Acute Insulin Resistance and Rapid Alterations in Neuronal Derived Blood Exosome Concentration After Branched Endovascular Aortic Aneurysm Repair.

OBJECTIVE: Hyperglycaemia following branched endovascular repair (BEVAR) of extensive aortic aneurysms is associated with post-operative lower extremity weakness (LEW). Insulin administration to maintain euglycaemia appears to decrease LEW rates. The purpose of this study was to examine changes in insulin receptor content of neuron derived blood exosomes (NDEs) after BEVAR. METHODS: Ten patients with a range of post-operative lower extremity neurological deficits after elective BEVAR were included in the study. Blood samples were collected pre-operatively, immediately after aneurysm repair, and on post-operative day 1. NDE insulin receptor substrate proteins were quantified by enzymevlinked immunosorbent assays. RESULTS: NDE levels of phosopho-serine312-type 1 insulin receptor substrate ([P-Ser312-IRS1], an inhibitor of insulin signalling) increased sevenfold in the immediate post-operative period (from 7.90 ± 0.89 to 58.54 ± 6.77 pg/mL; p < .001), whereas those of pan-tyrosine-phospho insulin receptor substrate ([P-panTyr-IRS1], which facilitates insulin signalling), rose only 50% (from 0.41 ± 0.07 to 0.63 ± 0.10 pg/mL; p = .03). As a result, the mean ratio of P-Ser312-IRS1 to P-panTyr-IRS1, which reflects the level of insulin resistance, increased fivefold immediately post-operatively (from 22.31 ± 3.28 to 106.33 ± 11.83; p < .001) and returned to normal levels by the next day (18.72 ± 1.87). CONCLUSION: BEVAR is associated with an acute state of insulin resistance within neuronal tissue. Further studies in a larger cohort of patients are needed to understand the potential interconnected processes of insulin resistance, hyperglycaemia, and spinal cord ischaemia after extensive endovascular aortic procedures.

Insulin resistance and exendin-4 treatment for multiple system atrophy.

See Stayte and Vissel (doi:10.1093/awx064) for a scientific commentary on this article. Multiple system atrophy is a fatal sporadic adult-onset neurodegenerative disorder with no symptomatic or disease-modifying treatment available. The cytopathological hallmark of multiple system atrophy is the accumulation of α-synuclein aggregates in oligodendrocytes, forming glial cytoplasmic inclusions. Impaired insulin/insulin-like growth factor-1 signalling (IGF-1) and insulin resistance (i.e. decreased insulin/IGF-1) have been reported in other neurodegenerative disorders such as Alzheimer's disease. Increasing evidence also suggests impaired insulin/IGF-1 signalling in multiple system atrophy, as corroborated by increased insulin and IGF-1 plasma concentrations in multiple system atrophy patients and reduced IGF-1 brain levels in a transgenic mouse model of multiple system atrophy. We here tested the hypothesis that multiple system atrophy is associated with brain insulin resistance and showed increased expression of the key downstream messenger insulin receptor substrate-1 phosphorylated at serine residue 312 in neurons and oligodendrocytes in the putamen of patients with multiple system atrophy. Furthermore, the expression of insulin receptor substrate 1 (IRS-1) phosphorylated at serine residue 312 was more apparent in inclusion bearing oligodendrocytes in the putamen. By contrast, it was not different between both groups in the temporal cortex, a less vulnerable structure compared to the putamen. These findings suggest that insulin resistance may occur in multiple system atrophy in regions where the neurodegenerative process is most severe and point to a possible relation between α-synuclein aggregates and insulin resistance. We also observed insulin resistance in the striatum of transgenic multiple system atrophy mice and further demonstrate that the glucagon-like peptide-1 analogue exendin-4, a well-tolerated and Federal Drug Agency-approved antidiabetic drug, has positive effects on insulin resistance and monomeric α-synuclein load in the striatum, as well as survival of nigral dopamine neurons. Additionally, plasma levels of exosomal neural-derived IRS-1 phosphorylated at serine residue 307 (corresponding to serine residue 312 in humans) negatively correlated with survival of nigral dopamine neurons in multiple system atrophy mice treated with exendin-4. This finding suggests the potential for developing this peripheral biomarker candidate as an objective outcome measure of target engagement for clinical trials with glucagon-like peptide-1 analogues in multiple system atrophy. In conclusion, our observation of brain insulin resistance in multiple system atrophy patients and transgenic mice together with the beneficial effects of the glucagon-like peptide-1 agonist exendin-4 in transgenic mice paves the way for translating this innovative treatment into a clinical trial.

Long-term effect of early postnatal overnutrition on insulin resistance and serum fatty acid profiles in male rats.

BACKGROUND: Increasing evidence suggests that overnutrition during the early postnatal period, a critical window of development, increases the risk of adult-onset obesity and insulin resistance. In this study, we investigated the impact of overnutrition during the suckling period on body weight, serum biochemistry and serum fatty acid metabolomics in male rats. METHODS: Rats raised in small litters (SL, 3 pups/dam) and normal litters (NL, 10 pups/dam) were used to model early postnatal overnutrition and control, respectively. Serum glucose, triglyceride, high-density lipoprotein-cholesterol, free fatty acid, insulin and leptin concentrations were assayed using standard biochemical techniques. Serum fatty acids were identified and quantified using a gas chromatography-mass spectrometry-based metabolomic approach. mRNA and protein levels of key components of the insulin receptor signaling pathway were measured in epididymal fat and gastrocnemius muscle by quantitative PCR and western blotting. RESULTS: SL rats were 37.3 % and 15.1 % heavier than NL rats at weaning and 16-weeks-old, respectively. They had increased visceral fat mass, adult-onset insulin resistance and glucose intolerance as well as elevated serum levels of free fatty acids and triglycerides. All detectable fatty acids were elevated in the serum of SL pups at weaning compared to NL controls, and significant increases in the levels of four fatty acids (palmitic acid, palmitoleic acid, oleic acid and arachidonic acid) persisted into adulthood. Moreover, a significantly positive correlation was identified between an insulin resistance index (HOMA-IR) and concentrations of myristic, palmitic, palmitoleic and oleic acid in serum at postnatal 16 weeks. Early postnatal overnutrition also resulted in a significant downregulation of insulin receptor substrate-1 (Irs-1), protein kinase B (Akt2) and glucose transporter 4 (Glut4) at the protein level in epididymal fat of SL rats at 16 weeks, accompanied by decreased mRNA levels for Irs-1 and Glut4. In gastrocnemius muscle, Akt2 and Glut4 mRNA and Glut4 protein levels were significantly decreased in SL rats. CONCLUSIONS: This study demonstrates that early postnatal overnutrition can have long-lasting effects on body weight and serum fatty acid profiles and can lead to impaired insulin signaling pathway in visceral white adipose tissue and skeletal muscle, which may play a major role in IR.

Circulating 25-hydroxyvitamin D, IRS1 variant rs2943641, and insulin resistance: replication of a gene-nutrient interaction in 4 populations of different ancestries.

BACKGROUND: Associations of either insulin receptor substrate 1 (IRS1) variants or circulating 25-hydroxyvitamin D [25(OH)D] with type 2 diabetes (T2D) and insulin resistance (IR) are inconsistent. This study sought to determine whether circulating 25(OH)D modulates the association of a potentially functional variant at IRS1 (rs2943641) with insulin resistance. METHOD: Interaction between IRS1 rs2943641 and circulating 25(OH)D on homeostasis model assessment for IR (HOMA-IR) was examined in the Boston Puerto Rican Health Study (BPRHS) (n = 1144). Replication was performed in the African-American (n = 1126), non-Hispanic white (n = 1967), and Hispanic (n = 1241) populations of the Multi-Ethnic Study of Atherosclerosis (MESA) with genotypes of 3 IRS1 variants, rs2972144, rs1515104, and rs2673142, which are tag single nucleotide polymorphisms (SNPs) and in strong linkage disequilibrium with rs2943641. RESULTS: Higher circulating 25(OH)D was associated with lower risk of T2D and IR in BPRHS women homozygous for minor allele rs2943641T. Consistently, in each of 3 MESA populations, HOMA-IR and insulin decreased more evidently with higher circulating 25(OH)D in women of the rs2943641TT genotype than in carriers of the major allele (rs2943641C). Metaanalysis indicated significant and consistent interactions between circulating 25(OH)D and IRS1 variants on HOMA-IR (log transformed) [pooled ß = -0.008, 95% CI: -0.016 to -0.001, P interaction = 0.004] and insulin (log transformed) (pooled ß = -0.006, 95% CI: -0.011 to -0.002, P interaction = 0.023) in 3065 women of the 4 populations. CONCLUSIONS: Participants with different genotypes of IRS1 rs2943641 exhibit differential benefit from high circulating 25(OH)D for the reduction of insulin resistance and T2D risk. This gene-nutrient interaction, which appears to be limited to women, warrants further examination in randomized controlled trials of vitamin D supplementation.

Impact of insulin-like growth factor 2, insulin-like growth factor receptor 2, insulin receptor substrate 2 genes polymorphisms on susceptibility and clinicopathological features of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death worldwide. Insulin-like growth factor-2 (IGF-2) is an important autocrine and paracrine growth factor which may induce cell proliferation and inhibit cell apoptosis leading to the transformation of normal cells into malignant cells. This study aimed to evaluate the possible roles of IGF-2, insulin-like growth factor-2 receptor (IGF-2R), and insulin receptor substrate (IRS)-2 genes polymorphisms in susceptibility and clinicopathological features of HCC in Egyptian population. MATERIALS AND METHODS: Four hundred and twenty-six HCC patients and 334 controls were enrolled in the study. Polymorphisms of IGF-2+3580, IGF-2+3123, IGF-2R 1619, and IRS-2 1057 gene were detected using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Serum IGF-2 were determined using ELISA. RESULTS: Serum IGF-2 levels were significantly lower in HCC patients than in healthy controls. IGF-2+3580 AA genotype, IGF-2+3123 GG genotype or G allele, IRS-2 1057 DD genotype and D allele were significantly associated with HCC risk. The combination of IGF-2+3580 AA homozygosity and IGF-2R 1619 GG homozygosity presented a significant protective effect against HCC (OR=0.16,95% CI=0. 08-0.34, P=0. 005). Serum IGF-2 concentrations were significantly increased in HCC patients with the IGF-2+3580 AA genotype. We also observed that increased alpha-fetoprotein (AFP), Child-Pugh grade, tumor size, and number of malignant lesions were accompanied by a significant increase of serum IGF-2 mean values of in HCC patients. CONCLUSION: IGF-2, IGF-2R, and IRS-2 genes polymorphisms and their combinations are associated with risk of HCC.

Insulin injection restored increased insulin receptor substrate (IRS)-2 protein during short-term protein restriction but did not affect reduced insulin-like growth factor (IGF)-I mRNA or increased triglyceride accumulation in the liver of rats.

Dietary protein restriction reduces insulin-like growth factor (IGF)-I synthesis and impairs growth. Moreover, insulin secretion is impaired and hepatic insulin signaling is activated presumably through upregulation of insulin receptor substrate (IRS)-2, which can stimulate lipogenesis thereby resulting in steatosis. In order to determine whether impaired insulin secretion is the primary cause of these changes, we injected insulin into protein-restricted rats and compensated for the reduction in insulin secretion for 1 and 7 d. Insulin infusion did not overcome the reduction in liver IGF-I mRNA nor the hepatic triglyceride accumulation. In contrast, it clearly suppressed the upregulation of hepatic IRS-2 on day 1, but not on day 7. Furthermore, insulin elimination increased IRS-2 in H4IIE-C3 cells. In summary, we found that reduced insulin secretion during protein restriction directly increased hepatic IRS-2 as a rapid response on day 1, while additional mechanisms contributed to the upregulation of IRS-2 on day 7.

Sex-dimorphic genetic effects and novel loci for fasting glucose and insulin variability.

Differences between sexes contribute to variation in the levels of fasting glucose and insulin. Epidemiological studies established a higher prevalence of impaired fasting glucose in men and impaired glucose tolerance in women, however, the genetic component underlying this phenomenon is not established. We assess sex-dimorphic (73,089/50,404 women and 67,506/47,806 men) and sex-combined (151,188/105,056 individuals) fasting glucose/fasting insulin genetic effects via genome-wide association study meta-analyses in individuals of European descent without diabetes. Here we report sex dimorphism in allelic effects on fasting insulin at IRS1 and ZNF12 loci, the latter showing higher RNA expression in whole blood in women compared to men. We also observe sex-homogeneous effects on fasting glucose at seven novel loci. Fasting insulin in women shows stronger genetic correlations than in men with waist-to-hip ratio and anorexia nervosa. Furthermore, waist-to-hip ratio is causally related to insulin resistance in women, but not in men. These results position dissection of metabolic and glycemic health sex dimorphism as a steppingstone for understanding differences in genetic effects between women and men in related phenotypes.

Improving effect of vitamin D supplementation on obesity-related diabetes in rats.

BACKGROUND: Vitamin D, a fat-soluble secosteroid, plays a key role in several metabolic diseases like diabetes. Diabetes is becoming a third leading chronic disease in the world, which seriously threatens human health. METHODS: In the current study, we found the beneficial effects of vitamin D supplementation in obesity-related diabetes rat. Enzyme-linked immunosorbent assay, biochemical testing, real-time PCR and western blot were carried to investigate the effect of vitamin D supplementation on diabetes. RESULTS: Successful modeling of obesity-related diabetes was determined by significant weight loss and elevated levels of fasting blood glucose, glycated hemoglobin and blood lipids at 12 weeks. Supplementation of vitamin D obviously increased body weight and decreased fasting blood glucose, glycated hemoglobin, and blood lipids, accompanied by increased 25-hydroxyvitamin D and decreased insulin, parathormone and adipocytokines. Furthermore, low expressed insulin receptor substrate-1 (IRS-1)/phosphorylation of IRS-1 (p-IRS-1), glucose transporter type 4 (GluT4) and vitamin D receptor (VDR) was increased. CONCLUSIONS: These suggested the beneficial effects of vitamin D supplementation in obesity-related diabetes rat, which may through VDR, IRS-1/p-IRS-1, and GluT4 signaling activation.

Factors in serum from type 2 diabetes patients can cause cellular insulin resistance.

This pilot study was aimed to investigate whether there are humoral factors in serum from type 2 diabetic subjects that, in addition to glucose, insulin and free fatty acids are able to induce or contribute to peripheral insulin resistance with respect to glucose transport. Isolated subcutaneous adipocytes from 11 type 2 diabetic subjects and 10 nondiabetic controls were incubated for 24-h in medium supplemented with 25 % serum from a control or a type 2 diabetic donor, in the presence of a low (5 mM) or a high (15 mM) glucose concentration, respectively. After the incubation period glucose uptake capacity was assessed. Serum from type 2 diabetic donors, compared to serum from controls, significantly reduced the maximal insulin eff ect to stimulate glucose uptake (approximately 40 %, p < 0.05) in adipocytes from control subjects, independent of surrounding glucose concentrations. Glucose uptake capacity in adipocytes isolated from type 2 diabetic subjects was similar regardless of culture condition. No significant alterations were found in cellular content of key proteins in the insulin signaling cascade (insulin receptor substrate-1 and -2, and glucose transporter 4) that could explain the impaired insulin-stimulated glucose transport in control adipocytes incubated with serum from type 2 diabetic donors. The present findings indicate the presence of biomolecules in the circulation of type 2 diabetic subjects, apart from glucose, insulin, and free fatty acids with the ability to induce peripheral insulin resistance. This further implies that even though normoglycemia is achieved other circulating factors can still negatively affect insulin sensitivity in type 2 diabetic patients.

Analysis of the association of preeclampsia with polymorphisms of the INS, INSR and IRS1 genes in Mexican women.

BACKGROUND/AIMS: It has been proposed that preeclampsia is a metabolic syndrome of pregnancy. The polymorphisms PstI and MaeIII of INS, NsiI of INSR and Ala513Pro and Gly972Arg of IRS1 have been associated with metabolic syndrome; moreover, the products of these genes are functionally contiguous during insulin signaling. The aim of this study was to assess whether these polymorphisms are associated with preeclampsia. METHODS: 46 normotensive pregnant women and 43 preeclamptic patients were included in the study to develop a clinical, biochemical and genotypic profile of preeclampsia. Clinical evaluation consisted of measurement of blood pressure, height and weight. Peripheral blood samples were collected for determination of fasting glucose and insulin concentrations and for extraction of genomic DNA. Proteinuria was determined. Polymorphisms were detected using PCR-RFLP. RESULTS: The normotensive and preeclampsia groups did not differ significantly in clinical and biochemical traits, except for systolic and diastolic blood pressure (p < 0.0001). Polymorphisms previously associated with metabolic syndrome in Mexican populations were not associated with preeclampsia in Mexican women (p > 0.05). CONCLUSION: The lack of an association between preeclampsia and the polymorphisms studied suggests that other genes whose products do not have direct functional interaction with metabolic syndrome or epigenetic factors may play a role in preeclampsia.

Reduced biliverdin reductase-A levels are associated with early alterations of insulin signaling in obesity.

Biliverdin reductase-A (BVR-A) is a serine/threonine/tyrosine kinase involved in the regulation of insulin signaling. In vitro studies have demonstrated that BVR-A is a substrate of the insulin receptor and regulates IRS1 by avoiding its aberrant activation, and in animal model of obesity the loss of hepatic BVR-A has been associated with glucose/insulin alterations and fatty liver disease. However, no studies exist in humans. Here, we evaluated BVR-A expression levels and activation in peripheral blood mononuclear cells (PBMC) from obese subjects and matched lean controls and we investigated the related molecular alterations of the insulin along with clinical correlates. We showed that BVR-A levels are significantly reduced in obese subjects and associated with a hyper-activation of the IR/IRS1/Akt/GSK-3ß/AS160/GLUT4 pathway. Low BVR-A levels also associate with the presence of obesity, metabolic syndrome, NASH and visceral adipose tissue inflammation. These data suggest that the reduction of BVR-A may be responsible for early alterations of the insulin signaling pathway in obesity and in this context may represent a novel molecular target to be investigated for the comprehension of the process of insulin resistance development in obesity.

Biomarkers Associated with Ischemic Stroke in Diabetes Mellitus Patients.

Diabetes is an established risk factor for ischemic stroke, but the associated molecular mechanisms remain to be fully elucidated. This study investigated the role of plasma and platelet microRNAs and their targeting proteins in the activation of platelets and their association with the occurrence of ischemic stroke in patients with type 2 diabetes mellitus (T2DM). Results showed that the expressions of platelet and plasma miR-144 and miR-223 were significantly altered in T2DM patients with or without ischemic stroke compared to that in healthy controls, but these changes were more significant in T2DM patients with ischemic stroke. The expressions of P2Y12 and IRS-1 as well as phosphorylation levels of IRS-1, PI3K, and Akt in platelets were significantly altered in T2DM patients with or without ischemic stroke. The expression of platelet miR-144 and miR-223 significantly correlated with their plasma levels, P2Y12 and IRS-1 expression, blood glucose concentration, and platelet activation rate. High glucose concentration significantly elevated P-selectin, miR-144 and P2Y12 expression and significantly reduced miR-223 and IRS-1 expression in UT-7 cells. Overexpression of miR-223 and blocking of miR-144 expression significantly normalized the effects of high glucose concentration in UT-7 cells. In conclusion, hyperglycemia may activate platelets through miR-144 and miR-223 to downregulate IRS-1 and upregulate P2Y12 expression in the platelets of T2DM patients through an IRS-1-PI3K-Akt signaling. Low platelet and plasma miR-223 expression in addition to high platelet and plasma miR-144 expression are risk factors for ischemic stroke in T2DM patients.

Maternal nicotine exposure leads to higher liver oxidative stress and steatosis in adult rat offspring.

Early nicotine exposure causes future obesity and insulin resistance. We evaluated the long-term effect of the maternal nicotine exposure during lactation in liver oxidative status, insulin sensitivity and morphology in adult offspring. Two days after birth, osmotic minipumps were implanted in the dams: nicotine (N), 6 mg/kg/day for 14 days or saline (C). Offspring were killed at 180 days. Protein content of superoxide dismutase, glutathione peroxidase, catalase, nitrotyrosine, 4HNE, IRS1, Akt1 and PPARs were measured. MDA, bound protein carbonyl content, SOD, GPx and catalase activities were determined in liver and plasma. Hepatic morphology and triglycerides content were evaluated. Albumin and bilirubin were determined. In plasma, N offspring had higher catalase activity, and SOD/GPx ratio, albumin and bilirubin levels but lower MDA content. In liver, they presented higher MDA and 4HNE levels, bound protein carbonyl content, SOD activity but lower GPx activity. N offspring presented an increase of lipid droplet, higher triglyceride content and a trend to lower PPARα in liver despite unchanged insulin signaling pathway. Early nicotine exposure causes oxidative stress in liver at adulthood, while protect against oxidative stress at plasma level. In addition, N offspring develop liver microsteatosis, which is related to oxidative stress but not to insulin resistance.

Novel link between inflammation and impaired glucose transport during equine insulin resistance.

Although insulin resistance (IR) has been increasingly recognized in horses, a clear understanding of its pathophysiology is lacking. The purpose of the present study was to determine the early pathologic changes in IR horses by characterizing alterations in proteins that play key roles in innate immunological responses and inflammatory pathways, and by identifying potential links with glucose transport and insulin signaling. Visceral (VIS) and subcutaneous (SC) adipose tissue and skeletal muscle (SM) biopsies were collected from horses, which were classified as insulin-sensitive (IS) or IR based on the results of an insulin-modified frequently sampled intravenous glucose tolerance test. Protein expression of Toll-like receptor 4 (TLR-4), suppressor of cytokine signaling 3 (SOCS-3) and tumor necrosis factor alpha (TNF-α) were quantified by Western blotting in VIS and SC adipose depots and SM, as well as insulin receptor substrate 1 (IRS-1). To better characterize the potential relationship between inflammation, IR and impaired glucose transport, we correlated active cell surface glucose transporter 4 (GLUT-4) content (measured by a cell surface biotinylated assay) with individual- and tissue-specific data related to inflammation. IR was associated with a significantly increased expression of TLR-4 and SOCS-3 in SM and VIS tissue, without a significant change in SC site. We also observed a significant increase in TNF-α in VIS, but not in SC, tissue of IR vs. IS horses. There was no difference in total content or serine phosphorylation of IRS-1 for any sampling site in IR compared to IS horses. We further observed a significant positive correlation between TLR-4 content and SOCS-3, as well as a significant negative correlation between SOCS-3 content and GLUT-4 trafficking. Taken together, the data suggested a pro-inflammatory state in SM and VIS, but not SC, adipose depot during compensated IR. In addition, SOCS-3 appears to be a novel link between inflammation and dysregulated glucose metabolism and insulin sensitivity during the early pathogenesis of insulin resistance.

Soluble and cell-associated insulin receptor dysfunction correlates with severity of HAND in HIV-infected women.

BACKGROUND: Blood sugar metabolism abnormalities have been identified in HIV-infected individuals and associated with HIV-associated neurocognitive disorders (HAND). These abnormalities may occur as a result of chronic HIV infection, long-term use of combined antiretroviral treatment (CART), aging, genetic predisposition, or a combination of these factors, and may increase morbidity and mortality in this population. OBJECTIVE: To determine if changes in soluble and cell-associated insulin receptor (IR) levels, IR substrate-1 (IRS-1) levels, and IRS-1 tyrosine phosphorylation are associated with the presence and severity of HAND in a cohort of HIV-seropositive women. METHODS AND RESULTS: This is a retrospective cross-sectional study using patient database information and stored samples from 34 HIV-seropositive women and 10 controls without history of diabetes from the Hispanic-Latino Longitudinal Cohort of Women. Soluble IR subunits [sIR, ectodomain (α) and full-length or intact (αß)] were assayed in plasma and CSF samples by ELISA. Membrane IR levels, IRS-1 levels, and IRS-1 tyrosine phosphorylation were analyzed in CSF white cell pellets (WCP) using flow cytometry. HIV-seropositive women had significantly increased levels of intact or full-length sIR in plasma (p<0.001) and CSF (p<0.005) relative to controls. Stratified by HAND, increased levels of full-length sIR in plasma were associated with the presence (p<0.001) and severity (p<0.005) of HAND. A significant decrease in IRS-1 tyrosine-phosphorylation in the WCP was also associated with the presence (p<0.02) and severity (p<0.02) of HAND. CONCLUSIONS: This study provides evidence that IR secretion is increased in HIV-seropositive women, and increased IR secretion is associated with cognitive impairment in these women. Thus, IR dysfunction may have a role in the progression of HAND and could represent a biomarker for the presence and severity of HAND.