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Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and in vivo disease models. Using asialoglycoprotein receptor 1 (ASGR) fused with Cypridina luciferase (CLuc) as reporter assay for folding capacity, we have screened a million small molecule library and identified APC655 as a potent activator of protein folding, that appears to act by promoting chaperone expression. Furthermore, APC655 improved pancreatic β cell viability and insulin secretion under ER stress conditions induced by thapsigargin or cytokines. APC655 was also effective in preserving β cell function and decreasing lipid accumulation in the liver of the leptin-deficient (ob/ob) mouse model. These results demonstrate a successful uHTS campaign that identified a modulator of UPR, which can provide a novel candidate for potential therapeutic development for a host of metabolic diseases., Graphical abstract A cell-based high throughput screening identified a small molecule that improves pancreatic beta cell viability and function through increased UPR chaperones and ER folding capacity.Image 1

Graphical abstract, Highlights • Interactions between sex, diet & Nat2 were investigated in rats congenic for Nat2. • Rapid Nat2 congenic rats developed greater dyslipidemia. • Total cholesterol (TC)-to-HDL ratios were significantly higher in rapid Nat2 rats. • HDL-to-LDL ratios were significantly lower in rapid Nat2 rats. • Rapid Nat2 rats may have increased risk towards metabolic & cardiovascular disease., Recent reports suggest that arylamine N-acetyltransferases (NAT1 and/or NAT2) serve important roles in regulation of energy utility and insulin sensitivity. We investigated the interaction between diet (control vs. high-fat diet) and acetylator phenotype (rapid vs. slow) using previously established congenic rat lines (in F344 background) that exhibit rapid or slow Nat2 (orthologous to human NAT1) acetylator genotypes. Male and female rats of each genotype were fed control or high-fat (Western-style) diet for 26 weeks. We then examined diet- and acetylator genotype-dependent changes in body and liver weights, systemic glucose tolerance, insulin sensitivity, and plasma lipid profile. Male and female rats on the high fat diet weighed approximately 10% more than rats on the control diet and the percentage liver to body weight was consistently higher in rapid than slow acetylator rats. Rapid acetylator rats were more prone to develop dyslipidemia overall (i.e., higher triglyceride; higher LDL; and lower HDL), compared to slow acetylator rats. Total cholesterol (TC)-to-HDL ratios were significantly higher and HDL-to-LDL ratios were significantly lower in rapid acetylator rats. Our data suggest that rats with rapid systemic Nat2 (NAT1 in humans) genotype exhibited higher dyslipidemia conferring risk for metabolic syndrome and cardiovascular dysfunction.

s Echigoshirayukidake is an edible mushroom found in Uonuma, Japan in 1994. It was assigned to a new species of Basidiomycetes (BDM-X) but is uniquely defect of forming bashidium. The high antioxidant activity and β-glucan content of BDM-X suggest possible functions preventing type 2 diabetes. In the present study, anti-obesity and insulin resistance preventive functions of BDM-X were examined using genetically defined obese model rat, OLETF (Otsuka Long Evans Tokushima Fatty) by feeding regular diet with and without supplementation of 5% dried BDM-X powder (BDMP) for 15 weeks. BDMP supplementation to the diet significantly (p, Graphical abstract Image 1, Highlights • A new mushroom, Echigoshirayukidake (BDM-X), ameliorates postprandial sugar and insulin spike enhancing insulin sensitivity. • BDM-X prevented body weight gain, hyperlipidemia, NEFA, and visceral fat deposition. • HOMA-IR was improved by BDM-X. • Anti-metabolic syndrome effect of BDM-X could be related to increase of adiponectin level.

Background & Aims Epidemiological and animal studies have indicated an inverse correlation between the rising prevalence of obesity and metabolic syndrome and exposure to helminths. Whether helminth-induced immune response contributes to microbiota remodeling in obesity remains unknown. The aim of this study is to explore the immune-regulatory role of helminth in the prevention of HFD-induced obesity through remodeling gut microbiome. Methods C57BL/6J WT and STAT6-/- mice were infected with Heligmosomoides polygyrus and followed by high fat diet (HFD) feeding for 6 weeks. The host immune response, body weight, and fecal microbiota composition were analyzed. We used adoptive transfer of M2 macrophages and microbiota transplantation approaches to determine the impact of these factors on HFD-obesity. We also examined stool microbiota composition and short chain fatty acids (SCFAs) concentration and determined the expression of SCFA-relevant receptors in the recipient mice. Results Helminth infection of STAT6-/- (Th2-deficient) mice and adoptive transfer of helminth-induced alternatively activated (M2) macrophages demonstrated that the helminth-associated Th2 immune response plays an important role in the protection against obesity and induces changes in microbiota composition. Microbiota transplantation showed that helminth-induced, Th2-dependent alterations of the gut microbiota are sufficient to confer protection against obesity. Collectively, these results indicate that helminth infection protects against HFD-induced obesity by Th2-dependent, M2 macrophage-mediated alterations of the intestinal microbiota. Conclusion Our findings provide new mechanistic insights into the complex interplay between helminth infection, the immune system and the gut microbiota in a HFD-induced obesity model and holds promise for gut microbiome-targeted immunotherapy in obesity prevention., Graphical abstract

Objective Dual amylin and calcitonin receptor agonists (DACRAs) are novel therapeutic agents that not only improve insulin sensitivity but also work as an adjunct to established T2DM therapies. DACRAs are currently administered once daily, though it is unknown whether DACRAs with increased plasma half-life can be developed as a once-weekly therapy. Methods The in vitro potencies of the KBP-066A and KBP-066 (non-acylated) were assessed using reporter assays. Acylation functionality was investigated by a combination of pharmacokinetics and acute food intake in rats. in vivo efficacies were investigated head-to-head in obese (HFD) and T2D (ZDF) models. Results In in vitro, KBP-066A activated the CTR and AMY-R potently, with no off-target activity. Acylation functionality was confirmed by acute tests, as KBP-066A demonstrated a prolonged PK and PD response compared to KBP-066. Both compounds induced potent and dose-dependent weight loss in the HFD rat model. In ZDF rats, fasting blood glucose/fasting insulin levels (tAUC) were reduced by 39%/50% and 36%/47% for KBP-066 and KBP-066A, respectively. This effect resulted in a 31% and 46% vehicle-corrected reduction in HbA1c at the end of the study for KBP-066 and KBP-066A, respectively. Conclusions Here, we present pre-clinical data on an acylated DACRA, KBP-066A. The in vivo efficacy of KBP-066A is significantly improved compared to its non-acylated variant regarding weight loss and glycemic control in obese (HFD) and obese diabetic rats (ZDF). This compendium of pre-clinical studies highlights KBP-066A as a promising, once-weekly therapeutic agent for treating T2DM and obesity., Highlights • DACRAs are promising once daily therapeutic candidates for metabolic diseases. • We here present a novel DACRA called KBP-066A optimized for weekly delivery. • KBP-066A potently reduced appetite and body weight in obese rats. • More importantly, KBP-066A was superior to the corresponding daily DACRA in terms of glucose control. • KBP-066A is a novel promising therapy for metabolic diseases.

Background: If a pregnant woman is overweight, this can evoke metabolic alterations that may have health consequences for both mother and child. Methods: Pregnant women with overweight/obesity (n = 358) received fish oil+placebo, probiotics+placebo, fish oil+probiotics or placebo+placebo from early pregnancy onwards. The serum metabolome was analysed from fasting samples with a targeted NMR-approach in early and late pregnancy. GDM was diagnosed by OGTT. Findings: The intervention changed the metabolic profile of the women, but the effect was influenced by their GDM status. In women without GDM, the changes in nine lipids (FDR

Objective Islets secrete neurotransmitters including glutamate which participate in fine regulation of islet function. The excitatory ionotropic glutamate receptor GluK2 of the kainate receptor family is widely expressed in brain and also found in islets, mainly in α and γ cells. α cells co-release glucagon and glutamate and the latter increases glucagon release via ionotropic glutamate receptors. However, neither the precise nature of the ionotropic glutamate receptor involved nor its role in glucose homeostasis is known. As isoform specific pharmacology is not available, we investigated this question in constitutive GluK2 knock-out mice (GluK2−/−) using adult and middle-aged animals to also gain insight in a potential role during aging. Methods We compared wild-type GluK2+/+ and knock-out GluK2−/− mice using adult (14–20 weeks) and middle-aged animals (40–52 weeks). Glucose (oral OGTT and intraperitoneal IPGTT) and insulin tolerance as well as pyruvate challenge tests were performed according to standard procedures. Parasympathetic activity, which stimulates hormones secretion, was measured by electrophysiology in vivo. Isolated islets were used in vitro to determine islet β-cell electrical activity on multi-electrode arrays and dynamic secretion of insulin as well as glucagon was determined by ELISA. Results Adult GluK2−/− mice exhibit an improved glucose tolerance (OGTT and IPGTT), and this was also apparent in middle-aged mice, whereas the outcome of pyruvate challenge was slightly improved only in middle-aged GluK2−/− mice. Similarly, insulin sensitivity was markedly enhanced in middle-aged GluK2−/− animals. Basal and glucose-induced insulin secretion in vivo was slightly lower in GluK2−/− mice, whereas fasting glucagonemia was strongly reduced. In vivo recordings of parasympathetic activity showed an increase in basal activity in GluK2−/− mice which represents most likely an adaptive mechanism to counteract hypoglucagonemia rather than altered neuronal mechanism. In vitro recording demonstrated an improvement of glucose-induced electrical activity of β-cells in islets obtained from GluK2−/− mice at both ages. Finally, glucose-induced insulin secretion in vitro was increased in GluK2−/− islets, whereas glucagon secretion at 2 mmol/l of glucose was considerably reduced. Conclusions These observations indicate a general role for kainate receptors in glucose homeostasis and specifically suggest a negative effect of GluK2 on glucose homeostasis and preservation of islet function during aging. Our observations raise the possibility that blockade of GluK2 may provide benefits in glucose homeostasis especially during aging., Highlights • KO of GluK2 improved glucose tolerance and lowered fasting glucagon levels in vivo. • KO of GluK2 reduced in vitro islet glucagon secretion and increased β-cell activity. • GluK2 contributes to the known impact of aging on glucose homeostasis.

Background: Metabolic syndrome is a cluster of metabolic risk factors. The clear causes of its development are not known yet and there is no comprehensive treatment of this disease. There is a trend to use natural substances in the treatment of various diseases, but their effects need to be well explored. We decided to test effect of rutin compared to the effect of the standard drug atorvastatin. Methods: As a model of metabolic syndrome we used males of hypertriacylglycerolemic rats in combination with high-fat-high-fructose diet. Rutin (100 mg/kg) and atorvastatin (50 mg/kg) were administered orally daily for 5 weeks. Results: We determined biochemical parameters from blood: HDL-cholesterol, LDL-cholesterol, total cholesterol, triacylglycerols. Relaxation and contraction response of aorta was measured to determine vessel dysfunctions and possible predisposition to cardiovascular disease. The negative influence on cognitive functions could be associated with the development of metabolic cognitive syndrome. Therefore we aimed to monitor spatial memory by Morris water maze test. Both rutin and atorvastatin had a tendency to decrease levels of serum triacylglycerols, but only atorvastatin significantly reduced levels od LDL-cholesterol and increased HDL-cholesterol levels. Both compounds significantly reduced the phenylephrine-induced contractile response of the aorta and improved the relaxation response. Further, treated animals learned better compared to untreated rats in the Morris water maze. Conclusion: Based on our results we can assume that atorvastatin and rutin had positive effect on spatial memory and vessel reactivity. Atorvastatin optimized lipid profile of blood serum. Keywords: Metabolic syndrome, Rutin, Atorvastatin, Dyslipidemia, Aorta, Spatial memory

Background & Aims The first-line treatment for non-alcoholic fatty liver disease (NAFLD) is weight reduction. Several diets have been proposed, with various effects specifically on liver steatosis. This trial compared the effects of intermittent calorie restriction (the 5:2 diet) and a low-carb high-fat diet (LCHF) on reduction of hepatic steatosis. Methods We conducted an open-label randomised controlled trial that included 74 patients with NAFLD randomised in a 1:1:1 ratio to 12 weeks’ treatment with either a LCHF or 5:2 diet, or general lifestyle advice from a hepatologist (standard of care; SoC). The primary outcome was reduction of hepatic steatosis as measured by magnetic resonance spectroscopy. Secondary outcomes included transient elastography, insulin resistance, blood lipids, and anthropometrics. Results The LCHF and 5:2 diets were both superior to SoC treatment in reducing steatosis (absolute reduction: LCHF: −7.2% [95% CI = −9.3 to −5.1], 5:2: −6.1% [95% CI = −8.1 to −4.2], SoC: −3.6% [95% CI = −5.8 to −1.5]) and body weight (LCHF: −7.3 kg [95% CI = −9.6 to −5.0]; 5:2: −7.4 kg [95% CI = −8.7 to −6.0]; SoC: −2.5 kg [95% CI =−3.5 to −1.5]. There was no difference between 5:2 and LCHF (p = 0.41 for steatosis and 0.78 for weight). Liver stiffness improved in the 5:2 and SoC but not in the LCHF group. The 5:2 diet was associated with reduced LDL levels and was tolerated to a higher degree than LCHF. Conclusions The LCHF and 5:2 diets were more effective in reducing steatosis and body weight in patients with NAFLD than SoC, suggesting dietary advice can be tailored to meet individual preferences. Lay summary For a person with obesity who suffers from fatty liver, weight loss through diet can be an effective treatment to improve the condition of the liver. Many popular diets that are recommended for weight reduction, such as high-fat diets and diets based on intermittent fasting, have not had their effects on the liver directly evaluated. This study shows that both a low-carb high-fat and the 5:2 diet are effective in treating fatty liver caused by obesity. Clinical Trials Registration This study is registered at Clinicaltrials.gov (NCT03118310)., Graphical abstract, Highlights • Weight reduction is the only generally available treatment for NAFLD today. • High-fat diets, such as the LCHF, are controversial in treating NAFLD. • The 5:2 diet has become popular and is widely used to achieve weight loss. • In this RCT, both the LCHF and 5:2 diets were highly effective in treating NAFLD. • The 5:2 diet reduced LDL and liver stiffness and was tolerated to a higher degree.

Irrespective of the definition and diagnostic criteria used, the term prediabetes denotes a state of dysmetabolism with a high risk of progression to diabetes mellitus. Although diabetes-related complications may already be evident among individuals with prediabetes, interventions at this stage primarily aim to hinder the development of overt hyperglycemia rather than to prevent complications. Current recommendations for prediabetes testing are common across all adult age categories. Recent evidence arising from the prospective investigation of the natural course of prediabetes among elderly individuals pose questions regarding the benefits of meticulous prediabetes screening in this age group. In view of this and due to the lack of sufficient data to concretely support a positive impact of further preventive strategies among older individuals, screening recommendations should be reevaluated to target selected elderly individuals who are most likely to benefit in terms of quality of life and prognosis. Further therapeutic measures should be tailored to the inherent features of this frail age group, in order to exert a meaningful effect on overall health status.

Background & Aims The paradox of hepatic insulin resistance describes the inability for liver to respond to bioenergetics hormones in suppressing gluconeogenesis whilst maintaining lipid synthesis. Here, we report the deficiency of miR-192-3p in the livers of mice with diabetes and its role in alleviating hepatic steatosis. Methods As conventional pre-microRNA (miRNA) stem-loop overexpression only boosts guiding strand (i.e. miR-192-5p) expression, we adopted an artificial AAV(DJ)-directed, RNA Pol III promoter-driven miRNA hairpin construct for star-strand-specific overexpression in the liver. Liver steatosis and insulin resistance markers were evaluated in primary hepatocytes, mice with diabetes, and mice with excessive carbohydrate consumption. Results Functional loss of miR-192-3p in liver exacerbated hepatic micro-vesicular steatosis and insulin resistance in either mice with diabetes or wild-type mice with excessive fructose consumption. Liver-specific overexpression of miR-192-3p effectively halted hepatic steatosis and ameliorated insulin resistance in these mice models. Likewise, hepatocytes overexpressing miR-192-3p exhibited improved lipid accumulation, accompanied with decreases in lipogenesis and lipid-accumulation-related transcripts. Mechanistically, glucocorticoid receptor (GCR, also known as nuclear receptor subfamily 3, group C, member 1 [NR3C1]) was demonstrated to be negatively regulated by miR-192-3p. The effect of miR-192-3p on mitigating micro-vesicular steatosis was ablated by the reactivation of NR3C1. Conclusions The star strand miR-192-3p was an undermined glycerolipid regulator involved in controlling fat accumulation and insulin sensitivity in liver through blockade of hepatic GCR signalling; this miRNA may serve as a potential therapeutic option for the common co-mobility of diabetic mellitus and fatty liver disease. Lay summary The potential regulatory activity of star strand microRNA (miRNA) species has been substantially underestimated. In this study, we investigate the role and mechanism of an overlooked star strand miRNA (miR-192-3p) in regulating hepatic steatosis and insulin signalling in the livers of mice with diabetes and mice under excessive carbohydrate consumption., Graphical abstract, Highlights • Liver-specific knockdown of miR-192-3p recapitulated functional loss of the miRNA as in mice with diabetes. • This knockdown was characterised by pronounced hepatic micro-vesicular steatosis coupled to insulin resistance. • In vivo overexpression of miR-192-3p alleviated hepatic steatosis in mice with diabetes and wild-type mice with excessive fructose consumption. • Glucocorticoid receptor (also known as NR3C1) was discovered as the immediate target of miR-192-3p in regulating hepatic lipid turnover and storage.

Objective A novel dual GIP and GLP-1 receptor agonist, LY3298176, was developed to determine whether the metabolic action of GIP adds to the established clinical benefits of selective GLP-1 receptor agonists in type 2 diabetes mellitus (T2DM). Methods LY3298176 is a fatty acid modified peptide with dual GIP and GLP-1 receptor agonist activity designed for once-weekly subcutaneous administration. LY3298176 was characterised in vitro, using signaling and functional assays in cell lines expressing recombinant or endogenous incretin receptors, and in vivo using body weight, food intake, insulin secretion and glycemic profiles in mice. A Phase 1, randomised, placebo-controlled, double-blind study was comprised of three parts: a single-ascending dose (SAD; doses 0.25–8 mg) and 4-week multiple-ascending dose (MAD; doses 0.5–10 mg) studies in healthy subjects (HS), followed by a 4-week multiple-dose Phase 1 b proof-of-concept (POC; doses 0.5–15 mg) in patients with T2DM (ClinicalTrials.gov no. NCT02759107). Doses higher than 5 mg were attained by titration, dulaglutide (DU) was used as a positive control. The primary objective was to investigate safety and tolerability of LY3298176. Results LY3298176 activated both GIP and GLP-1 receptor signaling in vitro and showed glucose-dependent insulin secretion and improved glucose tolerance by acting on both GIP and GLP-1 receptors in mice. With chronic administration to mice, LY3298176 potently decreased body weight and food intake; these effects were significantly greater than the effects of a GLP-1 receptor agonist. A total of 142 human subjects received at least 1 dose of LY3298176, dulaglutide, or placebo. The PK profile of LY3298176 was investigated over a wide dose range (0.25–15 mg) and supports once-weekly administration. In the Phase 1 b trial of diabetic subjects, LY3298176 doses of 10 mg and 15 mg significantly reduced fasting serum glucose compared to placebo (least square mean [LSM] difference [95% CI]: −49.12 mg/dL [−78.14, −20.12] and −43.15 mg/dL [−73.06, −13.21], respectively). Reductions in body weight were significantly greater with the LY3298176 1.5 mg, 4.5 mg and 10 mg doses versus placebo in MAD HS (LSM difference [95% CI]: −1.75 kg [−3.38, −0.12], −5.09 kg [−6.72, −3.46] and −4.61 kg [−6.21, −3.01], respectively) and doses of 10 mg and 15 mg had a relevant effect in T2DM patients (LSM difference [95% CI]: −2.62 kg [−3.79, −1.45] and −2.07 kg [−3.25, −0.88], respectively. The most frequent side effects reported with LY3298176 were gastrointestinal (vomiting, nausea, decreased appetite, diarrhoea, and abdominal distension) in both HS and patients with T2DM; all were dose-dependent and considered mild to moderate in severity. Conclusions Based on these results, the pharmacology of LY3298176 translates from preclinical to clinical studies. LY3298176 has the potential to deliver clinically meaningful improvement in glycaemic control and body weight. The data warrant further clinical evaluation of LY3298176 for the treatment of T2DM and potentially obesity., Highlights • LY3298176 activates both GIP and GLP-1 receptor signaling in vitro. • LY3298176 lowers blood glucose in mice through actions on both incretin receptors. • LY3298176 reduced fasting glucose in humans with type 2 diabetes. • Weight loss was greater with LY3298176 than the selective GLP-1 receptor agonist, dulaglutide in healthy humans. • Tolerability of LY3298176 was comparable to GLP-1 receptor agonists.

Objective Insulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D. Methods A novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice. Results In vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge. Conclusion Collectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology., Highlights • A novel anti-insulin receptor monoclonal antibody (IRAB-A) was identified that has both agonist and sensitizing activities. • IRAB-A increases the receptor's affinity for insulin by binding to an allosteric site and does not compete with insulin. • Mice injected once with IRAB-A show improved glycemia and reduced insulinemia, indicative of enhanced insulin sensitivity. • In diet induced obese mice, the insulin sensitizing effect of IRAB-A appears to depend on the degree of insulin resistance. • Chronic treatment of obese mice showed mixed effects on glucose homeostasis under normal fed or meal challenged conditions.

Transcription factor 7-like 2 (TCF7L2) is the main susceptibility gene for type 2 diabetes, primarily through impairing the insulin secretion by pancreatic β cells. However, the exact in vivo mechanisms remain poorly understood. We performed a family study and determined if the T risk allele of the rs7903146 in the TCF7L2 gene increases the risk of type 2 diabetes based on real-time stable isotope measurements of insulin synthesis during an Oral Glucose Tolerance Test. In addition, we performed oral minimal model (OMM) analyses to assess insulin sensitivity and β cell function indices. Compared to unaffected relatives, individuals with type 2 diabetes had lower OMM indices and a higher level of insulin synthesis. We found a T allele-dosage effect on insulin synthesis and on glucose tolerance status, therefore insulin synthesis was higher among T-allele carriers with type 2 diabetes than in wild-type individuals. These results suggest that hyperinsulinemia is not only an adaptation to insulin resistance, but also a direct cause of type 2 diabetes., Highlights • We developed a test to follow insulin synthesis in real-time in vivo and used it in type 2 diabetes high-risk families. • Insulin synthesis was increased in individuals with non-insulin treated type 2 diabetes. • A variant of the TCF7L2 gene linked insulin synthesis with type 2 diabetes. Transcription factor 7-like 2 (TCF7L2) is the main susceptibility gene for type 2 diabetes, predominantly by affecting insulin secretion of pancreatic β cells. However the exact in vivo mechanisms remain poorly understood. We investigated the relationship between the TCF7L2 rs7903146 variant and real-time insulin synthesis measurements in vivo. We found that genetically increased insulin synthesis contributed to development of type 2 diabetes. Our data implies that hyperinsulinemia is a sign not only of resistance to insulin but also of intrinsic β cell dysfunction. Our findings can help in the understanding and treatment of type 2 diabetes. The glucose-sensitive TFC7L2 pathway might be a target for intervention.

Objective While prostate cancer does not occur more often in men with diabetes, survival is markedly reduced in this patient group. Androgen signaling is a known and major driver for prostate cancer progression. Therefore, we analyzed major components of the androgen signaling chain and cell proliferation in relation to type 2 diabetes. Methods Tumor content of 70 prostate tissue samples of men with type 2 diabetes and 59 samples of patients without diabetes was quantified by an experienced pathologist, and a subset of 51 samples was immunohistochemically stained for androgen receptor (AR). mRNA expression of AR, insulin receptor isoform A (IR-A) and B (IR-B), IGF-1 receptor (IGF1R), Cyp27A1 and Cyp7B1, PSA gene KLK3, PSMA gene FOLH1, Ki-67 gene MKI67, and estrogen receptor beta (ESR2) were analyzed by RT-qPCR. Results AR mRNA and protein expression were associated with the tumor content only in men with diabetes. AR expression also correlated with downstream targets PSA (KLK3) and PSMA (FOLH1) and increased cell proliferation. Only in diabetes, AR expression was correlated to higher IR-A/IR-B ratio and lower IR-B/IGF1R ratio, thus, in favor of the mitogenic isoforms. Reduced Cyp27A1 and increased Cyp7B1 expressions in tumor suggest lower levels of protective estrogen receptor ligands in diabetes. Conclusions We report elevated androgen receptor signaling and activity presumably due to altered insulin/IGF-1 receptors and decreased levels of protective estrogen receptor ligands in prostate cancer in men with diabetes. Our results reveal new insights why these patients have a worse prognosis. These findings provide the basis for future clinical trials to investigate treatment response in patients with prostate cancer and diabetes., Highlights • Androgen receptor expression is elevated in prostate cancer in men with diabetes. • This correlates with altered IR and IGF-1R and protective estrogen receptor ligands. • Our results reveal new insights why these patients have worse prognosis.

Objective The incidence of depression is significantly compounded by obesity. Obesity arising from excessive intake of high-fat food provokes anxiodepressive behavior and elicits molecular adaptations in the nucleus accumbens (NAc), a region well-implicated in the hedonic deficits associated with depression and in the control of food-motivated behavior. To determine the etiology of diet-induced depression, we studied the impact of different dietary lipids on anxiodepressive behavior and metabolic and immune outcomes and the contribution of NAc immune activity. Methods Adult C57Bl/6 mice were subjected to isocaloric high-fat/high-sucrose diets (HFD), enriched in either saturated or monounsaturated fat, or a control low-fat diet (LFD). Metabolic responses, anxiodepressive behavior, and plasma and NAc inflammatory markers were assessed after 12 weeks. In separate experiments, an adenoviral construct inhibiting IKKβ, an upstream component of the nuclear factor kappa-b (NFkB) pathway, was a priori injected into the NAc. Results Both HFDs resulted in obesity and hyperleptinemia; however, the saturated HFD uniquely triggered anxiety-like behavior, behavioral despair, hyperinsulinemia, glucose intolerance, peripheral inflammation, and multiple pro-inflammatory signs in the NAc, including reactive gliosis, increased expression of cytokines, antigen-presenting markers and NFкB transcriptional activity. Selective NAc IKKβ inhibition reversed the upregulated expression of inflammatory markers, prevented anxiodepressive behavior and blunted compulsive sucrose-seeking in mice fed the saturated HFD. Conclusions Metabolic inflammation and NFкB-mediated neuroinflammatory responses in the NAc contribute to the expression of anxiodepressive behavior and heightened food cravings caused by a diet high in saturated fat and sugar., Graphical abstract, Highlights • Saturated HFD uniquely triggers metabolic impairments & depressive behavior. • Nucleus accumbens (NAc) inflammation & gliosis are distinct features of the saturated HFD. • Depressive behavior & NAc immune activation are mediated by IKKβ/NFκB signaling. • Inhibiting NAc IKKβ/NFκB suppresses compulsive sucrose seeking in mice fed the saturated HFD.

Objective Glucose-dependent insulinotropic polypeptide (GIP) is released during meals and promotes nutrient uptake and storage. GIP receptor knockout mice are protected from diet induced weight gain and thus GIP antagonists have been proposed as a treatment for obesity. In this study, we assessed the role of GIP in hyperphagia induced obesity and metabolic abnormalities in leptin deficient (Lepob/ob) mice. Methods We crossbred GIP-GFP knock-in homozygous mice (GIPgfp/gfp) that have complete GIP knockout, and mice heterozygous for the ob mutation (Lepob/+) mice to generate Lepob/+/GIP+/+, Lepob/ob/GIP+/+, and Lepob/ob/GIPgfp/gfp mice. Male animals were weighed weekly and both oral glucose and insulin tolerance testing were performed to assess glucose homeostasis and circulating profiles of GIP and insulin. Body composition was evaluated by computerized tomography (CT) scan and analyses of indirect calorimetry and locomotor activity were performed. Results Postprandial GIP levels were markedly elevated in Lepob/ob/GIP+/+ mice compared to Lepob/+/GIP+/+ controls and were undetectable in Lepob/ob/GIPgfp/gfp mice. Insulin levels were equivalently elevated in both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice compared to controls at 8 weeks of age but the hyperinsulinemia was marginally reduced in Lepob/ob/GIPgfp/gfp by 21 weeks, in association with amelioration of glucose intolerance. Both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice remained equivalently insulin resistant. Body weight gain and subcutaneous and visceral fat volume of both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice were significantly higher than that of Lepob/+/GIP+/+ mice, while no significant differences were seen between Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice. Locomotor activity and energy expenditure were decreased in both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice compared to control Lepob/+/GIP+/+ mice, while no significant differences were seen between Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice. There was no significant difference in fat oxidation among the three groups. Fat content in liver was significantly lower in Lepob/ob/GIPgfp/gfp compared to Lepob/ob/GIP+/+ mice, while that of control Lepob/+/GIP+/+ mice was the lowest. Conclusions Our results indicate that GIP knockout does not prevent excess weight gain and metabolic derangement in hyperphagic leptin deficient mice., Highlights • Complete absence of GIP does not prevent hyperinsulinemia and insulin resistance from developing in Lepob/ob mice. • GIP deficiency does not alleviate excess weight gain and fat accumulation in Lepob/ob mice. • Endogenous GIP is not involved in the development of obesity in mice with complete absence of leptin.

Obesity and its related metabolic disorders are closely correlated with gut dysbiosis. Montmorillonite is a common medicine used to treat diarrhea. We have previously found that dietary lipid adsorbent-montmorillonite (DLA-M) has an unexpected role in preventing obesity. The aim of this study was to further investigate whether DLA-M regulates intestinal absorption and gut microbiota to prevent obesity-related metabolic disorders. Here, we show that DLA-M absorbs free fatty acids (FFA) and endotoxins in vitro and in vivo. Moreover, the combination of fluorescent tracer technique and polarized light microscopy showed that DLA-M crystals immobilized BODIPY® FL C16 and FITC-LPS, respectively, in the digestive tract in situ. HFD-fed mice treated with DLA-M showed mild changes in the composition of the gut microbiota, particularly increases in short-chain fatty acids (SCFA)-producing Blautia bacteria and decreases in endotoxin-producing Desulfovibrio bacteria, these changes were positively correlated with obesity and inflammation. Our results indicated that DLA-M immobilizes FFA and endotoxins in the digestive tract via the adsorption-excretion axis and DLA-M may potentially be used as a prebiotic to prevent intestinal dysbiosis and obesity-associated metabolic disorders in obese individuals., Highlights • Montmorillonite prevents obesity-associated metabolic disorders in obese individuals. • Montmorillonite immobilizes triglycerides, cholesterol, FFA and endotoxin in the digestive tract via the adsorption-excretion axis. • Montmorillonite may potentially be used as a prebiotic to modulate the gut microbiota composition in HFD-fed mice. Obesity and its related insulin resistance, hepatic steatosis are closely correlated with low-grade inflammation and gut dysbiosis. Montmorillonite is initially characterized as a commonly gastrointestinal mucosal barrier protective agent for the treatment of diarrhea. The purpose of this work is to assess the ability of montmorillonite on preventing obesity-related metabolic disorders and the underlying mechanisms. The results of our experiments provide some theoretical reference for the instruction purpose of clinical medication on montmorillonite.

Glucose-stimulated insulin secretion (GSIS) by pancreatic β cells is biphasic. However, the physiological significance of biphasic GSIS and its relationship to diabetes are not yet fully understood. This study demonstrated that impaired first-phase GSIS follows fasting, leading to increased blood glucose levels and brain glucose distribution in humans. Animal experiments to determine a possible network between the brain and β cells revealed that fasting-dependent hyperactivation of AMP-activated protein kinase in the hypothalamus inhibited first-phase GSIS by stimulating the α-adrenergic pancreatic nerve. Furthermore, abnormal excitability of this brain-β cell neural axis was involved in diabetes-related impairment of first-phase GSIS in diabetic animals. Finally, pancreatic denervation improved first-phase GSIS and glucose tolerance and ameliorated severe diabetes by preventing β cell loss in diabetic animals. These results indicate that impaired first-phase GSIS is critical for brain distribution of dietary glucose after fasting. Furthermore, β cells in individuals with diabetes mistakenly sense that they are under conditions that mimic prolonged fasting. The present study provides additional insight into both β cell physiology and the pathogenesis of β cell dysfunction in type 2 diabetes., Highlights • Fasting-induced hypothalamic AMPK activation inhibited first-phase GSIS by stimulating the α-adrenergic nerve. • The brain-pancreas neural axis was involved in β cell dysfunction and glucose intolerance in diabetes. • Pancreatic denervation improved first-phase GSIS, glucose tolerance and β cell survival in type 2 diabetic animals. Glucose-stimulated insulin secretion (GSIS) from pancreatic β cells is biphasic. Furthermore, first-phase GSIS is inhibited in type 2 diabetes. This study revealed that fasting reduced first-phase GSIS by signaling via the brain-pancreatic β cell neural axis, which is essential for maintaining glucose supply to the brain at re-feeding after fasting. Abnormal excitability of this neural axis was also associated with impaired first-phase GSIS in type 2 diabetes. Surgical pancreatic denervation improved diabetes in an animal study. The present data reveal that diabetic β cells exist under conditions that mimic starvation and provide a therapeutic potency of pancreatic denervation against diabetes., Graphical Abstract Image 2

Objective Insulin release from pancreatic islet β cells should be tightly controlled to avoid hypoglycemia and insulin resistance. The cortical actin cytoskeleton is a gate for regulated exocytosis of insulin secretory granules (SGs) by restricting their mobility and access to the plasma membrane. Prior studies suggest that SGs interact with F-actin through their transmembrane cargo islet cell autoantigen 512 (Ica512) (also known as islet antigen 2/Ptprn). Here we investigated how Ica512 modulates SG trafficking and exocytosis. Methods Transcriptomic changes in Ica512−/− mouse islets were analyzed. Imaging as well as biophysical and biochemical methods were used to validate if and how the Ica512-regulated gene villin modulates insulin secretion in mouse islets and insulinoma cells. Results The F-actin modifier villin was consistently downregulated in Ica512−/− mouse islets and in Ica512-depleted insulinoma cells. Villin was enriched at the cell cortex of β cells and dispersed villin−/− islet cells were less round and less deformable. Basal mobility of SGs in villin-depleted cells was enhanced. Moreover, in cells depleted either of villin or Ica512 F-actin cages restraining cortical SGs were enlarged, basal secretion was increased while glucose-stimulated insulin release was blunted. The latter changes were reverted by overexpressing villin in Ica512-depleted cells, but not vice versa. Conclusion Our findings show that villin controls the size of the F-actin cages restricting SGs and, thus, regulates their dynamics and availability for exocytosis. Evidence that villin acts downstream of Ica512 also indicates that SGs directly influence the remodeling properties of the cortical actin cytoskeleton for tight control of insulin secretion., Highlights • Ica512-depletion reduces the genetic expression of the F-actin modifier villin. • Villin-depletion enhances basal insulin granule mobility and exocytosis. • Villin regulates the size of actin cages restraining insulin granules. • Villin acts downstream of insulin granule cargo Ica512. • The Ica512-villin genetic link enables granules to control cytoskeleton plasticity.

Pioglitazone, a thiazolidinedione (TZD), is widely used as an insulin sensitizer in the treatment of type 2 diabetes. However, body weight gain is frequently observed in TZD-treated patients. Fish oil improves lipid metabolism dysfunction and obesity. In this study, we demonstrated suppression of body weight gain in response to pioglitazone administration by combination therapy of pioglitazone and fish oil in type 2 diabetic KK mice. Male KK mice were fed experimental diets for 8 weeks. In safflower oil (SO), safflower oil/low-dose pioglitazone (S/PL), and safflower oil/high-dose pioglitazone (S/PH) diets, 20% of calories were provided by safflower oil containing 0%, 0.006%, or 0.012% (wt/wt) pioglitazone, respectively. In fish oil (FO), fish oil/low-dose pioglitazone (F/PL), and fish oil/high-dose pioglitazone (F/PH) diets, 20% of calories were provided by a mixture of fish oil and safflower oil. Increased body weight and subcutaneous fat mass were observed in the S/PL and S/PH groups; however, diets containing fish oil were found to ameliorate these changes. Hepatic mRNA levels of lipogenic enzymes were significantly decreased in fish oil-fed groups. These findings demonstrate that the combination of pioglitazone and fish oil decreases subcutaneous fat accumulation, ameliorating pioglitazone-induced body weight gain, through fish oil-mediated inhibition of hepatic de novo lipogenesis., Open Access.

Background and objective The Pro12Ala (exon 2) and His447His (exon 6) polymorphisms of PPAR-γ, and Gly972Arg polymorphism of IRS-1 have been implicated in insulin resistance (IR) and adiposity. Our aim was to investigate the influence of these polymorphisms on metabolic features of polycystic ovary syndrome (PCOS). Methods Fifty-three PCOS women and 26 control women underwent a clinical and biochemical evaluation, including a 75-g oral glucose tolerance test. Insulin secretion and insulin sensitivity indices were calculated. Results Frequencies of PPAR-γ polymorphisms did not differ from those predicted by the Hardy-Weinberg equilibrium. Instead, the IRS-1 Gly972Arg allele was significantly more frequent in the PCOS group compared to controls. The most frequent allelic combinations were IRS1+/exon2-/exon6- (which prevailed in PCOS) and IRS-1-/exon2-/exon6- (which prevailed in controls). Among PCOS women, compared with the wild type patients, carriers of the Gly972Arg IRS-1 allele had lower E2 levels, while carriers of the Pro12Ala PPAR-γ (exon 2) allele had lower free testosterone levels. No other significant relationships were noted. When compared with the wild type, in PCOS group IR and beta-cell function were: (i) trendwise greater in carriers of the variant IRS-1 allele; (ii) trendwise lower in carriers of the variant PPAR-γ exon 6 allele; (iii) significantly lower in carriers of the variant PPAR-γ exon 2 allele. Conclusions Our data support the protective influence of PPAR-γ-exon 2 and exon 6 variants on IR and beta cell function, whereas IRS-1 polymorphism is associated with an unfavorable metabolic profile. However, these associations do not fully explain the high metabolic risk associated with PCOS.

Background Caloric restriction and n-3 polyunsaturated fatty acid (PUFA) supplementation protect from some of the metabolic complications. The aim of this study was to assess the influence of a low calorie diet with or without n-3 PUFA supplementation on glucose dependent insulinotropic polypeptide (GIP) output and insulin sensitivity markers in obese subjects. Methods Obese, non-diabetic subjects (BMI 30–40 kg/m2) and aged 25–65 yr. were put on low calorie diet (1200–1500 kcal/day) supplemented with either 1.8 g/day n-3 PUFA (DHA/EPA, 5:1) (n = 24) or placebo capsules (n = 24) for three months in a randomized placebo controlled trial. Insulin resistance markers and GIP levels were analysed from samples obtained at fasting and during an oral glucose tolerance test (OGTT). Results Caloric restriction with n-3 PUFA led to a decrease of insulin resistance index (HOMA-IR) and a significant reduction of insulin output as well as decreased GIP secretion during the OGTT. These effects were not seen with caloric restriction alone. Changes in GIP output were inversely associated with changes in red blood cell EPA content whereas fasting GIP level positively correlated with HOMA-IR index. Blood triglyceride level was lowered by caloric restriction with a greater effect when n-3 PUFA were included and correlated positively with fasting GIP level. Conclusions Three months of caloric restriction with DHA + EPA supplementation exerts beneficial effects on insulin resistance, GIP and triglycerides. General significance Combining caloric restriction and n-3 PUFA improves insulin sensitivity, which may be related to a decrease of GIP levels., Highlights • Caloric restricted diet with n-3 PUFA improves insulin sensitivity in obese subjects • n-3 PUFA supplementation combined with low calorie diet decrease GIP output • Blood triglycerides level reduces after caloric restriction diet combined with n-3 PUFA • GIP level positively correlates with HOMA-IR index and triglycerides

Background Turner syndrome (TS) is the most common chromosomal abnormality in females and is associated with several co-morbidities. It commonly results from X monosomy which is diagnosed on a 30 cell karyotype. Congenital heart disease is a clinical feature in 30% of cases. It is becoming evident that TS patients have an increased risk of cardiovascular and cerebrovascular diseases. Scope of review This review provides a detailed overview of the literature surrounding cardiometabolic health in childhood and adolescent TS. In addition, the review also summarises the current data on the impact of growth hormone (GH) therapy on cardiometabolic risk in paediatric TS patients. Major conclusions Current epidemiological evidence suggests that young women and girls with TS have unfavourable cardiometabolic risk factors which predispose them to adverse cardiac and cerebrovascular outcomes in young adulthood. It remains unclear whether this risk is the result of unidentified factors which are intrinsic to TS, or whether modifiable risk factors (obesity, hypertension, hyperglycaemia) are contributing to this risk. General significance From a clinical perspective, this review highlights the importance of regular screening and pro-active management of cardiometabolic risk from childhood in TS cohorts and that future research should aim to address whether modification of these variables at a young age can alter the disease process and atherosclerotic outcomes in adulthood., Highlights • Increase in cardiovascular and cerebrovascular diseases in adult Turner syndrome • Cardiometabolic risk factors impact adult metabolic health and outcome. • Cardiometabolic risk factors in childhood Turner syndrome need careful assessment. • Future research should focus on early modification of these risk factors and outcome.