in intracellular Ca2+ increase in immunocytes and pancreatic β-cells. To clarify the involvement of TRPM2 in insulin secretion, we analyzed TRPM2 knockout (TRPM2-KO) mice.RESEARCH DESIGN AND METHODSOral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed in TRPM2-KO and wild-type mice. We also measured cytosolic free Ca2+ in single pancreatic cells using fura-2 microfluorometry and insulin secretion from pancreatic islets.RESULTSBasal blood glucose levels were higher in TRPM2-KO mice than in wild-type mice without any difference in plasma insulin levels. The OGTT and IPGTT demonstrated that blood glucose levels in TRPM2-KO mice were higher than those in wild-type mice, which was associated with an impairment in insulin secretion. In isolated β-cells, smaller intracellular Ca2+ increase was observed in response to high concentrations of glucose and incretin hormone in TRPM2-KO cells than in wild-type cells. Moreover, insulin secretion from the islets of TRPM2-KO mice in response to glucose and incretin hormone treatment was impaired, whereas the response to tolbutamide, an ATP-sensitive potassium channel inhibitor, was not different between the two groups.CONCLUSIONSThese results indicate that TRPM2 is involved in insulin secretion stimulated by glucose and that further potentiated by incretins. Thus, TRPM2 may be a new target for diabetes therapy.

Menin Controls Growth of Pancreatic ß-Cells in Pregnant Mice and Promotes Gestational Diabetes Mellitus

During pregnancy, maternal pancreatic islets grow to match dynamic physiological demands, but the mechanisms regulating adaptive islet growth in this setting are poorly understood. Here we show that menin, a protein previously characterized as an endocrine tumor suppressor and transcriptional regulator, controls islet growth in pregnant mice. Pregnancy stimulated proliferation of maternal pancreatic islet beta-cells that was accompanied by reduced islet levels of menin and its targets. Transgenic expression of menin in maternal beta-cells prevented islet expansion and led to hyperglycemia and impaired glucose tolerance, hallmark features of gestational diabetes. Prolactin, a hormonal regulator of pregnancy, repressed islet menin levels and stimulated beta-cell proliferation. These results expand our understanding of mechanisms underlying diabetes pathogenesis and reveal potential targets for therapy in diabetes.

Glucokinase-activating ureas

The synthesis, SAR and biological evaluation of a series of ureas that activate glucokinase, a target for diabetes therapy as a result of its critical role in the regulation of whole-body glucose homeostasis, are described. Some of the urea-containing glucokinase activators lowered blood glucose levels in vivo following oral dosing to C57BL/6J mice.