Regulation Of Insulin Gene Expression Research Articles

Glycolytic enzyme Enolase-1 regulates insulin gene expression in pancreatic β-cell

Enolase-1 (Eno1) plays a critical role in regulating glucose metabolism; however, its specific impact on pancreatic islet β-cells remains elusive. This study aimed to provide a preliminary exploration of Eno1 function in pancreatic islet β-cells. The findings revealed that the expression of ENO1 mRNA in type 2 diabetes donors was significantly increased and positively correlated with HbA1C and negatively correlated with insulin gene expression. A high level of Eno1 in human insulin-secreting rat INS-1832/13 cells with co-localization with intracellular insulin proteins was accordingly observed. Silencing of Eno1 using siRNA or inhibiting Eno1 protein activity with an Eno1 antagonist significantly reduced insulin secretion and insulin content in β-cells, while the proinsulin/insulin content ratio remained unchanged. This reduction in β-cells function was accompanied by a notable decrease in intracellular ATP and mitochondrial cytochrome C levels. Overall, our findings confirm that Eno1 regulates the insulin secretion process, particularly glucose metabolism and ATP production in the β-cells. The mechanism primarily involves its influence on insulin production, suggesting that Eno1 represents a potential target for β-cell protection and diabetes treatment.

TFEB and TFE3 control glucose homeostasis by regulating insulin gene expression.

To fulfill their function, pancreatic beta cells require precise nutrient-sensing mechanisms that control insulin production. Transcription factor EB (TFEB) and its homolog TFE3 have emerged as crucial regulators of the adaptive response of cell metabolism to environmental cues. Here, we show that TFEB and TFE3 regulate beta-cell function and insulin gene expression in response to variations in nutrient availability. We found that nutrient deprivation in beta cells promoted TFEB/TFE3 activation, which resulted in suppression of insulin gene expression. TFEB overexpression was sufficient to inhibit insulin transcription, whereas beta cells depleted of both TFEB and TFE3 failed to suppress insulin gene expression in response to amino acid deprivation. Interestingly, ChIP-seq analysis showed binding of TFEB to super-enhancer regions that regulate insulin transcription. Conditional, beta-cell-specific, Tfeb-overexpressing, and Tfeb/Tfe3 double-KO mice showed severe alteration of insulin transcription, secretion, and glucose tolerance, indicating that TFEB and TFE3 are important physiological mediators of pancreatic function. Our findings reveal a nutrient-controlled transcriptional mechanism that regulates insulin production, thus playing a key role in glucose homeostasis at both cellular and organismal levels.

Genetic and Functional Analyses of the Novel KLF11 Pro193Thr Variant in a Three-Generation Family with MODY7.

KLF11 regulates insulin gene expression through binding to the insulin promoter and has been reported as a causative gene for maturity-onset diabetes of the young 7 (MODY7). Here, we report a novel KLF11 variant associated with a three-generation family with early childhood-onset diabetes and explore its clinical and functional characteristics. The three-generational pedigree contains five patients affected by diabetes. The pathogenic variant identified by whole-exome sequencing was further confirmed by Sanger sequencing and pedigree verification. Luciferase reporter assays and glucose-stimulated insulin secretion were used to examine whether the KLF11 variant binds to the insulin promoter and regulate insulin secretion in vitro. The proband, his son, and his uncle exhibited hyperglycemia at ages 32, 13 and 71 years, respectively. All three patients showed characteristics of metabolic syndrome (obesity, dyslipidemia, and diabetes), but the insulin secretion of islet β-cells was impaired. A novel heterozygous missense variant, c.577 C>A (p.Pro193Thr) of the KLF11 gene was detected in all three patients. This variant co-segregates with the diabetes phenotype, consistent with an autosomal dominant disorder. The identified KLF11 p.Pro193Thr variant drastically decreased the transcriptional activity of KLF11, as demonstrated by luciferase reporter assay. Functional analyses revealed that the KLF11 Pro193Thr variant inhibited glucose-stimulated insulin secretion. We identified a novel KLF11 Pro193Thr variant in a three generation family with MODY7. These findings shed light on the molecular mechanisms underlying the pathogenesis of MODY7 and expand the genotype and clinical spectrum of MODY7.

YY1 Regulates Glucose Homeostasis Through Controlling Insulin Transcription in Pancreatic β-Cells.

To date, identification of nonislet-specific transcriptional factors in the regulation of insulin gene expression has been little studied. Here, we report that the expression level of the transcription factor YY1 is increased dramatically in both human and mouse pancreatic β-cells after birth. Nevertheless, the physiological role of YY1 during β-cell development and its regulatory mechanism in β-cell function remain largely unknown. After β-cell ablation of Yy1, we observed rapid onset of hyperglycemia, impaired glucose tolerance, and reduced β-cell mass in neonatal and adult mice. These mice also had hypoinsulinemia with normal insulin sensitivity compared with their wild-type littermates, manifesting as a type 1 diabetic phenotype. Mechanistically, genome-wide RNA sequencing has defined dysregulated insulin signaling and defective glucose responsiveness in β-cells devoid of YY1. Integrative analyses coupled with chromatin immunoprecipitation assays targeting YY1, and histone modifications, including H3K4me1, H3K27ac, and H3K27me3, have further identified Ins1 and Ins2 as direct gene targets of YY1. Luciferase reporter assays and loss- and gain-of-function experiments also demonstrated that YY1 binds to the enhancer regions in exon 2 of Ins1 and Ins2, activating insulin transcription and, therefore, proinsulin and insulin production in pancreatic β-cells. YY1 also directly interacts with RNA polymerase II, potentially stabilizing the enhancer-promoter interaction in the multiprotein-DNA complex during transcription initiation. Taken together, our findings suggest a role for YY1 as a transcriptional activator of insulin gene expression, assisting β-cell maturation and function after birth. These analyses may advance our understanding of β-cell biology and provide clinically relevant insights targeting the pathophysiological origins of diabetes.

Evidence from oyster suggests an ancient role for Pdx in regulating insulin gene expression in animals

Hox and ParaHox genes encode transcription factors with similar expression patterns in divergent animals. The Pdx (Xlox) homeobox gene, for example, is expressed in a sharp spatial domain in the endodermal cell layer of the gut in chordates, echinoderms, annelids and molluscs. The significance of comparable gene expression patterns is unclear because it is not known if downstream transcriptional targets are also conserved. Here, we report evidence indicating that a classic transcriptional target of Pdx1 in vertebrates, the insulin gene, is a likely direct target of Pdx in Pacific oyster adults. We show that one insulin-related gene, cgILP, is co-expressed with cgPdx in oyster digestive tissue. Transcriptomic comparison suggests that this tissue plays a similar role to the vertebrate pancreas. Using ATAC-seq and ChIP, we identify an upstream regulatory element of the cgILP gene which shows binding interaction with cgPdx protein in oyster hepatopancreas and demonstrate, using a cell culture assay, that the oyster Pdx can act as a transcriptional activator through this site, possibly in synergy with NeuroD. These data argue that a classic homeodomain-target gene interaction dates back to the origin of Bilateria.

The Role of Estrogens in Insulin Secretion. Implications for Aromatase Inhibitor Treatment

Introduction The main hormonal treatment of estrogen sensitive breast cancer includes the use of selective estrogen receptor modulators, aromatase inhibitors and GnRH-Analogs. It has been observed that administering aromatase inhibitor to breast cancer patients not only impairs their glucose metabolism but it can even cause frank diabetes mellitus. Moreover, it has been hypothesized that aromatase inhibitors may have an impact on glucose metabolism by their effect on estrogen levels. Therefore, we designed an experiment in order to assess the effect of estrogens on insulin secretion from rat beta pancreatic insulinoma INS-1 cells. Methods Experiments were conducted on an INS-1 cell line, a rodent beta cell line derived from a rat insulinoma induced by X-ray irradiation, which displays high insulin content, production of both proinsulin I and II and responsiveness to glucose and hormones. INS-1 cells were routinely cultured in 75cm2 flasks (T75) containing 10ml of appropriate culture media and then were transferred into 6-well plates and treated with 17β-E2. Proliferation, as well as insulin expression in the 17β-E2 treated cells in mRNA and protein level was then investigated. Cell cultures were treated with 12.5mM, 25mM, 50mM, 100mM and 200mM estradiol. After 24 hours, RNA as well as protein extraction was carried out. Gene expression was examined in mRNA and protein level, by real time quantitative reverse transcriptase PCR and by western blotting, respectively. Extraction of total RNA was achieved with the use of NucleoZOL (Macherey-Nagel, Duren, Germany). Protein concentration of samples was measured by using the bicinchoninic acid (BCA) assay and bovine serum albumin (BSA) as the standard (Thermo Scientific TM Pierce TM BCA Protein Assay Kit, USA). Results Real time PCR showed a dose-dependent up regulation of insulin gene expression by estradiol. The findings were consistent with the results from western blot, although the estradiol dose-dependent increase in gene expression was not so clear. Finally, both protein and mRNA expression of insulin increased in a dose dependent manner, with mRNA reaching a plateau at 100nM estradiol treatment. Conclusion The experimental data suggest that there might be a direct effect of estrogen on beta cells and insulin secretion.

Genetically engineered pigs manifesting pancreatic agenesis with severe diabetes

IntroductionPancreatic duodenum homeobox 1 (Pdx1) expression is crucial for pancreatic organogenesis and is a key regulator of insulin gene expression. Hairy and enhancer of split 1 (Hes1) controls tissue morphogenesis...

Naringin (4′,5,7-Trihydroxyflavanone 7-Rhamnoglucoside) Attenuates β-Cell Dysfunction in Diabetic Rats through Upregulation of PDX-1

Background: Pancreatic duodenal homeobox-1 (PDX-1) is a key transcription factor which regulates Insulin gene expression and insulin secretion in adult β-cells and helps to maintain β-cells mass. Naringin, a flavanone, owing to its anti­oxidant property, is reported to have antidiabetic effects. Objectives: The present study tries to evaluate the role of naringin on the β-cell-specific transcription factor PDX-1 in diabetic rats. Methods: Diabetes was induced in male rats using streptozotocin and treated with naringin (100 mg/kg) orally for 4 and 8 weeks. Serum insulin level, Pdx-1 and Insulin gene expression, and PDX-1 protein expression were assessed in the rat pancreas. Histopathological and ultrastructural changes in the islet and β-cells were observed. Results: Naringin prevented leukocytic infiltration in the pancreas of diabetic rats and recouped the β-cells with adequate secretory granules. Naringin-treated diabetic rats showed significantly increased mRNA expression of Pdx-1 and Insulin genes, increased expression of transcription factor PDX-1, and higher serum insulin levels than the diabetic control animals. These changes were more pronounced in the 8-week naringin-treated diabetic animals. Conclusions: Naringin was found to be an effective antidiabetic agent which increased Insulin gene expression and insulin secretion by upregulating the PDX-1 gene and protein expression.

Insulin Action Research and the Future of Diabetes Treatment: The 2017 Banting Medal for Scientific Achievement Lecture.

Diabetes is caused by combined abnormalities in insulin production and action. The pathophysiology of these defects has been studied extensively and is reasonably well understood. Their causes are elusive and their manifestations pleiotropic, likely reflecting the triple threat of genes, environment, and lifestyle. Treatment, once restricted to monotherapy with secretagogues or insulin, now involves complex combinations of expensive regimens that stem the progression but do not fundamentally alter the underlying causes of the disease. As advances in our understanding of insulin action and β-cell failure reach a critical stage, here I draw on lessons learned from our research on insulin regulation of gene expression and pancreatic β-cell dedifferentiation to address the question of how we can translate this exciting biology into mechanism-based interventions to reverse the course of diabetes.

Possible role of miR-2909 RNomics in arsenic mediated pancreatic β-cell dysfunction

Chronic exposure of humans to inorganic arsenic as a potential risk for the incidence of diabetes has received wide attention. However, the biological mechanism through which arsenic plays a role in the development of diabetes is still being evaluated. One of the hallmark of diabetes is the β-cell dysfunction followed by the changes in the insulin secretion. Pancreatic duodenal homeobox 1 (PDX1) has been widely recognized to play crucial role in the β-cell development, survival and its regulation of insulin gene expression. Many of the arsenic mediated cellular affects have been shown to be regulated by miR-2909 in vitro. Our present study provides evidence to reveal that arsenic affects miR-2909 expression in the pancreatic β-cell and this novel miRNA regulates PDX1 transcriptional expression indirectly through genes coding for c-Jun, MafA, PI3K and directly at the translational level by targeting the PDX1 mRNA. We provide further evidence for this miR-2909 RNomics in pancreatic tissue obtained from NOD mice where the expression of miR-2909 was high compared to the control mice. Keeping in view the fact that arsenic is known to cause β-cell dysfunction and most of the cellular effects of arsenic have been shown to be mediated through miR-2909 RNomics, our study revealed that arsenic employs miR-2909 (at low doses) and c-Jun (at high doses) to down regulate PDX1 in order to cause β-cell dysfunction leading to diabetic state.

Cross-talk among HMGA1 and FoxO1 in control of nuclear insulin signaling

As a mediator of insulin-regulated gene expression, the FoxO1 transcription factor represents a master regulator of liver glucose metabolism. We previously reported that the high-mobility group AT-hook 1 (HMGA1) protein, a molecular switch for the insulin receptor gene, functions also as a downstream target of the insulin receptor signaling pathway, representing a critical nuclear mediator of insulin function. Here, we investigated whether a functional relationship existed between FoxO1 and HMGA1, which might help explain insulin-mediated gene transcription in the liver. To this end, as a model study, we investigated the canonical FoxO1-HMGA1-responsive IGFBP1 gene, whose hepatic expression is regulated by insulin. By using a conventional GST-pull down assay combined with co-immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) analyses, we provide evidence of a physical interaction between FoxO1 and HMGA1. Further investigation with chromatin immunoprecipitation, confocal microscopy, and Fluorescence Recovery After Photobleaching (FRAP) technology indicated a functional significance of this interaction, in both basal and insulin-stimulated states, providing evidence that, by modulating FoxO1 transactivation, HMGA1 is essential for FoxO1-induced IGFBP1 gene expression, and thereby a critical modulator of insulin-mediated FoxO1 regulation in the liver. Collectively, our findings highlight a novel FoxO1/HMGA1-mediated mechanism by which insulin may regulate gene expression and metabolism.

GLIS3 rs7020673 and rs10758593 polymorphisms interact in the susceptibility for type 1 diabetes mellitus.

The transcription factor Gli-similar 3 (GLIS3) plays a key role in the development and maintenance of pancreatic beta cells as well as in the regulation of Insulin gene expression in adults. Accordingly, genome-wide association studies identified GLIS3 as a susceptibility locus for type 1 diabetes mellitus (T1DM) and glucose metabolism traits. Therefore, the aim of this study was to replicate the association of the rs10758593 and rs7020673 single nucleotide polymorphisms (SNPs) in the GLIS3 gene with T1DM in a Brazilian population. Frequencies of the rs7020673 (G/C) and rs10758593 (A/G) SNPs were analyzed in 503 T1DM patients (cases) and in 442 non-diabetic subjects (controls). Haplotypes constructed from the combination of these SNPs were inferred using a Bayesian statistical method. Genotype and allele frequencies of rs7020673 and rs10758593 SNPs did not differ significantly between case and control groups. However, the frequency of ≥3 minor alleles of the analyzed SNPs in haplotypes was higher in T1DM patients compared to non-diabetic subjects (6.2 vs. 1.6%; P=0.001). The presence of ≥3 minor alleles remained independently associated with risk of T1DM after adjustment for T1DM high-risk HLA DR/DQ haplotypes, age and ethnicity (OR=3.684 95% CI 1.220-11.124). Moreover, levels of glycated hemoglobin seem to be higher in T1DM patients with rs10758593 A/A genotype than patients carrying the G allele of this SNP (P=0.038), although this association was not kept after Bonferroni correction. Our results indicate that individually the rs7020673 and rs10758593 SNPs are not significantly associated with T1DM but seem to interact in the predisposition for this disease.

Effects of Taurine Supplementation on Neuronal Excitability and Glucose Homeostasis.

In this study we examined the role of chronic taurine supplementation on plasma glucose homeostasis and brain excitability through activation of the insulin receptor. FVB/NJ male mice were supplemented with taurine in drinking water (0.05% w/v) for 4weeks and subjected to a glucose tolerance test (7.5mg/kg BW) after 12h fasting. We found that taurine-fed mice were slightly hypoglycemic prior to glucose injection and showed significantly reduced plasma glucose at 30 and 60 min post-glucose injection when compared to control mice. Previously, we reported that taurine supplementation induces biochemical changes that target the GABAergic system. Those studies show that taurine-fed mice are hyperexcitable, have reduced GABAA receptors expression and increased GAD and somatostatin expression in the brain. In this study, we found that taurine-fed mice had a significant increase in insulin receptor (IR) immuno-reactivity in the pancreas and all brain regions examined. At the mRNA level, we found that the IR showed differential regional expression. Surprisingly, we found that neurons express the gene for insulin and that taurine had a significant role in regulating insulin gene expression. We propose that increased insulin production and secretion in taurine-fed mice cause an increase activation of the central IR and may be partially responsible for the increased neuronal excitability observed in taurine supplemented mice. Furthermore, the high levels of neuronal insulin expression and its regulation by taurine implicates taurine in the regulation of metabolic homeostasis.

PDX1 and ISL1 differentially coordinate with epigenetic modifications to regulate insulin gene expression in varied glucose concentrations

The mechanism of insulin gene transcription control in response to glucose concentration is poorly defined. The islet-restricted transcription factors PDX1 and ISL1 interact with BETA2, activating insulin gene expression. However, their contribution and hierarchical organization in insulin expression control based on glucose concentration remain unknown. We investigated PDX1 and ISL1 regulation of insulin gene expression in pancreatic β cells cultured in normal (5 mM/L) and high (25 mM/L) glucose conditions. ISL1 interacted with BETA2 to maintain basic insulin gene transcriptional activity under normal glucose. The ISL1-recruited cofactors SET9 and JMJD3 facilitated insulin gene histone modifications under normal glucose. In high-glucose concentrations, PDX1 formed a complex with BETA2 to enhance insulin gene expression. PDX1 also recruited SET9 and JMJD3 to promote the activation of histone modulation on the insulin promoter. This is the first evidence transcription factors orchestrate epigenetic modifications to control insulin gene expression based on glucose concentration.

Myotubes derived from human-induced pluripotent stem cells mirror in vivo insulin resistance

Induced pluripotent stem cells (iPS cells) represent a unique tool for the study of the pathophysiology of human disease, because these cells can be differentiated into multiple cell types in vitro and used to generate patient- and tissue-specific disease models. Given the critical role for skeletal muscle insulin resistance in whole-body glucose metabolism and type 2 diabetes, we have created a novel cellular model of human muscle insulin resistance by differentiating iPS cells from individuals with mutations in the insulin receptor (IR-Mut) into functional myotubes and characterizing their response to insulin in comparison with controls. Morphologically, IR-Mut cells differentiated normally, but had delayed expression of some muscle differentiation-related genes. Most importantly, whereas control iPS-derived myotubes exhibited in vitro responses similar to primary differentiated human myoblasts, IR-Mut myotubes demonstrated severe impairment in insulin signaling and insulin-stimulated 2-deoxyglucose uptake and glycogen synthesis. Transcriptional regulation was also perturbed in IR-Mut myotubes with reduced insulin-stimulated expression of metabolic and early growth response genes. Thus, iPS-derived myotubes from individuals with genetically determined insulin resistance demonstrate many of the defects observed in vivo in insulin-resistant skeletal muscle and provide a new model to analyze the molecular impact of muscle insulin resistance.

Higher protein kinase C ζ in fatty rat liver and its effect on insulin actions in primary hepatocytes.

We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF) rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ), a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL) rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.

A Novel Function of Onecut1 Protein as a Negative Regulator of MafA Gene Expression

The transcription factor MafA is a key regulator of insulin gene expression and maturation of islet β cells. Despite its importance, the regulatory mechanism of MafA gene expression is still unclear. To identify the transcriptional regulators of MafA, we examined various transcription factors, which are potentially involved in β cell differentiation. An adenovirus-mediated overexpression study clearly demonstrated that Onecut1 suppresses the promoter activity of MafA through the Foxa2-binding cis-element on the MafA enhancer region (named area A). However, ChIP analysis showed that Foxa2 but not Onecut1 could directly bind to area A. Furthermore, overexpression of Onecut1 inhibited the binding of Foxa2 onto area A upon ChIP analysis. Importantly, insertion of a mutation in the Foxa2-binding site of area A significantly decreased the promoter activity of MafA. These findings suggest that Onecut1 suppresses MafA gene expression through the Foxa2-binding site. In the mouse pancreas, MafA expression was first detected at the latest stage of β cell differentiation and was scarcely observed in Onecut1-positive cells during pancreas development. In addition, Onecut1 expression was significantly increased in the islets of diabetic db/db mice, whereas MafA expression was markedly decreased. The improved glucose levels of db/db mice with insulin injections significantly reduced Onecut1 expression and rescued the reduction of MafA expression. These in vivo experiments also suggest that Onecut1 is a negative regulator of MafA gene expression. This study implicates the novel role of Onecut1 in the control of normal β cell differentiation and its involvement in β cell dysfunction under diabetic conditions by suppressing MafA gene expression.

B-cell translocation gene 2 positively regulates GLP-1-stimulated insulin secretion via induction of PDX-1 in pancreatic β-cells

Glucagon-like peptide-1 (GLP-1) is a potent glucoincretin hormone and an important agent for the treatment of type 2 diabetes. Here we demonstrate that B-cell translocation gene 2 (BTG2) is a crucial regulator in GLP-1-induced insulin gene expression and insulin secretion via upregulation of pancreatic duodenal homeobox-1 (PDX-1) in pancreatic β-cells. GLP-1 treatment significantly increased BTG2, PDX-1 and insulin gene expression in pancreatic β-cells. Notably, adenovirus-mediated overexpression of BTG2 significantly elevated insulin secretion, as well as insulin and PDX-1 gene expression. Physical interaction studies showed that BTG2 is associated with increased PDX-1 occupancy on the insulin gene promoter via a direct interaction with PDX-1. Exendin-4 (Ex-4), a GLP-1 agonist, and GLP-1 in pancreatic β-cells increased insulin secretion through the BTG2–PDX-1–insulin pathway, which was blocked by endogenous BTG2 knockdown using a BTG2 small interfering RNA knockdown system. Finally, we revealed that Ex-4 and GLP-1 significantly elevated insulin secretion via upregulation of the BTG2–PDX-1 axis in pancreatic islets, and this phenomenon was abolished by endogenous BTG2 knockdown. Collectively, our current study provides a novel molecular mechanism by which GLP-1 positively regulates insulin gene expression via BTG2, suggesting that BTG2 has a key function in insulin secretion in pancreatic β-cells.

Glucose-responsive artificial promoter-mediated insulin gene transfer improves glucose control in diabetic mice

To investigate the effect of insulin gene therapy using a glucose-responsive synthetic promoter in type 2 diabetic obese mice. We employed a recently developed novel insulin gene therapy strategy using a synthetic promoter that regulates insulin gene expression in the liver in response to blood glucose level changes. We intravenously administered a recombinant adenovirus expressing furin-cleavable rat insulin under the control of the synthetic promoter (rAd-SP-rINSfur) into diabetic Lepr(db/db) mice. A recombinant adenovirus expressing β-galactosidase under the cytomegalovirus promoter was used as a control (rAd-CMV-βgal). Blood glucose levels and body weights were monitored for 50 d. Glucose and insulin tolerance tests were performed. Immunohistochemical staining was performed to investigate islet morphology and insulin content. Administration of rAd-SP-rINSfur lowered blood glucose levels and normoglycemia was maintained for 50 d, whereas the rAd-CMV-βgal control virus-injected mice remained hyperglycemic. Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice cleared exogenous glucose from the blood more efficiently than control virus-injected mice at 4 wk [area under the curve (AUC): 21 508.80 ± 2248.18 vs 62 640.00 ± 5014.28, P < 0.01] and at 6 wk (AUC: 29 956.60 ± 1757.33 vs 60 016.60 ± 3794.47, P < 0.01). In addition, insulin sensitivity was also significantly improved in mice treated with rAd-SP-rINSfur compared with rAd-CMV-βgal-treated mice (AUC: 9150.17 ± 1007.78 vs 11 994.20 ± 474.40, P < 0.05). The islets from rAd-SP-rINSfur-injected mice appeared to be smaller and to contain a higher concentration of insulin than those from rAd-CMV-βgal-injected mice. Based on these results, we suggest that insulin gene therapy might be one therapeutic option for remission of type 2 diabetes.

Proteasome activator PA28γ stimulates degradation of GSK3-phosphorylated insulin transcription activator MAFA

MAFA is a member of the MAF family of basic leucine zipper transcription factors and is a critical regulator of insulin gene expression and islet β-cell function. To be degraded by the proteasome, MAFA must be phosphorylated by GSK3 and MAP kinases at multiple serine and threonine residues (Ser49, Thr53, Thr57, Ser61, and Ser65) within its amino-terminal domain. In this study, we report that MAFA degradation is stimulated by PA28γ (REGγ and PSME3), a member of a family of proteasome activators that bind and activate the 20S proteasome. To date, only a few PA28γ-proteasome pathway substrates have been identified, including steroid receptor coactivator 3 (SRC3) and the cell cycle inhibitor p21(CIP1). PA28γ binds to MAFA, induces its proteasomal degradation, and thereby attenuates MAFA-driven transcriptional activation of the insulin promoter. Co-expression of GSK3 enhanced the PA28γ-mediated degradation of MAFA, but mutants that contained alanine substitutions at the MAFA phosphorylation sites did not bind PA28γ and were resistant to degradation. We also found that a PA28γ mutant (N151Y) that did not stimulate p21 degradation enhanced MAFA degradation, and another mutant (K188D) that promoted greater p21 degradation did not enhance MAFA degradation.These results suggest that PA28γ stimulates MAFA degradation through a novel molecular mechanism that is distinct from that for the degradation of p21.