Publications by authors named "Natalia Tamarina"

20 Publications

  • Page 1 of 1

Deletion of Protein Kinase D1 in Pancreatic β-Cells Impairs Insulin Secretion in High-Fat Diet-Fed Mice.

Diabetes 2018 01 16;67(1):71-77. Epub 2017 Oct 16.

Montreal Diabetes Research Center, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada

Ββ-Cell adaptation to insulin resistance is necessary to maintain glucose homeostasis in obesity. Failure of this mechanism is a hallmark of type 2 diabetes (T2D). Hence, factors controlling functional β-cell compensation are potentially important targets for the treatment of T2D. Protein kinase D1 (PKD1) integrates diverse signals in the β-cell and plays a critical role in the control of insulin secretion. However, the role of β-cell PKD1 in glucose homeostasis in vivo is essentially unknown. Using β-cell-specific, inducible PKD1 knockout mice (βPKD1KO), we examined the role of β-cell PKD1 under basal conditions and during high-fat feeding. βPKD1KO mice under a chow diet presented no significant difference in glucose tolerance or insulin secretion compared with mice expressing the Cre transgene alone; however, when compared with wild-type mice, both groups developed glucose intolerance. Under a high-fat diet, deletion of PKD1 in β-cells worsened hyperglycemia, hyperinsulinemia, and glucose intolerance. This was accompanied by impaired glucose-induced insulin secretion both in vivo in hyperglycemic clamps and ex vivo in isolated islets from high-fat diet-fed βPKD1KO mice without changes in islet mass. This study demonstrates an essential role for PKD1 in the β-cell adaptive secretory response to high-fat feeding in mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db17-0982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741145PMC
January 2018

Growth Hormone-Releasing Hormone in Diabetes.

Front Endocrinol (Lausanne) 2016 10;7:129. Epub 2016 Oct 10.

Department of Medicine, Kovler Diabetes Center, The University of Chicago, Chicago, IL, USA; Department of Pediatrics, The University of Chicago, Chicago, IL, USA.

Growth hormone-releasing hormone (GHRH) is produced by the hypothalamus and stimulates growth hormone synthesis and release in the anterior pituitary gland. In addition, GHRH is an important regulator of cellular functions in many cells and organs. Expression of GHRH G-Protein Coupled Receptor (GHRHR) has been demonstrated in different peripheral tissues and cell types, including pancreatic islets. Among the peripheral activities, recent studies demonstrate a novel ability of GHRH analogs to increase and preserve insulin secretion by beta-cells in isolated pancreatic islets, which makes them potentially useful for diabetes treatment. This review considers the role of GHRHR in the beta-cell and addresses the unique engineered GHRH agonists and antagonists for treatment of type 2 diabetes mellitus. We discuss the similarity of signaling pathways activated by GHRHR in pituitary somatotrophs and in pancreatic beta-cells and possible ways as to how the GHRHR pathway can interact with glucose and other secretagogues to stimulate insulin secretion. We also consider the hypothesis that novel GHRHR agonists can improve glucose metabolism in Type 2 diabetes by preserving the function and survival of pancreatic beta-cells. Wound healing and cardioprotective action with new GHRH agonists suggest that they may prove useful in ameliorating certain diabetic complications. These findings highlight the future potential therapeutic effectiveness of modulators of GHRHR activity for the development of new therapeutic approaches in diabetes and its complications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fendo.2016.00129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056186PMC
October 2016

The Brain-to-Pancreatic Islet Neuronal Map Reveals Differential Glucose Regulation From Distinct Hypothalamic Regions.

Diabetes 2016 09 12;65(9):2711-23. Epub 2016 Apr 12.

Kovler Diabetes Center, Department of Medicine, Section of Endocrinology, Diabetes and Metabolism, University of Chicago, Chicago, IL

The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db15-0629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5001176PMC
September 2016

TCF1 links GIPR signaling to the control of beta cell function and survival.

Nat Med 2016 Jan 7;22(1):84-90. Epub 2015 Dec 7.

Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada.

The glucagon-like peptide-1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor transduce nutrient-stimulated signals to control beta cell function. Although the GLP-1 receptor (GLP-1R) is a validated drug target for diabetes, the importance of the GIP receptor (GIPR) for the function of beta cells remains uncertain. We demonstrate that mice with selective ablation of GIPR in beta cells (MIP-Cre:Gipr(Flox/Flox); Gipr(-/-βCell)) exhibit lower levels of meal-stimulated insulin secretion, decreased expansion of adipose tissue mass and preservation of insulin sensitivity when compared to MIP-Cre controls. Beta cells from Gipr(-/-βCell) mice display greater sensitivity to apoptosis and markedly lower islet expression of T cell-specific transcription factor-1 (TCF1, encoded by Tcf7), a protein not previously characterized in beta cells. GIP, but not GLP-1, promotes beta cell Tcf7 expression via a cyclic adenosine monophosphate (cAMP)-independent and extracellular signal-regulated kinase (ERK)-dependent pathway. Tcf7 (in mice) or TCF7 (in humans) levels are lower in islets taken from diabetic mice and in humans with type 2 diabetes; knockdown of TCF7 in human and mouse islets impairs the cytoprotective responsiveness to GIP and enhances the magnitude of apoptotic injury, whereas restoring TCF1 levels in beta cells from Gipr(-/-βCell) mice lowers the number of apoptotic cells compared to that seen in MIP-Cre controls. Tcf7(-/-) mice show impaired insulin secretion, deterioration of glucose tolerance with either aging and/or high-fat feeding and increased sensitivity to beta cell injury relative to wild-type (WT) controls. Hence the GIPR-TCF1 axis represents a potential therapeutic target for preserving both the function and survival of vulnerable, diabetic beta cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm.3997DOI Listing
January 2016

Dynamin 2 regulates biphasic insulin secretion and plasma glucose homeostasis.

J Clin Invest 2015 Nov 28;125(11):4026-41. Epub 2015 Sep 28.

Alterations in insulin granule exocytosis and endocytosis are paramount to pancreatic β cell dysfunction in diabetes mellitus. Here, using temporally controlled gene ablation specifically in β cells in mice, we identified an essential role of dynamin 2 GTPase in preserving normal biphasic insulin secretion and blood glucose homeostasis. Dynamin 2 deletion in β cells caused glucose intolerance and substantial reduction of the second phase of glucose-stimulated insulin secretion (GSIS); however, mutant β cells still maintained abundant insulin granules, with no signs of cell surface expansion. Compared with control β cells, real-time capacitance measurements demonstrated that exocytosis-endocytosis coupling was less efficient but not abolished; clathrin-mediated endocytosis (CME) was severely impaired at the step of membrane fission, which resulted in accumulation of clathrin-coated endocytic intermediates on the plasma membrane. Moreover, dynamin 2 ablation in β cells led to striking reorganization and enhancement of actin filaments, and insulin granule recruitment and mobilization were impaired at the later stage of GSIS. Together, our results demonstrate that dynamin 2 regulates insulin secretory capacity and dynamics in vivo through a mechanism depending on CME and F-actin remodeling. Moreover, this study indicates a potential pathophysiological link between endocytosis and diabetes mellitus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI80652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4639984PMC
November 2015

Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects.

J Biol Chem 2015 Aug 2;290(34):20934-46. Epub 2015 Jul 2.

From the Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel,

The tumor suppressor liver kinase B1 (LKB1) is an important regulator of pancreatic β cell biology. LKB1-dependent phosphorylation of distinct AMPK (adenosine monophosphate-activated protein kinase) family members determines proper β cell polarity and restricts β cell size, total β cell mass, and glucose-stimulated insulin secretion (GSIS). However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood. We report here that in the absence of LKB1 in β cells, GSIS is dramatically and persistently improved. The enhancement is seen both in vivo and in vitro and cannot be explained by altered cell polarity, increased β cell number, or increased insulin content. Increased secretion does require membrane depolarization and calcium influx but appears to rely mostly on a distal step in the secretion pathway. Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway. Thus LKB1 is essential for mitochondrial homeostasis in β cells and in parallel is a powerful negative regulator of insulin secretion. This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.639237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543653PMC
August 2015

Characterization of mice expressing Ins1 gene promoter driven CreERT recombinase for conditional gene deletion in pancreatic β-cells.

Islets 2014 ;6(1):e27685

a Department of Medicine; University of Chicago; Chicago, IL USA.

Gene manipulation using Cre-loxP recombination has proven to be an important approach for studying the impact of gene expression on pancreatic β-cell biology. We report the generation of a transgenic mouse line that enables a highly specific system for conditional gene manipulation within β-cells and achieve tissue specific and temporally regulated deletion of the Ctnnb1 (β-catenin) gene in pancreatic β-cells. cDNA encoding Cre recombinase fused to modified estrogen receptor (CreERT) under control of mouse insulin 1 gene promoter (Ins1) was used to construct the mouse line Tg(Ins1-Cre/ERT)1Lphi, also termed MIP1-CreERT. In a cross of MIP1-CreERT with a ROSA26/LacZ reporter strain, tamoxifen [Tmx] - dependent β-galactosidase expression occurred within pancreatic β-cells but not in other organ systems. Intraperitoneal glucose tolerance tests and glucose-stimulated changes in β-cell cytoplasmic calcium concentration were not adversely affected in adult MIP1-CreERT. A mouse line with floxed Ctnnb1 gene (Ctnnb1f/f) was crossed with the MIP1-CreERT line to generate a mouse model for inducible β-cell specific deletion of β-catenin gene (Ctnnb1f/f:MIP1-CreERT). Ctnnb1f/f:MIP1-CreERT mice and Ctnnb1f/f littermate controls, were injected with Tmx as adults to knock down β-catenin production in the majority of pancreatic β-cells. These mice showed normal glucose tolerance, islet cyto-architecture and insulin secretion. A novel protein fraction of 50Kd, immunoreactive with anti-β-catenin was observed in islet extracts from Ctnnb1f/f:MIP1-CreERT[Tmx] mice but not MIP1-CreERT-negative Ctnnb1f/f[Tmx] controls, indicating possible presence of a cryptic protein product of recombined Ctnnb1 gene. The MIP1-CreERT mouse line is a powerful tool for conditional manipulation of gene expression in β-cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/isl.27685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114654PMC
August 2015

Functional role of serotonin in insulin secretion in a diet-induced insulin-resistant state.

Endocrinology 2015 Feb 26;156(2):444-52. Epub 2014 Nov 26.

Graduate School of Medical Science and Engineering (K.K., C.-M.O., S.P., J.N., H.K.), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea; Department of Biochemistry (M.O.-I., S.N.), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; Department of Biochemistry (S.P.), Catholic Kwandong University College of Medicine, Gangneung, Gangwondo 210-701, Republic of Korea; Department of Biochemistry (J.N.), Yonsei University Wonju College of Medicine, Wonju, Gangwondo 220-701, Republic of Korea; Department of Genetics and Development (V.K.Y., G.K.), Columbia University Medical Center, New York, New York 10032; Department of Medicine (N.A.T., L.H.P.), The University of Chicago, Chicago, Illinois 60637; Departments of Medicine and Cell and Developmental Biology (M.W.R.), State University of New York Upstate Medical University, Syracuse, New York 13210; and Diabetes Center (M.S.G.), Hormone Research Institute and Department of Medicine, University of California San Francisco, San Francisco, California 94143.

The physiological role of serotonin, or 5-hydroxytryptamine (5-HT), in pancreatic β-cell function was previously elucidated using a pregnant mouse model. During pregnancy, 5-HT increases β-cell proliferation and glucose-stimulated insulin secretion (GSIS) through the Gαq-coupled 5-HT2b receptor (Htr2b) and the 5-HT3 receptor (Htr3), a ligand-gated cation channel, respectively. However, the role of 5-HT in β-cell function in an insulin-resistant state has yet to be elucidated. Here, we characterized the metabolic phenotypes of β-cell-specific Htr2b(-/-) (Htr2b βKO), Htr3a(-/-) (Htr3a knock-out [KO]), and β-cell-specific tryptophan hydroxylase 1 (Tph1)(-/-) (Tph1 βKO) mice on a high-fat diet (HFD). Htr2b βKO, Htr3a KO, and Tph1 βKO mice exhibited normal glucose tolerance on a standard chow diet. After 6 weeks on an HFD, beginning at 4 weeks of age, both Htr3a KO and Tph1 βKO mice developed glucose intolerance, but Htr2b βKO mice remained normoglycemic. Pancreas perfusion assays revealed defective first-phase insulin secretion in Htr3a KO mice. GSIS was impaired in islets isolated from HFD-fed Htr3a KO and Tph1 βKO mice, and 5-HT treatment improved insulin secretion from Tph1 βKO islets but not from Htr3a KO islets. Tph1 and Htr3a gene expression in pancreatic islets was not affected by an HFD, and immunostaining could not detect 5-HT in pancreatic islets from mice fed an HFD. Taken together, these results demonstrate that basal 5-HT levels in β-cells play a role in GSIS through Htr3, which becomes more evident in a diet-induced insulin-resistant state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/en.2014-1687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298319PMC
February 2015

The role of β cell glucagon-like peptide-1 signaling in glucose regulation and response to diabetes drugs.

Cell Metab 2014 Jun 15;19(6):1050-7. Epub 2014 May 15.

Division of Endocrinology, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH 45267, USA; Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 45237, USA. Electronic address:

Glucagon-like peptide-1 (GLP-1), an insulinotropic gut peptide released after eating, is essential for normal glucose tolerance (GT). To determine whether this effect is mediated directly by GLP-1 receptors (GLP1R) on islet β cells, we developed mice with β cell-specific knockdown of Glp1r. β cell Glp1r knockdown mice had impaired GT after intraperitoneal (i.p.) glucose and did not secrete insulin in response to i.p. or intravenous GLP-1. However, they had normal GT after oral glucose, a response that was impaired by a GLP1R antagonist. β cell Glp1r knockdown mice had blunted responses to a GLP1R agonist but intact glucose lowering with a dipeptidylpeptidase 4 (DPP-4) inhibitor. Thus, in mice, β cell Glp1rs are required to respond to hyperglycemia and exogenous GLP-1, but other factors compensate for reduced GLP-1 action during meals. These results support a role for extraislet GLP1R in oral glucose tolerance and paracrine regulation of β cells by islet GLP-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cmet.2014.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109713PMC
June 2014

Effect of genetic variation in a Drosophila model of diabetes-associated misfolded human proinsulin.

Genetics 2014 Feb 26;196(2):557-67. Epub 2013 Nov 26.

Department of Ecology and Evolution, The University of Chicago, Chicago, Illinois 60637.

The identification and validation of gene-gene interactions is a major challenge in human studies. Here, we explore an approach for studying epistasis in humans using a Drosophila melanogaster model of neonatal diabetes mellitus. Expression of the mutant preproinsulin (hINS(C96Y)) in the eye imaginal disc mimics the human disease: it activates conserved stress-response pathways and leads to cell death (reduction in eye area). Dominant-acting variants in wild-derived inbred lines from the Drosophila Genetics Reference Panel produce a continuous, highly heritable distribution of eye-degeneration phenotypes in a hINS(C96Y) background. A genome-wide association study (GWAS) in 154 sequenced lines identified a sharp peak on chromosome 3L, which mapped to a 400-bp linkage block within an intron of the gene sulfateless (sfl). RNAi knockdown of sfl enhanced the eye-degeneration phenotype in a mutant-hINS-dependent manner. RNAi against two additional genes in the heparan sulfate (HS) biosynthetic pathway (ttv and botv), in which sfl acts, also modified the eye phenotype in a hINS(C96Y)-dependent manner, strongly suggesting a novel link between HS-modified proteins and cellular responses to misfolded proteins. Finally, we evaluated allele-specific expression difference between the two major sfl-intronic haplotypes in heterozygtes. The results showed significant heterogeneity in marker-associated gene expression, thereby leaving the causal mutation(s) and its mechanism unidentified. In conclusion, the ability to create a model of human genetic disease, map a QTL by GWAS to a specific gene, and validate its contribution to disease with available genetic resources and the potential to experimentally link the variant to a molecular mechanism demonstrate the many advantages Drosophila holds in determining the genetic underpinnings of human disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/genetics.113.157800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3914626PMC
February 2014

Genetic complexity in a Drosophila model of diabetes-associated misfolded human proinsulin.

Genetics 2014 Feb 26;196(2):539-55. Epub 2013 Nov 26.

Department of Medicine, University of Chicago, Chicago, Illinois 60637.

Drosophila melanogaster has been widely used as a model of human Mendelian disease, but its value in modeling complex disease has received little attention. Fly models of complex disease would enable high-resolution mapping of disease-modifying loci and the identification of novel targets for therapeutic intervention. Here, we describe a fly model of permanent neonatal diabetes mellitus and explore the complexity of this model. The approach involves the transgenic expression of a misfolded mutant of human preproinsulin, hINS(C96Y), which is a cause of permanent neonatal diabetes. When expressed in fly imaginal discs, hINS(C96Y) causes a reduction of adult structures, including the eye, wing, and notum. Eye imaginal discs exhibit defects in both the structure and the arrangement of ommatidia. In the wing, expression of hINS(C96Y) leads to ectopic expression of veins and mechano-sensory organs, indicating disruption of wild-type signaling processes regulating cell fates. These readily measurable "disease" phenotypes are sensitive to temperature, gene dose, and sex. Mutant (but not wild-type) proinsulin expression in the eye imaginal disc induces IRE1-mediated XBP1 alternative splicing, a signal for endoplasmic reticulum stress response activation, and produces global change in gene expression. Mutant hINS transgene tester strains, when crossed to stocks from the Drosophila Genetic Reference Panel, produce F1 adults with a continuous range of disease phenotypes and large broad-sense heritability. Surprisingly, the severity of mutant hINS-induced disease in the eye is not correlated with that in the notum in these crosses, nor with eye reduction phenotypes caused by the expression of two dominant eye mutants acting in two different eye development pathways, Drop (Dr) or Lobe (L), when crossed into the same genetic backgrounds. The tissue specificity of genetic variability for mutant hINS-induced disease has, therefore, its own distinct signature. The genetic dominance of disease-specific phenotypic variability in our model of misfolded human proinsulin makes this approach amenable to genome-wide association study in a simple F1 screen of natural variation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1534/genetics.113.157602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3914625PMC
February 2014

β-Cell-specific protein kinase A activation enhances the efficiency of glucose control by increasing acute-phase insulin secretion.

Diabetes 2013 May 24;62(5):1527-36. Epub 2013 Jan 24.

Kovler Diabetes Center, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, Department of Medicine, The University of Chicago, Chicago, Illinois, USA.

Acute insulin secretion determines the efficiency of glucose clearance. Moreover, impaired acute insulin release is characteristic of reduced glucose control in the prediabetic state. Incretin hormones, which increase β-cell cAMP, restore acute-phase insulin secretion and improve glucose control. To determine the physiological role of the cAMP-dependent protein kinase (PKA), a mouse model was developed to increase PKA activity specifically in the pancreatic β-cells. In response to sustained hyperglycemia, PKA activity potentiated both acute and sustained insulin release. In contrast, a glucose bolus enhanced acute-phase insulin secretion alone. Acute-phase insulin secretion was increased 3.5-fold, reducing circulating glucose to 58% of levels in controls. Exendin-4 increased acute-phase insulin release to a similar degree as PKA activation. However, incretins did not augment the effects of PKA on acute-phase insulin secretion, consistent with incretins acting primarily via PKA to potentiate acute-phase insulin secretion. Intracellular calcium signaling was unaffected by PKA activation, suggesting that the effects of PKA on acute-phase insulin secretion are mediated by the phosphorylation of proteins involved in β-cell exocytosis. Thus, β-cell PKA activity transduces the cAMP signal to dramatically increase acute-phase insulin secretion, thereby enhancing the efficiency of insulin to control circulating glucose.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db12-1013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636652PMC
May 2013

Conditional gene targeting in mouse pancreatic ß-Cells: analysis of ectopic Cre transgene expression in the brain.

Diabetes 2010 Dec 29;59(12):3090-8. Epub 2010 Aug 29.

Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Chicago, Chicago, llinois, USA.

Objective: Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell- or pancreas-specific recombination, also drive Cre expression in the brain.

Research Design And Methods: Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1(tm1Cvw) lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.

Results: All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)(89.1Dam) mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)(1Lphi) mice were the only line that lacked Cre activity in the brain.

Conclusions: Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db10-0624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2992770PMC
December 2010

Reversible translocation of EYFP-tagged STIM1 is coupled to calcium influx in insulin secreting beta-cells.

Cell Calcium 2008 Dec 2;44(6):533-44. Epub 2008 May 2.

Department of Medicine, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA.

Calcium (Ca(2+)) signaling regulates insulin secretion in pancreatic beta-cells. STIM1 has been proposed to function as an endoplasmic reticulum (ER) Ca(2+) sensor regulating store-operated Ca(2+) entry (SOCE). Here we studied the translocation of EYFP-STIM1 in response to ER calcium depletion in mouse insulinoma MIN6 cells by fluorescent microscopy. While in resting cells EYFP-STIM1 is co-localized with an ER marker, in thapsigargin (Tg)-stimulated cells it occupied highly defined areas of the peri-PM space in punctae adjacent to, but not entirely coincident with the ER. Co-staining with fluorescent phalloidin revealed that EYFP-STIM1 punctae was located in actin-poor areas. Use of the SOCE blocker in MIN6 cells, 2-aminoethoxy diphenylborate (2-APB), prevented store depletion-dependent translocation of EYFP-STIM1 to the PM in a concentration-dependent (3.75-100muM) and reversible manner. TIRF microscopy revealed that 2-APB treatment led to the reversible disappearance of peri-PM EYFP-STIM1 punctae, while the ER structure in this compartment remained grossly unaffected. We conclude from this data that in these cells EYFP-STIM1 is delivered to a peri-PM location from the ER upon store depletion and this trafficking is reversibly blocked by 2-APB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ceca.2008.03.007DOI Listing
December 2008

Downstream regulatory element antagonistic modulator regulates islet prodynorphin expression.

Am J Physiol Endocrinol Metab 2006 Sep 18;291(3):E587-95. Epub 2006 Apr 18.

Department of Medicine, University of Chicago, IL 60637, USA.

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and alpha-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM(-/-) islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM(-/-) islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (approximately 200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate kappa-opioid receptors expressed predominantly in alpha-cells of the islet, we determined the role of dynorphin A-(1-17) in glucagon secretion from the alpha-cell. Stimulation with dynorphin A-(1-17) caused alpha-cell calcium fluctuations and a significant increase in glucagon release. DREAM(-/-) islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of alpha-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpendo.00612.2005DOI Listing
September 2006

Inositol (1,4,5)-trisphosphate dynamics and intracellular calcium oscillations in pancreatic beta-cells.

Diabetes 2005 Nov;54(11):3073-81

Department of Medicine, MC 1027, University of Chicago, Chicago, IL 60637, USA.

Glucose-stimulated insulin secretion is associated with transients of intracellular calcium concentration ([Ca2+]i) in the pancreatic beta-cell. We tested the hypothesis that inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] [Ca2+]i release is incorporated in glucose-induced [Ca2+]i oscillations in mouse islets and MIN6 cells. We found that depletion of intracellular Ca2+ stores with thapsigargin increased the oscillation frequency by twofold and inhibited the slow recovery phase of [Ca2+]i oscillations. We employed a pleckstrin homology domain-containing fluorescent biosensor, phospholipase C partial differential pleckstrin homology domain-enhanced green fluorescent protein, to visualize Ins(1,4,5)P3 dynamics in insulin-secreting MIN6 cells and mouse islets in real time using a video-rate confocal system. In both types of cells, stimulation with carbamoylcholine (CCh) and depolarization with KCl results in an increase in Ins(1,4,5)P3 accumulation in the cytoplasm. When stimulated with glucose, the Ins(1,4,5)P3 concentration in the cytoplasm oscillates in parallel with oscillations of [Ca2+]i. Maximal accumulation of Ins(1,4,5)P3 in these oscillations coincides with the peak of [Ca2+]i and tracks changes in frequencies induced by the voltage-gated K+ channel blockade. We show that Ins(1,4,5)P3 release in insulin-secreting cells can be stimulated by depolarization-induced Ca2+ flux. We conclude that Ins(1,4,5)P3 concentration oscillates in parallel with [Ca2+]i in response to glucose stimulation, but it is not the driving force for [Ca2+]i oscillations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/diabetes.54.11.3073DOI Listing
November 2005

Delayed-rectifier (KV2.1) regulation of pancreatic beta-cell calcium responses to glucose: inhibitor specificity and modeling.

Am J Physiol Endocrinol Metab 2005 Oct 12;289(4):E578-85. Epub 2005 Jul 12.

Dept. of Medicine, University of Chicago, IL 60637, USA.

The delayed-rectifier (voltage-activated) K(+) conductance (K(V)) in pancreatic islet beta-cells has been proposed to regulate plasma membrane repolarization during responses to glucose, thereby determining bursting and Ca(2+) oscillations. Here, we verified the expression of K(V)2.1 channel protein in mouse and human islets of Langerhans. We then probed the function of K(V)2.1 channels in islet glucose responses by comparing the effect of hanatoxin (HaTx), a specific blocker of K(V)2.1 channels, with a nonspecific K(+) channel blocker, tetraethylammonium (TEA). Application of HaTx (1 microM) blocked delayed-rectifier currents in mouse beta-cells, resulting in a 40-mV rightward shift in threshold of activation of the voltage-dependent outward current. In the presence of HaTx, there was negligible voltage-activated outward current below 0 mV, suggesting that K(V)2.1 channels form the predominant part of this current in the physiologically relevant range. We then employed HaTx to study the role of K(V)2.1 in the beta-cell Ca(2+) responses to elevated glucose in comparison with TEA. Only HaTx was able to induce slow intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in cells stimulated with 20 mM glucose, whereas TEA induced an immediate rise in [Ca(2+)](i) followed by rapid oscillations. In human islets, HaTx acted in a similar fashion. The data were analyzed using a detailed mathematical model of ionic flux and Ca(2+) regulation in beta-cells. The results can be explained by a specific HaTx effect on the K(V) current, whereas TEA affects multiple K(+) conductances. The results underscore the importance of K(V)2.1 channel in repolarization of the pancreatic beta-cell plasma membrane and its role in regulating insulin secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpendo.00054.2005DOI Listing
October 2005

Small-conductance calcium-activated K+ channels are expressed in pancreatic islets and regulate glucose responses.

Diabetes 2003 Aug;52(8):2000-6

Department of Medicine, University of Chicago, Chicago, Illinois, USA.

Glucose-stimulated insulin secretion is associated with transients of intracellular Ca(2+) concentration [Ca(2+)](i) in the pancreatic beta-cell. We identified the expression and function of specific small-conductance Ca(2+)-activated K(+) (SK) channel genes in insulin-secreting cells. The presence of mRNA for SK1, -2, -3, and -4 (intermediate-conductance Ca(2+)-activated K(+) 1 [IK1]) channels was demonstrated by RT-PCR in rodent islets and insulinoma cells. SK2 and -3 proteins in mouse islets were detected by immunoblot and immunocytochemistry. In the tTA-SK3 tet-off mouse, a normal amount of SK3 protein was present in islets, but it became undetectable after exposure to doxycycline (DOX), which inhibits the transcription of the tTA-SK3 gene. The SK/IK channel-blockers apamin, dequalinium, and charybdotoxin caused increases in average [Ca(2+)](i) levels and in frequency of [Ca(2+)](i) oscillations in wild-type mouse islets. In SK3-tTA tet-off mice, the addition of apamin with glucose and tetraethylammonium (TEA) caused a similar elevation in [Ca(2+)](i), which was greatly diminished after DOX suppression of SK3 expression. We conclude that SK1, -2, -3, and IK1 (SK4) are expressed in islet cells and insulin-secreting cells and are able to influence glucose-induced calcium responses, thereby regulating insulin secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/diabetes.52.8.2000DOI Listing
August 2003

Modeling of Ca2+ flux in pancreatic beta-cells: role of the plasma membrane and intracellular stores.

Am J Physiol Endocrinol Metab 2003 Jul 18;285(1):E138-54. Epub 2003 Mar 18.

Department of Medicine, University of Chicago, Illinois 60637, USA.

We have developed a detailed mathematical model of ionic flux in beta-cells that includes the most essential channels and pumps in the plasma membrane. This model is coupled to equations describing Ca2+, inositol 1,4,5-trisphosphate (IP3), ATP, and Na+ homeostasis, including the uptake and release of Ca2+ by the endoplasmic reticulum (ER). In our model, metabolically derived ATP activates inward Ca2+ flux by regulation of ATP-sensitive K+ channels and depolarization of the plasma membrane. Results from the simulations support the hypothesis that intracellular Na+ and Ca2+ in the ER can be the main variables driving both fast (2-7 osc/min) and slow intracellular Ca2+ concentration oscillations (0.3-0.9 osc/min) and that the effect of IP3 on Ca2+ leak from the ER contributes to the pattern of slow calcium oscillations. Simulations also show that filling the ER Ca2+ stores leads to faster electrical bursting and Ca2+ oscillations. Specific Ca2+ oscillations in isolated beta-cell lines can also be simulated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpendo.00194.2002DOI Listing
July 2003

TRP genes: candidates for nonselective cation channels and store-operated channels in insulin-secreting cells.

Diabetes 2002 Feb;51 Suppl 1:S183-9

Department of Neurobiology, Physiology, and Pharmacology, the University of Chicago, Chicago, Illinois, USA.

Nonselective cation channels may play a role in insulin secretion by regulating pancreatic beta-cell plasma membrane potential, Ca(2+) homeostasis, and thereby glucose signaling. Transient receptor potential channel (TRPC)-related genes encode nonselective cation channels, some of which are similar to those described for beta-cells. Some TRPC-like channels are activated via G-protein--coupled mechanisms, some have been reported to be calcium-store-operated channels (SOC), and others are activated by novel signaling molecules or are sensitive to pressure and osmotic strength. Here we report the cloning and expression of mSTRPC4 from a mouse insulinoma cDNA library. mSTRPC4 encoded a protein of 97 kd, expressed in both endocrine cells and the brain. Stable cell lines expressing mSTRPC4 showed abundant mSTRPC4 protein, but no reproducible currents could be detected. mSTRPC4 therefore probably functions as a heteromultimer. We also report that LTRPC2, a G-protein and adenosine 5'-diphosphoribose (ADPR)-activated nonselective cation channel, is also expressed in human islets. TRPC-like channels may provide a pathway for depolarization or Ca(2+) entry in beta-cells and may be interesting targets for manipulating beta-cell function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/diabetes.51.2007.s183DOI Listing
February 2002