The Novel Phosphate and Bile Acid Sequestrant Polymer SAR442357 Delays Disease Progression in a Rat Model of Diabetic Nephropathy.

As a gut-restricted, nonabsorbed therapy, polymeric bile acid sequestrants (BAS) play an important role in managing hyperlipidemia and hyperglycemia. Similarly, nonabsorbable sequestrants of dietary phosphate have been used for the management of hyperphosphatemia in end-stage renal disease. To evaluate the potential utility of such polymer sequestrants to treat type 2 diabetes (T2D) and its associated renal and cardiovascular complications, we synthesized a novel polymeric sequestrant, SAR442357, possessing optimized bile acid (BA) and phosphate sequestration characteristics. Long-term treatment of T2D obese cZucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) with SAR442357 resulted in enhanced sequestration of BAs and phosphate in the gut, improved glycemic control, lowering of serum cholesterol, and attenuation of diabetic kidney disease (DKD) progression. In comparison, colesevelam, a BAS with poor phosphate binding properties, did not prevent DKD progression, whereas losartan, an angiotensin II receptor blocker that is widely used to treat DKD, showed no effect on hyperglycemia. Analysis of hepatic gene expression levels of the animals treated with SAR442357 revealed upregulation of genes responsible for the biosynthesis of cholesterol and BAs, providing clear evidence of target engagement and mode of action of the new sequestrant. Additional hepatic gene expression pathway changes were indicative of an interruption of the enterohepatic BA cycle. Histopathological analysis of ZSF1 rat kidneys treated with SAR442357 further supported its nephroprotective properties. Collectively, these findings reveal the pharmacological benefit of simultaneous sequestration of BAs and phosphate in treating T2D and its associated comorbidities and cardiovascular complications. SIGNIFICANCE STATEMENT: A new nonabsorbed polymeric sequestrant with optimum phosphate and bile salt sequestration properties was developed as a treatment option for DKD. The new polymeric sequestrant offered combined pharmacological benefits including glucose regulation, lipid lowering, and attenuation of DKD progression in a single therapeutic agent.

A novel dual glucagon-like peptide and glucagon receptor agonist SAR425899: Results of randomized, placebo-controlled first-in-human and first-in-patient trials.

AIMS: To evaluate the safety, pharmacokinetics and pharmacodynamics of SAR425899, a novel polypeptide, active as an agonist at both the glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCR), in healthy volunteers and in overweight/obese patients with type 2 diabetes (T2D). METHODS: Subcutaneous administrations of SAR425899 were tested in two randomized, placebo-controlled, double-blind clinical trials. In the first trial, healthy overweight volunteers (body mass index [BMI] 25-30 kg/m2 ; n = 32) received single-ascending doses (0.01-0.1 mg) of SAR425899 or placebo. In the second, a multiple-ascending-dose trial (NCT02411825), healthy normal- to overweight volunteers (BMI 20-30 kg/m2 ; n = 40) and overweight/obese patients with T2D (BMI 28-42 kg/m2 ; n = 36) received daily doses of SAR425899 or placebo over 21 or 28 days, respectively. RESULTS: The most frequently reported adverse events were gastrointestinal; gastrointestinal side effects were less pronounced in patients with T2D compared with healthy volunteers. SAR425899 significantly reduced levels of fasting plasma glucose (P < 0.05 vs. placebo) and glycated haemoglobin (P < 0.001 versus placebo) in patients with T2D. Additionally, SAR425899 led to reductions in body weight, with a maximal reduction of 5.32 kg in healthy volunteers and 5.46 kg in patients with T2D (P < 0.001 vs. placebo) at end of treatment. CONCLUSIONS: SAR425899 was well tolerated and led to favourable glycaemic effects in patients with T2D and weight reduction in both healthy volunteers and patients. Whether dual GLP-1R/GCR agonism represents a treatment method that is superior to pure GLP-1R agonists for obesity and diabetes treatment remains to be confirmed.

Running on mixed fuel-dual agonistic approach of GLP-1 and GCG receptors leads to beneficial impact on body weight and blood glucose control: A comparative study between mice and non-human primates.

AIM: We performed acute and chronic studies in healthy and diet-induced obese animals using mouse-specific or monkey-specific dual GLP-1R/GCGR agonists to investigate their effects on food intake, body weight, blood glucose control and insulin secretion. The selective GLP-1R agonist liraglutide was used as comparator. METHODS: The mouse-specific dual agonist and liraglutide were tested in lean wild type, GLP-1R knockout and diet-induced obese mice at different doses. A chronic study was performed in DIO mice to investigate the effect on body weight, food consumption and total energy expenditure (TEE) in obese and diabetic monkeys with a focus on body weight and energy intake. RESULTS: The mouse-specific dual agonist and liraglutide similarly affected glycaemic control. A higher loss in body weight was measured in dual agonist-treated obese mice. The dual agonist significantly enhanced plasma glucose excursion in overnight fed GLP-1R-/- mice, probably reflecting a potent GCGR agonist activity. It increased TEE and enhanced fat and carbohydrate oxidation, while liraglutide produced no effect on TEE. In obese and diabetic monkeys, treatment with the monkey-specific dual agonist reduced total energy intake to 60%-70% of baseline TEI during chronic treatment. A decrease in body weight and significant improvement in glucose tolerance was observed. CONCLUSIONS: In DIO mice and non-human primates, dual agonists elicited robust glycaemic control, similar to the marketed GLP-1R agonist, while eliciting greater effects on body weight. Results from DIO mice suggest that the increase in TEE is caused not only by increased fat oxidation but also by an increase in carbohydrate oxidation.

Glucagonlike Peptide-1 Receptor Imaging in Individuals with Type 2 Diabetes.

The glucagonlike peptide-1 receptor (GLP1R) is a gut hormone receptor, intricately linked to regulation of blood glucose homeostasis via several mechanisms. It is an established and emergent drug target in metabolic disease. The PET radioligand 68Ga-DO3A-VS-exendin4 (68Ga-exendin4) has the potential to enable longitudinal studies of GLP1R in the human pancreas. Methods:68Ga-exendin4 PET/CT examinations were performed on overweight-to-obese individuals with type 2 diabetes (n = 13) as part of a larger target engagement study (NCT03350191). A scanning protocol was developed to optimize reproducibility (target amount of 0.5 MBq/kg [corresponding to peptide amount of <0.2 µg/kg], blood sampling, and tracer stability assessment). The pancreas and abdominal organs were segmented, and binding was correlated with clinical parameters. Results: Uptake of 68Ga-exendin4 in the pancreas, but not in other abdominal tissues, was high but variable between individuals. There was no evidence of self-blocking of GLP1R by the tracer in this protocol, despite the high potency of exendin4. The results showed that a full dynamic scan can be simplified to a short static scan, potentially increasing throughput and reducing patient discomfort. The 68Ga-exendin4 concentration in the pancreas (i.e., GLP1R density) correlated inversely with the age of the individual and tended to correlate positively with body mass index. However, the total GLP1R content in the pancreas did not. Conclusion: In summary, we present an optimized and simplified 68Ga-exendin4 scanning protocol to enable reproducible imaging of GLP1R in the pancreas. 68Ga-exendin4 PET may enable quantification of longitudinal changes in pancreatic GLP1R during the development of type 2 diabetes, as well as target engagement studies of novel glucagonlike peptide-1 agonists.

ADD1/SREBP1c activates the PGC1-alpha promoter in brown adipocytes.

Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and beta-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor alpha (LXRalpha), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein-1c (SREBP1c) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC1alpha) in brown and inguinal white adipose tissues, but not in epididymal white adipose tissue. Using in vitro models of white and brown adipocytes we demonstrate that beta-adrenergic stimulation induced expression of LXRalpha, ADD1/SREBP1c and PGC1alpha in cells with a brown-like adipose phenotype. We demonstrate that ADD1/SREBP1c is a powerful inducer of PGC1alpha expression via a conserved E box in the proximal promoter and that beta-adrenergic stimulation led to recruitment of ADD1/SREBP1c to this E box. The ability of ADD1/SREBP1c to activate the PGC1alpha promoter exhibited a striking cell type dependency, suggesting that additional cell type-restricted factors contribute to ADD1/SREBP1c-mediated activation. In conclusion, our data demonstrate a novel role of ADD1/SREBP1c as a regulator of PGC1alpha expression in brown adipose tissue.

Drug Occupancy Assessment at the Glucose-Dependent Insulinotropic Polypeptide Receptor by Positron Emission Tomography.

Targeting of the glucose-dependent insulinotropic polypeptide receptor (GIPR) is an emerging strategy in antidiabetic drug development. The aim of this study was to develop a positron emission tomography (PET) radioligand for the GIPR to enable the assessment of target distribution and drug target engagement in vivo. The GIPR-selective peptide S02-GIP was radiolabeled with 68Ga. The resulting PET tracer [68Ga]S02-GIP-T4 was evaluated for affinity and specificity to human GIPR (huGIPR). The in vivo GIPR binding of [68Ga]S02-GIP-T4 as well as the occupancy of a drug candidate with GIPR activity were assessed in nonhuman primates (NHPs) by PET. [68Ga]S02-GIP-T4 bound with nanomolar affinity and high selectivity to huGIPR in overexpressing cells. In vivo, pancreatic binding in NHPs could be dose-dependently inhibited by coinjection of unlabeled S02-GIP-T4. Finally, subcutaneous pretreatment with a high dose of a drug candidate with GIPR activity led to a decreased pancreatic binding of [68Ga]S02-GIP-T4, corresponding to a GIPR drug occupancy of almost 90%. [68Ga]S02-GIP-T4 demonstrated a safe dosimetric profile, allowing for repeated studies in humans. In conclusion, [68Ga]S02-GIP-T4 is a novel PET biomarker for safe, noninvasive, and quantitative assessment of GIPR target distribution and drug occupancy.

Imaging of the Glucagon Receptor in Subjects with Type 2 Diabetes.

Despite the importance of the glucagon receptor (GCGR) in disease and in pharmaceutical drug development, there is a lack of specific and sensitive biomarkers of its activation in humans. The PET radioligand 68Ga-DO3A-VS-Tuna-2 (68Ga-Tuna-2) was developed to yield a noninvasive imaging marker for GCGR target distribution and drug target engagement in humans. Methods: The biodistribution and dosimetry of 68Ga-Tuna-2 was assessed by PET/CT in 13 individuals with type 2 diabetes as part of a clinical study assessing the occupancy of the dual GCGR/glucagon like peptide-1 receptor agonist SAR425899. Binding of 68Ga-Tuna-2 in liver and reference tissues was evaluated and correlated to biometrics (e.g., weight or body mass index) or other biomarkers (e.g., plasma glucagon levels). Results:68Ga-Tuna-2 binding was seen primarily in the liver, which is in line with the strong expression of GCGR on hepatocytes. The kidneys demonstrated high excretion-related retention, whereas all other tissue demonstrated rapid washout. The SUV55 min (SUV during the last 10-min time frame, 50-60 min after administration) uptake endpoint was sensitive to endogenous levels of glucagon. 68Ga-Tuna-2 exhibited a safe dosimetry profile and no adverse events after intravenous administration. Conclusion:68Ga-Tuna-2 can be used for safe and accurate assessment of the GCGR in human. It may serve as an important tool in understanding the in vivo pharmacology of novel drugs engaging the GCGR.

Stereological assessment of pancreatic beta-cell mass development in male Zucker Diabetic Fatty (ZDF) rats: correlation with pancreatic beta-cell function.

The present study was initiated to improve our understanding of pancreatic beta-cell dynamics in male Zucker Diabetic Fatty (ZDF) rats and hence provide a framework for future diabetes studies in this animal model. Male ZDF rats from 6, 8, 10, 12, 14, 16, 20 and 26 weeks of age were subjected to an oral glucose tolerance test (OGTT). The animals were then euthanized and pancreases were removed for morphometric analyses of pancreatic beta-cell mass. As evident by a marked fourfold increase in insulin secretion, insulin resistance developed rapidly from 6 to 8 weeks of age. Simultaneously, the pancreatic beta-cell mass expanded from 6.17 ± 0.41 mg at 6 weeks of age, reaching a maximum of 16.5 ± 2.5 mg at 16 weeks of age, at which time pancreatic beta-cell mass gradually declined. The corresponding changes in glucose/insulin homeostasis were analysed using a standard insulin sensitivity index (ISI), an area under the curve (AUC) glucose-insulin index, or simple semi-fasted glucose levels. The study demonstrated that male ZDF rats underwent rapid changes in pancreatic beta-cell mass from the onset of insulin resistance to frank diabetes coupled directly to marked alterations in glucose/insulin homeostasis. The study underscores the need for a critical co-examination of glucose homeostatic parameters in studies investigating the effects of novel anti-diabetic compounds on pancreatic beta-cell mass in the male ZDF rat. A simple assessment of fasting glucose levels coupled with information about age can provide a correct indication of the actual pancreatic beta-cell mass and the physiological state of the animal.

Gut Mucosal Gene Expression and Metabolic Changes After Roux-en-Y Gastric Bypass Surgery.

OBJECTIVE: Changes in the secretion of gut-derived peptide hormones have been associated with the metabolic benefits of Roux-en-Y gastric bypass (RYGB) surgery. In this study, the effects of RYGB on anthropometrics, postprandial plasma hormone responses, and mRNA expression in small intestinal mucosa biopsy specimens before and after RYGB were evaluated. METHODS: In a cross-sectional study, 20 individuals with obesity undergoing RYGB underwent mixed meal tests and upper enteroscopy with retrieval of small intestinal mucosa biopsy specimens 3 months before and after surgery. Concentrations of circulating gut and pancreatic hormones during mixed meal tests as well as full mRNA sequencing of biopsy specimens were evaluated. RESULTS: RYGB-induced improvements of body weight and composition, insulin resistance, and circulating cholesterols were accompanied by significant changes in postprandial plasma responses of pancreatic and gut hormones. Global gene expression analysis of biopsy specimens identified 2,437 differentially expressed genes after RYGB, including changes in genes that encode prohormones and G protein-coupled receptors. CONCLUSIONS: RYGB affects the transcription of a wide range of genes, indicating that the observed beneficial metabolic effects of RYGB may rely on a changed expression of several genes in the gut. RYGB-induced changes in the expression of genes encoding signaling peptides and G protein-coupled receptors may disclose new gut-derived treatment targets against obesity and diabetes.

Rimonabant induced anorexia in rodents is not mediated by vagal or sympathetic gut afferents.

The selective CB1 receptor antagonist rimonabant is a novel weight control agent. Although CB1 receptors and binding sites are present in both the rodent central and peripheral nervous systems, including the afferent vagus nerve, the role of gut afferents in mediating anorexia following CB1R blockade is still debated. In the present study we examined rimonabant-induced anorexia in male C57BL/6J mice with subdiaphragmatic vagotomy (VGX) as well as in male Sprague-Dawley rats subjected to either subdiaphragmatic vagal deafferentation (SDA) alone or in combination with a complete celiac-superior mesenteric ganglionectomy (CGX). Irrespective of the operational procedure, rimonabant (10mg/kg) effectively reduced standard chow as well as palatable diet (ensure) intake. In conclusion, the data clearly demonstrate that neither vagal gut afferents, nor gut afferents traveling via the sympathetic nervous system, are required for rimonabant to inhibit food intake leading to the hypothesis that centrally located CB1 receptors are the prime mediators of rimonabant-induced anorexia.

The role of C16:0 ceramide in the development of obesity and type 2 diabetes: CerS6 inhibition as a novel therapeutic approach.

OBJECTIVE: Ectopic fat deposition is associated with increased tissue production of ceramides. Recent genetic mouse studies suggest that specific sphingolipid C16:0 ceramide produced by ceramide synthase 6 (CerS6) plays an important role in the development of insulin resistance. However, the therapeutic potential of CerS6 inhibition not been demonstrated. Therefore, we pharmacologically investigated the selective ablation of CerS6 using antisense oligonucleotides (ASO) in obese insulin resistance animal models. METHODS: We utilized ASO as therapeutic modality, CerS6 ASO molecules designed and synthesized were initially screened for in-vitro knock-down (KD) potency and cytotoxicity. ASOs with >85% inhibition of CerS6 mRNA were selected for further investigations. Most promising ASOs verified for in-vivo KD efficacy in healthy mice. CerS6 ASO (AAGATGAGCCGCACC) was found most active with hepatic reduction of CerS6 mRNA expression. Prior to longitudinal metabolic studies, we performed a dose titration target engagement analysis with CerS6 ASO in healthy mice to select the optimal dose. Next, we utilized leptin deficiency ob/ob and high fat diet (HFD) induced obese mouse models for pharmacological efficacy study. RESULTS: CerS6 expression were significantly elevated in the liver and brown adipose, this was correlated with significantly elevated C16:0 ceramide concentrations in plasma and liver. Treatment with CerS6 ASO selectively reduced CerS6 expression by ∼90% predominantly in the liver and this CerS6 KD resulted in a significant reduction of C16:0 ceramide by about 50% in both liver and plasma. CerS6 KD resulted in lower body weight gain and accompanied by a significant reduction in whole body fat and fed/fasted blood glucose levels (1% reduction in HbA1c). Moreover, ASO-mediated CerS6 KD significantly improved oral glucose tolerance (during oGTT) and mice displayed improved insulin sensitivity. Thus, CerS6 appear to play an important role in the development of obesity and insulin resistance. CONCLUSIONS: Our investigations identified specific and selective therapeutic valid ASO for CerS6 ablation in in-vivo. CerS6 should specifically be targeted for the reduction of C16:0 ceramides, that results in amelioration of insulin resistance, hyperglycemia and obesity. CerS6 mediated C16:0 ceramide reduction could be a potentially attractive target for the treatment of insulin resistance, obesity and type 2 diabetes.

Assessment of glucagon receptor occupancy by Positron Emission Tomography in non-human primates.

The glucagon receptor (GCGR) is an emerging target in anti-diabetic therapy. Reliable biomarkers for in vivo activity on the GCGR, in the setting of dual glucagon-like peptide 1/glucagon (GLP-1/GCG) receptor agonism, are currently unavailable. Here, we investigated [68Ga]Ga-DO3A-S01-GCG as a biomarker for GCGR occupancy in liver, the tissue with highest GCGR expression, in non-human primates (NHP) by PET. [68Ga]Ga-DO3A-S01-GCG was evaluated by dynamic PET in NHPs by a dose escalation study design, where up to 67 µg/kg DO3A-S01-GCG peptide mass was co-injected. The test-retest reproducibility of [68Ga]Ga-DO3A-S01-GCG binding in liver was evaluated. Furthermore, we investigated the effect of pre-treatment with acylated glucagon agonist 1-GCG on [68Ga]Ga-DO3A-S01-GCG binding in liver. [68Ga]Ga-DO3A-S01-GCG bound to liver in vivo in a dose-dependent manner. Negligible peptide mass effect was observed for DO3A-S01-GCG doses <0.2 µg/kg. In vivo Kd for [68Ga]Ga-DO3A-S01-GCG corresponded to 0.7 µg/kg, which indicates high potency. The test-retest reproducibility for [68Ga]Ga-DO3A-S01-GCG binding in liver was 5.7 ± 7.9%. Pre-treatment with 1-GCG, an acylated glucagon agonist, resulted in a GCGR occupancy of 61.5 ± 9.1% in liver. Predicted human radiation dosimetry would allow for repeated annual [68Ga]Ga-DO3A-S01-GCG PET examinations. In summary, PET radioligand [68Ga]Ga-DO3A-S01-GCG is a quantitative biomarker of in vivo GCGR occupancy.

First-in-class positron emission tomography tracer for the glucagon receptor.

The glucagon receptor (GCGR) is emerging as an important target in anti-diabetic therapy, especially as part of the pharmacology of dual glucagon-like peptide-1/glucagon (GLP-1/GCG) receptor agonists. However, currently, there are no suitable biomarkers that reliably demonstrate GCG receptor target engagement. METHODS: Two potent GCG receptor peptide agonists, S01-GCG and S02-GCG, were labeled with positron emission tomography (PET) radionuclide gallium-68. The GCG receptor binding affinity and specificity of the resulting radiopharmaceuticals [68Ga]Ga-DO3A-S01-GCG and [68Ga]Ga-DO3A-S02-GCG were evaluated in HEK-293 cells overexpressing the human GCG receptor and on frozen hepatic sections from human, non-human primate, and rat. In in vivo biodistribution, binding specificity and dosimetry were assessed in rat. RESULTS: [68Ga]Ga-DO3A-S01-GCG in particular demonstrated GCG receptor-mediated binding in cells and liver tissue with affinity in the nanomolar range required for imaging. [68Ga]Ga-DO3A-S01-GCG binding was not blocked by co-incubation of a GLP-1 agonist. In vivo binding in rat liver was GCG receptor specific with low non-specific binding throughout the body. Moreover, the extrapolated human effective doses, predicted from rat biodistribution data, allow for repeated PET imaging potentially also in combination with GLP-1R radiopharmaceuticals. CONCLUSION: [68Ga]Ga-DO3A-S01-GCG thus constitutes a first-in-class PET tracer targeting the GCG receptor, with suitable properties for clinical development. This tool has potential to provide direct quantitative evidence of GCG receptor occupancy in humans.

The preprohormone expression profile of enteroendocrine cells following Roux-en-Y gastric bypass in rats.

OBJECTIVE: Roux-en-Y gastric bypass (RYGB) leads to rapid remission of type 2 diabetes (T2D) and sustained body weight loss, but the underlying molecular mechanisms are still not fully understood. To further elucidate these mechanisms and identify potentially novel preprohormone encoding genes with anti-diabetic and/or anti-obesity properties, we performed a comprehensive analysis of gene expression changes in enteroendocrine cells after RYGB in diet-induced obese (DIO) rats. METHODS: The mRNA expression profiles of enteroendocrine cell enriched samples were characterized at 9, 22 and 60 days after RYGB surgery in a DIO rat model. Enteroendocrine cells were identified by chromogranin A immunohistochemistry and isolated by laser capture microdissection (LCM) from five regions covering the full rostro-caudal extension of the gastrointestinal (GI) tract. RNA sequencing and bioinformatic analyses were subsequently applied to identify differentially expressed preprohormone encoding genes. RESULTS: From the analysis of enteroendocrine cell mRNA expression profiles, a total of 54 preprohormones encoding genes were found to be differentially regulated at one or more time-points following RYGB. These included well-known RYGB associated preprohormone genes (e.g. Gcg, Cck, Gip, Pyy and Sct) and less characterized genes with putative metabolic effects (e.g. Nmu, Guca2a, Guca2b, Npw and Adm), but also 16 predicted novel preprohormone genes. Among the list of gene transcripts, Npw, Apln and Fam3d were further validated using in situ mRNA hybridization and corresponding peptides were characterized for acute effects on food intake and glucose tolerance in mice. CONCLUSION: We present a comprehensive mRNA expression profile of chromogranin A positive enteroendocrine cells following RYGB in rats. The data provides a region-specific characterization of all regulated preprohormone encoding genes in the rat GI tract including 16 not hitherto known. The comprehensive catalogue of preprohormone expression changes may support our understanding of hormone mediated effects of RYGB on diabetes remission and body weight reduction.

Hepatic transcriptomic signatures of statin treatment are associated with impaired glucose homeostasis in severely obese patients.

BACKGROUND: Clinical data identified an association between the use of HMG-CoA reductase inhibitors (statins) and incident diabetes in patients with underlying diabetes risk factors such as obesity, hypertension and dyslipidemia. The molecular mechanisms however are unknown. METHODS: An observational cross-sectional study included 910 severely obese patients, mean (SD) body mass index (BMI) 46.7 (8.7), treated with or without statins (ABOS cohort: a biological atlas of severe obesity). Data and sample collection took place in France between 2006 and 2016. Transcriptomic signatures of statin treatment in human liver obtained from genome-wide transcriptomic profiling of five different statin drugs using microarrays were correlated to clinico-biological phenotypes and also assigned to biological pathways and mechanisms. Patients from the non-statin-users group were matched to patients in the statin users group by propensity score analysis to minimize confounding effects from age, gender, parental familial history of diabetes, BMI, waist circumference, systolic and diastolic blood pressure and use of anti-hypertensive drugs as pre-specified covariates. RESULTS: We determined the hepatic, statin-related gene signature from genome-wide transcriptomic profiling in severely obese patients with varying degrees of glucose tolerance and cardio-metabolic comorbidities. One hundred and fifty seven patients on statin treatment in the matched cohort showed higher diabetes prevalence (OR = 2.67; 95%CI, 1.60-4.45; P = 0.0002) and impairment of glucose homeostasis. This phenotype was associated with molecular signatures of increased hepatic de novo lipogenesis (DNL) via activation of sterol regulatory element-binding protein 1 (SREBP1) and concomitant upregulation of the expression of key genes in both fatty acid and triglyceride metabolism. CONCLUSIONS: A DNL gene activation profile in response to statins is associated with insulin resistance and the diabetic status of the patients. Identified molecular signatures thus suggest that statin treatment increases the risk for diabetes in humans at least in part via induction of DNL. TRIAL REGISTRATION: NCT01129297 . Registered May 242,010 (retrospectively registered).

Investigating Intestinal Glucagon After Roux-en-Y Gastric Bypass Surgery.

CONTEXT: After Roux-en-Y gastric bypass (RYGB) surgery, postprandial plasma glucagon concentrations have been reported to increase. This occurs despite concomitant improved glucose tolerance and increased circulating plasma concentrations of insulin and the glucagon-inhibiting hormone glucagon-like peptide 1 (GLP-1). OBJECTIVE: To investigate whether RYGB-induced hyperglucagonemia may be derived from the gut. DESIGN AND SETTING: Substudy of a prospective cross-sectional study at a university hospital in Copenhagen, Denmark. PARTICIPANTS: Morbidly obese individuals undergoing RYGB (n = 8) with or without type 2 diabetes. INTERVENTIONS: Three months before and after RYGB, participants underwent upper enteroscopy with retrieval of gastrointestinal mucosal biopsy specimens. Mixed-meal tests were performed 1 week and 3 months before and after RYGB. MAIN OUTCOME MEASURES: The 29-amino acid glucagon concentrations in plasma and in mucosal gastrointestinal biopsy specimens were assessed using mass spectrometry-validated immunoassays, and a new monoclonal antibody reacting with immunoreactive glucagon was used for immunohistochemistry. RESULTS: Postprandial plasma concentrations of glucagon after RYGB were increased. Expression of the glucagon gene in the small intestine increased after surgery. Glucagon was identified in the small-intestine biopsy specimens obtained after, but not before, RYGB. Immunohistochemically, mucosal biopsy specimens from the small intestine harbored cells costained for GLP-1 and immunoreactive glucagon. CONCLUSION: Increased concentrations of glucagon were observed in small-intestine biopsy specimens and postprandially in plasma after RYGB. The small intestine harbored cells immunohistochemically costaining for GLP-1 and glucagon-like immunoreactivity after RYGB. Glucagon derived from small-intestine enteroendocrine l cells may contribute to postprandial plasma concentrations of glucagon after RYGB.

The once-daily human GLP-1 analog, liraglutide, reduces olanzapine-induced weight gain and glucose intolerance.

Therapeutic use of atypical antipsychotic agents is often associated with weight gain and impaired glucose tolerance. The once-daily human GLP-1 analog liraglutide improves glycemic control and reduces body weight. We have investigated the ability of liraglutide to improve olanzapine-induced metabolic effects in female rats. Female Sprague-Dawley rats were implanted with subcutaneous osmotic mini pumps for delivery of olanzapine (1.75 mg/24 h) or vehicle for 28 days (n=20). After 14 days, ten animals from each group were given liraglutide (0.2 mg/kg) or vehicle twice daily for the remainder of the study. Compared to vehicle treated animals, olanzapine infusion for 4 weeks significantly increased end point cumulated food intake (667.3+/-7.0 versus 593.2+/-13.2g, p<0.01), body weight (306.6+/-4.2 versus 276.4+/-3.6 g, p<0.001), subcutaneous inguinal fat (3.4+/-0.3 versus 1.9+/-0.1 g, p<0.001), mesenteric fat (3.1+/-0.2 versus 1.7+/-0.2g, p<0.001), retroperitoneal fat (6.2+/-0.6 versus 2.8+/-0.3 g, p<0.001), and impaired glucose tolerance, measured as total area under the glucose curve during an oral glucose tolerance test (1906+/-66 versus 1770+/-28 mMxmin, p<0.05). These olanzapine-induced elevations were significantly reduced by liraglutide (cumulated food intake: 601.8+/-20.4 g, p<0.01; body weight: 280.2+/-5.6 g, p<0.001; subcutaneous inguinal fat: 2.4+/-0.2 g, p<0.001; mesenteric fat: 1.8+/-0.1 g, p<0.001; retroperitoneal fat: 3.5+/-0.4 g, p<0.001; AUC: 1764+/-32 mMxmin, p<0.05). In conclusion, subcutaneous olanzapine infusion in female rats leads to weight gain and metabolic changes of which several are reversed following liraglutide treatment. It may therefore be relevant to study these effects of liraglutide in patients treated with atypically antipsychotics.

Upregulation of the brainstem preproglucagon system in the obese Zucker rat.

A group of neurons in the caudal nucleus of the solitary tract (NTS) processes preproglucagon to glucagon-like peptide 1 (GLP-1), GLP-2 and oxyntomodulin. Whereas the anorectic capacity of all three neuropeptides has been demonstrated, only relatively little is known of preproglucagon mRNA regulation in the brain stem. Using in situ hybridization and fluorescence immunohistochemistry, we examined hindbrain preproglucagon expression in lean and obese Zucker rats under different metabolic perturbations. First, the effect of an acute 48-h fast was examined in male Sprague-Dawley as well as in lean and obese Zucker rats. Whereas fasting had no effect on preproglucagon expression in either genotype, mRNA levels were strongly up regulated in obese Zucker rats. Using a direct immunostaining procedure and a monoclonal GLP-2 antibody, we found a doubling of the immunofluorescence signal emanating from the preproglucagon neurons in caudal brainstem suggesting that indeed the high mRNA levels observed using in situ hybridization histochemistry also reflect a higher translational activity. To investigate the effects of long-term body weight perturbations, lean and obese Zucker rats were either free-fed, voluntarily overfed (chocolate spread enriched chow) or food restricted for 35 days. Preproglucagon levels remained high in the obese Zucker rats irrespective of diet. Finally, in order to functionally validate the apparent hyperactivity in the preproglucagon system in the Zucker rat, we examined the effect of central GLP-1 receptor blockade. ICV administration of 20 microg of the GLP-1 receptor antagonist Des-His-Exendin-9-39 in the morning increased 4-h food intake in obese but not in lean Zucker rats, pointing to an increased activity in central preproglucagon containing pathways in leptin receptor deficient rats. Our data suggest that the preproglucagon neurons in the brainstem are influenced by leptin signaling and point to a role of preproglucagon neurons in the integration of metabolic signals that occurs in the nucleus of the solitary tract.

Dissociation of antihyperglycaemic and adverse effects of partial perioxisome proliferator-activated receptor (PPAR-gamma) agonist balaglitazone.

Balaglitazone is a novel thiazolidinedione in clinical development for the treatment of type 2 diabetes. Common side effects associated with PPARgamma receptor agonists are weight gain, oedema and adipogenesis. Balaglitazone is a selective partial PPARgamma agonist and it has been speculated that such compounds have a more favourable safety margin than full agonists. We have compared impact of equi-efficacious antihyperglycaemic doses of balaglitazone with full PPARgamma agonist rosiglitazone on body fluid accumulation, cardiac enlargement, and adipogenesis. Equi-efficacious antihyperglycaemic doses (ED(90)) of balaglitazone (3 mg/kg/day) and rosiglitazone (6 mg/kg/day) were determined in male diabetic db/db mice. In adult male rats treated for up to 42 days, feeding, drinking, anthropometry, and plasma volumes were measured. Total plasma volume was measured with dye dilution technique. Compared to vehicle, rosiglitazone consistently increased food intake throughout the 42 day treatment period. In contrast, balaglitazone increased food intake in the last week of the experiment. However, both rosiglitazone and balaglitazone increased water intake. After 42 days, rosiglitazone treated rats displayed significantly elevated adiposity. Rosiglitazone increased total blood and plasma volumes throughout the treatment. Twenty-one days of balaglitazone treatment had no significant impact on blood or plasma volumes, whilst 42 days of balaglitazone increased plasma volume but to a significantly lesser extent than seen for rosiglitazone (vehicle: 46.1+/-1.5; balaglitazone: 50.8+/-1.21; rosiglitazone: 54.6+/-1.6 ml/kg). Heart weight was significantly elevated only in rosiglitazone treated animals. At doses inducing comparable antihyperglycaemic control, the full PPARgamma agonist, rosiglitazone, induces more pronounced body fluid retention and heart enlargement than seen for the partial PPARgamma agonist, balaglitazone. Thus, partial agonists may pose safer alternative to current anti-diabetic therapy with full PPARgamma agonist.

Team Players or Opponents: Coadministration of Selective Glucagon and GLP-1 Receptor Agonists in Obese Diabetic Monkeys.

We assessed the therapeutic contribution of the individual components of glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) agonists alone and in combination upon energy homeostasis and glycemic control in diet-induced obese, diabetic nonhuman primates. The pharmacological active dose ranges of selective agonists were established through a dose-finding study, followed by a 6-week chronic study. Repeated subcutaneous administration of a selective GCGR agonist (30 µg/kg once daily) did not affect food intake or body weight, whereas the selective GLP-1R agonist (3 µg/kg once daily) alone decreased energy intake by 18% and body weight by 3.8% ± 0.9%. Combination of both agonists reduced significantly cumulative food intake by 27% and body weight by 6.6% ± 0.9%. Fasting plasma glucose (FPG) was improved by GLP-1R agonist (baseline vs end of study, 176.7 ± 34.0 vs 115.9 ± 16.1 mg/dL). In contrast, groups exposed to GCGR agonist experienced nonsignificant elevations of FPG. More accurate assessment of therapeutic interventions on glucose homeostasis was tested by an IV glucose tolerance test. Glucose excursion was significantly elevated by chronic GCGR agonist administration, whereas it was significantly decreased in GLP-1R agonist-treated monkeys. In the combination group, a nonsignificant increase of glucose excursion was seen, concomitantly with significantly increased insulin secretion. We conclude that chronic glucagon agonism does not affect energy homeostasis in nonhuman primates. In combination with GLP-1R agonism, glucagon agonism synergistically enhances negative energy balance with resulting larger body weight loss. However, adding GCGR to GLP-1R agonism diminishes glycemic control in diabetic monkeys. Therefore, long-term therapeutic implications of using GLP-1R/GCGR coagonists for weight management in diabetes warrants further scrutiny.