Insulin Binding Research Articles

The Effects of Systemic and Local Acidosis on Insulin Resistance and Signaling.

Most pathological conditions that cause local or systemic acidosis by overcoming the buffering activities of body fluids overlap with those diseases that are characterized by glucose metabolic disorders, including diabetes mellitus, inflammation, and cancer. This simple observation suggests the existence of a strong relationship between acidosis and insulin metabolism or insulin receptor signaling. In this review, we summarized the current knowledge on the activity of insulin on the induction of acidosis and, vice versa, on the effects of changes of extracellular and intracellular pH on insulin resistance. Insulin influences acidosis by promoting glycolysis. Although with an unclear mechanism, the lowering of pH, in turn, inhibits insulin sensitivity or activity. In addition to ketoacidosis that is frequently associated with diabetes, other important and more complex factors are involved in this delicate feedback mechanism. Among these, in this review we discussed the acid-mediated inhibiting effects on insulin binding affinity to its receptor, on glycolysis, on the recycling of glucose transporters, and on insulin secretion via transforming growth factor β (TGF-β) activity by pancreatic β-cells. Finally, we revised current data available on the mutual interaction between insulin signaling and the activity of ion/proton transporters and pH sensors, and on how acidosis may enhance insulin resistance through the Nuclear Factor kappa B (NF-κB) inflammatory pathway.

Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1.

Study objectives were to determine the effects of chromium (Cr) propionate (Cr propionate 0.04%; 0.5 g/kg of feed to deliver 200 parts per billion Cr/d; KemTRACE Cr, Kemin Industries, Inc., Des Moines, IA) on growth performance, metabolism, and health biomarkers in heat-stressed and nutrient-restricted pigs. Crossbred barrows (n = 96; 105 ± 1 kg BW) were enlisted in an experiment conducted in two replicates, blocked by initial BW, and randomly assigned to one of six dietary-environmental treatments: (i) thermoneutral (TN) and fed ad libitum a control diet (TNCtl), (ii) TN and fed ad libitum a Cr supplemented diet (TNCr), (iii) TN and pair-fed a control diet (PFCtl), (iv) TN and pair-fed a Cr supplemented diet (PFCr), (v) heat stress (HS) and ad libitum fed a control diet (HSCtl), or (vi) HS and ad libitum fed a Cr supplemented diet (HSCr). The study consisted of three experimental periods (P). During P0 (5 d), all pigs were housed in TN conditions (21.3 ± 0.1 °C, 56.8 ± 0.3% relative humidity [RH]) and fed the control diet ad libitum. During P1 (5 d), pigs were fed their respective dietary treatments ad libitum and kept in TN conditions. During P2 (35 d), HSCtl and HSCr-treated pigs were fed ad libitum and exposed to progressive cyclical HS conditions (27 to 31 °C, 50 ± 0.3% RH), while TNCtl, TNCr, PFCtl, and PFCr pigs remained in TN conditions and were fed ad libitum or pair-fed to their respective HSCtl and HSCr counterparts to eliminate the confounding effects of dissimilar feed intake. Overall, HS pigs had increased (P < 0.01) rectal temperature, skin temperature, and respiration rate (0.3 °C, 3.8 °C, and 32 breaths per minute, respectively) relative to TN pigs. Overall, HS decreased ADFI and ADG (20 and 21%, respectively; P < 0.01) compared with TN controls. Final BW tended to be increased in HSCr (2.7 kg, P = 0.06) compared with HSCtl pigs. Similarly, ADG tended to be increased during P2 in HSCr relative to HSCtl-treatment (0.77 vs. 0.72 kg/d; P = 0.10). There were no effects of Cr on most production parameters, but ADFI tended to be increased in Cr relative to Ctl-fed pigs (3.19 vs. 3.09 kg/d; P = 0.08). No effects of Cr supplementation were detected on circulating glucose, insulin, NEFA, cholesterol, triglycerides, or lipopolysaccharide binding protein. However, blood neutrophils were increased in HSCr (37%; P < 0.01) relative to HSCtl pigs. In summary, these results suggest Cr supplementation may benefit growth performance during HS.

Primary molecular basis of androgenic gland endocrine sex regulation revealed by transcriptome analysis in Eriocheir sinensis

In crustaceans, the male sexual differentiation and maintenance are specially regulated by androgenic gland (AG). However, little is known about the genes involved in the regulation process. RNA-Seq was performed on AG with ejaculatory duct (AG_ED) and ejaculatory duct (ED) as control in Eriocheir sinensis, one of the most important economic and fishery crabs with typically sex dimorphism. A total of 925 unigenes were identified as differentially expressed genes (DEGs) and the expression of nine genes randomly selected was confirmed by qRT-PCR. 667 unigenes were up-regulated in AG_ED, being supposed to be AG preferential genes. Among them, the full length of insulin-like androgenic gland factor (IAG) cDNA named as Es-IAG was obtained as a logo gene of AG, which together with the genes insulin-like receptor (INR), and single insulin binding domain protein (SIBD), might constitute the sex regulation pathway. Several sex related genes were identified, and their function will have to be investigated. Also, the identification of juvenile hormone epoxide hydrolase 1 (JHEH1), ecdysteroid 22-hydroxylase (DIB) and ecdysone receptor (ECR) preliminarily clarified the molecular regulation mechanism of eyestalk-AG-testis axis, which plays important roles in molting and reproduction. The results will enhance our understanding for the molecular basis of the AG involved in male sex regulation in crabs.

A novel recombinant peptide INSR-IgG4Fc (Yiminsu) restores insulin sensitivity in experimental insulin resistance models

Type 2 diabetes mellitus (T2DM) is a chronic degenerative endocrine and metabolic disease with high mortality and morbidity, yet lacks effective therapeutics. We recently generated a novel fusion peptide INSR-IgG4Fc, Yiminsu (YMS), to facilitate the high-affinity binding and transportation of insulin. Thus, the aim of the present study was to determine whether the novel recombinant peptide, YMS, could contribute to restoring insulin sensitivity and glycaemic control in insulin resistance models and revealing its underlying mechanism. Palmitic acid (PA)-treated LO2 cells and high fat diet (HFD)-fed mice were treated with YMS. Therapeutic effects of YMS were measured using Western blotting, ELISA, qPCR, Histology and transmission electron microscopy. We observed that YMS treatment effectively improved insulin signaling in PA-treated LO2 cells and HFD-fed mice. Notably, YMS could significantly reduce serum levels of glucose, triglycerides, fatty acids and cholesterol without affecting the serum insulin levels. Moreover, our data demonstrated that YMS could restore glucose and lipid homeostasis via facilitating insulin transportation and reactivating PI3K/Akt signaling in both PA-treated cells and liver, gastrocnemius and brown fat of HFD-fed mice. Additionally, we noticed that the therapeutic effects of YMS was similar as rosiglitazone, a well-recognized insulin sensitizer. Our findings suggested that YMS is a potentially candidate for pharmacotherapy for metabolic disorders associated with insulin resistance, particularly in T2DM.

Hepatic Insulin Clearance in Regulation of Systemic Insulin Concentrations-Role of Carbohydrate and Energy Availability.

Hyperinsulinemia is the hallmark of insulin resistance in obesity, and the relative importance of insulin clearance, insulin resistance, and insulin hypersecretion has been widely debated. On the basis of recent experimental evidence, we summarize existing evidence to suggest hepatic insulin clearance as a major and immediate regulator of systemic insulin concentrations responding within days to altered dietary energy and, in particular, carbohydrate intake. Hepatic insulin clearance seems to be closely associated with opposite alterations in hepatic lipid content and glucose production, providing a potential mechanistic link to hepatic insulin sensitivity. The molecular regulation of insulin clearance in the liver is likely to involve changes in insulin binding and receptor internalization in response to the dietary alterations, the molecular mechanisms of which await further research.

Insulin transport across the blood-brain barrier can occur independently of the insulin receptor.

Insulin enters the brain from the blood via a saturable transport system. It is unclear how insulin is transported across the blood-brain barrier (BBB). Using two models of the signalling-related insulin receptor loss or inhibition, we show insulin transport can occur in vivo without the signalling-related insulin receptor. Insulin in the brain has multiple roles including acting as a metabolic regulator and improving memory. Understanding how insulin is transported across the BBB will aid in developing therapeutics to further increase CNS concentrations. A saturable system transports insulin from blood across the blood-brain barrier (BBB) and into the central nervous system. Whether or not the classic or signalling-related insulin receptor plays a role in mediating this transport in vivo is controversial. Here, we employed kinetics methods that distinguish between transport across the brain endothelial cell and reversible luminal surface receptor binding. Using a previously established line of mice with endothelial-specific loss of the signalling-related insulin receptor (EndoIRKO) or inhibiting the insulin receptor with the selective antagonist S961, we show insulin transport across the BBB is maintained. Rates of insulin transport were similar in all groups and transport was still saturable. Unlike transport, binding of insulin to the brain endothelial cell was decreased with the loss or inhibition of the signalling-related insulin receptor. These findings demonstrate that the signalling-related insulin receptor is not required for insulin transport across the BBB.

Phosphorylation of protein kinase B, the key enzyme in insulin-signaling cascade, is enhanced in linoleic and arachidonic acid-treated HT29 and HepG2 cells.

Defects in the insulin-signaling pathway have been implicated in the pathogenesis of impaired glucose uptake, insulin resistance, and type 2 diabetes. However, the specific defects that precipitate these abnormalities are yet to be fully elucidated. After binding to insulin, the plasma membrane-embedded insulin receptor transmembrane protein initiates a cascade of phosphorylation that leads to the activation of protein kinase B (AKT) and subsequently to the initiation of some metabolic actions of insulin. The activities of this receptor, insulin binding, and tyrosine kinase activation is dependent on its plasma lipid environment. Published data on the influence of omega-3 and -6 polyunsaturated fatty acids on insulin response are scarce. Moreover, the findings of the published investigations, most of which used omega-3 and -6, polyunsaturated fatty-acid blends, have been inconclusive. Hence, further, well thought out research is needed. The aim of the current study was to elucidate the effect of treatments with linoleic acid (LNA), arachidonic acid (ARA), alpha-linolenic acid (ALA), docoshexaenoic acid (DHA), and eicosapentaenoic acid (EPA) on cell membrane composition and consequently on the insulin-signaling pathway and specifically AKT phosphorylation. Human colon adenocarcinoma (HT29) and liver hepatocellular (HepG2) cells were treated with or without 40 µM of LNA, ARA, ALA, EPA, or DHA for 48 h, the fatty-acid composition of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from the treated cells by capillary gas liquid chromatograph. Cells were incubated for 30 min with or without human insulin (50 ng/mL), and the phosphorylation of AKT was assessed with the use of Western blotting. The fatty acids were incorporated in the PtdCho and PtdEtn of both cell lines, but the level of incorporation was higher in HT29. Phosphorylation of AKT increased when HT29 was treated with LNA (P < 0.05) and ARA (P < 0.01) but not with ALA, EPA, or DHA. A similar but non-significant increase in AKT phosphorylation was observed in LNA- and ARA- treated HepG2 cells. The finding of this investigation demonstrates that plasma membrane lipid bilayer enrichment with LNA or ARA treatment enhances insulin action by AKT activation.

Performance optimisation of porous silicon rugate filter biosensor for the detection of insulin

A porous silicon rugate filter (pSiRF) insulin biosensor has been constructed and systematically optimised by investigating the effects of specific experimental parameters. The pSiRF biosensor is based on an optical sensing platform using interferometric reflectance spectroscopy (IRS) to monitor insulin binding to a surface-anchored insulin binding aptamer (IBA) within the pores. The aim of this study was to determine the optimal fabrication to elicit the maximum sensor response, lowest limit of detection (LOD) and fastest response time. Thermal hydrosilylation with undecylenic acid was used as a base surface modification, with IBA attached through formation of an amide bond. The following pSiRF fabrication conditions were investigated; porous film thickness and IBA concentration in the conjugation solution. The effect of solution flow speed over the pSiRF during insulin biosensing was also investigated in an effort to improve the performance. The optimised pSiRF biosensor was successfully applied for the detection of insulin secreted from human islets on stimulation with glucose. Finally, as a proof of concept, insulin sensing in the presence of human islets on the pSiRF surface was performed to demonstrate the robust performance of the biosensor and the potential for in-situ insulin biosensing.

IgM Immunotherapy Restores Immune Homeostasis and Reverses Hyperglycemia in New-Onset type 1 Diabetes

Goal To determine the mechanism by which IgM immunotherapy restores immune homeostasis in Type 1 Diabetes (T1D). Background IgM immunotherapy prevents the onset and progression of T1D. Methods 1) 5wks old non-obese diabetic (NOD) mice received human IgM (hIgM, 50ugs/wk) or saline, beginning at 5wks of age until 18wks. 2) For diabetes reversal, NOD mice were treated after the onset of diabetes with two doses (100ug) of either prediabetic NOD IgM or C57BL/6 IgM on Days 1 and 4, and their blood glucose monitored serially. 3) 5wks-old C57BL/6 and NOD mice, C57BL/6 and NOD VH125 mice, and humanized BLT mice received IgM (100ug igM on Day 1 followed by 50ug on Days 3,5,7 and 10) followed by spleen and bone-marrow cell harvest on Day 13. VH125 mice possess a heavy chain specific for human insulin knocked into the endogenous IgM locus. This heavy chain combines with endogenous light chains to produce insulin-reactive B lymphocytes. Humanized BLT mice are NOD/SCIDs that have been cotransplanted with human liver and thymus tissues, along with autologous CD34+ hematopoietic stem cells. Flow cytometry (FC) or Time of flight mass cytometry (CyTOF) was performed. Results 80% of saline-injected NOD mice became diabetic by 18-20 weeks of age (n=30). In contrast, none of the hIgM treated mice developed diabetes (n=10). Two doses of IgM (100ug) reversed hyperglycemia in new-onset diabetic mice and maintained BG<200mg/dL in 63% of mice for the entire duration of time (2 months) that they were monitored post treatment (n=11). In contrast, IgM derived from pre-diabetic NOD donors did not reverse diabetes. IgM therapy expanded both, myeloid derived suppressor cell and peripheral regulatory T cell (Treg) populations. Thymic Tregs were also expanded in a regulatory B cell-dependent manner. IgM therapy diminished autoreactivity in NOD mice by A) reducing the percentage of marginal zone B cells, a subset associated with perpetuating autoimmunity (p<0.05); B) increasing transitional B cell proportions (p<0.01), indicating normalization of B cell homeostatic defects; and C) inhibiting plasma insulin autoantibody levels (p<0.0001). In NODVH125 mice, IgM eliminated insulin binding B cell population (p<0.0001) indicating a reduction in autoreactive B cell activation. In humanized BLT mice, hIgM therapy expanded the Helios+Foxp3+Treg population. Conclusions IgM therapy reverses new onset T1D by restoring immune homeostasis and diminishing autoreactivity. The clinical relevance of IgM therapy is confirmed in the humanized BLT mouse model wherein hIgM therapy induces the expansion of Tregs. This beneficial effect may be translatable to diabetic patients or islet graft recipients.

Anti-Human Herpesvirus 8 antibodies affect both insulin and glucose uptake by virus-infected human endothelial cells

Human Herpesvirus 8 (HHV8) is known to be the cause of the malignant tumour named Kaposi's sarcoma. It is believed to induce an intense modification of cell metabolism in endothelial cells. In this work we analysed the role of anti-HHV8 antibodies in both the insulin and glucose uptake of HHV8-infected primary human endothelial cells (HUVEC). Western blotting, immunofluorescence and radiolabelled glucose were employed to assess the pPI3K expression, insulin binding and glucose-uptake by HUVEC cells, respectively. We confirmed that HHV8-infection is able to enhance both insulin binding and glucose-uptake in HHV8-infected primary endothelial cells; in addition, we found that anti-HHV8 specific antibodies are able to further increase both insulin and glucose uptake during the late latent phase of HHV8-infection in vitro. These findings suggest that a specific immune response to HHV8-infection may cooperate in boosting the cell metabolism, further enhancing the already increased insulin binding and glucose-uptake in HHV8-infected cells, which is a peculiar property of several oncogenic viruses.

Insulin Receptor Levels Regulated by the Receptor- Associated Protein Progesterone Receptor Membrane Component 1 (PGRMC1)

As of 2014, 29.1 million Americans suffer from diabetes, creating a severe socioeconomic and medical burdenon society. Impaired insulin signaling is key to the development of type 2 diabetes, presenting a unique therapeutic challenge. Obese individuals demonstrate decreased insulin binding due to a reduction in IR levels, without an alteration in ligand-receptor binding affinity. The progesterone receptor membrane component 1 (PGRMC1) is an endosomal protein that promotes cellular signaling via altered receptor trafficking. A recent translational study determined that PGRMC1 was decreased in patients with insulin-resistant disease, suggesting a role in insulin signaling. In the present study, we hypothesized that PGRMC1 affects the levels of IR? (insulin receptor-? sub-unit) in adipocytes. Indeed, we show that treatment with PGRMC1 ligands significantly increase IR? protein levels in fully differentiated human subcutaneous adipocytes. Protein levels are likely affected through the direct interaction of PGRMC1 and IR?, as we demonstrate their co immunoprecipitation in differentiated 3T3-L1 cells. Notably, PGRMC1 ligand treatment significantly reduced IR? protein levels in two rodent model systems, indicating a pharmacological difference across species.

Characterization of insulin and IGF-1 receptor binding in equine liver and lamellar tissue: implications for endocrinopathic laminitis

Although it is well established that equine laminitis can be triggered by extreme hyperinsulinemia, the mechanism of insulin action is not known. High concentrations of insulin lead to separation of the weight-bearing apparatus from the hoof wall and are associated with an increased cycle of cell death and proliferation in the lamellae. Gene expression and immunohistochemistry studies have indicated that the lamellae are sparsely populated with insulin receptors, whereas IGF-1 receptors (IGF-1R) are abundant, suggesting that the action of insulin may be mediated by insulin binding to the IGF-1R. To investigate this possibility, cell membrane fragments containing IGF-1R were extracted from the livers of 6 horses and the lamellae of >50 horses euthanized for nonresearch purposes at an abattoir. Radioligand-binding studies using 125I-IGF-1 and 125I-insulin confirmed an abundance of high-affinity IGF-1R in the liver (KD 0.11 nM, Bmax 223 fmol/mg protein) and lamellae (KD 0.16 nM, Bmax 243 fmol/mg protein). However, the affinity of insulin for binding to the lamellar IGF-1R (Ki 934 nM) was >5,800 fold less than that of IGF-1, suggesting that insulin is unlikely to bind to equine IGF-1R at physiological concentrations. Although insulin receptors could be detected in the liver (KD 0.48 nM, Bmax 123 fmol/mg protein), they were barely detectable in lamellae (estimated Bmax 14 fmol/mg protein). There was no evidence to support the presence of insulin/IGF-1 hybrid receptors in either tissue. These findings suggest that insulin does not act directly through IGF-1 receptors and that an alternative theory is required to explain the mechanism of insulin action in laminitis.

The Effects of Metformin and Weight Loss on Biomarkers Associated With Breast Cancer Outcomes.

This study investigated the effects of metformin and weight loss on biomarkers associated with breast cancer prognosis. Overweight/obese postmenopausal breast cancer survivors (n = 333) were randomly assigned to metformin vs placebo and to a weight loss intervention vs control (ie, usual care). The 2 × 2 factorial design allows a single randomized trial to investigate the effect of two factors and interactions between them. Outcomes were changes in fasting insulin, glucose, C-reactive protein (CRP), estradiol, testosterone, and sex-hormone binding globulin (SHBG). The trial was powered for a main effects analysis of metformin vs placebo and weight loss vs control. All tests of statistical significance were two-sided. A total of 313 women (94.0%) completed the six-month trial. High prescription adherence (ie, ≥80% of pills taken) ranged from 65.9% of participants in the metformin group to 81.3% of those in the placebo group (P < .002). Mean percent weight loss was statistically significantly higher in the weight loss group (-5.5%, 95% confidence interval [CI] = -6.3% to -4.8%) compared with the control group (-2.7%, 95% CI = -3.5% to -1.9%). Statistically significant group differences (ie, percent change in metformin group minus placebo group) were -7.9% (95% CI = -15.0% to -0.8%) for insulin, -10.0% (95% CI = -18.5% to -1.5%) for estradiol, -9.5% (95% CI = -15.2% to -3.8%) for testosterone, and 7.5% (95% CI = 2.4% to 12.6%) for SHBG. Statistically significant group differences (ie, percent change in weight loss group minus placebo group) were -12.5% (95% CI = -19.6% to -5.3%) for insulin and 5.3% (95% CI = 0.2% to 10.4%) for SHBG. As adjuvant therapy, weight loss and metformin were found to be a safe combination strategy that modestly lowered estrogen levels and advantageously affected other biomarkers thought to be on the pathway for reducing breast cancer recurrence and mortality.

Insulin Receptor Plasma Membrane Levels Increased by the Progesterone Receptor Membrane Component 1

The insulin receptor (IR) is a ligand-activated receptor tyrosine kinase that has a key role in metabolism, cellular survival, and proliferation. Progesterone receptor membrane component 1 (PGRMC1) promotes cellular signaling via receptor trafficking and is essential for some elements of tumor growth and metastasis. In the present study, we demonstrate that PGRMC1 coprecipitates with IR. Furthermore, we show that PGRMC1 increases plasma membrane IR levels in multiple cell lines and decreases insulin binding at the cell surface. The findings have therapeutic applications because a small-molecule PGRMC1 ligand, AG205, also decreases plasma membrane IR levels. However, PGRMC1 knockdown via short hairpin RNA expression and AG205 treatment potentiated insulin-mediated phosphorylation of the IR signaling mediator AKT. Finally, PGRMC1 also increased plasma membrane levels of two key glucose transporters, GLUT-4 and GLUT-1. Our data support a role for PGRMC1 maintaining plasma membrane pools of the receptor, modulating IR signaling and function.

Towards the Development of a Real‐time Insulin Biosensor

Disease management for Type 1 Diabetes (T1D) patients can be challenging, and more than two-thirds of the diabetic population in the United States fails to maintain optimal blood glucose levels. The artificial pancreas (AP), automates disease management by automatically delivering insulin with dosage adjusted in real-time as a response to blood glucose levels. However, despite automation using advanced control algorithms, patients reliant on the AP risk hyper and hypoglycemia, which can result in fatality or other less serious complications. Such mis-dosing can result from discrepancies between the administered and the circulating insulin levels. As such, a direct continuous monitoring of circulating insulin could tighten glucose regulation while improving safety and patient response to treatment. Thus, we aim to develop a real-time insulin biosensor for APs using nature's own insulin sensor, the insulin receptor (InR). We plan to engineer our biosensor modeled upon the InR ectodomain using phage-displayed libraries with 109 different InR variants and iterative rounds of selections. FRET and polarization assays will be used to assess InR conformational change, binding kinetics of insulin to InR variants, and device sensitivity. Support or Funding Information Juvenile Diabetes Research Foundation, The Helmsley Charitable Trust Insulin binding (kon) and release (koff) kinetics determine sensitivity of insulin monitoring over a dynamic range of concentrations. Conformational changes of the native InR are used to develop a FRET-based optical detection system for insulin binding using fusion proteins covalently attached to fluorophores. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Control of hyperglycemia in male mice by leflunomide: mechanisms of action.

p70 S6 kinase (S6K1) is a serine/threonine kinase that phosphorylates the insulin receptor substrate-1 (IRS-1) at serine 1101 and desensitizes insulin receptor signaling. S6K1 hyperactivation due to overnutrition leads to hyperglycemia and type 2 diabetes. Our recent study showed that A77 1726, the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide, is an inhibitor of S6K1. Whether leflunomide can control hyperglycemia and sensitize the insulin receptor has not been tested. Here we report that A77 1726 increased AKTS473/T308 and S6K1T389 phosphorylation but decreased S6S235/236 and IRS-1S1101 phosphorylation in 3T3-L1 adipocytes, C2C12 and L6 myotubes. A77 1726 increased insulin receptor tyrosine phosphorylation and binding of the p85 subunit of the PI-3 kinase to IRS-1. A77 1726 enhanced insulin-stimulated glucose uptake in L6 myotubes and 3T3-L1 adipocytes, and enhanced insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the plasma membrane of L6 cells. Finally, we investigated the anti-hyperglycemic effect of leflunomide on ob/ob and high-fat diet (HFD)-induced diabetes mouse models. Leflunomide treatment normalized blood glucose levels and overcame insulin resistance in glucose and insulin tolerance tests in ob/ob and HFD-fed mice but had no effect on mice fed a normal chow diet (NCD). Leflunomide treatment increased AKTS473/T308 phosphorylation in the fat and muscle of ob/ob mice but not in normal mice. Our results suggest that leflunomide sensitizes the insulin receptor by inhibiting S6K1 activity in vitro, and that leflunomide could be potentially useful for treating patients with both RA and diabetes.

Regulation of Insulin Sensitivity by Skeletal Muscle Specific Modulation Of p21‐Activated Kinase 1 (PAK1)

Impaired skeletal muscle (skm) insulin signaling results in insulin resistance and the development of type 2 diabetes (T2D). Insulin binding to its receptors on skm cells leads to an intracellular signaling cascade resulting in glucose transporter type 4 (GLUT4) translocation to the plasma membrane (PM) and glucose uptake. The p21–activated kinase 1 (PAK1) is a required element for skm insulin sensitivity. It has been shown that whole body PAK1−/− knock‐out (KO) mice exhibit glucose intolerance and insulin resistance. In clonal skm cells (L6‐GLUT4myc) PAK1 inhibition was shown to impair insulin‐induced filamentous actin (F‐actin) remodeling and GLUT4 translocation; coupled with a loss of normal insulin‐stimulated cofilin dephosphorylation (i.e. activation). Recently, we have shown that PAK1 is essential in the process of actin polymerization via p41‐ARC (Actin‐related protein 2/3 complex subunit 1B). However, the effect of skm specific PAK1 modulations on in vivo glucose homeostasis, and downstream effectors of PAK1 are yet to be fully determined.We hypothesize that PAK1 is essential for insulin signaling in skm, and that upon diabetogenic stress, i) reduced PAK1 levels in skm will adversely affect whole body insulin sensitivity, whereas ii) PAK1 enrichment can preserve or restore insulin sensitivity. To study the effects of PAK1 modulation on insulin sensitivity in vivo, state‐of‐the‐art inducible skm‐specific PAK1 KO and overexpressing mouse models are used. L6‐GLUT4myc clonal cells are used to identify key downstream effectors of PAK1 using cutting‐edge imaging, mass spectrometry, and metabolomic analyzes.Importantly, both PAK1 and p41‐ARC protein levels were found to be significantly reduced in the skm of human T2D donors compared to healthy human donors (n=3–4). We have generated a doxycycline‐inducible skm‐specific, i) PAK1 depletion mouse model (skm‐PAK1‐KO) and ii) PAK1 overexpressing transgenic mouse model (skm‐PAK1‐Tg). PAK1 deletion in skm resulted into impaired in vivo insulin sensitivity in skm‐PAK1‐KO mice compared to control mice (n=10, per group), studied by intraperitoneal insulin tolerance test (IPITT). Moreover, this was coupled with reduced insulin‐induced GLUT4 translocation in skm of skm‐PAK1‐KO mice compared to the control mice (n=2). Conversely, overexpression of PAK1 in L6‐GLUT4myc cells significantly improved GLUT4 translocation under insulin resistance condition (5 nM of insulin for 12 h) compared to control cells (n=4). Altogether, these findings emphasize the i) importance of PAK1 in skm and thereby whole body glucose homeostasis and ii) potential protective effect of PAK1 enrichment on skm insulin sensitivity under diabetogenic conditions. Moreover, this study in future is aimed at determining key downstream effectors of PAK1, which might serve as insulin sensitizers.Support or Funding InformationThis study was supported by grants from the National Institutes of Health (DK067912 and DK102233 to D.C.T.), the American Heart Association (15PRE21970002 to RT, 17POST33661194 to JZ), as well a gift from the Ruth and Robert Lanman Endowment (to D.C.T.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.

Periodic food shortages are a major challenge faced by organisms in natural habitats. Cave-dwelling animals must withstand long periods of nutrient deprivation, as-in the absence of photosynthesis-caves depend on external energy sources such as seasonal floods. Here we show that cave-adapted populations of the Mexican tetra, Astyanax mexicanus, have dysregulated blood glucose homeostasis and are insulin-resistant compared to river-adapted populations. We found that multiple cave populations carry a mutation in the insulin receptor that leads to decreased insulin binding in vitro and contributes to hyperglycaemia. Hybrid fish from surface-cave crosses carrying this mutation weigh more than non-carriers, and zebrafish genetically engineered to carry the mutation have increased body weight and insulin resistance. Higher body weight may be advantageous in caves as a strategy to cope with an infrequent food supply. In humans, the identical mutation in the insulin receptor leads to a severe form of insulin resistance and reduced lifespan. However, cavefish have a similar lifespan to surface fish and do not accumulate the advanced glycation end-products in the blood that are typically associated with the progression of diabetes-associated pathologies. Our findings suggest that diminished insulin signalling is beneficial in a nutrient-limited environment and that cavefish may have acquired compensatory mechanisms that enable them to circumvent the typical negative effects associated with failure to regulate blood glucose levels.

Insulin-like receptors and carbohydrate metabolism in gills of the euryhaline crab Neohelice granulata: Effects of osmotic stress

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG.

Hypomagnesemia As A Marker Of Diabetic Nephropathy

Magnesium is an essential element and has a fundamental role in carbohydrate metabolism in general and in the insulin action in particular. Magnesium is involved in multiple levels in insulin secretion, binding and activity. Cellular magnesium deficiency can alter the activity of the membrane bound Na+K+ ATPase, which is involved in the maintenance of gradients of sodium and potassium and in glucose transport(1). Magnesium depletion has a negative impact on glucose homeostasis and insulin sensitivity in patients with type 2 diabetes as well as on the evolution of complications such as retinopathy, arterial atherosclerosis and nephropathy. Moreover, low serum magnesium is a strong, independent predictor of development of type 2 diabetes(2). A cross sectional study included 105 type 2 diabetic patients. Twenty nine were males (27.6%) and seventy six were females (72.4%).Their ages ranged from 30-77 with a mean of 49.7± 10.6. All patients were subjected to full clinical examination, and investigations which included: serum creatinine, HbA1c, albumin creatinine ratio and serum magnesium. Their mean BMI was 23 kg/m2 and mean waist/hip ratio was 0.9.Their mean HbA1c was 8.55 %. Of all patients, 13 of them had normal level of A/C ratio (control group) and 92 were albuminuric with a mean A/C ratio 238.26 ± 727.9 with a range of 33.7 (0.09-4700), mean s.creatinine was 1.29 ± 1.16 and their mean s.magnesium level was 2.04 ± 0.49 with a range of 1.9 (0.8-3.9). We observed significant negative correlation between A/C ratio and serum creatinine with a p-value of <0.0001. The study shows negative correlation between serum magnesium and A/C ratio(r=-0.202, p=0.039).