Insulin Receptor Protein Levels Research Articles

HT22 cell differentiation reduces insulin receptor levels

Purpose: The brain is an insülin-sensitive organ and has widespread insulin receptor (IR) expression. IR signaling in the brain is essential for neuronal development, feeding behavior, body weight, and cognitive processes such as attention, learning, and memory. HT22 cells, which are derived from parent HT4 cells that are immortalized from primary mouse hippocampal neuronal cells are used in research related to insulin signaling. However, the role of these cells in insulin signaling is not known. In this study, we aimed to examine IR levels in cells differentiated using neurobasal medium. Material and methods: For the study, briefly, the cells were seeded in 6-well plates at 2x105 cells/well for 24 h. After the cells reached 80% confluence, the normal growth medium was replaced with a differentiation medium and the cells were incubated for 72 hours at 37 0C in 5% CO2. Western blot procedure was used to determine the expression of the IR. Result: Our results show that differentiation of HT22 cells stimulates neurite outgrowth. Furthermore, IR protein levels were significantly downregulated in differentiated HT22 cells. Conclusion: This finding may require careful consideration of the use of neurobasal medium in conditions where IR signaling is important.

Effect of Aerobic Exercise Intervention on Renal Insulin Signaling Pathway in db/db Mice

Objective: To study the effects on insulin signaling pathway in db/db mice during aerobic exercise and discuss the related mechanisms. Methods: Ten BKS mice were selected as the quiet control group (WT group), and 20 BKS-db/db mice were randomly divided into two groups as the diabetic quiet control group (C group) and the diabetic simple exercise group (E group), with 10 mice in each group. The mice in E group were acclimatized to the low-intensity criterion (2~7m/min, 10min/day, 5 days/week) for one week, and then subjected to 8 weeks of aerobic endurance exercise training on a running platform (0° incline, 7~12m/min, 30~40min/day, 5 days/week), while the mice in groups WT and C were kept quietly. At the end of the experiment, the fasting blood glucose content, glucose tolerance, insulin tolerance of each group of mice were detected respectively, and the expression levels of AKT protein and insulin receptor protein were detected by Western Blot technique. Results: (1) Compared with the diabetic quiet control group (Group C), the glucose tolerance and insulin tolerance in the quiet control group (Group WT) showed significant changes (P＜0.05); in the diabetic simple exercise group (Group E), there was no significant change in glucose tolerance and insulin tolerance (P＞0.05). (2) Compared with the diabetic quiet control group (Group C), the expression level of insulin receptor protein was down-regulated (P<0.05) and AKT protein expression was up-regulated (P<0.05) in the diabetic simple exercise group (Group E); compared with the quiet control group (Group WT), the expression level of insulin receptor protein was increased (P<0.05) and AKT protein was decreased (P<0.05) in the diabetic quiet control group (Group C), expression level decreased (P<0.05). Conclusion: Exercise alone could not improve insulin resistance in db/db mice; eight weeks of aerobic exercise improved and corrected renal insulin signaling pathway and alleviated renal insulin resistance in db/db mice.

200-OR: Role of Insulin-Degrading Enzyme (IDE) in the Tubulin–Primary Cilium Axis to Regulate Glucagon Secretion

Hyperglucagonemia is a hallmark of T2D, although molecular mechanisms underlying glucagon secretion dysregulation are poorly understood. We have recently reported that genetic ablation of IDE in α-cells (A-IDE-KO mouse) leads to constitutive glucagon secretion, hypertrophy, hyperplasia and increased proliferation. αTC1.9-KD-IDE cells (~30% IDE-knock-down) displayed reduced tubulin and acetylated-α-tubulin protein levels, impaired primary cilium and increased proliferation. Thus, we aimed to elucidate the role of IDE in α-cells tubulin network and ciliogenesis, and how this axis may be part of the glucagon secretion machinery. We have studied by western-blot IDE, tubulin, acetylated-α-tubulin and Arl13b (primary cilium marker) levels in αTC1.9 cells under low- and high-glucose conditions. We have found that high-glucose lowers IDE protein levels by 50% compared to low-glucose conditions, leading to a similar decrease in tubulin, acetylated-α-tubulin and Arl13b protein levels. These findings agree with our previous results using αTC1.9-KD-IDE cells, suggesting a relevant role of IDE in microtubule dynamics to control glucagon granules exocytosis. On the other hand, αTC1.9-KD-Arl13b cells (~70% Arl13b-knock-down) were used as a model of impaired ciliogenesis, showing 50% decrease in tubulin, and dysregulated glucagon secretion, mimicking αTC1.9-KD-IDE cells phenotype. In parallel, insulin-mediated inhibition of glucagon secretion is lost and insulin receptor protein levels were decreased by 40% in both, αTC1.9-KD-IDE and αTC1.9-KD-Arl13b cells, showing the relevance of IDE and primary cilium in the paracrine regulation of α-cells. Furthermore, these results have been reproduced in A-IDE-KO mice isolated islets. In conclusion, our results show that IDE-tubulin-primary cilium axis is regulated during glucose-stimulated glucagon secretion, this axis is also required for the paracrine action of insulin on α-cells to inhibit glucagon secretion. Disclosure E.Casanueva-alvarez: None. A.Sanz-gonzález: None. B.Merino: None. G.Perdomo: None. I.Cozar-castellano: None. Funding Ministerio de Ciencia e Innovación-Spain (PID2019-110496RB-C21)

Effect of β-sitosterol on insulin resistance & protein expression of insulin signalling molecules in quadriceps muscle of high fat diet-induced type-2 diabetic rats.

Changes in life style, such as high-calorie diet intake and lack of exercise, have increased the global prevalence of obesity and diabetes. The major pathophysiological event contributing to the development of type 2 diabetes mellitus is the target tissues to insulin action. Currently available drugs are unsuccessful for the treatment of type 2 diabetes due to their unwanted side effects. Hence, search drugs, from plant sources. β-sitosterol is plant sterols with structurally almost like that of cholesterol. It is widely present in various medicinal plants. It has been reported to elicit multitude of bioactivities including anti-lipidemic and anti-hyperglycemic activity. However, specific effect of β-sitosterol on insulin signalling molecules of quadriceps muscle remains unclear. Hence, the study aimed to assess the beneficial role of β-sitosterol on the expression of insulin signalling molecules in quadriceps muscle of high fat diet and sucrose-induced type-2 diabetic rats. The oral effective dose of β-sitosterol (25 mg/kg body weight) was given once each day until the end of the study (30 days post‑induction of diabetes) to HFD- fed diabetic rats. At the end of the experimental period, fasting blood sugar (FBG), oral glucose (OGTT) and insulin tolerances (IT), Tissue glycogen, Serum insulin, Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) and Quantitative Insulin Sensitivity Check Index (QUICKI), as well as the levels of insulin signalling molecules like insulin receptor (IR), glucose transporter subtype 4 (GLUT4) proteins and glycogen concentration within the quadriceps muscle were assessed. A diabetic rat indicates impaired glucose and insulin tolerances and insulin signalling molecules (IR and GLUT4) proteins and glycogen concentration. Serum insulin, were found to be increased in diabetic rats. The treatment with β-sitosterol normalized the altered levels of blood glucose, serum insulin, and IR and GLUT4 protein levels. Thus, we concluded that β-sitosterol enhances glycemic control through activation of IR and GLUT4 in the quadriceps muscle of high fat diet and sucrose‑induced type2 diabetic rats.

441-P: Oleate Prevents Palmitate-Induced Abnormalities in Insulin Signaling in Human Cardiac Progenitor Cells by Inhibiting p38 MAPK and c-Jun Phosphorylation

Elevated saturated fatty acid deposition in the heart results in insulin resistance, stress kinase activation, and increased cardiovascular risk in humans. The viability of human cardiac progenitor cells (hCPC) is essential for myocardium homeostasis. This study investigates the ability of palmitate, a saturated fatty acid, to impair insulin signaling in hCPC isolated from right auricle biopsies, and the potential protective effects of oleate, a mono-unsaturated fatty acid. hCPC were found to express both insulin receptor (IR) isoforms, IR-A and IR-B, with higher IR-A:IR-B ratio. Exposure of hCPC to 100 nM insulin for 15 min induced Akt (S473) phosphorylation (p<0.05) . However, treatment with 0.25 mM palmitate, but not with 0.1 mM oleate, for 24 h resulted in impaired insulin-induced Akt phosphorylation (p<0.05) with no effect on total Akt protein levels. Palmitate, but not oleate, also induced p38 MAPK (T180/Y182) and c-Jun (S63) phosphorylation (p<0.05) . Pretreatment for 1 h with 15 µM SB202190, a p38 MAPK inhibitor, or with 20 µM SP600125, a JNK inhibitor, significantly inhibited the ability of palmitate to impair insulin-induced Akt phosphorylation (p<0.05) . Interestingly, co-incubation of palmitate with oleate abolished the palmitate-induced p38 MAPK and c-Jun phosphorylation (p<0.05) and inhibition of insulin-induced Akt phosphorylation (p<0.05) . In addition, palmitate, but not oleate, was associated with downregulation of IR protein levels (p<0.05) , although with increased expression of total IR mRNA, IR-A, IR-B, and increased IR-A/IR-B ratio (p<0.05) . Co-incubation with oleate also prevented the palmitate-induced changes in IR (p<0.05) . In conclusion, oleate prevents the palmitate-induced abnormalities in insulin signaling in hCPC, largely by counteracting p38 MAPK and JNK activation. Hence, oleate supplementation might limit lipotoxicity, thus contributing to cardiac protection. Disclosure R.Doria: None. F.Giorgino: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk, Consultant; Lilly Diabetes, Sanofi, Research Support; Lilly Diabetes, Roche Diabetes Care, Takeda Pharmaceutical Company Limited. L.Laviola: Advisory Panel; Lilly Diabetes, Novo Nordisk, Roche Diabetes Care, Sanofi, Speaker's Bureau; A. Menarini Diagnostics, Abbott Diabetes, Medtronic, Terumo Corporation. I.Calderoni: None. C.Caccioppoli: None. V.Genchi: None. G.Santarpino: None. A.Leonardini: None. A.Natalicchio: Consultant; Novo Nordisk, Other Relationship; AstraZeneca, Lilly Diabetes, Sanofi. S.Perrini: None. A.Cignarelli: None.

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.

Hypoglycemic and hypolipidemic effects of sugar beet (Beta vulgaris L.) extract on C57BL/KsJ db/db mice

Sugar beet (Beta vulgaris L. (BV)) is one of the most important economic crops. The purpose of this study was to examine the effects and mechanism of BV on the glucolipid metabolism. The study was conducted on C57BL/KsJ db/db mice, and the experiments were divided into four groups: diabetic control (distilled water, db/db-Veh), metformin treatment (metformin, db/db-Met, 250 mg/kg), and BV treatment (BV, db/db, 1.8 and 3.6 g/kg of BV). The metformin and BV were intragastrically administrated into mice. The results showed that the treatment with BV resulted in the decreased levels of the fasting blood glucose (FBG), uric acid (UA), and lipid, as well as improving the abnormalities of liver and pancreas. The protein levels of insulin receptor substrate‐1 (IRS‐1)/PI3K/AKT and AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) were quantified by Western blotting. The results showed that the hypoglycemic effect was partly due to the activation of the IRS‐1/PI3K/AKT signaling pathway, and the AMPK/ACC signaling pathway was responsible for the hypolipidemic activity. This study demonstrated that BV possesses hypoglycemic and hypolipidemic effects on diabetic db/db mice, indicating that it can be contributed to the treatment of type 2 diabetes.

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.

Activation of Hippocampal IR/IRS-1 Signaling Contributes to the Treatment with Zuogui Jiangtang Jieyu Decoction on the Diabetes-Related Depression.

Background Zuogui Jiangtang Jieyu decoction (ZJJ) is mainly used for the treatment of diabetes-related depression in current clinical applications and research. This study aims to investigate whether the brain IR/IRS-1 signaling pathway is involved in the therapeutic effect of ZJJ on depression-like behavior in diabetic rats. Methods Sprague–Dawley rats were fed with high-fat diet and subjected to streptozotocin injection to establish the diabetes animal model. After treatment with different doses of ZJJ (20.530 g/kg or 10.265 g/kg) for 4 weeks, the blood glucose level and peripheral insulin resistance were measured. The forced-swimming test (FST) and Morris water maze test (MWMT) were applied for the mood and cognitive function assessment. Then, the Western blot method was used to analyze the protein levels of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylonositol-3-kinase (PI3K), and protein kinase B (PKB, also as known as AKT) in the hippocampus of diabetic rats. Meanwhile, the immunofluorescence method was performed to analyze the above proteins' expression in the neuron and astrocyte. At last, the levels of glycogen, lactate, and ATP were tested by the ELISA method. Additionally, the insulin-sensitive glucose transporter 4 (GLUT4) and the lactate transporter monocarboxylate transporter 4 (MCT4) were analyzed by the Western blot method. Results ZJJ administration significantly decreased the level of blood glucose and improved the peripheral insulin resistance in diabetic rats. Besides, ZJJ attenuated the depression-like behavior and the cognitive dysfunction in rats with diabetes. Furthermore, we found the upregulation of protein expression of phospho-IR, phospho-IRS-1, phospho-PI3K, and phospho-AKT in the hippocampus of diabetic rats after being treated with ZJJ. Moreover, the above proteins are increased not only in the neuron but also in the astrocyte after ZJJ administration. In addition, ZJJ increased the content of ATP, glycogen, and lactate, as well as the expression of GLUT4 and MCT4 in the hippocampus of diabetic rats. Conclusions These findings suggest that ZJJ improves the depression-like behavior of diabetic rats by activating the IR/IRS-1 signaling pathway in both hippocampal neuron and astrocyte. And the brain IR/IRS-1 signaling pathway plays an important role in astrocyte-neuron metabolic coupling, providing a potential mechanism by which the IR/IRS-1 signaling pathway may contribute to the treatment of ZJJ on diabetes-related depression.

374-P: Oleate Prevents Palmitate-Induced Abnormalities in Insulin Signaling in Human Cardiac Progenitor Cells

Myocardial ectopic lipid deposition has been associated with insulin resistance and cardiovascular risk. Palmitate, one of the most abundant saturated fatty acids, has been shown to impair insulin signaling in multiple cell types. Human cardiac progenitor cells (CPC) represent a compartment of multipotent stem cells essential for constant tissue repair and renewal in the adult heart. In this study, the ability of palmitate to impair insulin signaling, and the modulatory effects of oleate, a mono-unsaturated fatty acid, on palmitate-induced abnormalities were investigated in human CPC isolated from control subjects undergoing cardiac surgery. Human CPC express insulin receptor (IR), as assessed by quantitative RT-PCR and immunoblotting. Specifically, in human CPC both IR isoforms, A (IR-A) and B (IR-B), were identified, and IR-A was more expressed than IR-B. Exposure of human CPC to 100 nM insulin for 15 minutes resulted in increased Akt and p44/p42 MAPK phosphorylation (p&amp;lt;0.05), evidenced by immunoblotting. Treatment of human CPC with 0.25 mM palmitate for 24 h impaired the ability of insulin, added for the last 15 minutes, to phosphorylate both Akt and p44/p42 MAPK (p&amp;lt;0.05), without no effect on total Akt and p44/p42 MAPK protein levels. Interestingly, palmitate was able to increase both IR-A and IR-B mRNA levels (p&amp;lt;0.05), and to reduce IR protein expression (p&amp;lt;0.05). Co-incubation of palmitate with 0.1 mM oleate prevented the ability of palmitate to reduce insulin-induced Akt phosphorylation (p&amp;lt;0.05), but did not rescue the impaired p44/p42 MAPK phosphorylation. In addition, co-treatment with oleate also prevented both palmitate-induced upregulation of IR-A and IR-B mRNA (p&amp;lt;0.05), and downregulation of IR protein levels (p&amp;lt;0.05). In conclusion, oleate prevents some of the palmitate-induced impairments in insulin signaling in human CPC, thus contributing to cardiac protection from lipotoxicity-induced metabolic alterations. Disclosure R. Doria: None. F. Giorgino: Consultant; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Novo Nordisk, Roche Diabetes Care, Sanofi, Research Support; Self; Lilly Diabetes, Roche Diabetes Care. C. Caccioppoli: None. R. Schipani: None. V. Genchi: None. A. Leonardini: None. A. Natalicchio: Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Sanofi. S. Perrini: None. A. Cignarelli: None. L. Laviola: Advisory Panel; Self; A. Menarini Diagnostics, Abbott, AstraZeneca, Lilly Diabetes, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis, Speaker’s Bureau; Self; Boehringer Ingelheim International GmbH, Medtronic.

Developmental programming: gestational testosterone excess disrupts LH secretion in the female sheep fetus

BackgroundPrenatal testosterone (T) excess results in reproductive and metabolic perturbations in female sheep that closely recapitulate those seen in women with polycystic ovary syndrome (PCOS). At the neuroendocrine level, prenatal T-treated sheep manifest increased pituitary sensitivity to GnRH and subsequent LH hypersecretion. In this study, we investigated the early effects of gestational T-treatment on LH secretion and pituitary function in the female sheep fetus. Additionally, because prenatal T effects can be mediated via the androgen receptor or due to changes in insulin homeostasis, prenatal co-treatment with an androgen antagonist (flutamide) or an insulin sensitizer (rosiglitazone) were tested.MethodsPregnant sheep were treated from gestational day (GD) 30 to 90 with either: 1) vehicle (control); 2) T-propionate (~ 1.2 mg/kg); 3) T-propionate and flutamide (15 mg/kg/day); and 4) T-propionate and rosiglitazone (8 mg/day). At GD 90, LH concentrations were determined in the uterine artery (maternal) and umbilical artery (fetal), and female fetuses were euthanized. Pituitary glands were collected, weighed, and protein level of several key regulators of LH secretion was determined.ResultsFetal pituitary weight was significantly reduced by prenatal T-treatment. Flutamide completely prevented the reduction in pituitary weight, while rosiglitazone only partially prevented this reduction. Prenatal T markedly reduced fetal LH concentrations and flutamide co-treatment partially restored LH to control levels. Prenatal T resulted in a marked reduction in LH-β protein level, which was associated with a reduction in GnRH receptor and estrogen receptor-α levels and an increase in androgen receptor. With the exception of androgen receptor, flutamide co-treatment completely prevented these alterations in the fetal pituitary, while rosiglitazone largely failed to prevent these changes. Prenatal T-treatment did not alter the protein levels of insulin receptor-β and activation (phosphorylation) of the insulin signaling pathways.ConclusionsThese findings demonstrate that prenatal T-treatment results in reduced fetal LH secretion, reduced fetal pituitary weight, and altered protein levels of several regulators of gonadotropin secretion. The observations that flutamide co-treatment prevented these changes suggest that programming during fetal development likely occurs via direct androgen actions.

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.

Role of Grb10 in mTORC1-dependent regulation of insulin signaling and action in human skeletal muscle cells.

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that binds to the insulin receptor, upon which insulin signaling and action are thought to be inhibited. Grb10 is also a substrate for the mechanistic target of rapamycin complex 1 (mTORC1) that mediates its feedback inhibition on phosphatidylinositide 3-kinase (PI3K)/Akt signaling. To characterize the function of Grb10 and its regulation by mTORC1 in human muscle, primary skeletal muscle cells were isolated from healthy lean young men and then induced to differentiate into myotubes. Knockdown of Grb10 enhanced insulin-induced PI3K/Akt signaling and glucose uptake in myotubes, reinforcing the notion underlying its function as a negative regulator of insulin action in human muscle. The increased insulin responsiveness in Grb10-silenced myotubes was associated with a higher abundance of the insulin receptor. Furthermore, insulin and amino acids independently and additively stimulated phosphorylation of Grb10 at Ser476. However, acute inhibition of mTORC1 with rapamycin blocked Grb10 Ser476 phosphorylation and repressed a negative-feedback loop on PI3K/Akt signaling that increased myotube responsiveness to insulin. Chronic rapamycin treatment reduced Grb10 protein abundance in conjunction with increased insulin receptor protein levels. Based on these findings, we propose that mTORC1 controls PI3K/Akt signaling through modulation of insulin receptor abundance by Grb10. These findings have potential implications for obesity-linked insulin resistance, as well as clinical use of mTORC1 inhibitors.

Afadin is a scaffold protein repressing insulin action via HDAC6 in adipose tissue.

Insulin orchestrates metabolic homeostasis through a complex signaling network for which the precise mechanisms controlling its fine-tuning are not completely understood. Here, we report that Afadin, a scaffold protein, is phosphorylated on S1795 (S1718 in humans) in response to insulin in adipocytes, and this phosphorylation is impaired with obesity and insulin resistance. In turn, loss of Afadin enhances the response to insulin in adipose tissues via upregulation of the insulin receptor protein levels. This happens in a cell-autonomous and phosphorylation-dependent manner. Insulin-stimulated Afadin-S1795 phosphorylation modulates Afadin binding with interaction partners in adipocytes, among which HDAC6 preferentially interacts with phosphorylated Afadin and acts as a key intermediate to suppress insulin receptor protein levels. Adipose tissue-specific Afadin depletion protects against insulin resistance and improves glucose homeostasis in diet-induced obese mice, independently of adiposity. Altogether, we uncover a novel insulin-induced cellular feedback mechanism governed by the interaction of Afadin with HDAC6 to negatively control insulin action in adipocytes, which may offer new strategies to alleviate insulin resistance.

Reduced Insulin Receptor Expression and Altered DNA Methylation in Fat Tissues and Blood of Women With GDM and Offspring.

Altered expression of the insulin receptor (IR) in adipose tissue (AT) could contribute to gestational diabetes mellitus (GDM) etiopathogenesis. Transcriptional regulation via epigenetic mechanisms (e.g., DNA methylation) may play a critical role. However, the human IR promoter DNA methylation patterns and involvement in gene expression are unknown. We evaluated IR mRNA and protein expression accompanied by targeted DNA methylation analyses in AT and blood cells of women with GDM and their offspring. Prospective observational study. Academic clinic and research unit. GDM-affected (n = 25) and matched control (n = 30) mother-child dyads. Maternal IR gene and protein expression in paired subcutaneous (SAT) and visceral adipose tissue samples (VAT). DNA methylation levels in IR promoter and intronic regions in maternal AT and blood cells of mother-offspring pairs. In SAT and VAT, IR mRNA/protein expressions were significantly reduced in women with GDMs (P < 0.05). The decrease in VAT was more pronounced and independent of maternal body mass index. VAT IR protein levels were inversely associated with key maternal and neonatal anthropometric and metabolic parameters (P < 0.05). DNA methylation patterns were similar across tissues, with significant yet small size alterations between groups in mothers and offspring (P < 0.05). Decreased IR levels in AT may be a relevant pathogenic factor in GDM, affecting materno-fetal metabolism. Further investigation of causal factors for IR dysregulation is necessary, especially in VAT. Potential functional and/or clinical roles of altered DNA methylation also should be evaluated.

Hyperinsulinemia precedes insulin resistance in offspring rats exposed to angiotensin II type 1 autoantibody in utero.

Insulin resistance is highly associated with an adverse intrauterine environment. We previously reported that fetal rats exposed to angiotensin II type 1 receptor (AT1R) autoantibody (AT1-AA) displayed increased susceptibility to metabolic diseases during middle age. However, the timing of the onset of insulin resistance remains unknown. In this study, we examined the offspring of AT1-AA-positive rats, tracking the development of insulin resistance. Pregnant rats were intravenously injected with AT1-AA. Afterwards, we collected serum samples and liver tissues of the offspring at various stages, including gestation day 18, 3 weeks (weaning period), 18 weeks (young adulthood), and 48 weeks (middle age) after birth. Compared with saline control group, hepatic vacuolar degeneration was visible in AT1-AA offspring rats as early as 3 weeks; hyperinsulinemia and impaired glucose tolerance occurred at 18 weeks of age, however, insulin resistance was not observed until 48 weeks. At 18 weeks we detected suppressed protein levels of insulin receptor (IR) but increased levels of IR substrate 1 (IRS1) in the liver of AT1-AA group rats. Interestingly, both IR and IRS1/2 were significantly decreased at 48 weeks. Liver proteomic analysis indicated that the differences in protein expression between the AT1-AA and control rats became more pronounced with age, particularly in terms of mitochondrial energy metabolism. Rats exposed to AT1-AA in utero developed hyperinsulinemia from young adulthood which subsequently progressed to insulin resistance, and was linked with abnormal hepatic structure and impaired IR signaling. Additionally, dysregulation of energy metabolism may play a fundamental role in predisposing offspring to insulin resistance.

Insulin receptor signaling in the subfornical organ protects against the development of metabolic syndrome

Metabolic syndrome encompasses a combination of conditions including obesity, diabetes, dyslipidemia, and hypertension. Brain insulin resistance has emerged as a contributor to the development of metabolic syndrome, although the neural regions involved remain unclear. While most investigations have focused on insulin action in the hypothalamus, recent evidence suggests that the insulin receptor (IR) gene is also expressed in the subfornical organ (SFO); a circumventricular organ well known for cardiovascular/fluid regulation and recently recognized as a metabolic nucleus. We therefore hypothesized that IR signaling in the SFO is involved in metabolic regulation. We first examined protein levels of SFO IR in male C57Bl/6 mice (n=3) using immunohistochemistry, and observed that insulin receptor expressing cells are rich in the medial to caudal SFO (Figure), while robust IR‐immunoreactivity (ir) on fibers is detected in the rostral SFO. Co‐immunohistochemistry further revealed heterogeneous cellular expression of the SFO IR, with 11.9 ± 2.2% of IR‐ir detected on astrocytes (GFAP), 57.2 ± 2.6% on endothelial cells (TIE2), and 18.3 ± 0.8% on neurons (NeuN). Interestingly, neuronal expression of IR in the SFO was restricted to glutamatergic cells (CAMKII co‐localization), but absent in GABAergic cells (GAD67‐mCherry reporter mice). To test the functional role of SFO IR, we next utilized mice harboring a conditional allele of the IR gene (IRfl/fl), and selectively knocked down the SFO IR via SFO‐targeted delivery of an adeno‐associated virus encoding Cre‐recombinase (AAV‐Cre‐eGFP; n=4), or control vector (AAV‐eGFP; n=3). Both groups remained on normal chow, and metabolic parameters were continuously monitored using indirect calorimetry for 12 weeks. Selective removal of SFO IR did not influence food and water intake, but resulted in a greater increase in body weight (e.g. 12 weeks: 27.9 ± 1.5 vs. 31.4 ± 1.4 g, AAV‐eGFP vs. AAV‐CreeGFP, ANOVA interaction p=0.0005). This was associated with a significantly lower energy expenditure (e.g. 12 week average: 12.5 ± 0.6 vs. 11.7 ± 0.2 kcal/hr/kg, AAV‐eGFP vs. AAV‐Cre‐eGFP, ANOVA interaction p=0.013) and a slight reduction in ambulatory activity in AAV‐Cre‐eGFP mice relative to controls. Examination of regional adipose tissue also revealed an ~40% increase in overall adiposity following ablation of SFO IR (total adipose: 1.4 ± 0.4 vs. 2.2 ± 0.3 g, AAV‐eGFP vs. AAV‐Cre‐eGFP, p=0.1). Glucose and insulin tolerance testing in a separate cohort indicated that whole body glucose clearance and insulin sensitivity were comparable between groups. SFO‐specific reductions in IR were confirmed by anatomical and molecular verification. These data demonstrate that ablation of SFO IRs under normal diet conditions results in a deleterious metabolic state. Moreover, these findings indicate a tonic metabolic regulatory role for SFO IR, and suggest that impairments in IR signaling in the SFO may contribute to a development of metabolic syndrome.Support or Funding InformationHL116776This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

P41 Exploring the biological role of insulin and insulin-like growth factor-1 hybrid receptors in the endothelium

Endothelial cells co-express insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) homodimers. Furthermore, association of insulin αβ half receptors and IGF-1 αβ half receptors also occur resulting in insulin/IGF-1 hybrid receptors. An abundance of hybrid receptors has been associated with reduced insulin sensitivity in metabolic tissue. The aim of this project is to study the differential expression of vascular derived insulin receptors, IGF-1 receptors and insulin/IGF-1 hybrids during insulin resistance in a diet-induced model of obesity (DIO). C57BL/6 mice were fed on high-fat or low-fat diets ad libitum for 16-weeks. Obese mice showed significant metabolic abnormalities indicative of insulin resistance when compared with lean littermates. A significant increase in hybrid receptor levels were observed in aorta from obese mice (immunoprecipitation and Western blotting). Total IR and IGF-1R protein levels were reduced in obese mice (Western blotting) however, no change was observed in IR and IGF-1R mRNA levels (real time-PCR). Insulin-stimulated (4.5 nmol/kg injected) in vivo serine phosphorylation of Akt was significantly blunted in aortae of obese mice, whereas IGF-1-stimulated (90 nmol/kg injected) remained unaffected. We have shown that IR and IGF1R homodimers are preferentially titrated into hybrid receptors in a model of DIO and this is accompanied with a reduction in insulin sensitivity but not IGF-1 sensitivity in the aorta. Hybrid receptors may provide a novel target for the prevention of CVD progression in vulnerable patients.

Reduced Insulin Receptor Expression Enhances Proximal Tubule Gluconeogenesis.

Reduced insulin receptor protein levels have been reported in the kidney cortex from diabetic humans and animals. We recently reported that, targeted deletion of insulin receptor (IR) from proximal tubules (PT) resulted in hyperglycemia in non-obese mice. To elucidate the mechanism, we examined human proximal tubule cells (hPTC) and C57BL/6 mice fed with high-fat diet (HFD, 60% fat for 20 weeks). Immunoblotting revealed a significantly lower protein level of IR in HFD compare to normal chow diet (NCD). Furthermore, a blunted rise in p-AKT308 levels in the kidney cortex of HFD mice was observed in response to acute insulin (0.75 IU/kg body weight, i.p) relative to NCD n = 8/group, P < 0.05). Moreover, we found significantly higher transcript levels of phosphoenolpyruvate carboxykinase (PEPCK, a key gluconeogenic enzyme) in the kidney cortex from HFD, relative to mice on NCD. The higher level of PEPCK in HFD was confirmed by immunoblotting. However, no significant differences were observed in cortical glucose-6-phosphatase (G6Pase) or fructose-1,6, bisphosphosphatase (FBPase) enzyme transcript levels. Furthermore, we demonstrated insulin inhibited glucose production in hPTC treated with cyclic AMP and dexamethasone (cAMP/DEXA) to stimulate gluconeogenesis. Transcript levels of the gluconeogenic enzyme PEPCK were significantly increased in cAMP/DEXA-stimulated hPTC cells (n = 3, P < 0.05), and insulin attenuated this upregulation Furthermore, the effect of insulin on cAMP/DEXA-induced gluconeogenesis and PEPCK induction was significantly attenuated in IR (siRNA) silenced hPTC (n = 3, P < 0.05). Overall the above data indicate a direct role for IR expression as a determinant of PT-gluconeogenesis. Thus reduced insulin signaling of the proximal tubule may contribute to hyperglycemia in the metabolic syndrome via elevated gluconeogenesis. J. Cell. Biochem. 118: 276-285, 2017. © 2016 Wiley Periodicals, Inc.