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Background: Insulin feedback is a critical mechanism responsible for the poor clinical efficacy of phosphatidylinositol 3-kinase (PI3K) inhibition in cancer, and hyperglycemia is an independent factor associated with poor prognosis in glioblastoma (GBM). We investigated combination anti-hyperglycemic therapy in a mouse model of GBM and evaluated the association of glycemic control in clinical trial data from patients with GBM., Methods: The effect of the anti-hyperglycemic regimens, metformin and the ketogenic diet, was evaluated in combination with PI3K inhibition in patient-derived GBM cells and in an orthotopic GBM mouse model. Insulin feedback and the immune microenvironment were retrospectively evaluated in blood and tumor tissue from a Phase 2 clinical trial of buparlisib in patients with recurrent GBM., Results: We found that PI3K inhibition induces hyperglycemia and hyperinsulinemia in mice and that combining metformin with PI3K inhibition improves the treatment efficacy in an orthotopic GBM xenograft model. Through examination of clinical trial data, we found that hyperglycemia was an independent factor associated with poor progression-free survival in patients with GBM. We also found that PI3K inhibition increased insulin receptor activation and T-cell and microglia abundance in tumor tissue from these patients., Conclusion: Reducing insulin feedback improves the efficacy of PI3K inhibition in GBM in mice, and hyperglycemia worsens progression-free survival in patients with GBM treated with PI3K inhibition. These findings indicate that hyperglycemia is a critical resistance mechanism associated with PI3K inhibition in GBM and that anti-hyperglycemic therapy may enhance PI3K inhibitor efficacy in GBM patients., (Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2023.)

Interleukin 33 (IL-33) signaling regulates most of the key processes of pregnancy, including decidualization, trophoblast proliferation and invasion, vascular remodeling, and placental growth. Accordingly, dysregulation of IL-33, its membrane-bound receptor (ST2L, transducer of IL-33 signaling), and its soluble decoy receptor (sST2, inhibitor of IL-33 signaling) has been linked to a wide range of adverse pregnancy outcomes that are common in women with obesity and polycystic ovary syndrome, that is, conditions associated with hyperandrogenism, insulin resistance, and compensatory hyperinsulinemia. To reveal if androgens and insulin might modulate uteroplacental IL-33 signaling, we investigated the effect of dihydrotestosterone (DHT) and/or insulin on the expression of ST2L and sST2 (along with the activity of their promoter regions), IL-33 and sIL1RAP (heterodimerization partner of sST2), during in vitro decidualization of endometrial stromal cells from 9 healthy women. DHT and insulin markedly upregulated sST2 secretion, in addition to the upregulation of its messenger RNA (mRNA) expression, while the proximal ST2 promoter, from which the sST2 transcript originates, was upregulated by insulin, and in a synergistic manner by DHT and insulin combination treatment. On the other hand, sIL1RAP was slightly downregulated by insulin and IL-33 mRNA expression was not affected by any of the hormones, while ST2L mRNA expression and transcription from its promoter region (distal ST2 promoter) could not be detected or showed a negligibly low level. We hypothesize that high levels of androgens and insulin might lead to subfertility and pregnancy complications, at least partially, through the sST2-dependent downregulation of uteroplacental IL-33 signaling., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Objectives: To compare the therapeutic potential of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) preconditioned ex-vivo with resveratrol (MCR) and BM-MSCs isolated from resveratrol-pre-treated rats (MTR) in type-1 diabetic rats., Methods: Type-1 diabetes was induced by a single streptozotocin injection (50 mg/kg; ip) in 24 rats. Following the confirmation of T1DM, diabetic rats were randomly divided into four groups: diabetic control (DC), diabetic rats treated with insulin subcutaneous (7.5 IU/kg/day), diabetic rats treated with MCR cells (3 × 106cells/rat, intravenous) and diabetic rats treated with MTR cells (3 × 106cells/rat, intravenous). Rats were sacrificed 4 weeks following cellular transplantation., Key Findings: Untreated diabetic rats suffered from pancreatic cell damage, had high blood glucose levels, increased apoptotic, fibrosis, and oxidative stress markers and decreased survival and pancreatic regeneration parameters. Both MSCs preconditioned ex-vivo with RES and MSCs isolated from rats pre-treated with RES homed successfully in injured pancreas and showed therapeutic potential in the treatment of STZ-induced T1DM. MCR cells showed better efficiency than MTR cells., Conclusions: The pre-conditioning of BM-MSCs with resveratrol may be a promising therapeutic possibility in T1DM. Resveratrol-preconditioned BM-MSCs encouraged effects almost comparable to that of exogenous insulin with the advantages of cured pancreas and restored islets not attained by insulin., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Exogenous ketone body administration lowers circulating glucose levels but the underlying mechanisms are uncertain., Objective: We tested the hypothesis that administration of the ketone body β-hydroxybutyrate (βOHB) acutely increases insulin sensitivity via feedback suppression of circulating free fatty acid (FFA) levels., Methods: In a randomized, single-blinded crossover design, 8 healthy men were studied twice with a growth hormone (GH) infusion to induce lipolysis in combination with infusion of either βOHB or saline. Each study day comprised a basal period and a hyperinsulinemic-euglycemic clamp combined with a glucose tracer and adipose tissue and skeletal muscle biopsies., Results: βOHB administration profoundly suppressed FFA levels concomitantly with a significant increase in glucose disposal and energy expenditure. This was accompanied by a many-fold increase in skeletal muscle content of both βOHB and its derivative acetoacetate., Conclusion: Our data unravel an insulin-sensitizing effect of βOHB, which we suggest is mediated by concomitant suppression of lipolysis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Central response to insulin is suspected to be defective in Alzheimer's disease. As most insulin is secreted in the bloodstream by the pancreas, its capacity to regulate brain functions must, at least partly, be mediated through the cerebral vasculature. However, how insulin interacts with the blood-brain barrier and whether alterations of this interaction could contribute to Alzheimer's disease pathophysiology both remain poorly defined. Here, we show that human and murine cerebral insulin receptors (INSRs), particularly the long isoform INSRα-B, are concentrated in microvessels rather than in the parenchyma. Vascular concentrations of INSRα-B were lower in the parietal cortex of subjects diagnosed with Alzheimer's disease, positively correlating with cognitive scores, leading to a shift towards a higher INSRα-A/B ratio, consistent with cerebrovascular insulin resistance in the Alzheimer's disease brain. Vascular INSRα was inversely correlated with amyloid-β plaques and β-site APP cleaving enzyme 1, but positively correlated with insulin-degrading enzyme, neprilysin and P-glycoprotein. Using brain cerebral intracarotid perfusion, we found that the transport rate of insulin across the blood-brain barrier remained very low (<0.03 µl/g·s) and was not inhibited by an insulin receptor antagonist. However, intracarotid perfusion of insulin induced the phosphorylation of INSRβ that was restricted to microvessels. Such an activation of vascular insulin receptor was blunted in 3xTg-AD mice, suggesting that Alzheimer's disease neuropathology induces insulin resistance at the level of the blood-brain barrier. Overall, the present data in post-mortem Alzheimer's disease brains and an animal model of Alzheimer's disease indicate that defects in the insulin receptor localized at the blood-brain barrier strongly contribute to brain insulin resistance in Alzheimer's disease, in association with β-amyloid pathology., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

(Pro)renin receptor [(P)RR] is related to both the renin-angiotensin system and V-ATPase with various functions including stimulation of cell proliferation. (P)RR is implicated in the pathophysiology of diabetes mellitus and cancer. Hyperinsulinemia is observed in obesity-related breast cancer. However, the relationship between (P)RR and insulin has not been clarified. We have therefore studied the effect of insulin on (P)RR expression, cell viability and AKT phosphorylation under the conditions with and without (P)RR knockdown. Effects of insulin were studied in a human breast cancer cell line, MCF-7. Cell proliferation assay was performed by WST-8 assay. (P)RR expression was suppressed by (P)RR-specific siRNAs. The treated cells were analysed by western blotting and reverse transcriptase-quantitative polymerase chain reaction analysis. Insulin stimulated proliferation of MCF-7 cells and increased (P)RR protein expression, but not (P)RR mRNA levels. Moreover, autophagy flux was suppressed by insulin. Suppression of (P)RR expression reduced cell number of MCF-7 cells and AKT phosphorylation significantly in both the presence and the absence of insulin, indicating that (P)RR is important for cell viability and AKT phosphorylation. In conclusion, insulin upregulates the level of (P)RR protein, which is important for cell viability, proliferation, AKT phosphorylation and autophagy in breast cancer cells., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Arylamine N-acetyltransferase 2 (NAT2) is well-known for its role in phase II metabolism of xenobiotics and drugs. More recently, genome wide association studies and murine models implicated NAT2 in regulation of insulin sensitivity and plasma lipid levels. However, the mechanism remains unknown. Transcript levels of human NAT2 varied dynamically in HepG2 (hepatocellular) cells, depending on the nutrient status of the culture media. Culturing the cells in the presence of glucose induced NAT2 mRNA expression as well as its N-acetyltransferase activity significantly. In addition, insulin or acetate treatment also significantly induced NAT2 mRNA. We examined and compared the glucose- and acetate-dependent changes in NAT2 expression to those of genes involved in glucose and lipid metabolism, including FABP1, CPT1A, ACACA, SCD, CD36, FASN, ACLY, G6PC, and PCK1. Genes that are involved in fatty acid transport and lipogenesis, such as FABP1 and CD36, shared a similar pattern of expression with NAT2. In silico analysis of genes co-expressed with NAT2 revealed an enrichment of biological processes involved in lipid and cholesterol biosynthesis and transport. Among these, A1CF (APOBEC1 complementation factor) showed the highest correlation with NAT2 in terms of its expression in normal human tissues. The current study shows, for the first time, that human NAT2 is transcriptionally regulated by glucose and insulin in liver cancer cell lines and that the gene expression pattern of NAT2 is similar to that of genes involved in lipid metabolism and transport., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: In this study, we investigated whether scopoletin stimulated the secretion of insulin in pancreatic β cells as well as the underlying mechanism involved in this process., Methods: We incubated the INS-1 pancreatic β cells with various concentrations of glucose (1.1, 5.6 or 16.7 mM) in the presence or absence of scopoletin. We then analysed the secretion of insulin in the cells treated with insulin secretion inhibitors or secretagogues. The intracellular influx of calcium induced by scopoletin was also analysed using the Fluo-2 AM dye., Key Findings: We found that scopoletin (1-20 µM) markedly induced the secretion of insulin in a glucose concentration-dependent manner compared with the control. At depolarizing concentrations of potassium chloride (KCl), scopoletin markedly enhanced the insulin secretion compared with the cells which were treated only with KCl. Moreover, the treatment with diazoxide-opening K+ATP channel and verapamil blocking Ca2+ channel significantly decreased the scopoletin-induced increase in insulin secretion. After the pre-treatment of cells with a Ca2+ fluorescent dye, treatment with 20 µM scopoletin resulted in a significant increase in the influx of intracellular Ca2+, exhibiting fluorescence changes in various spectra., Conclusions: Scopoletin stimulates the secretion of insulin via a K+ATP channel-dependent pathway in the INS-1 pancreatic β cells., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The ventromedial hypothalamic (VMH) nucleus is a well-established hub for energy and glucose homeostasis. In particular, VMH neurons are thought to be important for initiating the counterregulatory response to hypoglycemia, and ex vivo electrophysiology and immunohistochemistry data indicate a clear role for VMH neurons in sensing glucose concentration. However, the temporal response of VMH neurons to physiologically relevant changes in glucose availability in vivo has been hampered by a lack of available tools for measuring neuronal activity over time. Since the majority of neurons within the VMH are glutamatergic and can be targeted using the vesicular glutamate transporter Vglut2, we expressed cre-dependent GCaMP7s in Vglut2 cre mice and examined the response profile of VMH to intraperitoneal injections of glucose, insulin, and 2-deoxyglucose (2DG). We show that reduced available glucose via insulin-induced hypoglycemia and 2DG-induced glucoprivation, but not hyperglycemia induced by glucose injection, inhibits VMH Vglut2 neuronal population activity in vivo. Surprisingly, this inhibition was maintained for at least 45 minutes despite prolonged hypoglycemia and initiation of a counterregulatory response. Thus, although VMH stimulation, via pharmacological, electrical, or optogenetic approaches, is sufficient to drive a counterregulatory response, our data suggest VMH Vglut2 neurons are not the main drivers required to do so, since VMH Vglut2 neuronal population activity remains suppressed during hypoglycemia and glucoprivation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Design of "first-generation" insulin analogues over the past 3 decades has provided pharmaceutical formulations with tailored pharmacokinetic (PK) and pharmacodynamic (PD) properties. Application of a molecular tool kit-integrating protein sequence, chemical modification, and formulation-has thus led to improved prandial and basal formulations for the treatment of diabetes mellitus. Although PK/PD changes were modest in relation to prior formulations of human and animal insulins, significant clinical advantages in efficacy (mean glycemia) and safety (rates of hypoglycemia) were obtained. Continuing innovation is providing further improvements to achieve ultrarapid and ultrabasal analogue formulations in an effort to reduce glycemic variability and optimize time in range. Beyond such PK/PD metrics, next-generation insulin analogues seek to exploit therapeutic mechanisms: glucose-responsive ("smart") analogues, pathway-specific ("biased") analogues, and organ-targeted analogues. Smart insulin analogues and delivery systems promise to mitigate hypoglycemic risk, a critical barrier to glycemic control, whereas biased and organ-targeted insulin analogues may better recapitulate physiologic hormonal regulation. In each therapeutic class considerations of cost and stability will affect use and global distribution. This review highlights structural principles underlying next-generation design efforts, their respective biological rationale, and potential clinical applications., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor β subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRβ-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Members of the enhancer of split- and hairy-related protein (SHARP) family, SHARP-1 and SHARP-2, are basic helix-loop-helix transcriptional repressors and belong to the clock genes. In this study, an effect of retinoic acid (RA) on the SHARP family gene expression in the differentiated cells was examined. RA rapidly and temporarily induced the SHARP-2 mRNA expression in hepatic H4IIE cells. Then, whether the SHARP-2 mRNA expression is altered by dexamethasone (Dex), insulin, and the combination of RA and Dex or RA and insulin was examined. Dex had different effects on the expression of SHARP-2 mRNA in the presence or absence of RA. Then, the molecular mechanisms were investigated using inhibitors of various signaling molecules. The RA-induction of SHARP-2 mRNA level was mainly inhibited by LY294002, staurosporine, and actinomycin D, respectively. Finally, whether RA acts on the transcriptional regulatory region of the SHARP-2 gene was analysed using luciferase reporter gene assay. At least two RA-responsive regions were mapped at the nucleotide sequences between -3,700 and -1,600 of the SHARP-2 gene. In addition, this effect was dependent on the RA receptor and retinoid X receptor. Thus, we conclude that RA stimulated transcription of the SHARP-2 gene via multiple pathways., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Objectives: The aim of this study was to evaluate the effects of fasudil on insulin resistance (IR) in HTR-8/SVneo cells., Methods: HTR-8/SVneo cells were treated with insulin or/and fasudil. Cell proliferation, apoptosis, inflammation and related signalling pathways were assessed., Key Findings: Insulin treatment significantly enhanced the protein expressions of RhoA and Rho kinase (ROCK1 and ROCK2), but decreased glucose consumption. Administration of fasudil effectively promoted glucose uptake. Moreover, fasudil enhanced cell viability and the level of proliferating cell nuclear antigen (PCNA). Insulin-mediated cell apoptosis was inhibited by fasudil via the down-regulation of bax and cleaved-caspase-3, and the up-regulation of bcl-2. At the same time, fasudil led to the reduction of IL-1β, TNF-α, IL-6 and IL-8 mRNA levels in insulin-treated cells. In addition, RhoA, ROCK2 and phosphorylated myosin phosphatase target subunit-1 (p-MYPT-1) expressions were down-regulated by fasudil. Importantly, fasudil activated insulin receptor substrate-1 (IRS-1) through increasing p-IRS-1 (Tyr612) and p-Akt expressions. The nuclear NF-κB p65 and p-IκB-α levels were reduced via the administration of fasudil in insulin-treated cells., Conclusions: Fasudil mitigated IR by the promotion of cell proliferation, inhibition of apoptosis and inflammation and regulation of RhoA/ROCK/insulin/NF-κB signalling pathway through in vitro studies., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The molecular interactions between the maternal environment and the developing embryo are key for early pregnancy success and are influenced by factors such as maternal metabolic status. Our understanding of the mechanism(s) through which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success is, however, limited. Here we report, for the first time, the use of an endometrium-on-a-chip microfluidics approach to produce a multicellular endometrium in vitro. Isolated endometrial cells (epithelial and stromal) from the uteri of nonpregnant cows in the early luteal phase (Days 4-7) were seeded in the upper chamber of the device (epithelial cells; 4-6 × 104 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 × 104 cells/mL). Exposure of cells to different concentrations of glucose (0.5, 5.0, or 50 mM) or insulin (Vehicle, 1 or 10 ng/mL) was performed at a flow rate of 1 µL/minute for 72 hours. Quantitative differences in the cellular transcriptome and the secreted proteome of in vitro-derived uterine luminal fluid were determined by RNA-sequencing and tandem mass tagging mass spectrometry, respectively. High glucose concentrations altered 21 and 191 protein-coding genes in epithelial and stromal cells, respectively (P < .05), with a dose-dependent quantitative change in the protein secretome (1 and 23 proteins). Altering insulin concentrations resulted in limited transcriptional changes including transcripts for insulin-like binding proteins that were cell specific but altered the quantitative secretion of 196 proteins. These findings highlight 1 potential mechanism by which changes to maternal glucose and insulin alter uterine function., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Objectives: The main objective of the present study was to develop a nanoparticulate drug delivery system that can protect insulin against harsh conditions in the gastrointestinal (GI) tract. The effects of the following employed techniques, including lyophilisation, cross-linking and nanoencapsulation, on the physicochemical properties of the formulation were investigated., Methods: We herein developed a nanocarrier via ionotropic gelation by using positively charged chitosan and negatively charged Dz13Scr. The lyophilised nanoparticles with optimal concentrations of tripolyphosphate (cross-linking agent) and β-cyclodextrin (stabilising agent) were characterised by using physical and cellular assays., Key Findings: The addition of cryoprotectants (1% sucrose) in lyophilisation improved the stability of nanoparticles, enhanced the encapsulation efficiency, and ameliorated the pre-mature release of insulin at acidic pH. The developed lyophilised nanoparticles did not display any cytotoxic effects in C2C12 and HT-29 cells. Glucose consumption assays showed that the bioactivity of entrapped insulin was maintained post-incubation in the enzymatic medium., Conclusions: Freeze-drying with appropriate cryoprotectant could conserve the physiochemical properties of the nanoparticles. The bioactivity of the entrapped insulin was maintained. The prepared nanoparticles could facilitate the permeation of insulin across the GI cell line., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As advances are made in the field of developmental origins of health and disease, there is an emphasis on long-term influence of maternal environmental factors on offspring health. Maternal high-fat diet (HFD) consumption has been suggested to exert detrimental effects on cognitive function in offspring, but whether HFD-dependent brain remodeling can be transmitted to the next generations is still unclear. This study tested the hypothesis that HFD consumption during rat pregnancy and lactation multigenerationally influences male offspring hippocampal synaptic plasticity and cognitive function. We observed that hippocampus-dependent learning and memory was impaired in 3 generations from HFD-fed maternal ancestors (referred as F1-F3), as assessed by novel object recognition and Morris water maze tests. Moreover, maternal HFD exposure also affected electrophysiological and ultrastructure measures of hippocampal synaptic plasticity across generations. We observed that intranasal insulin replacement partially rescued hippocampal synaptic plasticity and cognitive deficits in F3 rats, suggesting central insulin resistance may play an important role in maternal diet-induced neuroplasticity impairment. Furthermore, maternal HFD exposure enhanced the palmitoylation of GluA1 critically involved in long-term potentiation induction, while palmitoylation inhibitor 2-bromopalmitate counteracts GluA1 hyperpalmitoylation and partially abolishes the detrimental effects of maternal diet on learning and memory in F3 offspring. Importantly, maternal HFD-dependent GluA1 hyperpalmitoylation was reversed by insulin replacement. Taken together, our data suggest that maternal HFD exposure multigenerationally influences adult male offspring hippocampal synaptic plasticity and cognitive performance, and central insulin resistance may serve as the cross-talk between maternal diet and cognitive impairment across generations., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Podocytes, the principal component of the glomerular filtration barrier, regulate glomerular permeability to albumin via their contractile properties. Both insulin- and high glucose (HG)-dependent activation of protein kinase G type Iα (PKGIα) cause reorganization of the actin cytoskeleton and podocyte disruption. Vasodilator-stimulated phosphoprotein (VASP) is a substrate for PKGIα and involved in the regulation of actin cytoskeleton dynamics. We investigated the role of the PKGIα/VASP pathway in the regulation of podocyte permeability to albumin. We evaluated changes in high insulin- and/or HG-induced transepithelial albumin flux in cultured rat podocyte monolayers. Expression of PKGIα and downstream proteins was confirmed by western blot and immunofluorescence. We demonstrate that insulin and HG induce changes in the podocyte contractile apparatus via PKGIα-dependent regulation of the VASP phosphorylation state, increase VASP colocalization with PKGIα, and alter the subcellular localization of these proteins in podocytes. Moreover, VASP was implicated in the insulin- and HG-dependent dynamic remodelling of the actin cytoskeleton and, consequently, increased podocyte permeability to albumin under hyperinsulinaemic and hyperglycaemic conditions. These results indicate that insulin- and HG-dependent regulation of albumin permeability is mediated by the PKGIα/VASP pathway in cultured rat podocytes. This molecular mechanism may explain podocytopathy and albuminuria in diabetes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)

Pancreatic islet microcirculation, consisting of pancreatic islet microvascular endothelial cells (IMECs) and pericytes (IMPCs), provides crucial support for the physiological function of pancreatic islet. Emerging evidence suggests that pancreatic islet microcirculation is impaired in type 1 diabetes mellitus (T1DM). Here, we investigated the potential ultrastructural protective effects of insulin against streptozotocin (STZ)-induced ultrastructural abnormalities of the pancreatic islet microcirculation in T1DM mouse model. For this purpose, pancreatic tissues were collected from control, STZ-induced T1DM and insulin-treated mice, and a pancreatic IMECs cell line (MS1) was cultured under control, 35 mM glucose with or without 10-8 M insulin conditions. Transmission and scanning electron microscopies were employed to evaluate the ultrastructure of the pancreatic islet microcirculation. We observed ultrastructural damage to IMECs and IMPCs in the type 1 diabetic group, as demonstrated by destruction of the cytoplasmic membrane and organelles (mainly mitochondria), and this damage was substantially reversed by insulin treatment. Furthermore, insulin inhibited collagenous fiber proliferation and alleviated edema of the widened pancreatic islet exocrine interface in T1DM mice. We conclude that insulin protects against T1DM-induced ultrastructural abnormalities of the pancreatic islet microcirculation., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Insulin is a peptide hormone produced by the pancreas. The physiological role of insulin is the regulation of glucose metabolism. Under certain pathological conditions the insulin levels can be reduced leading to the metabolic disorder diabetes mellitus (DM). For type 1 DM and, dependent on the disease progression for type 2 DM, insulin substitution becomes indispensable. To relieve insulin substitution therapy for patients, novel insulin analogs with pharmacokinetic and pharmacodynamic profiles aiming for long-lasting or fast-acting insulins have been developed. The next step in the evolution of novel insulins should be insulin analogs with a time action profile beyond 1-2 days, preferable up to 1 week. Nowadays, insulin is produced in a recombinant manner. This approach facilitates the design and production of further insulin-analogs or insulin-fusion proteins. The usage of the Fc-domain from immunoglobulin as a fusion partner for therapeutic proteins and peptides is widely used to extend their plasma half-life. Insulin consists of two chains, the A- and B-chain, which are connected by two disulfide-bridges. To produce a novel kind of Fc-fusion protein we have fused the A-chain as well as the B-chain to Fc-fragments containing either 'knob' or 'hole' mutations. The 'knob-into-hole' technique is frequently used to force heterodimerization of the Fc-domain. Using this approach, we were able to produce different variants of two-chain-insulin-Fc-protein (tcI-Fc-protein) variants. The tcI-Fc-fusion variants retained activity as shown in in vitro assays. Finally, prolonged blood glucose lowering activity was demonstrated in normoglycemic rats. Overall, we describe here the production of novel insulin-Fc-fusion proteins with prolonged times of action., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

To act on tissues, circulating insulin must perfuse the relevant organ and then leave the bloodstream by crossing the endothelium-a process known as insulin delivery. It has been postulated that the continuous endothelium is a rate-limiting barrier to insulin delivery but existing data are contradictory. This conflict is in part due to the limitations of current models, including the inability to maintain a constant blood pressure in animals and the absence of shear stress in cultured cells. We developed a murine cardiac ex vivo perfusion model that delivers insulin to the heart in situ at a constant flow. We hypothesized that if the endothelial barrier were rate-limiting to insulin delivery, increasing endothelial permeability would accelerate insulin action. The kinetics of myocardial insulin action were determined in the presence or absence of agents that increased endothelial permeability. Permeability was measured using Evans Blue, which binds with high affinity to albumin. During our experiments, the myocardium remained sensitive to insulin and the vasculature retained barrier integrity. Perfusion with insulin induced Akt phosphorylation in myocytes but not in the endothelium. Infusion of platelet-activating factor or vascular endothelial growth factor significantly increased permeability to albumin without altering insulin action. Amiloride, an inhibitor of fluid-phase uptake, also did not alter insulin action. These data suggest that the endothelial barrier is not rate limiting to insulin's action in the heart; its passage out of the coronary circulation is consistent with diffusion or convection. Modulation of transendothelial transport to overcome insulin resistance is unlikely to be a viable therapeutic strategy., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High-fat diet (HFD) induced hepatic endoplasmic reticulum (ER) stress drives insulin resistance (IR) and steatosis. NK cells in adipose tissue play an important role in the pathogenesis of IR in obesity. Whether NK cells in the liver can induce hepatic ER stress and thus promote IR in obesity is still unknown. We demonstrate that HFD-fed mice display elevated production of proinflammatory cytokine osteopontin (OPN) in hepatic NK cells, especially in CD49a + DX5 - tissue-resident NK (trNK) cells. Obesity-induced ER stress, IR, and steatosis in the liver are ameliorated by ablating NK cells with neutralizing antibody in HFD-fed mice. OPN treatment enhances the expression of ER stress markers, including p-PERK, p-eIF2, ATF4, and CHOP in both murine liver tissues and HL-7702, a human liver cell line. Pretreatment of HL-7702 cells with OPN promotes hyperactivation of JNK and subsequent decrease of tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), resulting in impaired insulin signaling, which can be reversed by inhibiting ER stress. Collectively, we demonstrate that hepatic NK cells induce obesity-induced hepatic ER stress, and IR through OPN production., (©2019 Society for Leukocyte Biology.)

Prolonged exposure to glucocorticoids (GCs) causes various metabolic derangements. These include obesity and insulin resistance, as inhibiting glucose utilization in adipose tissues is a major function of GCs. Although adipose tissue distribution and glucose homeostasis are sex-dependently regulated, it has not been evaluated whether GCs affect glucose metabolism and adipose tissue functions in a sex-dependent manner. In this study, high-dose corticosterone (rodent GC) treatment in C57BL/6J mice resulted in nonfasting hyperglycemia in male mice only, whereas both sexes displayed hyperinsulinemia with normal fasting glucose levels, indicative of insulin resistance. Metabolic testing using stable isotope-labeled glucose techniques revealed a sex-specific corticosterone-driven glucose intolerance. Corticosterone treatment increased adipose tissue mass in both sexes, which was reflected by elevated serum leptin levels. However, female mice showed more metabolically protective adaptations of adipose tissues than did male mice, demonstrated by higher serum total and high-molecular-weight adiponectin levels, more hyperplastic morphological changes, and a stronger increase in mRNA expression of adipogenic differentiation markers. Subsequently, in vitro studies in 3T3-L1 (white) and T37i (brown) adipocytes suggest that the increased leptin and adiponectin levels were mainly driven by the elevated insulin levels. In summary, this study demonstrates that GC-induced insulin resistance is more severe in male mice than in female mice, which can be partially explained by a sex-dependent adaptation of adipose tissues., (Copyright © 2019 Endocrine Society.)

Insulin is present in most maintenance media for human embryonic stem cells (hESCs), but little is known about its essential role in the cell survival of individualized cells during passage. In this article, we show that insulin suppresses caspase cleavage and apoptosis after dissociation. Insulin activates insulin-like growth factor (IGF) receptor and PI3K/AKT cascade to promote cell survival and its function is independent of rho-associated protein kinase regulation. During niche reformation after passaging, insulin activates integrin that is essential for cell survival. IGF receptor colocalizes with focal adhesion complex and stimulates protein phosphorylation involved in focal adhesion formation. Insulin promotes cell spreading on matrigel-coated surfaces and suppresses myosin light chain phosphorylation. Further study showed that insulin is also required for the cell survival on E-cadherin coated surface and in suspension, indicating its essential role in cell-cell adhesion. This work highlights insulin's complex roles in signal transduction and niche re-establishment in hESCs. Stem Cells 2019;37:1030-1041., (© 2019 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2019.)

Context and Objective: Not only type 2 diabetes (T2D), but also type 1 diabetes (T1D), can be associated with insulin resistance, as assessed using insulin-stimulated whole-body glucose disposal (M-value). We hypothesized that different factors would affect the M-value at the onset of T1D and T2D., Design and Patients: We examined 132 patients with T1D or T2D matched for sex, age, and body mass index with a known diabetes duration of <12 months. Multivariable linear regression analyses were applied to test the associations between glycemic control, blood lipid levels, adiponectin, and proinflammatory immune mediators and the M-value, obtained from the hyperinsulinemic-euglycemic clamp., Results: Despite comparable age, body mass index, and near-normoglycemic control, the mean M-value was lower in those with T2D than in those with T1D. Patients with T1D had a lower waist/hip ratio and serum triglycerides but higher serum adiponectin than patients with T2D. However, the circulating proinflammatory markers were not different. Even with adjustments for glucose-lowering treatments, the fasting blood glucose correlated negatively with the M-value in both groups. However, gamma-glutamyl transferase-independently of any treatments-correlated negatively only in T2D. In contrast, serum adiponectin correlated positively with the M-values., Conclusions: Fasting glycemia correlated with insulin-stimulated glucose disposal in both diabetes types. However, altered liver and adipose tissue function were associated with insulin-stimulated glucose disposal only in T2D, underpinning the specific differences between these diabetes types., (Copyright © 2019 Endocrine Society.)

Objectives: The present study was designed to assess the pharmacokinetic and pharmacodynamic performance of inhaled recombinant human insulin (rh-insulin) dry powders together with their safety profiles after 14-day inhalation., Methods: In the pharmacokinetic and pharmacodynamic study, pulmonary surfactant (PS)-loaded and phospholipid hexadecanol tyloxapol (PHT)-loaded rh-insulin dry powders were intratracheally administered to male rats at the dose of 20 U/kg. Novolin R was used as control. Serum glucose and rh-insulin concentrations were determined by glucose oxidase method and human rh-insulin CLIA kit, respectively. For the safety study, rats were exposed to rh-insulin dry powders or air for 14-day by nose-only inhalation chambers. Bronchoalveolar lavage and histopathology examinations were performed after inhalation., Key Findings: There were no significant differences in the major pharmacokinetic and pharmacodynamic parameters between PS-loaded and PHT-loaded rh-insulin dry powders. The relative bioavailabilities and pharmacodynamic availabilities were 39.9%, 25.6% for PS-loaded dry powders and 30.1%, 23% for PHT-loaded dry powders, respectively. Total protein was the only injury marker that was significantly altered. Histopathology examinations showed the ranking of irritations (from slight to severe) were PHT-loaded rh-insulin, negative air control and PS-loaded rh-insulin., Conclusions: Both PS- and PHT-loaded rh-insulin dry powders were able to deliver rh-insulin systemically with appropriate pharmacokinetic, pharmacodynamic and safety profiles., (© 2018 Royal Pharmaceutical Society.)

Objective: To quantify glucose-mediated glucose disposal with and without basal insulin replacement and insulin-mediated glucose disposal in subjects with impaired fasting glucose (IFG)., Research Design and Methods: We used the hyperglycemic/pancreatic clamp and stepped euglycemic clamp techniques to quantify glucose disposal and suppression of endogenous glucose production (EGP) in those with normal glucose tolerance (NGT; n = 14) and those with IFG (n = 14)., Results: Total body glucose-mediated glucose uptake, measured with the hyperglycemic/pancreatic clamp, was not significantly affected by the basal plasma insulin levels in subjects with IFG and those with NGT. Compared with subjects with NGT, those with IFG had significantly lower glucose-mediated glucose uptake (by 15%) during the hyperglycemic clamp performed with and without basal insulin replacement. In contrast, insulin-mediated glucose disposal was comparable in both groups. The suppression of EGP by hyperglycemia was similar in both groups. However, the suppression of EGP by insulin was attenuated in those with IFG compared with those with NGT., Conclusions: The results of the present study have demonstrated that (i) glucose-mediated glucose disposal is impaired in those with IFG; (ii) insulin-mediated glucose uptake in IFG is normal; and (iii) insulin action to suppress EGP is impaired.

Controlled changes in mitochondrial biogenesis and morphology are required for cell survival and homeostasis, but the molecular mechanisms are largely unknown. Here, male and female prepubertal mice (P21) with insulin and IGF1 receptors deletions in steroidogenic tissues (Insr/Igf1r-DKO) were used to investigate transcription of the key regulators of mitochondrial biogenesis (Ppargc1a, Ppargc1b, Pparg, Nrf1, Tfam) and architecture in Leydig cells, ovaries, and adrenals. Results showed that the expression of PGC1, a master regulator of mitochondrial biogenesis and integrator of environmental signals, and its downstream target Tfam, significantly decreased in androgen-producing Leydig cells. This is followed by reduction of Mtnd1, a mitochondrial DNA encoded transcript whose core subunit belongs to the minimal assembly required for catalysis. The same markers remained unchanged in ovaries. In contrast, in adrenals, the pattern of transcripts for mitochondrial biogenesis markers was the same in both sexes, but opposite from that observed in Leydig cells. The level of transcripts for markers of mitochondrial architecture (Mfn1, Mfn2) significantly increased in Leydig cells from Insr/Igf1r-DKO, but not in ovaries. This was followed by mitochondrial morphology disturbance, suggesting that the mitochondrial phase of steroidogenesis could be affected. Indeed, basal and pregnenolone stimulated progesterone productions in the mitochondria of Leydig cells from Insr/Igf1r-DKO decreased more than androgen production, and were barely detectable. Our results are the first to show that INSR/IGF1R are important for mitochondrial biogenesis in gonadal steroidogenic cells of prepubertal males, but not females and they serve as important regulators of mitochondrial architecture and biogenesis markers in Leydig cells.

Context: Glycogen synthesis is a critical metabolic function of the endometrium to prepare for successful implantation and sustain embryo development. Yet, regulation of endometrial carbohydrate metabolism is poorly characterized. Whereas glycogen synthesis is attributed to progesterone, we previously found that the metabolic B isoform of the insulin receptor is maximally expressed in secretory-phase endometrium, indicating a potential role of insulin in glucose metabolism., Objective: We sought to determine whether insulin or progesterone regulates glycogen synthesis in human endometrium., Design, Participants, Outcome Measurements: Endometrial epithelial cells were isolated from 28 healthy women and treated with insulin, medroxyprogesterone (MPA), or vehicle. Intracellular glycogen and the activation of key enzymes were quantified., Results: In epithelia, insulin induced a 4.4-fold increase in glycogen, whereas MPA did not alter glycogen content. Insulin inactivated glycogen synthase (GS) kinase 3α/β (GSK3α/β), relieving inhibition of GS. In a regulatory mechanism, distinct from liver and muscle, insulin also increased GS by 3.7-fold through increased GS 2 (GYS2) gene expression., Conclusions: We demonstrate that insulin, not progesterone, directly regulates glycogen synthesis through canonical acute inactivation of GSK3α/β and noncanonical stimulation of GYS2 transcription. Persistently elevated GS enables endometrium to synthesize glycogen constitutively, independent of short-term nutrient flux, during implantation and early pregnancy. This suggests that insulin plays a key, physiological role in endometrial glucose metabolism and underlines the need to delineate the effect of maternal obesity and hyperinsulinemia on fertility and fetal development.

Context: The regulation of bone mass by the testis is a well-recognized mechanism, but the role of Leydig-specific marker insulin-like 3 peptide (INSL3) on the most abundant bone cell population, osteocytes, is unknown. In this study, we aimed to investigate the relationship between INSL3 and sclerostin, an osteocyte-specific protein that negatively regulates bone formation., Design: Serum sclerostin and INSL3 levels were evaluated in Klinefelter syndrome (KS) and healthy controls. In vitro effect of INSL3 on sclerostin production was evaluated in human cultured osteocytes., Patients: A total of 103 KS patients and 60 age- and sex-matched controls were recruited., Main Outcome Measures: Serum sclerostin and INSL3 levels were assessed by enzyme-linked immunosorbent assay. Osteocytes were isolated by fluorescence-assisted cell sorting. Sclerostin expression was evaluated by western blot, immunofluorescence, and reverse transcription polymerase chain reaction. Measurement of bone mineral density was done by dual-energy X-ray absorptiometry at lumbar spine (L1-L4) and femoral neck., Results: Sclerostin levels were significantly increased in KS subjects, and negatively correlated with INSL3 levels in both cohorts and with bone mineral density in the KS group. Stimulation of cultured osteocytes with INSL3 at 10-7 M significantly decreased both sclerostin messenger RNA and protein expression., Conclusions: We report a negative association between the testicular hormone INSL3 and the osteocytic negative regulator of bone formation, sclerostin. We further explored this association in vitro and showed that INSL3 was able to reduce sclerostin expression. These results add further knowledge on the emerging role of sclerostin as a therapeutic target for osteoporosis treatment.

Objective: Insulin resistance is reflected by the rates of reduced glucose uptake (GU) into the key insulin-sensitive tissues, skeletal muscle, liver and adipose tissue. It is unclear whether insulin resistance occurs simultaneously in all these tissues or whether insulin resistance is tissue specific., Design and Methods: We measured GU in skeletal muscle, adipose tissue and liver and endogenous glucose production (EGP), in a single session using 18 F-fluorodeoxyglucose with positron emission tomography (PET) and euglycemic-hyperinsulinemic clamp. The study population consisted of 326 subjects without diabetes from the CMgene study cohort., Results: Skeletal muscle GU less than 33 µmol/kg tissue/min and subcutaneous adipose tissue GU less than 11.5 µmol/kg tissue/min characterized insulin-resistant individuals. Men had considerably worse insulin suppression of EGP compared to women. By using principal component analysis (PCA), BMI inversely and skeletal muscle, adipose tissue and liver GU positively loaded on same principal component explaining one-third of the variation in these measures. The results were largely similar when liver GU was replaced by EGP in PCA. Liver GU and EGP were positively associated with aging., Conclusions: We have provided threshold values, which can be used to identify tissue-specific insulin resistance. In addition, we found that insulin resistance measured by GU was only partially similar across all insulin-sensitive tissues studied, skeletal muscle, adipose tissue and liver and was affected by obesity, aging and gender., (© 2018 The authors.)

A hyperglycemic and hyperinsulinemic environment characteristic of type 2 diabetes causes insulin resistance. In adipocytes, defects in both insulin sensitivity and maximum response of glucose transport have been demonstrated. To investigate the molecular mechanisms, freshly isolated rat adipocytes were incubated in control (5.6 mM glucose, no insulin) and high glucose (20 mM)/high insulin (100 nM) (HG/HI) for 18 hours to induce insulin resistance. Insulin-resistant adipocytes manifested decreased sensitivity of glucose uptake associated with defects in insulin receptor substrate (IRS)-1 Tyr phosphorylation, association of p85 subunit of phosphatidylinositol-3-kinase, Akt Ser473 and Thr308 phosphorylation, accompanied by impaired glucose transporter 4 translocation. In contrast, protein kinase C (PKC)-ζ activity was augmented by chronic HG/HI. Inhibition of PKC-ζ with a specific cell-permeable peptide reversed the signaling defects and insulin sensitivity of glucose uptake. Transfection of dominant-negative, kinase-inactive PKC-ζ blocked insulin resistance, whereas constitutively active PKC-ζ recapitulated the defects. The HG/HI incubation was associated with stimulation of IRS-1 Ser318 and Akt Thr34 phosphorylation, targets of PKC-ζ. Transfection of IRS-1 S318A and Akt T34A each partially corrected insulin signaling, whereas combined transfection of both completely normalized insulin signaling. In vivo hyperglycemia/hyperinsulinemia in rats for 48 hours similarly resulted in activation of PKC-ζ and increased phosphorylation of IRS-1 Ser318 and Akt Thr34. These data indicate that impairment of insulin signaling by chronic HG/HI is mediated by dual defects at IRS-1 and Akt mediated by PKC-ζ.

Context: The proinflammatory cytokine TNFα is a key player in insulin resistance (IR). The role of miRNAs in inflammation associated with IR is poorly understood., Objective: To investigate miR-181a-5p and miR-23a-3p expression profiles in obesity and to study their role in TNFα-induced IR in adipocytes., Design: Two separate cohorts were used. Cohort 1 was used in adipose tissue (AT) expression studies and included 28 subjects with body mass index (BMI) <30 kg/m2 and 30 with BMI ≥30 kg/m2. Cohort 2 was used in circulating serum miRNA studies and included 101 subjects with 4 years of follow-up (48 case subjects and 53 control subjects). miR-181a-5p and miR-23a-3p expression was assessed in subcutaneous and visceral AT. Functional analysis was performed in adipocytes, using miRNA mimics and inhibitors. Key molecules of the insulin pathway, AKT, PTEN, AS160, and S6K, were analyzed., Results: Expression of miR-181a-5p and miR-23a-3p was reduced in adipose tissue from obese and diabetic subjects and was inversely correlated to adiposity and homeostasis model assessment of IR index. Overexpression of miR-181a-5p and miR-23a-3p in adipocytes upregulated insulin-stimulated AKT activation and reduced TNFα-induced IR, regulating PTEN and S6K expression. Serum levels of miR-181a-5p were reduced in case vs control subjects at baseline, suggesting a prognostic value. Variable importance in projection scores revealed miR-181a-5p had more effect on the model than insulin or glucose at 120 minutes., Conclusion: miR-181a-5p and miR-23a-3p may prevent TNFα-induced IR in adipocytes through modulation of PTEN and S6K expression.

Context: Insulin administration to the central nervous system inhibits food intake, but this effect has been found to be less pronounced in female compared with male organisms. This sex-specific pattern has been suggested to arise from a modulating influence of estrogen signaling on the insulin effect., Objective: We assessed in healthy young men whether pretreatment with transdermal estradiol interacts with the hypophagic effect of central nervous insulin administration via the intranasal pathway., Design, Setting, Participants, and Intervention: According to a 2×2 design, two groups of men (n = 16 in each group) received a 3-day transdermal estradiol (100 µg/24 h) or placebo pretreatment and on two separate mornings were intranasally administered 160 IU regular human insulin or placebo., Main Outcome Measures: We assessed free-choice ad libitum calorie intake from a rich breakfast buffet and relevant blood parameters in samples collected before and after breakfast., Results: Estrogen treatment induced a 3.5-fold increase in serum estradiol concentrations and suppressed serum testosterone concentrations by 70%. Independent of estradiol administration, intranasal insulin reduced the intake of carbohydrates during breakfast, attenuating in particular the consumption of sweet, palatable foods. Estradiol treatment per se decreased protein consumption. We did not find indicators of eating-related interactions between both hormones., Conclusions: Results indicate that, in an acute setting, estrogen does not interact with central nervous insulin signaling in the control of eating behavior in healthy men. Insulin and estradiol rather exert independent inhibiting effects on macronutrient intake.

Grb10 is an adaptor-type signaling protein most highly expressed in tissues involved in insulin action and glucose metabolism, such as muscle, pancreas, and adipose. Germline deletion of Grb10 in mice creates a phenotype with larger muscles and improved glucose homeostasis. However, it has not been determined whether Grb10 ablation specifically in muscle is sufficient to induce hypermuscularity or affect whole body glucose metabolism. In this study we generated muscle-specific Grb10-deficient mice (Grb10-mKO) by crossing Grb10flox/flox mice with mice expressing Cre recombinase under control of the human α-skeletal actin promoter. One-year-old Grb10-mKO mice had enlarged muscles, with greater cross-sectional area of fibers compared with wild-type (WT) mice. This degree of hypermuscularity did not affect whole body glucose homeostasis under basal conditions. However, hyperinsulinemic/euglycemic clamp studies revealed that Grb10-mKO mice had greater glucose uptake into muscles compared with WT mice. Insulin signaling was increased at the level of phospho-Akt in muscle of Grb10-mKO mice compared with WT mice, consistent with a role of Grb10 as a modulator of proximal insulin receptor signaling. We conclude that ablation of Grb10 in muscle is sufficient to affect muscle size and metabolism, supporting an important role for this protein in growth and metabolic pathways.

Purpose: We previously demonstrated the anti-inflammatory and antioxidant effects of exenatide. We now hypothesized that exenatide also increases the plasma concentration of interleukin-1 receptor antagonist (IL-1RA), an endogenous anti-inflammatory protein, and modulates the nuclear factor erythroid 2‒related factor‒Kelchlike ECH-associated protein 1‒antioxidant response element (Nrf-2‒Keap-1‒ARE) system to induce key antioxidant enzymes to suppress inflammatory and oxidative stress., Methods: Twenty-four patients with obesity and type 2 diabetes receiving combined oral and insulin therapy were randomly assigned to receive either exenatide 10 μg or placebo twice a day for 12 weeks., Results: Exenatide increased IL-1RA concentration by 61% (from 318 ± 53 to 456 ± 88 pg/mL; P < 0.05). Exenatide treatment also suppressed Keap-1 protein (P < 0.05) and increased messenger RNA expression of NQO-1, glutathione S-transferase PI, heme oxygenase-1, and p21 and increased NAD(P)H dehydrogenase [quinone] 1 protein (P < 0.05) in mononuclear cells., Conclusions: Because IL-1RA protects, maintains, and stimulates β-cell function in humans and Nrf-2‒Keap-1‒ARE protects β cells in animals with experimental diabetes, these actions of exenatide may contribute to a potential protective effect on β cells in diabetes.

Insulin coordinates the complex response to feeding, affecting numerous metabolic and hormonal pathways. Forkhead box protein O1 (FoxO1) is one of several signaling molecules downstream of insulin; FoxO1 drives gluconeogenesis and is suppressed by insulin. To determine the role of FoxO1 in mediating other actions of insulin, we studied mice with hepatic deletion of the insulin receptor, FoxO1, or both. We found that mice with deletion of the insulin receptor alone showed not only hyperglycemia but also a 70% decrease in plasma insulin-like growth factor 1 and delayed growth during the first 2 months of life, a 24-fold increase in the soluble leptin receptor and a 19-fold increase in plasma leptin levels. Deletion of the insulin receptor also produced derangements in fatty acid metabolism, with a decrease in the expression of the lipogenic enzymes, hepatic diglycerides, and plasma triglycerides; in parallel, it increased expression of the fatty acid oxidation enzymes. Mice with deletion of both insulin receptor and FoxO1 showed a much more modest phenotype, with normal or near-normal glucose levels, growth, leptin levels, hepatic diglycerides, and fatty acid oxidation gene expression; however, lipogenic gene expression remained low. Taken together, these data reveal the pervasive role of FoxO1 in mediating the effects of insulin on not only glucose metabolism but also other hormonal signaling pathways and even some aspects of lipid metabolism., (Copyright © 2018 Endocrine Society.)

The goal of this study was to characterize the function of microRNA-27a-3p (miR-27a-3p) in polycystic ovary syndrome (PCOS). miR-27a-3p expression was analyzed in excised granulosa cells (GCs) from 21 patients with PCOS and 12 normal patients undergoing in vitro fertilization cycle treatments and in 17 nontreated cuneiform ovarian resection PCOS samples and 13 control ovarian samples from patients without PCOS. We found that the expression of miR-27a-3p was significantly increased in both excised GCs and the ovaries of patients with PCOS compared with the controls. Insulin treatment of the human granulosa-like tumor cell line (KGN) resulted in decreased downregulated expression of miR-27a-3p, and this effect appeared to be mediated by signal transducer and activator of transcription STAT1 and STAT3. The overexpression of miR-27a-3p in KGN cells inhibited SMAD5, which in turn decreased cell proliferation and promoted cell apoptosis. After KGN cells were stimulated with insulin for 48 hours, there was increased expression of SMAD5 protein and decreased apoptosis. Additionally, knockdown/overexpression of SMAD5 in KGN cells reduced/increased cell number and promoted/inhibited cell apoptosis. Insulin-stimulated primary GCs isolated from patients with PCOS, in contrast to normal GCs or KGN cells, did not exhibit decreased miR-27a-3p expression. The differences in the expression levels in KGN cells and human PCOS GCs are likely explained by increased miR-27a-3p expression in the GCs caused by insulin resistance in PCOS. Taken together, our data provided evidence for a functional role of miR-27a-3p in the GCs' dysfunction that occurs in patients with PCOS., (Copyright © 2018 Endocrine Society.)

Context: Abnormal glucagon concentrations contribute to hyperglycemia, but the mechanisms of α-cell dysfunction in prediabetes are unclear., Objective: We sought to determine the relative contributions of insulin secretion and action to α-cell dysfunction in nondiabetic participants across the spectrum of glucose tolerance., Design: This was a cross-sectional study. A subset of participants (n = 120) was studied in the presence and absence of free fatty acid (FFA) elevation, achieved by infusion of Intralipid (Baxter Healthcare, Deerfield, IL) plus heparin, to cause insulin resistance., Setting: An inpatient clinical research unit at an academic medical center., Participants: A total of 310 nondiabetic persons participated in this study., Interventions: Participants underwent a seven-sample oral glucose tolerance test. Subsequently, 120 participants were studied on two occasions. On one day, infusion of Intralipid plus heparin raised FFA. On the other day, participants received glycerol as a control., Main Outcome Measure(s): We examined the relationship of glucagon concentration with indices of insulin action after adjusting for the effects of age, sex, and weight. Subsequently, we sought to determine whether an acute decrease in insulin action, produced by FFA elevation, altered glucagon concentrations in nondiabetic participants., Results: Fasting glucagon concentrations correlated positively with fasting insulin and C-peptide concentrations and inversely with insulin action. Fasting glucagon was not associated with any index of β-cell function in response to an oral challenge. As expected, FFA elevation decreased insulin action and also raised glucagon concentrations., Conclusions: In nondiabetic participants, glucagon secretion was altered by changes in insulin action., (Copyright © 2017 Endocrine Society)

As part of the adaptation of maternal glucose regulation during pregnancy to ensure glucose provision to the fetus, maternal insulin concentrations become elevated. However, increased central actions of insulin, such as suppression of appetite, would be maladaptive during pregnancy. We hypothesized that central nervous system targets of insulin become less responsive during pregnancy to prevent overstimulation by the increased circulating insulin concentrations. To test this hypothesis, we have measured insulin-induced phosphorylation of Akt (pAkt) in specific hypothalamic nuclei as an index of hypothalamic insulin responsiveness. Despite higher endogenous insulin concentrations following feeding, arcuate nucleus pAkt levels were significantly lower in the pregnant group compared with the nonpregnant group. In response to an intracerebroventricular injection of insulin, insulin-induced pAkt was significantly reduced in the arcuate nucleus and ventromedial nucleus of pregnant rats compared with nonpregnant rats. Similar levels of insulin receptor β and PTEN, a negative regulator of the phosphoinositide 3-kinase/Akt pathway, were detected in hypothalamic areas of nonpregnant and pregnant rats. In the ventromedial nucleus, however, levels of phosphorylated PTEN were significantly lower in pregnancy, suggesting that reduced inactivation of PTEN may contribute to the attenuated insulin signaling in this area during pregnancy. In conclusion, these results demonstrate region-specific changes in responsiveness to insulin in the hypothalamus during pregnancy that may represent an adaptive response to minimize the impact of elevated circulating insulin on the maternal brain., (Copyright © 2017 Endocrine Society.)

Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4 R24C/R24C ). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs. Stem Cells 2017;35:2403-2416., (© 2017 AlphaMed Press.)

Insulin can stimulate hepatic expression of carbohydrate-responsive element-binding protein (ChREBP). As recent studies revealed potential metabolic beneficial effects of ChREBP, we asked whether its expression can also be regulated by the dietary polyphenol curcumin. We also aimed to determine mechanisms underlying ChREBP stimulation by insulin and curcumin. The effect of insulin on ChREBP expression was assessed in mouse hepatocytes, while the effect of curcumin was assessed in mouse hepatocytes and with curcumin gavage in mice. Chemical inhibitors for insulin signaling molecules were utilized to identify involved signaling molecules, and the involvement of p21-activated protein kinase 1 (Pak1) was determined with its chemical inhibitor and Pak1-/- hepatocytes. We found that both insulin and curcumin-stimulated ChREBP expression in Akt-independent but MEK/ERK-dependent manner, involving the inactivation of the transcriptional repressor Oct-1. Aged Pak1-/- mice showed reduced body fat volume. Pak1 inhibition or its genetic deletion attenuated the stimulatory effect of insulin or curcumin on ChREBP expression. Our study hence suggests the existence of a novel signaling cascade Pak1/MEK/ERK/Oct-1 for both insulin and curcumin in exerting their glucose-lowering effect via promoting hepatic ChREBP production, supports the recognition of beneficial functions of ChREBP, and brings us a new overview on dietary polyphenols., (© The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

The increased prevalence of obesity and its cardiometabolic implications demonstrates the imperative to identify novel therapeutic targets able to effect meaningful metabolic changes in this population. Antibody-mediated targeting of fibroblast growth factor receptor 1c isoform (FGFR1c) has been shown to ameliorate hyperglycemia and protect from diet- and genetically-induced obesity in rodents and nonhuman primates. However, it is currently unknown which tissue(s) contribute to this glucose-lowering effect. Thus, to elucidate this effect, we treated euglycemic mice with H7, a monoclonal antibody that selectively targets FGFR1c, and used whole-body positron emission computed tomography with a glucose tracer (18F-fluorodeoxyglucose). Treatment with H7 increased basal glucose uptake in white adipose tissue (WAT), brown adipose tissue (BAT), the brain, and liver but reduced it in the quadriceps muscles. Consequentially, blood glucose was significantly reduced in response to treatment. Under insulin-stimulated conditions, the effects of H7 were maintained in WAT, BAT, liver, and muscle. Treatment with H7 decreased triglyceride (TG) content and increased adipose TG lipase content in white adipose tissue, while increasing activation of acetyl coenzyme A carboxylase, suggesting futile cycling of TGs, albeit favoring net hydrolysis. We demonstrated, in vitro, this is a direct effect of treatment in adipose tissue, as basal cellular respiration and glucose uptake were increased in response to treatment. Taken together, these data suggest that antibody-mediated targeting of FGFR1c exerts its powerful glucose-lowering efficacy primarily due to increased glucose uptake in adipose tissue., (Copyright © 2017 Endocrine Society.)

The role of the intestinal insulin receptor (IR) is not well understood. We therefore explored the effect of insulin (300 nmol/kg per day for 12 days) on the intestine in sex-matched C57Bl/6J mice. The intestinal and metabolic profiles were also characterized in male and female intestinal-epithelial IR knockout (IE-irKO) mice compared with all genetic controls on a chow diet or Western diet (WD) for 4 to 12 weeks. Insulin treatment did not affect intestinal size, intestinal resistance, or metabolic genes, but it reduced proximal-colon crypt depth and acutely increased colonic serine/threonine-specific protein kinase B (AKT) activation. Feeding with a WD increased body weight and fasting insulin level and decreased oral glucose tolerance in C57Bl/6J and IE-irKO mice. However, although the overall responses of the IE-irKO mice were not different from those of Villin-Cre (Vil-Cre):IRfl/+ and IRfl/fl controls, profound differences were found for female control Vil-Cre mice, which demonstrated reduced food intake, body weight, jejunal glucose transport, oral glucose tolerance, and fasting insulin and cholesterol levels. Vil-Cre mice also had smaller intestines compared with those of IE-irKO and IRfl/fl mice and greater insulin-mediated activation of jejunal IR and AKT. In summary, gain- and loss-of-function studies, with and without caloric overload, indicate that insulin did not exert remarkable effects on intestinal metabolic or morphologic phenotype except for a small effect on the colon. However, the transgenic control Vil-Cre mice displayed a distinct phenotype compared with other control and knockout animals, emphasizing the importance of thoroughly characterizing genetically modified mouse models., (Copyright © 2017 Endocrine Society.)

Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid. Despite recent studies implicating adipose-derived ATX in metabolic disorders including obesity and insulin resistance, the nutritional and hormonal regulation of ATX in adipocytes remains unclear. The current study examined the regulation of ATX in adipocytes by glucose and insulin and the role of ATX in adipocyte metabolism. Induction of insulin resistance in adipocytes with high glucose and insulin concentrations increased ATX secretion, whereas coincubation with the insulin sensitizer, rosiglitazone, prevented this response. Moreover, glucose independently increased ATX messenger RNA (mRNA), protein, and activity in a time- and concentration-dependent manner. Glucose also acutely upregulated secreted ATX activity in subcutaneous adipose tissue explants. Insulin elicited a biphasic response. Acute insulin stimulation increased ATX activity in a PI3Kinase-dependent and mTORC1-independent manner, whereas chronic insulin stimulation decreased ATX mRNA, protein, and activity. To examine the metabolic role of ATX in 3T3-L1 adipocytes, we incubated cells with the ATX inhibitor, PF-8380, for 24 hours. Whereas ATX inhibition increased the expression of peroxisome proliferator-activated receptor-γ and its downstream targets, insulin signaling and mitochondrial respiration were unaffected. However, ATX inhibition enhanced mitochondrial H2O2 production. Taken together, this study suggests that ATX secretion from adipocytes is differentially regulated by glucose and insulin. This study also suggests that inhibition of autocrine/paracrine ATX-lysophosphatidic acid signaling does not influence insulin signaling or mitochondrial respiration, but increases reactive oxygen species production in adipocytes., (Copyright © 2017 Endocrine Society.)

Oxidative stress induces endogenous antioxidants via nuclear factor erythroid 2-related factor 2 (Nrf2), potentially preventing tissue injury. We investigated whether insulin affects renal Nrf2 expression in type 1 diabetes (T1D) and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension, oxidative stress, and renal injury; inhibited renal Nrf2 and angiotensinogen (Agt) gene expression; and upregulated heterogeneous nuclear ribonucleoprotein F and K (hnRNP F and hnRNP K) expression in Akita mice with T1D. In immortalized rat renal proximal tubular cells, insulin suppressed Nrf2 and Agt but stimulated hnRNP F and hnRNP K gene transcription in high glucose via p44/42 mitogen-activated protein kinase signaling. Transfection with small interfering RNAs of p44/42 MAPK, hnRNP F, or hnRNP K blocked insulin inhibition of Nrf2 gene transcription. Insulin curbed Nrf2 promoter activity via a specific DNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailed Nrf2 promoter activity. In hyperinsulinemic-euglycemic mice, renal Nrf2 and Agt expression was downregulated, whereas hnRNP F/K expression was upregulated. Thus, the beneficial actions of insulin in diabetic nephropathy appear to be mediated, in part, by suppressing renal Nrf2 and Agt gene transcription and preventing Nrf2 stimulation of Agt expression via hnRNP F/K. These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets in diabetes., (Copyright © 2017 Endocrine Society.)

Gestational low-protein (LP) diet causes hyperglycemia and insulin resistance in adult offspring, but the mechanism is not clearly understood. In this study, we explored the role of insulin signaling in gastrocnemius muscles of gestational LP-exposed female offspring. Pregnant rats were fed a control (20% protein) or an isocaloric LP (6%) diet from gestational day 4 until delivery. Normal diet was given to mothers after delivery and to pups after weaning until necropsy. Offspring were euthanized at 4 months, and gastrocnemius muscles were treated with insulin ex vivo for 30 minutes. Messenger RNA and protein levels of molecules involved in insulin signaling were assessed at 4 months. LP females were smaller at birth but showed rapid catchup growth by 4 weeks. Glucose tolerance test in LP offspring at 3 months showed elevated serum glucose levels (P < 0.01; glycemia Δ area under the curve 342 ± 28 in LP vs 155 ± 23 in controls, mmol/L * 120 minutes) without any change in insulin levels. In gastrocnemius muscles, LP rats showed reduced tyrosine phosphorylation of insulin receptor substrate 1 upon insulin stimulation due to the overexpression of tyrosine phosphatase SHP-2, but serine phosphorylation was unaffected. Furthermore, insulin-induced phosphorylation of Akt, glycogen synthase kinase (GSK)-3α, and GSK-3β was diminished in LP rats, and they displayed an increased basal phosphorylation (inactive form) of glycogen synthase. Our study shows that gestational protein restriction causes peripheral insulin resistance by a series of phosphorylation defects in skeletal muscle in a mechanism involving insulin receptor substrate 1, SHP-2, Akt, GSK-3, and glycogen synthase causing dysfunctional GSK-3 signaling and increased stored glycogen, leading to distorted glucose homeostasis., (Copyright © 2017 Endocrine Society.)

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

Background: Hypomagnesemia (plasma magnesium (Mg 2+ ) concentration <0.7 mmol/L) has been described in patients with type 2 diabetes. Polypharmacy is inevitable when treating a complex disease such as type 2 diabetes and could explain disturbances in the plasma Mg 2+ concentration. In this study, we aimed to establish the extent of hypomagnesemia in a cohort of type 2 diabetes patients and to identify the determinants of plasma Mg 2+ levels., Methods: Patient data and samples of 395 type 2 diabetes patients were investigated. Plasma Mg 2+ concentrations were measured using a spectrophotometric assay. Using Pearson correlation analyses, variables were correlated to plasma Mg 2+ levels. After excluding confounding variables, all parameters correlating (P < 0.1) with plasma Mg 2+ were included in a stepwise backward regression model., Results: The mean plasma Mg 2+ concentration in this cohort was 0.74 ± 0.10 mmol/L. In total, 121 patients (30.6%) suffered from hypomagnesemia. Both plasma triglyceride (r = -0.273, P < 0.001) and actual glucose levels (r = -0.231, P < 0.001) negatively correlated with the plasma Mg 2+ concentration. Patients using metformin (n = 251, 62%), proton pump inhibitors (n = 179, 45%) or β-adrenergic receptor agonists (n = 31, 8%) displayed reduced plasma Mg 2+ levels. Insulin use (n = 299, 76%) positively correlated with plasma Mg 2+ levels. The model predicted (R 2 ) 20% of all variance in the plasma Mg 2+ concentration., Conclusions: Hypomagnesemia is highly prevalent in type 2 diabetes patients. Plasma triglycerides and glucose levels are major determinants of the plasma Mg 2+ concentration, whereas only a minor part (<10%) of hypomagnesemia can be explained by drug intake, excluding polypharmacy as a major cause for hypomagnesemia in type 2 diabetes., (© 2017 European Society of Endocrinology.)

Context: Skeletal muscle protein synthesis is highly responsive to food intake. It has been suggested that the postprandial increase in circulating insulin modulates the muscle protein synthetic response to feeding., Objective: The objective of the study was to investigate whether a greater postprandial rise in circulating insulin level increases amino acid uptake in muscle and augments postprandial muscle protein synthesis rates., Participants and Design: Forty-eight healthy young (age 22 ± 1 y; body mass index 22.0 ± 0.3 kg/m 2 ) and older males (age 68 ± 1 y; body mass index 26.3 ± 0.4 kg/m 2 ) ingested 20 g intrinsically L-[1- 13 C]-leucine- and L-[1- 13 C]-phenylalanine-labeled casein protein with or without local insulin infusion. Primed continuous infusions of L-[1- 13 C]-leucine and L-[ring- 2 H 5 ]-phenylalanine were applied, with arterial and venous blood samples and muscle biopsies being collected during a 5-hour postprandial period., Results: Insulin administration did not increase overall leg blood flow (P = .509) but increased amino acid uptake over the leg in both young and older subjects (P = .003). The greater amino acid uptake over the leg did not further increase postprandial muscle protein synthesis rates (0.050% ± 0.006% and 0.037% ± 0.004% per hour vs 0.044% ± 0.004% and 0.037% ± 0.002% per hour in the insulin-stimulated vs control condition in the young and older groups, respectively; P = .804) and did not affect postprandial deposition of dietary protein-derived amino acids in de novo muscle protein (P = .872)., Conclusion: Greater postprandial plasma insulin availability stimulates amino acid uptake over the leg but does not further augment postprandial muscle protein synthesis rates or stimulate the postprandial deposition of protein derived amino acids into de novo muscle protein in healthy young and older men.