Insulin Receptor mRNA Levels Research Articles

Effects of heat stress on the feeding preference of yellow-feathered broilers and its possible mechanism

Heat stress is the most critical factor affecting animal feeding in summer. This experiment was conducted to investigate the effects of heat stress on the feeding preference of yellow-feathered broilers and its possible mechanism. As a result, the preference of yellow-feathered broilers for Tenebrio molitor was significantly decreased, and the fear response and serum corticosterone of broilers were significantly increased when the ambient temperatures are 35 °C (P < 0.05). In the central nervous system, consistent with the change in feeding preference, decreased dopamine in the nucleus accumbens (NAc) and increased mRNA levels of MAO-B in the ventral tegmental area (VTA) and NAc were found in yellow-feathered broilers (P < 0.05). In addition, we found significantly increased mRNA levels of corticotropin-releasing hormone receptor 1, corticotropin-releasing hormone receptor 2 and glucocorticoid receptor in the VTA and NAc of female broilers (P < 0.05). However, no similar change was found in male broilers. On the other hand, the serum levels of insulin and glucagon-like peptide-1 were increased only in male broilers (P < 0.05). Accordingly, the mRNA levels of insulin receptor and glucagon-like peptide-1 receptor in the VTA and the phosphorylation of mTOR and PI3K were increased only in male broilers (P < 0.05). In summary, the preference of yellow-feathered broilers for Tenebrio molitor feed decreased under heat stress conditions, and hedonic feeding behavior was significantly inhibited. However, the mechanism by which heat stress affects hedonic feeding behavior may contain gender differences. The insulin signaling pathway may participate in the regulation of heat stress on the male broiler reward system, while stress hormone-related receptors in the midbrain may play an important role in the effect of heat stress on the reward system of female broilers.

Decreased Serum Insulin Receptor Messenger RNA Level in H. pylori IgG Seropositive Type 2 Diabetic Patients.

Helicobacter pylori (H. pylori) is a known gastro-intestinal pathogen but implicated in extra-gastric diseases. The relationship between H. pylori infection and type 2 diabetes (T2DM) remains insufficiently elucidated, particularly in terms of molecular mediators such as microRNAs (miRNAs) and messenger RNAs (mRNAs). We aimed to characterize expression pattern of insulin signalling mRNAs and targeted miRNAs in T2DM patients exposed to H. pylori infection. We conducted a cross-sectional study among patients diagnosed with type 2 diabetes mellitus and were aged 18 to 60 years. Overnight fasting blood samples were collected and processed for plasma and serum. The plasma samples were used for glucose estimation and the serum used for H. pylori IgG screening. Total RNA was extracted from the serum with commercial kit, and mRNAs and miRNAs quantified by RT-qPCR with specific primers and under predetermined amplification conditions. Clinical data were obtained from medical records of patients. Among 351 patients enrolled, 267 (76.1%) were females, 224 (63.8%) were married, and 79 (22.5%) had tertiary education. Expression level of insulin receptor mRNA was significantly lower in H. pylori positive T2DM patients compared to H. pylori negative (P < .05). There was no evidence of a difference in insulin receptor substrate 1 mRNA level (P > .05). Although not statistically significant, the expression levels of miRNA-222 and miRNA-155 in the patients exposed to H. pylori were higher than that of the unexposed group (P > .05). We found a significantly reduced serum insulin receptor messenger RNA level and higher levels of miRNA-222 and miRNA-155 in H. pylori exposed T2DM patients. The findings suggest a possible role of the infection in insulin signalling alteration in the patients.

Development of Type 1 Diabetes may occur through a Type 2 Diabetes mechanism.

At diagnosis of Type 1 Diabetes (T1D), 30% of the beta cells are dormant, i.e. alive, but inactive. This could reduce beta cell destruction, as cellular stress contributes to beta cell damage. However, the beta cells, that are still active, must produce more insulin and are therefore more vulnerable. The inactive beta cells represent a potential for restoring the insulin secretion. We analyzed the expression of selected genes in islets from live, newly diagnosed T1D patients from the DiViD study and organ doners with longer duration of T1D, type 2 diabetes (T2D), or no diabetes from the nPOD study. Additionally, analysis of polymorphisms was performed on all the investigated genes. Various possibilities were considered for the inactivity of the beta cells: secretion defect, fetal state, hibernation, and insulin resistance. We analyzed genes related to the ceramide and sphingomyelin synthesis and degradation, secretion, circadian rhythm and insulin action, and found changes in T1D islets that resemble fetal dedifferentiation and asynchrony. Furthermore, we found low levels of insulin receptor mRNA in the islets. No polymorphisms were found. Our findings suggest a secretion defect, but also fetal dedifferentiation and desynchronization in the inactive beta cells. Together with previous evidence, that predisposing factors for T2D are also present for T1D development, we raise the idea to treat individuals with ongoing T1D development prophylactically with T2D medicine like GLP-1 receptor agonists, metformin, or others, combined with anti-inflammatory compounds, in order to reactivate the dormant beta cells, and to prevent autoimmune destruction. T2D mechanisms during T1D development should be investigated further.

CD36 gene deletion reduces muscle insulin sensitivity in mice by up-regulating PTP1B expression

To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. Wild-type (WT) mice and systemic CD36 knockout (CD36-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. CD36-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36-/- mice (P>0.05). In the muscle tissue of CD36-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36-/- mice. CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.

Prenatal Chronic Hypoxia in Rats Leads to Insulin Resistance and Hypertension in Adult Offspring

Abstract Background To investigate the effects of chronic intrauterine hypoxia on insulin resistance, hypertension, and the correlation between them in adult offspring rats. Methods A total of 25 pregnant Sprague Dawley rats were randomly assigned into 4 prenatal chronic hypoxia (H) groups (10% ± 1% oxygen) and a control group (21% oxygen). The H groups were divided into whole (1–21 day), early (1–7 day), mid (8–14 day), and late (15–21 day) gestational hypoxia groups (H1, H2, H3, and H4, respectively) with pregnant rats being housed in a hypoxia box for 3 hours per day. Five male and 5 female offspring in each group were studied at 1 day, 3 months, and 6 months old. Blood pressure, fasting blood glucose, fasting serum insulin, and insulin resistance index were determined. Results The mean blood pressure of offspring rats in H groups was higher at 3 months, and further increased at 6 months old compared to the control group (P &amp;lt; 0.05). The fasting blood glucose and homeostasis model insulin resistance index (HOMA-IR) of male and female offspring in the whole pregnancy (H1) and early pregnancy (H2) hypoxia groups were significantly higher than those in the control group at 6 months (P &amp;lt; 0.05). Fasting blood glucose and HOMA-IR were positively correlated with mean blood pressure (P &amp;lt; 0.05). The renal mass index in H2 group was lower at 3 months, and further decreased at 6 months compared to controls (P &amp;lt; 0.05). The mRNA and protein levels of insulin receptor, insulin receptor substrate (IRS-1 and IRS-2) in the kidneys in hypoxia groups were significantly decreased at 6 months in H1 and H2 hypoxia groups when compared with controls (P &amp;lt; 0.05). Conclusions Chronic intrauterine hypoxia causes insulin resistance and hypertension in adult offspring rats through poor intrauterine growth environment, and insulin resistance is positively associated with hypertension.

The Role of Zinc Status on Spatial Memory, Hippocampal Synaptic Plasticity, and Insulin Signaling in icv-STZ-Induced Sporadic Alzheimer's-Like Disease in Rats.

Alzheimer's disease (AD), especially its sporadic form (sAD), is of multifactorial nature. Brain insulin resistance and disrupted zinc homeostasis are two key aspects of AD that remain to be elucidated. Here, we investigated the effects of dietary zinc deficiency and supplementation on memory, hippocampal synaptic plasticity, and insulin signaling in intracerebroventricular streptozotocin (icv-STZ)-induced sAD in rats. The memory performance was evaluated by Morris water maze. The expression of hippocampal protein and mRNA levels of targets related to synaptic plasticity and insulin pathway was assessed by Western blot and real-time quantitative PCR. We found memory deficits in icv-STZ rats, which were fully recovered by zinc supplementation. Western blot analysis revealed that icv-STZ treatment significantly reduced hippocampal PSD95 and p-GSK3β, and zinc supplementation restored the normal protein levels. mRNA levels of BDNF, PSD95, SIRT1, GLUT4, insulin receptor, and ZnT3 were found to be reduced by icv-STZ and reestablished by zinc supplementation. Our data suggest that zinc supplementation improves cognitive deficits and rescues the decline in key molecular targets of synaptic plasticity and insulin signaling in hippocampus caused by icv-STZ induced sAD in rats.

Hyperandrogenic Milieu Dysregulates the Expression of Insulin Signaling Factors and Glucose Transporters in the Endometrium of Patients With Polycystic Ovary Syndrome

Purpose: Subfertility associated with polycystic ovary syndrome (PCOS) mainly originates from oligoovulation/anovulation. Although insulin resistance and androgen excess are known to cause PCOS-associated implantation failure, the consequences of PCOS on endometrial homeostasis and pathophysiology have not been comprehensively understood. In this study, we examined whether the pathophysiologic milieu of PCOS intrinsically affects expression profiles of genes related to insulin signaling and facilitative glucose transporters (GLUTs) in the human endometrium and/or during in vitro decidualization. Study Design: Seven healthy women with regular menstrual cycles and 13 patients with PCOS were recruited for this study. To mimic the hyperandrogenic or hyperinsulinemic milieu in the endometrium of patient with PCOS (PCOSE) in vitro, human endometrial stromal cells (hESCs) were treated with dihydrotestosterone (DHT) or insulin, respectively. Results: In PCOSE, messenger RNA (mRNA) levels of insulin receptor (IR), IR substrate (IRS) 1, and IRS2 were significantly increased. Furthermore, GLUT1 and GLUT12 were aberrantly increased. Chronic exposure to insulin or DHT aberrantly increased IRS1/IRS2 phosphorylation and protein levels of GLUT1 and GLUT12 in hESCs, suggesting that not only hyperinsulinemic but also hyperandrogenic conditions affect insulin signaling and glucose metabolism. The mRNA microarrays demonstrated that DHT dysregulates various gene sets, including cell cycle and glucose metabolism, in hESCs. Furthermore, DHT suppressed the expression of GLUT1 and GLUT12 as well as decidualization markers, IGFBP1 and prolactin, during in vitro decidualization. Conclusions: The hyperandrogenic milieu affects gene expression profiles, including gene sets associated with insulin signaling, cell cycle, glucose metabolism, and/or glucose transport, in human endometrium and during in vitro decidualization.

Dynamic changes of blood glucose, serum biochemical parameters and gene expression in response to exogenous insulin in Arbor Acres broilers and Silky fowls

Silky chicken is a breed of chickens with black skin and slow growth rate used in Chinese traditional medicine, whereas Arbor Acres broiler is a well-known commercial breed in the poultry industry, it is featured by a large size, rapid-growth rate, high feed-conversion rate and strong adaptability. The difference in their rate of growth may be primarily related to different mechanism for glucose metabolism. Here we compared the insulin sensitivity of the two breeds; we investigated the temporal changes (at 0 min, 120 min and 240 min) of serum insulin and other biochemical parameters and determined the spatio-temporal changes of gene mRNA abundance in response to exogenous insulin (80 μg/kg body weight). The results indicated that: (1) Silky chickens showed stronger blood glucose recovery than broilers in the insulin resistance test. (2) The serum urea level in Silky chickens was twice of broilers; exogenous insulin significantly up-regulated serum uric acid level in Silky fowls in a time-dependent manner and increased serum cholesterol content at 120 min. (3) Two breeds showed distinctly different temporal changed in serum insulin in response to exogenous insulin stimulation. The fasting serum insulin concentration of broilers was three-fold of Silky chickens at the basal state; it decreased significantly after insulin injection and the levels at 120 min and 240 min of broilers were only 23% (P < 0.01) and 14% (P < 0.01) of the basal state, respectively. Whereas the serum insulin content in Silky chickens showed stronger recovery, and the 240 min level was close to the 0 min level. (4) GLUT2, GLUT12, neuropeptide Y and insulin receptor (IR) were predominantly expressed in the liver, pectoralis major, olfactory bulb and pancreas, respectively, where these genes presented stronger insulin sensitivity. In addition, the IR mRNA level was strongly positively with the GLUT12 level. In conclusion, our findings suggested that Silky chickens have a stronger ability to regulate glucose homeostasis than broilers, owing to their higher IR levels in the basal state, stronger serum insulin homeostasis and candidate genes functioning primarily in their predominantly expressed tissue in response to exogenous insulin.

Detrimental Effects of Sodium Fluoride on the Expression of Insulin Receptor in the Olfactory Bulb and Hippocampus of Male Mice.

Fluoride exposure is associated with lowered cognitive function ability, intelligence quotient, and mental decline, especially in children. The brain insulin receptor (IR) signaling system is related to neuronal plasticity and consequent cognitive ability. In our previous study, NaF exposure decreased IR expressions in olfactory bulb (OB) and hippocampus after Y-maze test in male mice. In order to further explore whether the Y-maze test affected IR gene and protein expression levels in the OB and hippocampus under the NaF exposure, healthy male mice were randomly allotted into four groups and challenged with 0, 50, 100, and 150mg/L NaF for three continuous months. The results showed that femur fluorine content of the NaF-exposed groups increased significantly in a dose-dependent manner. NaF significantly decreased brain protein content and organ coefficient of the treated male mice. The protein and mRNA expression levels of the IR were significantly decreased in the OB and hippocampus of the NaF-treated mice. Interestingly, indicators (brain protein content and organ coefficient) measured in the present study were significantly lower than our previous study indicators (mice tested Y-maze test), especially the expression levels of IR protein and mRNA in the same concentration groups. Taken together, these results indicated that Y-maze test could promote the expression levels of IR protein and mRNA in the OB and hippocampus, while NaF had a stronger inhibitory effect, which resulted in adverse effects on the expression levels of IR in the OB and hippocampus of male mice.

Bitter melon fruit extract has a hypoglycemic effect and reduces hepatic lipid accumulation in ob/ob mice.

Bitter melon (Momordica charantia L.) is a vegetable and has been used as traditional medicine. Recently, we reported that bitter melon fruit extracts and its ethyl acetate (EtOAc)-soluble fraction markedly suppressed the expression of proinflammatory genes, including the inducible nitric oxide synthase gene. However, it is unclear whether bitter melon exhibits antidiabetic effects. In this study, we showed that cucurbitacin B, a cucurbitane-type triterpenoid, was present in an EtOAc-soluble fraction and suppressed nitric oxide production in hepatocytes. When the EtOAc-soluble fraction was administered for 7 days to ob/ob mice, a type 2 diabetes mellitus model, the mice fed with this fraction exhibited a significant decrease in body weight and blood glucose concentrations compared with the mice fed without the fraction. The administration of the fraction resulted in significant increases in serum insulin concentrations and the levels of both insulin receptor mRNA and protein in the ob/ob mouse liver. The EtOAc-soluble fraction decreased the interleukin-1β mRNA expression, as well as hepatic lipid accumulation in hepatocytes. Taken together, these results indicate that administration of an EtOAc-soluble fraction improved hyperglycemia and hepatic steatosis, suggesting that this fraction may be responsible for both the antidiabetic and anti-inflammatory effects of bitter melon fruit.

1762-P: Insulin Receptor Isoforms in Human Adipose Tissue and Adipose Stem Cells

Insulin regulates the development and metabolism of adipose tissue (AT) by activating its tyrosine kinase receptor in this tissue. The insulin receptor (IR) consists of two isoforms, a short isoform (IR-A) with prevalent mitogenic property and a long isoform (IR-B) with prevalent metabolic activity. We investigated the expression of IR-A and IR-B in human AT and adipocyte precursors. Biopsies of abdominal subcutaneous AT (S-AT) and visceral AT (V-AT) were obtained from subjects with different BMI. Adipose-derived stem cells (ASC) were also isolated from both AT depots, and were analyzed before and after differentiation into adipocytes (dASC). mRNA levels of IR-A were 2-fold and 3-fold higher compared to IR-B in S-AT and V-AT, respectively. Total IR and IR-A mRNA levels were also 2-fold higher in S-ASC compared to V-ASC. Accordingly, S-ASC exhibited a greater insulin-induced Akt phosphorylation (4-fold) as compared to V-ASC. IR-A expression displayed a positive correlation with BMI in S-ASC (r = 0.50; p = 0.04) but not in V-ASC, in which an inverse correlation was noted (r = -0.66; p = 0.01). Adipocyte differentiation in the presence of human insulin was associated with increased mRNA expression of IR-A in both S-dASC (3-fold) and V-dASC (2.8-fold), and of total IR (5-fold) and IR-B (2-fold) only in S-dASC. Moreover, S-dASC showed higher IR-A mRNA expression (2.5-fold) when compared to V-dASC, similarly to S-ASC and S-AT. In V-dASC, but not in S-dASC, the extent of lipogenesis assessed by oil-Red-O staining was positively correlated with basal expression of total IR (r = 0.94; p = 0.04), while adipogenesis measured by Nile red fluorescent staining showed a correlation with the pAkt/pErk ratio (r = 0.95; p &amp;lt; 0.01). In conclusion, expression of total IR, IR-A and IR-B differs in relation to the stage of tissue development (ASC, dASC, AT) and in a depot-related manner (S vs. V). IR-A appears to be the prevalent IR isoform in human AT and adipocyte precursors and to be particularly involved in the expansion of S-AT in human obesity. Disclosure V. Genchi: None. A. Cignarelli: Consultant; Self; Eli Lilly and Company. Speaker's Bureau; Self; Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S. S. Perrini: None. S. Porro: None. C. Caccioppoli: None. A. Natalicchio: Other Relationship; Self; Novo Nordisk Inc., Sanofi-Aventis. L. Laviola: Advisory Panel; Self; Abbott, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Novo Nordisk Inc. Board Member; Self; AstraZeneca, Roche Diabetes Care, Sanofi-Aventis. Speaker's Bureau; Self; Medtronic, Mundipharma, Takeda Pharmaceutical Company Limited. F. Giorgino: Advisory Panel; Self; Aegerion Pharmaceuticals, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, MedImmune, Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S, Roche Diabetes Care, Sanofi. Consultant; Self; Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Pharmaceutical Company Limited. Other Relationship; Self; AstraZeneca, Eli Lilly and Company, Sanofi.

Prepartum body conditions affect insulin signaling pathways in postpartum adipose tissues in transition dairy cows

BackgroundOverconditioned dairy cows are susceptible to excessive lipolysis and increased insulin resistance during the transition period. The associations among body fat reserve, insulin resistance, and lipolysis in adipose tissues (AT) remain to be elucidated. Therefore, this study aimed to investigate whether excessive fat reserves influence the insulin signaling pathway in AT postpartum.ResultsTwenty multiparous dairy cows were selected and assigned to one of two groups, according to prepartum body condition score (BCS): Control group (BCS = 3.0–3.5; n = 10) and Overconditioned group (BCS ≥ 4.0; n = 10). Blood samples were collected on days −14, −7, −4, −2, −1, 0, 1, 2, 4, 7, and 14 relative to parturition. Subcutaneous AT were collected on day 2 following parturition for quantitative real-time polymerase chain reaction and western blot analyses. No differences were observed between the two groups in serum glucose, non-esterified fatty acids, β-hydroxybutyric acid, tumor necrosis factor (TNF) α, insulin, or leptin concentrations during the experimental period. Compared with the control cows, the overconditioned cows had lower serum triglyceride levels and higher adiponectin concentrations. In the AT postpartum, insulin receptor mRNA and protein levels were lower in the overconditioned cows than in the control cows, and no differences were found in glucose transporter 4 mRNA. Compared with the control cows, the overconditioned cows had lower mRNA levels of TNFα and higher mRNA levels of peroxisome proliferator-activated receptor gamma (PPARγ) in AT postpartum. The phosphorylated protein kinase B (AKT) content and phosphorylation rate of AKT were increased in the overconditioned cows compared with the control cows, which suggested that the downstream insulin signaling in AT was affected.ConclusionsIn the present study, transition dairy cows with higher BCS did not show more fat mobilization. The changes of insulin signaling pathway in AT postpartum of overconditioned cows may be partly related to the expression of PPARγ and TNFα, and the secretion of adiponectin.

Hyperandrogenic Milieu Dysregulates the Expression of Insulin Signaling Factors and Glucose Transporters in the Endometrium of Patients With Polycystic Ovary Syndrome.

Subfertility associated with polycystic ovary syndrome (PCOS) mainly originates from oligoovulation/anovulation. Although insulin resistance and androgen excess are known to cause PCOS-associated implantation failure, the consequences of PCOS on endometrial homeostasis and pathophysiology have not been comprehensively understood. In this study, we examined whether the pathophysiologic milieu of PCOS intrinsically affects expression profiles of genes related to insulin signaling and facilitative glucose transporters (GLUTs) in the human endometrium and/or during in vitro decidualization. Seven healthy women with regular menstrual cycles and 13 patients with PCOS were recruited for this study. To mimic the hyperandrogenic or hyperinsulinemic milieu in the endometrium of patient with PCOS (PCOSE) in vitro, human endometrial stromal cells (hESCs) were treated with dihydrotestosterone (DHT) or insulin, respectively. In PCOSE, messenger RNA (mRNA) levels of insulin receptor (IR), IR substrate (IRS) 1, and IRS2 were significantly increased. Furthermore, GLUT1 and GLUT12 were aberrantly increased. Chronic exposure to insulin or DHT aberrantly increased IRS1/IRS2 phosphorylation and protein levels of GLUT1 and GLUT12 in hESCs, suggesting that not only hyperinsulinemic but also hyperandrogenic conditions affect insulin signaling and glucose metabolism. The mRNA microarrays demonstrated that DHT dysregulates various gene sets, including cell cycle and glucose metabolism, in hESCs. Furthermore, DHT suppressed the expression of GLUT1 and GLUT12 as well as decidualization markers, IGFBP1 and prolactin, during in vitro decidualization. The hyperandrogenic milieu affects gene expression profiles, including gene sets associated with insulin signaling, cell cycle, glucose metabolism, and/or glucose transport, in human endometrium and during in vitro decidualization.

Lean maternal hyperglycemia alters offspring lipid metabolism and susceptibility to diet-induced obesity in mice†.

We previously developed a model of gestational diabetes mellitus (GDM) in which dams exhibit glucose intolerance, insulin resistance, and reduced insulin response to glucose challenge only during pregnancy, without accompanying obesity. Here, we aimed to determine how lean gestational glucose intolerance affects offspring risk of metabolic dysfunction. One cohort of offspring was sacrificed at 19 weeks, and one at 31 weeks, with half of the second cohort placed on a high-fat, high-sucrose diet (HFHS) at 23 weeks. Exposure to maternal glucose intolerance increased weights of HFHS-fed offspring. Chow-fed offspring of GDM dams exhibited higher body fat percentages at 4, 12, and 20 weeks of age. At 28 weeks, offspring of GDM dams fed the HFHS but not the chow diet (CD) also had higher body fat percentages than offspring of controls (CON). Exposure to GDM increased the respiratory quotient (Vol CO2/Vol O2) in offspring. Maternal GDM increased adipose mRNA levels of peroxisome proliferator-activated receptor gamma (Pparg) and adiponectin (Adipoq) in 31-week-old CD-fed male offspring, and increased mRNA levels of insulin receptor (Insr) and lipoprotein lipase (Lpl) in 31-week-old male offspring on both diets. In liver at 31 weeks, mRNA levels of peroxisome proliferator-activated receptor alpha (Ppara) were elevated in CD-fed male offspring of GDM dams, and male offspring of GDM dams exhibited higher mRNA levels of Insr on both diets. Neither fasting insulin nor glucose tolerance was affected by exposure to GDM. Our findings show that GDM comprising glucose intolerance only during pregnancy programs increased adiposity in offspring, and suggests increased insulin sensitivity of subcutaneous adipose tissue.

Developmental exposure to DEHP alters hepatic glucose uptake and transcriptional regulation of GLUT2 in rat male offspring

Type-2-diabetes (T2D) is a long term metabolic disorder characterized by high blood glucose and insulin resistance. It has become an alarming issue globally due to tremendous increase in number of new subjects every year. Apart from the classical factors, there are few non-classical factors such as environmental pollutants, endocrine disrupting chemicals (EDCs) which also play a major role in pathogenesis of T2D. Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer which is an endocrine disrupting chemical. It is used in the plastic industry to give flexibility and durability. Its widespread use resulted in constant presence in the environment and human are under high risk of exposure to this compound. There are literature available stating that DEHP has an impact on glucose homeostasis. Glucose transporter 2 (GLUT2) is a principal transporter of glucose in liver and it is a bi-directional transporter. We investigated whether DEHP exposure during gestation and lactation alters transcriptional regulation of GLUT2 and epigenetics changes in the rat F1 male offspring at adulthood. Pregnant rats were divided into three groups and administered with DEHP (10 and 100 mg /kg /day) or olive oil from gestational day (GD) 9– to postnatal day (PND) 21 through oral gavage. DEHP treated rats showed decreased glucose uptake and oxidation, decreased mRNA levels of insulin receptor (IR), GLUT2 and reduced GLUT2 protein in cytosol but unaltered level in plasma membrane. There are three main transcription factors (SREBP1c, HNF3β and HNF1α) involved in the regulation of GLUT2 gene and all these proteins were reduced in DEHP exposed groups. A weak interaction of the transcription factors (SREBP1c & HNF1α) with GLUT2 gene promoter was observed in DEHP-treated groups. Hyper- methylation of IR and GLUT2 gene promoter was observed in both the DEHP-exposed groups compared to control. The present study reveals that DEHP exposure alters transcriptional regulation of GLUT2 and imposes epigenetic alteration in IR and GLUT2 gene promoters which plays a significant role in the development of metabolic abnormality in F1 male offspring at adulthood.

Pancreatic Adipose Tissue in Diet-Induced Type 2 Diabetes

Obesity represents one of the most important health problems. Proper adipose tissue expandability is crucial for avoiding metabolic disorders. Furthermore, not all fat depots are functionally equal. Our previous work indicates that the expansion of the mesenteric adipose tissue (MWAT) plays a role in the intestinal barrier. Whether pancreas-associated adipose tissue (PAT) has a specific function is currently unknown. We hypothesize that storage of excess nutrients in PAT and PAT-derived adipokines (such as adiponectin) protect the pancreas against metabolic dysfunction during weight gain/obesity. An initial morphological study showed that PAT has an increased number of small, but fewer large adipocytes relative the adjacent MWAT. We compared the response to a 16-week high-fat diet (HFD)/obesity between PAT, other fat depots and pancreas. We found higher mRNA expression of inflammation markers in MWAT, inguinal (IWAT) and gonadal fat (GWAT), but not in PAT in response to HFD/obesity. HFD also downregulated the expression of insulin receptor and adiponectin in all fat depots, but not in PAT. The pancreas showed an increase in inflammatory and insulin resistance markers only after long-term (16 weeks) HFD exposure, a time point when also PAT displayed a reduction in insulin receptor and adiponectin expression. Smaller adipocytes are typically more functional than large adipocytes. Thus, these data suggest that PAT have an increased capacity for energy storage. This in turn may protect the pancreas against ectopic lipid accumulation and lipotoxicity. Moreover, our data show that PAT is protected against the deleterious effects of HFD, maintaining normal mRNA levels of adiponectin, insulin receptor and markers of inflammation. In summary, we believe that PAT protects the pancreatic beta-cells from lipotoxicity through its increased capacity for energy storage, sustained insulin sensitivity and possibly also increased insulin exposure because of its anatomical location. Disclosure B. Chanclon Garcia: None. Y. Wu: None. E. Banke Nordbeck: None. S. Musovic: None. C.S. Olofsson: None. P. Rorsman: None. I. Wernstedt Asterholm: None.

Expression of a Constitutively Active Human Insulin Receptor in Hippocampal Neurons Does Not Alter VGCC Currents.

Memory and cognitive decline are the product of numerous physiological changes within the aging brain. Multiple theories have focused on the oxidative, calcium, cholinergic, vascular, and inflammation hypotheses of brain aging, with recent evidence suggesting that reductions in insulin signaling may also contribute. Specifically, a reduction in insulin receptor density and mRNA levels has been implicated, however, overcoming these changes remains a challenge. While increasing insulin receptor occupation has been successful in offsetting cognitive decline, alternative molecular approaches should be considered as they could bypass the need for brain insulin delivery. Moreover, this approach may be favorable to test the impact of continued insulin receptor signaling on neuronal function. Here we used hippocampal cultures infected with lentivirus with or without IRβ, a constitutively active, truncated form of the human insulin receptor, to characterize the impact continued insulin receptor signaling on voltage-gated calcium channels. Infected cultures were harvested between DIV 13 and 17 (48h after infection) for Western blot analysis on pAKT and AKT. These results were complemented with whole-cell patch-clamp recordings of individual pyramidal neurons starting 96h post-infection. Results indicate that while a significant increase in neuronal pAKT/AKT ratio was seen at the time point tested, effects on voltage-gated calcium channels were not detected. These results suggest that there is a significant difference between constitutively active insulin receptors and the actions of insulin on an intact receptor, highlighting potential alternate mechanisms of neuronal insulin resistance and mode of activation.

Study of insulin vascular sensitivity in aortic rings and endothelial cells from aged rats subjected to caloric restriction: Role of perivascular adipose tissue.

The prevalence of metabolic syndrome is dramatically increasing among elderly population. Metabolic syndrome in aged individuals is associated with hyperinsulinemia and insulin resistance both in metabolic tissues and in the cardiovascular system, with this fact being associated with the cardiometabolic alterations associated to this condition. Caloric restriction (CR) improves insulin sensitivity and is one of the dietetic strategies most commonly used to enlarge life and to prevent aging induced cardiovascular alterations. The aim of this study was to analyze the possible beneficial effects of CR in aging-induced vascular insulin resistance both in aortic rings and in primary culture of endothelial cells. In addition, the inflammatory profile of perivascular adipose tissue (PVAT) and its possible role in the impairment of vascular insulin sensitivity associated with aging was also assessed. Three experimental groups of male Wistar rats were used: 3 (3m), 24 (24m) fed ad libitum and 24months old rats subjected to 20% CR during their three last months of life (24m-CR). Aorta rings surrounded or not by PVAT were mounted in an organ bath and precontracted with phenylephrine (10-7.5M). Changes in isometric tension were recorded in response to cumulative insulin concentrations (10-8-10-5.5M) in the presence or absence of L-NAME (10-4M). Aortic rings and primary aortic endothelial cells were incubated in presence/absence of insulin (10-7M) and the activation of the PI3K/Akt and MAPK pathways as well as nitrite and nitrates concentrations and the mRNA levels of eNOS, insulin receptor, and GLUT-4 were assessed. CR prevented the aging-induced decrease in the vasodilator response to insulin and the aging-induced increase in the vasoconstrictor response to high insulin concentrations. Changes between 24m and 24m-CR aorta rings were abolished in the presence of L-NAME. CR induced-improvement in insulin vascular sensitivity was related with activation of the PI3K/Akt both in aortic rings and in aortic endothelial cells in response to insulin. CR attenuated the overexpression of iNOS, TNF-α and IL-1β in the PVAT of aged rats although aortic rings surrounded by PVAT from 24m rats showed and increased vasorelaxation in response to insulin compared to aortic rings from 3m and 24m-CR rats. In conclusion, a moderate protocol of CR improves insulin vascular sensitivity and prevents the aging induced overexpression of pro-inflammatory cytokines in PVAT.

Abstract 181: Insulin Receptor Signaling Regulates ApoA-I Secretion from Hepatocytes

The aim of the study was to assess the effect of insulin receptor (IR) signaling on hepatic apoA-I metabolism. The experimental approach was to compare hepatic apoA-I expression, secretion and cellular localization in IRfl/fl mice and LIRKO mice in which hepatic insulin receptors were specifically deleted by AAV delivered Cre-recombinase. Results showed that IR mRNA and protein levels were markedly reduced in the livers of LIRKO mice compared to control IRfl/fl mice. As expected, LIRKO mice exhibited decreased glucose tolerance and reduced hepatic insulin signaling. Knockdown of hepatic IR decreased plasma HDL cholesterol and apoA-I levels. Whereas apoA-I mRNA levels were similar in LIRKO and control hepatocytes, apoA-I protein levels were increased in both the liver and primary hepatocytes isolated from LIRKO mice. In contrast to apoA-I, apoE and apoB protein levels in the liver and in cultured hepatocytes, as well as in the plasma, were similar in LIRKO and control mice. ApoA-I accumulation in LIRKO hepatocytes was associated with a decreased rate of apoA-I secretion. Immunofluorescence staining demonstrated that apoA-I accumulated in LIRKO hepatocytes in membrane bound inclusions. These inclusions shared markers characteristic of early, late and recycling endosomes, and of lysosomes. We conclude that IR-mediated insulin signaling plays an important role in hepatic apoA-I secretion and consequent nascent HDL formation. Reduced apoA-I secretion from liver into the circulation may contribute to the lower HDL levels typically associated with insulin resistance.

High-sucrose diet induces diabetic-like phenotypes and oxidative stress in Drosophila melanogaster: Protective role of Syzygium cumini and Bauhinia forficata.

Diet is a key component for development and longevity of organisms. Here, the fruit fly was used to evaluate the detrimental effects caused by consumption of high-sucrose diets (HSD), namely phenotypic responses linked to insulin signaling and oxidative stress. The protective effects of extracts from medicinal plants Syzygium cumini and Bauhinia forficata were investigated. HSD intake (15% and 30%) delayed the time to pupation and reduced the number of white pupae. In adult flies, the intake of diets was associated with mortality and increased levels of glucose+trehalose, triacylglycerols and hydrogen peroxide. Indeed, 30% HSD induced body-weight loss, mitochondrial dysfunction and changes in acetylcholinesterase, δ-aminolevulinate dehydratase and antioxidant enzymes activity. Catalase, superoxide dismutase, keap1, HSP70, dILP-5 and Insulin receptor mRNA levels were over-expressed in flies emerged from 30% HSD. The extract treatments blunted the developmental alterations elicited by diets. Syzygium cumini extract was more efficient than B. forficata in reducing hyperglycaemia, redox disturbances and the changes in mRNA expression of insulin receptor.