Susan L. Samson

Gene Therapy for Diabetes: Metabolic Effects of Helper-dependent Adenoviral Exendin 4 Expression in a Diet-induced Obesity Mouse Model

Exendin 4 (Ex4) is a glucagon-like peptide-1 receptor (GLP- 1R) agonist which is available as a short-acting injectable treatment for type 2 diabetes. Our aim was to characterize the long-term effects of elevated steady-state levels of Ex4 provided by in vivo gene therapy. We constructed a helper-dependent adenoviral (HDAd) vector for long-term expression of Ex4 in vivo. A high-fat diet (HFD)-induced obesity (DIO) mouse model was chosen to approximate the metabolic derangements seen in obese patients. Mice were treated with a single injection of HDAd-Ex4 and were monitored for 15 weeks. Both hepatic Ex4 RNA and plasma Ex4 were detectable at the end of the study. HDAd-Ex4 treatment improved glucose homeostasis without increasing insulin levels. However, there was evidence of enhanced insulin action and decreased gluconeogenic enzyme expression. HDAd-Ex4 caused decreased weight gain without detectable changes in food intake, in part, due to increases in energy expenditure (EE). HDAd-Ex4 DIO mice also had reduced hepatic fat and an improved adipokine profile. In the liver, there was decreased expression of genes that were involved in de novo fatty acid synthesis. These observations are important in considering the development of longer acting GLP-1R agonists for the treatment of type 2 diabetes.

The Krüppel-like zinc finger protein Glis3 directly and indirectly activates insulin gene transcription

Glis3 is a member of the Krüppel-like family of transcription factors and is highly expressed in islet β cells. Mutations in GLIS3 cause the syndrome of neonatal diabetes and congenital hypothyroidism (NDH). Our aim was to examine the role of Glis3 in β cells, specifically with regard to regulation of insulin gene transcription. We demonstrate that insulin 2 (Ins2) mRNA expression in rat insulinoma 832/13 cells is markedly increased by wild-type Glis3 overexpression, but not by the NDH1 mutant. Furthermore, expression of both Ins1 and Ins2 mRNA is downregulated when Glis3 is knocked down by siRNA. Glis3 binds to the Ins2 promoter in the cell, detected by chromatin immunoprecipitation. Deletion analysis of Ins2 promoter identifies a sequence (5′-GTCCCCTGCTGTGAA-3′) from −255 to −241 as the Glis3 response element and binding occur specifically via the Glis3 zinc finger region as revealed by mobility shift assays. Moreover, Glis3 physically and functionally interacts with Pdx1, MafA and NeuroD1 to modulate Ins2 promoter activity. Glis3 also may indirectly affect insulin promoter activity through upregulation of MafA and downregulation of Nkx6-1. This study uncovers a role of Glis3 for regulation of insulin gene expression and expands our understanding of its role in the β cell.

Ghrelin: much more than a hunger hormone.

Purpose of reviewGhrelin is a multifaceted gut hormone that activates its receptor, growth hormone secretagogue receptor (GHS-R). Ghrelins hallmark functions are its stimulatory effects on growth hormone release, food intake and fat deposition. Ghrelin is famously known as the ‘hunger hormone’. However, ample recent literature indicates that the functions of ghrelin go well beyond its role as an orexigenic signal. Here, we have reviewed some of the most recent findings on ghrelin and its signalling in animals and humans. Recent findingsGhrelin regulates glucose homeostasis by inhibiting insulin secretion and regulating gluconeogenesis/glycogenolysis. Ghrelin signalling decreases thermogenesis to regulate energy expenditure. Ghrelin improves the survival prognosis of myocardial infarction by reducing sympathetic nerve activity. Ghrelin prevents muscle atrophy by inducing muscle differentiation and fusion. Ghrelin regulates bone formation and metabolism by modulating proliferation and differentiation of osteoblasts. SummaryIn addition to ghrelins effects on appetite and adiposity, ghrelin signalling also plays crucial roles in glucose and energy homeostasis, cardioprotection, muscle atrophy and bone metabolism. These multifaceted roles of ghrelin make ghrelin and GHS-R highly attractive targets for drug development. Ghrelin mimetics may be used to treat heart diseases, muscular dystrophy/sarcopenia and osteoporosis; GHS-R antagonists may be used to treat obesity and insulin resistance.

Effects of combined exenatide and pioglitazone therapy on hepatic fat content in type 2 diabetes.

We examined the effects of combined pioglitazone (peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) agonist) and exenatide (GLP‐1 receptor agonist) therapy on hepatic fat content and plasma adiponectin levels in patients with type 2 diabetes (T2DM). Twenty‐one T2DM patients (age = 52 ± 3 years, BMI = 32.0 ± 1.5, hemoglobin A1c (HbA1c) = 8.2 ± 0.4%) on diet and/or metformin received additional treatment with either pioglitazone 45 mg/day for 12 months (n = 10) or combined therapy with pioglitazone (45 mg/day) and exenatide (10 µg subcutaneously twice daily) for 12 months (n = 11). At baseline, hepatic fat content and plasma adiponectin levels were similar between the two treatment groups. Pioglitazone reduced fasting plasma glucose (FPG) (P < 0.05), fasting free fatty acid (FFA) (P < 0.05), and HbA1c (Δ = 1.0%, P < 0.01), while increasing plasma adiponectin concentration by 86% (P < 0.05). Hepatic fat (magnetic resonance spectroscopy (MRS)) was significantly reduced following pioglitazone treatment (11.0 ± 3.1 to 6.5 ± 1.9%, P < 0.05). Plasma triglyceride concentration decreased by 14% (P < 0.05) and body weight increased significantly (Δ = 3.7 kg). Combined pioglitazone and exenatide therapy was associated with a significantly greater increase in plasma adiponectin (Δ = 193%) and a significantly greater decrease in hepatic fat (12.1 ± 1.7 to 4.7 ± 1.3%) and plasma triglyceride (38%) vs. pioglitazone therapy despite the lack of a significant change in body weight (Δ = 0.2 kg). Hepatic injury biomarkers aspartate aminotransferase and alanine aminotransferase (ALT) were significantly decreased by both treatments; however, the reduction in ALT was significantly greater following combined pioglitazone and exenatide therapy. We conclude that combined in patients with T2DM, pioglitazone and exenatide therapy is associated with a greater reduction in hepatic fat content as compared to the addition of pioglitazone therapy (Δ = 61% vs. 41%, P < 0.05).

Ectopic corticotropin-releasing hormone (CRH) syndrome from metastatic small cell carcinoma: a case report and review of the literature.

BackgroundCushings Syndrome (CS) which is caused by isolated Corticotropin-releasing hormone (CRH) production, rather than adrenocorticotropin (ACTH) production, is extremely rare.MethodsWe describe the clinical presentation, course, laboratory values and pathologic findings of a patient with isolated ectopic CRH causing CS. We review the literature of the types of tumors associated with this unusual syndrome and the behavior of these tumors by endocrine testing.ResultsA 56 year old woman presented with clinical and laboratory features consistent with ACTH-dependent CS. Pituitary imaging was normal and cortisol did not suppress with a high dose dexamethasone test, consistent with a diagnosis of ectopic ACTH. CT imaging did not reveal any discrete lung lesions but there were mediastinal and abdominal lymphadenopathy and multiple liver lesions suspicious for metastatic disease. Laboratory testing was positive for elevated serum carcinoembryonic antigen and the neuroendocrine marker chromogranin A. Serum markers of carcinoid, medullary thyroid carcinoma, and pheochromocytoma were in the normal range. Because the primary tumor could not be identified by imaging, biopsy of the presumed metastatic liver lesions was performed. Immunohistochemistry was consistent with a neuroendocrine tumor, specifically small cell carcinoma. Immunostaining for ACTH was negative but was strongly positive for CRH and laboratory testing revealed a plasma CRH of 10 pg/ml (normal 0 to 10 pg/ml) which should have been suppressed in the presence of high cortisol.ConclusionsThis case illustrates the importance of considering the ectopic production of CRH in the differential diagnosis for presentations of ACTH-dependent Cushings Syndrome.

Gene therapy for diabetes: reinventing the islet

A cure for type 1 (insulin dependent) diabetes might be found in generating surrogate insulin-producing cells to replace beta cells. A gene therapy strategy using constructs designed to allow glucose-regulated insulin transcription when delivered to non-pancreatic tissues has not fully recreated the stringent control of blood glucose provided by the beta cell. A more promising gene therapy approach has been to express pancreatic endocrine developmental factors, such as PDX-1, NeuroD/BETA2 and Neurogenin 3, to promote differentiation of non-endocrine cells towards a beta cell or islet phenotype, enabling these cells to synthesize and secrete insulin in a glucose-regulated manner. Further research is necessary, however, to better define the most effective pro-endocrine factors and the most amenable cell types to achieve transdifferentiation for beta cell replacement.

GLP-1R agonist therapy for diabetes: benefits and potential risks.

Purpose of review Glucagon-like peptide 1 receptor (GLP-1R) agonists provide good glycemic control combined with low hypoglycemia risk and weight loss. Here, we summarize the recently published data for this therapy class, focusing on sustainability of action, use in combination with basal insulin, and the efficacy of longer acting agents currently in development. The safety profile of GLP-1R agonists is also examined. Recent findings GLP-1R agonists provide sustained efficacy and their combination with basal insulin is well tolerated, providing additional glycemic control and weight benefits compared with basal insulin alone. Data suggest that the convenience of longer acting agents may be at the expense of efficacy. Despite the initial concerns, most evidence indicates that GLP-1R agonists do not increase the risk of pancreatitis or thyroid cancer. However, the extremely low incidence of these events means further investigations are required before a causal link can be eliminated. Large-scale clinical trials investigating the long-term cardiovascular safety of this therapy class are ongoing and may also provide important insights into pancreatic and thyroid safety. Summary GLP-1R agonists offer sustained glycemic efficacy, weight loss benefits, and a low risk of hypoglycemia. The results of ongoing trials should help to clarify the safety of this therapy class.

Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice

Insulin deficiency in type 1 diabetes leads to disruptions in glucose, lipid, and ketone metabolism with resultant hyperglycemia, hyperlipidemia, and ketonemia. Exogenous insulin and hepatic insulin gene therapy cannot mimic the robust glucose-stimulated insulin secretion (GSIS) from native pancreatic islets. Gene therapy of streptozotocin-diabetic mice with neurogenin 3 (Ngn3) and betacellulin (Btc) leads to the induction of periportal oval cell-derived neo-islets that exhibit GSIS. We hence hypothesized that this gene therapy regimen may lead to a complete correction of the glucose and lipid metabolic abnormalities associated with insulin deficiency; we further hypothesized that the neo-islets formed in response to Ngn3-Btc gene delivery may display an ultrastructure and transcription profile similar to that of pancreatic islets. We injected streptozotocin-diabetic mice with helper-dependent adenoviral vectors carrying Ngn3 and Btc, which restored GSIS and reversed hyperglycemia in these animals. The treatment also normalized hepatic glucose secretion and reversed ketonemia. Furthermore, it restored hepatic glycogen content and reinstated hepatic lipogenesis-related gene transcripts back to nondiabetic levels. By transmission electron microscopy, the neo-islets displayed electron-dense granules that were similar in appearance to those in pancreatic islets. Finally, using RNA obtained by laser capture microdissection of the periportal neo-islets and normal pancreatic islets, we found that the neo-islets and pancreatic islets exhibited a very similar transcription profile on microarray-based transcriptome analysis. Taken together, this indicates that Ngn3-Btc gene therapy corrects the underlying dysregulated glucose and lipid metabolism in insulin-deficient diabetic mice by inducing neo-islets in the liver that are similar to pancreatic islets in structure and gene expression profile.

Impaired mitochondrial fatty acid oxidation and insulin resistance in aging: novel protective role of glutathione

Aging is associated with impaired fasted oxidation of nonesterified fatty acids (NEFA) suggesting a mitochondrial defect. Aging is also associated with deficiency of glutathione (GSH), an important mitochondrial antioxidant, and with insulin resistance. This study tested whether GSH deficiency in aging contributes to impaired mitochondrial NEFA oxidation and insulin resistance, and whether GSH restoration reverses these defects. Three studies were conducted: (i) in 82‐week‐old C57BL/6 mice, the effect of naturally occurring GSH deficiency and its restoration on mitochondrial 13C1‐palmitate oxidation and glucose metabolism was compared with 22‐week‐old C57BL/6 mice; (ii) in 20‐week C57BL/6 mice, the effect of GSH depletion on mitochondrial oxidation of 13C1‐palmitate and glucose metabolism was studied; (iii) the effect of GSH deficiency and its restoration on fasted NEFA oxidation and insulin resistance was studied in GSH‐deficient elderly humans, and compared with GSH‐replete young humans. Chronic GSH deficiency in old mice and elderly humans was associated with decreased fasted mitochondrial NEFA oxidation and insulin resistance, and these defects were reversed with GSH restoration. Acute depletion of GSH in young mice resulted in lower mitochondrial NEFA oxidation, but did not alter glucose metabolism. These data suggest that GSH is a novel regulator of mitochondrial NEFA oxidation and insulin resistance in aging. Chronic GSH deficiency promotes impaired NEFA oxidation and insulin resistance, and GSH restoration reverses these defects. Supplementing diets of elderly humans with cysteine and glycine to correct GSH deficiency could provide significant metabolic benefits.

Potential of incretin-based therapies for non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is becoming an epidemic, paralleling the increased prevalence of obesity and diabetes, which are risk factors. In this review, we present the current pre-clinical evidence showing that GLP-1 analogues and DPP4 inhibitors can improve hepatic steatosis. Although some of the effects could be due to overall improvement in metabolic parameters, there are data to support improvements independent of weight loss, as well as direct effects on the hepatocyte in vitro. Multiple hepatocyte signal transduction pathways appear to be activated by GLP-1 and its analogues, with both AMP-activated protein kinase and Akt proposed to be key players in improving hepatic steatosis. However, it is controversial as to whether the pancreatic-type GLP-1 receptor is present or responsible for conferring the GLP-1 signal in the hepatocyte. In total, the data support the need for more rigorous prospective clinical trials to further investigate the potential of incretin therapies for treatment of NAFLD.