Dhananjay Gupta

Peroxisome proliferator-activated receptor-γ regulates expression of PDX-1 and NKX6.1 in INS-1 cells

In the 60% pancreatectomy (Px) rat model of β-cell adaptation, normoglycemia is maintained by an initial week of β-cell hyperplasia that ceases and is followed by enhanced β-cell function. It is unknown how this complex series of events is regulated. We studied isolated islets and pancreas sections from 14-day post-Px versus sham-operated rats and observed a doubling of β-cell nuclear peroxisome proliferator–activated receptor (PPAR)-γ protein, along with a 2-fold increase in nuclear pancreatic duodenal homeobox (Pdx)-1 protein and a 1.4-fold increase in β-cell nuclear Nkx6.1 immunostaining. As PPAR-γ activation is known to both lower proliferation and have prodifferentiation effects in many tissues, we studied PPAR-γ actions in INS-1 cells. A 3-day incubation with the PPAR-γ agonist troglitazone reduced proliferation and increased Pdx-1 and Nkx6.1 immunostaining, along with glucokinase and GLUT2. Also, a 75% knockdown of PPAR-γ using RNA interference lowered the mRNA levels of Pdx-1, glucokinase, GLUT2, and proinsulin II by more than half. Our results show a dual effect of PPAR-γ in INS-1 cells: to curtail proliferation and promote maturation, the latter via enhanced expression of Pdx-1 and Nkx6.1. Additional studies are needed to determine whether there is a regulatory role for PPAR-γ signaling in the β-cell adaptation following a 60% Px in rats.

In Vivo and in Vitro Studies of a Functional Peroxisome Proliferator-activated Receptor γ Response Element in the Mouse pdx-1 Promoter

We reported that peroxisome proliferator-activated receptor γ (PPARγ) transcriptionally regulates the β-cell differentiation factor pancreatic duodenal homeobox (PDX)-1 based on in vitro RNA interference studies. We have now studied mice depleted of PPARγ within the pancreas (PANC PPARγ-/-) created by a Cre/loxP recombinase system, with Cre driven by the pdx-1 promoter. Male PANC PPARγ-/- mice were hyperglycemic at 8 weeks of age (8.1 ± 0.2 mm versus 6.4 ± 0.3 mm, p = 0.009) with islet cytoarchitecture and pancreatic mass of islet β-cells that were indistinguishable from the controls. Islet PDX-1 mRNA (p = 0.001) and protein levels (p = 0.003) were lowered 60 and 40%, respectively, in tandem with impaired glucose-induced insulin secretion and loss of thiazolidinedione-induced increase in PDX-1 expression. We next identified a putative PPAR-response element (PPRE) in the mouse pdx-1 promoter with substantial homology to the corresponding region of the human PDX-1 promoter. Electrophoretic mobility supershift assays with nuclear extracts from β-cell lines and mouse islets, also in vitro translated PPARγ and retinoid X receptor, and chromatin immunoprecipitation analysis demonstrated specific binding of PPARγ and retinoid X receptor to the human and mouse pdx-1 × PPREs. Transient transfection assays of β-cells with reporter constructs of mutated PPREs showed dramatically reduced pdx-1 promoter activity. In summary, we have presented in vivo and in vitro evidence showing PPARγ regulation of pdx-1 transcription in β-cells, plus our results support an important regulatory role for PPARγ in β-cell physiology and thiazolidinedione pharmacology of type 2 diabetes.

Physiologic and Pharmacologic Modulation of Glucose-Dependent Insulinotropic Polypeptide (GIP) Receptor Expression in β-Cells by Peroxisome Proliferator–Activated Receptor (PPAR)-γ Signaling Possible Mechanism for the GIP Resistance in Type 2 Diabetes

OBJECTIVE We previously showed that peroxisome proliferator–activated receptor (PPAR)-γ in β-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARγ in GIP-R transcription. RESEARCH DESIGN AND METHODS Chromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARγ knockout mice (PANC PPARγ−/−), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone. RESULTS In vitro studies of INS-1 cells confirmed that PPAR-γ binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARγ−/− mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress β-cell PPARγ. Thiazolidinedione activation (72 h) of this pathway in normal mouse islets caused a threefold increase of GIP-R protein and a doubling of insulin secretion to 16.7 mmol/l glucose/10 nmol/l GIP. Islets from obese normoglycemic ZF rats had twofold increased PPARγ and GIP-R protein levels versus lean rats, with both lowered by two-thirds in ZF rats made hyperglycemic by 60% Px. CONCLUSIONS Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in β-cells by PPARγ signaling. Also disruption of this signaling pathway may account for the lowered β-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

Physiologic and Pharmacologic Modulation of GIP Receptor Expression in ß- cells by PPARγ Signaling: Possible Mechanism for the GIP Resistance in Type 2 Diabetes

OBJECTIVE We previously showed that peroxisome proliferator–activated receptor (PPAR)-γ in β-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARγ in GIP-R transcription. RESEARCH DESIGN AND METHODS Chromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARγ knockout mice (PANC PPARγ−/−), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone. RESULTS In vitro studies of INS-1 cells confirmed that PPAR-γ binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARγ−/− mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress β-cell PPARγ. Thiazolidinedione activation (72 h) of this pathway in normal mouse islets caused a threefold increase of GIP-R protein and a doubling of insulin secretion to 16.7 mmol/l glucose/10 nmol/l GIP. Islets from obese normoglycemic ZF rats had twofold increased PPARγ and GIP-R protein levels versus lean rats, with both lowered by two-thirds in ZF rats made hyperglycemic by 60% Px. CONCLUSIONS Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in β-cells by PPARγ signaling. Also disruption of this signaling pathway may account for the lowered β-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

Peroxisome Proliferator-activated Receptor γ (PPARγ) and Its Target Genes Are Downstream Effectors of FoxO1 Protein in Islet β-Cells MECHANISM OF β-CELL COMPENSATION AND FAILURE

Background: The molecular mechanisms for islet β-cell compensation and failure are not fully known. Results: FoxO1/PPARγ signaling regulates key β-cell genes, with this network being up-regulated in nondiabetic insulin-resistant rats and impaired in rodents with diabetes. Conclusion: We examine the potential for the FoxO1/PPARγ network as a feature of β-cell compensation and failure. Significance: We identify targets for prevention of type 2 diabetes. The molecular mechanisms and signaling pathways that drive islet β-cell compensation and failure are not fully resolved. We have used in vitro and in vivo systems to show that FoxO1, an integrator of metabolic stimuli, inhibits PPARγ expression in β-cells, thus transcription of its target genes (Pdx1, glucose-dependent insulinotropic polypeptide (GIP) receptor, and pyruvate carboxylase) that are important regulators of β-cell function, survival, and compensation. FoxO1 inhibition of target gene transcription is normally relieved when upstream activation induces its translocation from the nucleus to the cytoplasm. Attesting to the central importance of this pathway, islet expression of PPARγ and its target genes was enhanced in nondiabetic insulin-resistant rats and markedly reduced with diabetes induction. Insight into the impaired PPARγ signaling with hyperglycemia was obtained with confocal microscopy of pancreas sections that showed an intense nuclear FoxO1 immunostaining pattern in the β-cells of diabetic rats in contrast to the nuclear and cytoplasmic FoxO1 in nondiabetic rats. These findings suggest a FoxO1/PPARγ-mediated network acting as a core component of β-cell adaptation to metabolic stress, with failure of this response from impaired FoxO1 activation causing or exacerbating diabetes.

Islet amyloid and type 2 diabetes: overproduction or inadequate clearance and detoxification?

A hallmark of type 2 diabetes is the reduction of pancreatic islet β cell mass through induction of apoptosis and lack of regeneration. In most patients, β cell dysfunction is associated with the presence of extracellular amyloid plaques adjacent to β cells and intracellular toxic oligomers that are comprised of islet amyloid polypeptide (IAPP). In this issue of the JCI, three independent research groups reveal that a functional autophagy system normally prevents the accumulation of toxic IAPP oligomers in human IAPP-expressing murine models. Furthermore, mice expressing human IAPP but deficient for β cell autophagy through genetic deletion of the autophagy initiator ATG7 developed β cell apoptosis and overt diabetes. Together, these studies indicate that autophagy protects β cells from the accumulation of toxic IAPP oligomers and suggest that enhancing autophagy may be a novel target for prevention of type 2 diabetes.

Temporal Characterization of β-cell Adaptive and Maladaptive Mechanisms During Chronic High Fat Feeding in C57BL/6NTac Mice

The onset of type 2 diabetes is characterized by transition from successful to failed insulin secretory compensation to obesity-related insulin resistance and dysmetabolism. Energy-rich diets in rodents are commonly studied models of compensatory increases in both insulin secretion and β cell mass. However, the mechanisms of these adaptive responses are incompletely understood, and it is also unclear why these responses eventually fail. We measured the temporal trends of glucose homeostasis, insulin secretion, β cell morphometry, and islet gene expression in C57BL/6NTac mice fed a 60% high-fat diet (HFD) or control diet for up to 16 weeks. A 2-fold increased hyperinsulinemia was maintained for the first 4 weeks of HFD feeding and then further increased through 16 weeks. β cell mass increased progressively starting at 4 weeks, principally through nonproliferative growth. Insulin sensitivity was not significantly perturbed until 11 weeks of HFD feeding. Over the first 8 weeks, we observed two distinct waves of increased expression of β cell functional and prodifferentiation genes. This was followed by activation of the unfolded protein response at 8 weeks and overt β cell endoplasmic reticulum stress at 12–16 weeks. In summary, β cell adaptation to an HFD in C57BL/6NTac mice entails early insulin hypersecretion and a robust growth phase along with hyperexpression of related genes that begin well before the onset of observed insulin resistance. However, continued HFD exposure results in cessation of gene hyperexpression, β cell functional failure, and endoplasmic reticulum stress. These data point to a complex but not sustainable integration of β cell-adaptive responses to nutrient overabundance, obesity development, and insulin resistance.