Burton M. Wice

Targeted Ablation of Glucose-dependent Insulinotropic Polypeptide-producing Cells in Transgenic Mice Reduces Obesity and Insulin Resistance Induced by a High Fat Diet

The K cell is a specific sub-type of enteroendocrine cell located in the proximal small intestine that produces glucose-dependent insulinotropic polypeptide (GIP), xenin, and potentially other unknown hormones. Because GIP promotes weight gain and insulin resistance, reducing hormone release from K cells could lead to weight loss and increased insulin sensitivity. However, the consequences of coordinately reducing circulating levels of all K cell-derived hormones are unknown. To reduce the number of functioning K cells, regulatory elements from the rat GIP promoter/gene were used to express an attenuated diphtheria toxin A chain in transgenic mice. K cell number, GIP transcripts, and plasma GIP levels were profoundly reduced in the GIP/DT transgenic mice. Other enteroendocrine cell types were not ablated. Food intake, body weight, and blood glucose levels in response to insulin or intraperitoneal glucose were similar in control and GIP/DT mice fed standard chow. In contrast to single or double incretin receptor knock-out mice, the incretin response was absent in GIP/DT animals suggesting K cells produce GIP plus an additional incretin hormone. Following high fat feeding for 21-35 weeks, the incretin response was partially restored in GIP/DT mice. Transgenic versus wild-type mice demonstrated significantly reduced body weight (25%), plasma leptin levels (77%), and daily food intake (16%) plus enhanced energy expenditure (10%) and insulin sensitivity. Regardless of diet, long term glucose homeostasis was not grossly perturbed in the transgenic animals. In conclusion, studies using GIP/DT mice demonstrate an important role for K cells in the regulation of body weight and insulin sensitivity.

TCF7L2 Variant rs7903146 Affects the Risk of Type 2 Diabetes by Modulating Incretin Action

OBJECTIVE Common variants in the gene TCF7L2 confer the largest effect on the risk of type 2 diabetes. The present study was undertaken to increase our understanding of the mechanisms by which this gene affects type 2 diabetes risk. RESEARCH DESIGN AND METHODS Eight subjects with risk-conferring TCF7L2 genotypes (TT or TC at rs7903146) and 10 matched subjects with wild-type genotype (CC) underwent 5-h oral glucose tolerance test (OGTT), isoglycemic intravenous glucose infusion, and graded glucose infusion (GGI). Mathematical modeling was used to quantify insulin-secretory profiles during OGTT and glucose infusion protocols. The incretin effect was assessed from ratios of the insulin secretory rates (ISR) during oral and isoglycemic glucose infusions. Dose-response curves relating insulin secretion to glucose concentrations were derived from the GGI. RESULTS β-cell responsivity to oral glucose was 50% lower (47 ± 4 vs. 95 ± 15 × 109 min−1; P = 0.01) in the group of subjects with risk-conferring TCF7L2 genotypes compared with control subjects. The incretin effect was also reduced by 30% (32 ± 4 vs. 46 ± 4%; P = 0.02) in the at-risk group. The lower incretin effect occurred despite similar glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) responses to oral glucose. The ISR response to intravenous glucose over a physiologic glucose concentration range (5–9 mmol/l) was similar between groups. CONCLUSIONS The TCF7L2 variant rs7903146 appears to affect risk of type 2 diabetes, at least in part, by modifying the effect of incretins on insulin secretion. This is not due to reduced secretion of GLP-1 and GIP but rather due to the effect of TCF7L2 on the sensitivity of the β-cell to incretins. Treatments that increase incretin sensitivity may decrease the risk of type 2 diabetes.

Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation

There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1+/− mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1+/− mice to Becn1+/− mice. After 1 week on a high fat diet, 4-week-old Pdx1+/− Becn1+/− mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1+/− mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention.

Sucralose Affects Glycemic and Hormonal Responses to an Oral Glucose Load

OBJECTIVE Nonnutritive sweeteners (NNS), such as sucralose, have been reported to have metabolic effects in animal models. However, the relevance of these findings to human subjects is not clear. We evaluated the acute effects of sucralose ingestion on the metabolic response to an oral glucose load in obese subjects. RESEARCH DESIGN AND METHODS Seventeen obese subjects (BMI 42.3 ± 1.6 kg/m2) who did not use NNS and were insulin sensitive (based on a homeostasis model assessment of insulin resistance score ≤2.6) underwent a 5-h modified oral glucose tolerance test on two separate occasions preceded by consuming either sucralose (experimental condition) or water (control condition) 10 min before the glucose load in a randomized crossover design. Indices of β-cell function, insulin sensitivity (SI), and insulin clearance rates were estimated by using minimal models of glucose, insulin, and C-peptide kinetics. RESULTS Compared with the control condition, sucralose ingestion caused 1) a greater incremental increase in peak plasma glucose concentrations (4.2 ± 0.2 vs. 4.8 ± 0.3 mmol/L; P = 0.03), 2) a 20 ± 8% greater incremental increase in insulin area under the curve (AUC) (P < 0.03), 3) a 22 ± 7% greater peak insulin secretion rate (P < 0.02), 4) a 7 ± 4% decrease in insulin clearance (P = 0.04), and 5) a 23 ± 20% decrease in SI (P = 0.01). There were no significant differences between conditions in active glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, glucagon incremental AUC, or indices of the sensitivity of the β-cell response to glucose. CONCLUSIONS These data demonstrate that sucralose affects the glycemic and insulin responses to an oral glucose load in obese people who do not normally consume NNS.

FORCED EXPRESSION OF ID-1 IN THE ADULT MOUSE SMALL INTESTINAL EPITHELIUM IS ASSOCIATED WITH DEVELOPMENT OF ADENOMAS

Ids are dominant-negative helix-loop-helix (HLH) proteins that play overlapping yet distinct roles in antagonizing basic HLH transcription factors. Although Ids affect myogenesis, neurogenesis, and B-cell development, little is known about theirin vivo functions in epithelia. We have examined the effects of forced expression of Id-1 in the small intestinal epithelium of adult chimeric mice. 129/Sv embryonic stem cells, transfected with DNA containing Id-1 under the control of transcriptional regulatory elements that function in all intestinal epithelial cell lineages, were introduced into C57Bl/6 (B6) blastocysts heterozygous for theROSA26 marker. The B6 ROSA26/+ intestinal epithelium of the resulting adult chimeras produces Escherichia coli β-galactosidase, allowing identification of this internal control cell population. Chimeras produced from nontransfected embryonic stem cells served as additional controls. Immunohistochemical studies of the control chimeras indicated that the small intestinal epithelium supports a complex pattern of endogenous Id expression. Id-1 is restricted to the cytoplasm; levels do not decrease as descendants of multipotent intestinal stem cells differentiate. Id-2 and Id-3 are only detectable in nuclei; levels increase markedly as epithelial cells differentiate. Forced expression of Id-1 in the 129/Sv epithelium results in a decline in Id-2 and Id-3 to below the limits of immunodetection. A subset of chimeric-transgenic mice lacked growth factor- and defensin-producing Paneth cells in their 129/Sv epithelium and also developed intestinal adenomas. These changes were not present in normal control chimeras. Adenomas were composed of proliferating β-Gal-positive and -negative epithelial cells, suggesting that they arose through cooperative interactions between 129/Sv(Id-1) and B6ROSA26/+ cells. These chimeras provide a model for studying how perturbations in Id expression affect tumorigenesis.

Activation of Serum Response Factor in the Depolarization Induction of Egr-1 Transcription in Pancreatic Islet β-Cells

The results of the current studies define the major elements whereby glucose metabolism in islet β-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2–20 mm) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that β-cell depolarization and Ca2+ influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca2+/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal −198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca2+-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser103-phosphorylated SRF in nuclear extracts, indicated that the SRE·SRF complexes contribute to the Ca2+-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.

A Tetraspan Membrane Glycoprotein Produced in the Human Intestinal Epithelium and Liver That Can Regulate Cell Density-dependent Proliferation

The human cell line HT-29 provides a model system for studying regulation of proliferation and differentiation in intestinal epithelial cell lineages: (i) HT-29 cells cultured in glucose resemble undifferentiated multipotent transit cells located in the lower half of intestinal crypts; (ii) proliferating HT-29 cells cultured in inosine resemble committed cells located in the upper half of the crypt; (iii) nonproliferating, confluent HT-29-inosine cells have features of differentiated enterocytes and goblet cells that overlie small intestinal villi. A cDNA library prepared from HT-29-inosine cells was screened with a series of subtracted cDNA probes to identify proteins that regulate proliferation/differentiation along the crypt-villus axis. A cDNA was recovered that encodes a 202-amino acid protein with four predicted membrane spanning domains and two potential sites for N-linked glycosylation. Levels of this new member of the superfamily of tetraspan membrane proteins (TMPs) increase dramatically as nondividing epithelial cells exit the proliferative compartment of the crypt-villus unit and migrate onto the villus. The protein is also produced in nondividing hepatocytes that have the greatest proliferative potential within liver acini. Three sets of observations indicate that in the appropriate cellular context, intestinal and liver (il)-TMP can mediate density-associated inhibition of proliferation. (i) Accumulation of il-TMP glycoforms precedes terminal differentiation of HT-29-inosine cells and occurs as they undergo density-dependent cessation of growth. il-TMP levels are lower and glycosylation less extensive in HT-29-glucose cells, which do not undergo growth arrest at confluence. (ii) HeLa cells normally do not produce il-TMP. Forced expression of il-TMP inhibits proliferation as cells approach confluence. The extent of il-TMP glycosylation in the transfected cells is similar to that observed in HT-29-inosine cells and greater than in HT-29-glucose cells. (iii) SW480 cells are derived from a human colon adenocarcinoma and do not express il-TMP. Like nontransfected HeLa cells, they do not stop dividing at confluence, whether grown in medium containing glucose or inosine. Expression of il-TMP has no effect on the growth properties of SW480 cells. The extent of il-TMP glycosylation in SW480-glucose cells is similar to that noted in HT-29-glucose cells, lending further support to the notion that il-TMPs activity is related to its state of N-glycosylation.

Novel insulin/GIP co‐producing cell lines provide unexpected insights into Gut K‐cell function in vivo

Enteroendocrine (EE) cells represent complex, rare, and diffusely‐distributed intestinal epithelial cells making them difficult to study in vivo. A specific sub‐population of EE cells called Gut K‐cells produces and secretes glucose‐dependent insulinotropic peptide (GIP), a hormone important for glucose homeostasis. The factors that regulate hormone production and secretion, as well as the timing of peptide release, are remarkably similar for K‐cells and islet β‐cells suggesting engineering insulin production by K‐cells is a potential gene therapeutic strategy to treat diabetes. K‐cell lines could be used to study the feasibility of this potential therapy and to understand Gut K‐cell physiology in general. Heterogeneous STC‐1 cells were transfected with a plasmid (pGIP/Neo) encoding neomycin phosphotransferase, driven by the GIP promoter‐only cells in which the GIP promoter was active survived genetic selection. Additional clones expressing pGIP/Neo plus a GIP promoter/insulin transgene were isolated—only doubly transfected cells produced preproinsulin mRNA. Bioactive insulin was stored and then released following stimulation with arginine, peptones, and bombesin—physiological GIP secretagogues. Like K‐cells in vivo, the GIP/insulin‐producing cells express the critical glucose sensing enzyme, glucokinase. However, glucose did not regulate insulin or GIP secretion or mRNA levels. Conversely, glyceraldehyde and methyl‐pyruvate were secretagogues, indicating cells depolarized in response to changes in intracellular metabolite levels. Potassium channel opening drugs and sulphonylureas had little effect on insulin secretion by K‐cells. The K‐cell lines also express relatively low levels of Kir 6.1, Kir 6.2, SUR1, and SUR2 suggesting secretion is independent of KATP channels. These results provided unexpected insights into K‐cell physiology and our experimental strategy could be easily modified to isolate/characterize additional EE cell populations.

Xenin-25 Potentiates Glucose-dependent Insulinotropic Polypeptide Action via a Novel Cholinergic Relay Mechanism

The intestinal peptides GLP-1 and GIP potentiate glucose-mediated insulin release. Agents that increase GLP-1 action are effective therapies in type 2 diabetes mellitus (T2DM). However, GIP action is blunted in T2DM, and GIP-based therapies have not been developed. Thus, it is important to increase our understanding of the mechanisms of GIP action. We developed mice lacking GIP-producing K cells. Like humans with T2DM, “GIP/DT” animals exhibited a normal insulin secretory response to exogenous GLP-1 but a blunted response to GIP. Pharmacologic doses of xenin-25, another peptide produced by K cells, restored the GIP-mediated insulin secretory response and reduced hyperglycemia in GIP/DT mice. Xenin-25 alone had no effect. Studies with islets, insulin-producing cell lines, and perfused pancreata indicated xenin-25 does not enhance GIP-mediated insulin release by acting directly on the β-cell. The in vivo effects of xenin-25 to potentiate insulin release were inhibited by atropine sulfate and atropine methyl bromide but not by hexamethonium. Consistent with this, carbachol potentiated GIP-mediated insulin release from in situ perfused pancreata of GIP/DT mice. In vivo, xenin-25 did not activate c-fos expression in the hind brain or paraventricular nucleus of the hypothalamus indicating that central nervous system activation is not required. These data suggest that xenin-25 potentiates GIP-mediated insulin release by activating non-ganglionic cholinergic neurons that innervate the islets, presumably part of an enteric-neuronal-pancreatic pathway. Xenin-25, or molecules that increase acetylcholine receptor signaling in β-cells, may represent a novel approach to overcome GIP resistance and therefore treat humans with T2DM.

Loss of Nix in Pdx1-deficient mice prevents apoptotic and necrotic β cell death and diabetes

Mutations in pancreatic duodenal homeobox (PDX1) are linked to human type 2 diabetes and maturity-onset diabetes of the young type 4. Consistent with this, Pdx1-haploinsufficient mice develop diabetes. Both apoptosis and necrosis of β cells are mechanistically implicated in diabetes in these mice, but a molecular link between Pdx1 and these 2 forms of cell death has not been defined. In this study, we introduced an shRNA into mouse insulinoma MIN6 cells to deplete Pdx1 and found that expression of proapoptotic genes, including NIP3-like protein X (Nix), was increased. Forced Nix expression in MIN6 and pancreatic islet β cells induced programmed cell death by simultaneously activating apoptotic and mitochondrial permeability transition-dependent necrotic pathways. Preventing Nix upregulation during Pdx1 suppression abrogated apoptotic and necrotic β cell death in vitro. In Pdx1-haploinsufficient mice, Nix ablation normalized pancreatic islet architecture, β cell mass, and insulin secretion and eliminated reactive hyperglycemia after glucose challenge. These results establish Nix as a critical mediator of β cell apoptosis and programmed necrosis in Pdx1-deficient diabetes.