InsR Expression Research Articles

Effects of Metformin on Reproductive, Endocrine, and Metabolic Characteristics of Female Offspring in a Rat Model of Letrozole-Induced Polycystic Ovarian Syndrome With Insulin Resistance.

The beneficial effects of metformin, especially its capacity to ameliorate insulin resistance (IR) in polycystic ovary syndrome (PCOS), explains why it is widely prescribed. However, its effect on the offspring of patients with PCOS remains uncertain. This study investigated the impact of metformin treatment on the first- and second-generation female offspring born to letrozole-induced PCOS-IR rats. Forty-five female Wistar rats were implanted with continuous-release letrozole pellets or placebo and treated with metformin or vehicle control. Rats exposed to letrozole showed PCOS-like reproductive, endocrine, and metabolic phenotypes in contrast to the controls. Metformin significantly decreased the risk of body weight gain and increased INSR expression in F1 female offspring in PCOS-IR rats, contributing to the improvement in obesity, hyperinsulinemia, and IR. Decreased FSHR expression and increased LHCGR expression were observed in F1 female rats of the PCOS-IR and PCOS-IR+Metformin groups, suggesting that FSHR and LHCGR dysfunction might promote the development of PCOS. Nevertheless, we found no significant differences in INSR, FSHR, and LHCGR expression or other PCOS phenotypes in F2 female offspring of PCOS-IR rats. These findings indicated widespread reproductive, endocrine, and metabolic changes in the PCOS-IR rat model, but the PCOS phenotypes could not be stably inherited by the next generations. Metformin might have contributed to the improvement in obesity, hyperinsulinemia, and IR in F1 female offspring. The results of this study could be used as a theoretical basis in support of using metformin in the treatment of PCOS-IR patients.

Altered immunometabolism in adipose tissue: A major contributor to the ageing process?

Altered immunometabolism in adipose tissue: A major contributor to the ageing process?

An evidence update on the protective mechanism of tangeretin against neuroinflammation based on network pharmacology prediction and transcriptomic analysis

Although the protective effects of tangeretin on neuroinflammation have been proven in cell and animal experiments, few studies explore its underlying molecular mechanism. In this study, we used the network pharmacology method combined with the transcriptome approach to investigate its underlying anti-inflammatory mechanism in human microglial cells. Based on network pharmacology analysis, four putative target proteins and ten potential pathways were identified. Among them, vascular endothelial growth factor A (VEGFA), epidermal growth factor receptor (EGFR) and the related phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), the mitogen-activated protein kinase (MAPK), mechanistic target of rapamycin (mTOR) signaling pathway were well-supported by transcriptome data. Meanwhile, transcriptome analysis supplemented two crucial targets: the insulin receptor (InsR) and insulin-like growth factor-I (IGF-1) receptor. Subsequently, VEGFA, EGFR, IGF-1 receptor, and InsR were further verified on the protein level. Taken together, we assumed that tangeretin could exert protective effects on neuroinflammation by decreasing the expression of VEGFA, EGFR, InsR, and IGF-1 receptor in the PI3K-AKT, MAPK, mTOR signaling pathway. More importantly, it is for the first time to show that the anti-neuroinflammatory effects of tangeretin through VEGFA, EGFR, IGF-1 receptor, InsR, and mTOR signaling pathway. These works offer new insight into the anti-neuroinflammatory functions of tangeretin and propose novel information on further anti-inflammatory mechanism studies.

Hyoscyamus albus nortropane alkaloids reduce hyperglycemia and hyperinsulinemia induced in HepG2 cells through the regulation of SIRT1/NF-kB/JNK pathway

BackgroundChronic superphysiological glucose and insulin concentrations are known to trigger several tissue and organ failures, including insulin resistance, oxidative stress and chronic low-grade inflammation. Hence, the screening for molecules that may counteract such conditions is essential in current existing therapeutic strategies, thereby the use of medicinal plant derivatives represents a promising axis in this regard.MethodsIn this study, the effect of a selected traditional medicinal plant, Hyoscyamus albus from which, calystegines have been isolated, was investigated in an experimental model of hyperinsulinemia and hyperglycemia induced on HepG2 cells. The mRNA and protein expression levels of different insulin signaling, gluconeogenic and inflammatory pathway- related molecules were examined. Additionally, cell viability and apoptosis, oxidative stress extent and mitochondrial dysfunctions were assayed using flow cytometric and qRT-PCR techniques.ResultsTreatment of IR HepG2 cells with calystegines strongly protected the injured cells from apoptosis, oxidative stress and mitochondrial integrity loss. Interestingly, nortropane alkaloids efficiently regulated the impaired glucose metabolism in IR HepG2 cells, through the stimulation of glucose uptake and the modulation of SIRT1/Foxo1/G6PC/mTOR pathway, which is governing the hepatic gluconeogenesis. Furthermore, the alkaloidal extract restored the defective insulin signaling pathway, mainly by promoting the expression of Insr at the mRNA and protein levels. What is more, treated cells exhibited significant mitigated inflammatory response, as evidenced by the modulation and the regulation of the NF- κB/JNK/TLR4 axis and the downstream proinflammatory cytokines recruitment.ConclusionOverall, the present investigation demonstrates that calystegines from Hyoscyamus albus provide cytoprotection to the HepG2 cells against insulin/glucose induced insulin resistance and apoptosis due to the regulation of SIRT1/Foxo1/G6PC/mTOR and NF-κB/JNK/TLR4 signaling pathways.1oVDx9MMsFkfL9sCGFfm2tVideo

Postnatal Catch-Up Growth Programs Telomere Dynamics and Glucose Intolerance in Low Birth Weight Mice.

Low birth weight and rapid postnatal weight gain are independent predictors of obesity and diabetes in adult life, yet the molecular events involved in this process remain unknown. In inbred and outbred mice, this study examines natural intrauterine growth restriction (IUGR) in relation to body weight, telomere length (TL), glucose tolerance, and growth factor gene (Igf1, Igf2, Insr, Igf1r, and Igf2r) mRNA expression levels in the brain, liver, and muscle at 2- and 10 days of age and then at 3- and 9 months of age. At birth, ~15% of the animals showed IUGR, but by 3 and 9 months, half of these animals had regained the same weight as controls without IUGR (recuperated group). At 10 days, there was no difference in TL between animals undergoing IUGR and controls. However, by 3 and 9 months of age, the recuperated animals had shorter TL than the control and IUGR-non recuperated animals and also showed glucose intolerance. Further, compared to controls, Igf1 and Igf2 growth factor mRNA expression was lower in Day 2-IUGR mice, while Igf2r and Insr mRNA expression was higher in D10-IUGR animals. Moreover, at 3 months of age, only in the recuperated group were brain and liver Igf1, Igf2, Insr, and Igf2r expression levels higher than in the control and IUGR-non-recuperated groups. These data indicate that catch-up growth but not IUGR per se affects TL and glucose tolerance, and suggest a role in this latter process of insulin/insulin-like growth signaling pathway gene expression during early development.

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes.

The steroid hormone 20-hydroxyecdysone counteracts insulin signaling via insulin receptor dephosphorylation

The insulin receptor (INSR) binds insulin to promote body growth and maintain normal blood glucose levels. While it is known that steroid hormones such as estrogen and 20-hydroxyecdysone counteract insulin function, the molecular mechanisms responsible for this attenuation remain unclear. In the present study, using the agricultural pest lepidopteran Helicoverpa armigera as a model, we proposed that the steroid hormone 20-hydroxyecdysone (20E) induces dephosphorylation of INSR to counteract insulin function. We observed high expression and phosphorylation of INSR during larval feeding stages that decreased during metamorphosis. Insulin upregulated INSR expression and phosphorylation, whereas 20E repressed INSR expression and induced INSR dephosphorylation in vivo. Protein tyrosine phosphatase 1B (PTP1B, encoded by Ptpn1) dephosphorylated INSR in vivo. PTEN (phosphatase and tensin homolog deleted on chromosome 10) was critical for 20E-induced INSR dephosphorylation by maintaining the transcription factor Forkhead box O (FoxO) in the nucleus, where FoxO promoted Ptpn1 expression and repressed Insr expression. Knockdown of Ptpn1 using RNA interference maintained INSR phosphorylation, increased 20E production, and accelerated pupation. RNA interference of Insr in larvae repressed larval growth, decreased 20E production, delayed pupation, and accumulated hemolymph glucose levels. Taken together, these results suggest that a high 20E titer counteracts the insulin pathway by dephosphorylating INSR to stop larval growth and accumulate glucose in the hemolymph.

Cord Blood from SGA Preterm Infants Exhibits Increased GLUT4 mRNA Expression.

Insulin and insulin-like growth factor (IGF) signaling plays an important role in prenatal and postnatal growth and glucose metabolism. Both small-for-gestational age (SGA) and preterm infants have abnormal growth and glucose metabolism. However, the underlying mechanism remains unknown. Recently, we showed that term SGA infants have abnormal insulin/IGF signaling in cord blood. In this study, we examined whether preterm infants show similar aberrations in cord blood insulin/IGF signaling. A total of 41 preterm cord blood samples were collected. Blood glucose, insulin, IGF-1, and C-peptide concentrations were measured, and mRNA expression of IGF1R, INSR, IRS1, IRS2, and SLC2A4 (i.e., GLUT4) was analyzed by quantitative reverse-transcription PCR. This study included 34 appropriate-for-gestational age (AGA) and 7 SGA preterm neonates. No hyperinsulinemia or any differences in IGF1R or INSR mRNA expression were detected between the two groups. However, GLUT4 mRNA levels were increased in preterm SGA. Moreover, the expression level in hypoglycemic preterm SGA was significantly higher than that in hypoglycemic preterm AGA. IRS2 mRNA expression did not show a statistically significant difference between preterm SGA and AGA neonates. SGA preterm birth does not induce hyperinsulinemia; however, it modifies insulin/IGF signaling components such as GLUT4 in umbilical cord blood. Our study suggests that prematurity or adaptation to malnutrition alters the insulin/IGF signaling pathway.

HOW WERE STRESS FAMILY AND INSR (INSULIN RECEPTOR) EXPRESSION IN POLYCYSTIC OVARY SYNDROME (PCOS) INSULIN RESISTANT IN MADURESE TRIBE?: INDONESIA

Introduce: Polycystic ovary syndrome (PCOS) is characterized by menstrual disorders (amenorrhea/oligomenorrhea), hirsutism, the appearance of acne, alopecia, and biochemical examination results showed an increase in androgen (testosterone). This study aimed to analyze family stress and expression of the gene INSR in insulin-resistant polycystic ovary syndrome (PCOS) in Madura. Methods: This is an analytic observational study with a study design case-control, the sample of 50 patients (PCOS cases) and 50 healthy women (Non-PCOS) in Madura Tribe, the research instrument used Kempe Family Stress Inventory (KFSI) and using in-depth interviews, followed by screening for SGOT, SGPT, BUN, Creatinin and GDA, PCR and RFLP on the INSR gene and continued sequencing on 10 samples. Result: The results of family stress reaction family with isolation, low self-esteem or depression average score was 56.4 (SD=4.23). The gene INSR had TT genotype 16.67%, TC 63.33%, and CC 20.00%, the control group had TT genotype 38.24%, TC 44.12%, and CC 17.65%, while in the case group T allele 48.33%, C allele 51.67% and T allele control group 56.67% and C allele 43.33%, based on statistical test chi-square in case and control groups, both genotype and allele had no significant difference (P-value> 0.05). Conclusion: The results showed family stress reaction with isolation, low self-esteem or depression. The expression result of INSR gene yielded was no difference between PCOS and healthy women. It needs to be continued because of racial differences between ethnic groups, especially in Indonesia.

The Biological Role of Optimized Recombinant Oral Long-Acting Glucagon Like Peptide-1 and Its Impact on the Expression of Genes Associated with Glucose Metabolism of Diabetes

Previously published studies have described optimized rolGLP-1 (Opt-rolGLP-1) exhibited good hypoglycemic efficacy. As such, additional studies were conducted aimed at exploring the therapeutic utility of Opt-rolGLP-1 in a murine model of diabetes and examining the mechanistic basis for such efficacy. A murine model of type 2 diabetes was constructed via a combination of an intraperitoneal STZ injection (STZ) together with a high fat/sugar diet. These model mice were then orally administered Opt-rolGLP-1. Treatment was associated with a reduction in average food and water intake. Notably, diabetic model animals tended to exhibit a reduction in overall body weight that was not evident in mice treated using Opt-rolGLP-1. Treatment with this compound was additionally associated with significant decreases in fasting plasma glucose, cholesterol, triglycerides, and glycosylated hemoglobin levels, whereas oral glucose tolerance and hyperinsulinemia improved significantly. At the gene expression level, we found that Opt-rolGLP-1 treatment increased the expression of GLUT2, INSR, and GCK in the liver, whereas the expression of PGC-1α fell significantly. These changes in gene expression may be associated with the mechanistic benefits of Opt-rolGLP-1 in this model of type 2 diabetes. Together these results highlight the value of further studying the potential clinical utility of Opt-rolGLP-1.

Changes in the transcriptional activity of the entero-insular axis genes in streptozotocin-induced diabetes and after the administration of TNF-α non-selective blockers.

The aim of the present study was to investigate the transcriptional activity of the GLP-1R, DPP-4, SGLT-1, INSR, and IGF-1R genes in GALT cells of rats with streptozotocin-induced diabetes in both untreated and treated with pentoxifylline, as a non-specific blocker of TNF-α. The expression of GLP-1R, DPP-4, SGLT-1, INSR, and IGF-1R genes in GALT cells of rats was studied by real time quantitative polymerase chain reaction. It was shown that the development of diabetes was accompanied by the decrease of GLP-1R and an increase of DPP-4 genes expression in rat ileum. The administration of pentoxifyl-line to diabetic animals led to an increase in the transcriptional activity of GLP-1R on the 4th week and decrease in transcriptional activity of DPP-4 on the 2nd and 4th weeks of the experiment. An increase in the normalized expression of SGLT-1 on the 4th week of the experimental diabetes was also noted, while the administration of pentoxifylline to diabetic animals did not lead to significant changes in this index. The transcriptional activity of the INSR and IGF-1R genes was reduced in diabetic rats and the administration of the non-specific TNF-α blocker - pentoxifylline led to a significant increase only for INSR gene in animals on the 4th week of the experimental diabetes. The expression of incretins, glucose transporters, and pro-inflammatory cytokines (e.g. TNF-α) in immune cells may be used as markers of several autoimmune pathologies progression such as type 1 diabetes due to their effect on the balance of pro- and anti-inflammatory factors.

Abstract D118: Insulin receptor splicing regulation as a potential target for improved prostate cancer disparity outcomes

Abstract Prostate Cancer is the third most commonly diagnosed cancer in the US, with 174,650 new cases predicted in 2019. Rates of incidence and mortality are 2 to 3 times higher among men from African American (AA) descent. These statistics necessitate unconventional and out-of-the box treatment approach to impede this cancer, specifically in AA men that are most likely to succumb to the cancer. Alternative splicing (AS) is a process in which a pre-mRNA can be processed into different mature mRNAs with the aid of the splice regulatory machinery. AS contributes to structural transcript variation and proteome diversity and is tightly regulated during development and differentiation. It has been shown that deregulated splicing can give rise to protein isoforms that contribute to tumor initiation, progression and resistance to therapy. The insulin receptor gene (INSR) has uniquely evolved to undergo AS to produce two isoforms: the full-length INR-B and exon 11 skipped INR-A isoform. INR-B primarily mediates the metabolic effects of insulin, whereas INR-A triggers growth-promoting effects. This increased INR-A expression promotes the proliferation of the cells because it encodes for a receptor which has high affinity for both insulin and IGF2 growth hormones and it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks like proliferation and angiogenesis. Published work as well as preliminary data from our own lab suggest that there is a significantly increased expression of INR-A levels in prostate cancer. This atypical increased expression was further confirmed in multiple prostate cancer cell-lines. Our data further shows that this conversion of INR-B to INR-A takes place in the presence of stress conditions such as hypoxia. Furthermore, the INSR expression and splicing is particularly relevant in prostate cancer since Hif1a has been shown to be significantly elevated and associated with worse prognosis in prostate cancer. Hypoxic signaling has also been found to be a key driver of malignancy as well a reason for treatment failure. Strikingly, the INR-A isoform is expressed more often in prostate cancer samples from AA males than those from European descent (EA). Our goal is to understand the role of AS in cancer and to target the splicing pathway as a therapeutic intervention. To this end, we used a novel hypoxia-induced splicing system that recapitulates the IN-R splicing seen in tumors to identify splice regulatory proteins and their respective binding sites capable of regulating INSR splicing in prostate cancer. In collaboration with Ionis Pharmaceuticals we have shown that splice-switching oligonucleotides (SSOs) can target the splicing enhancer and repressor sequences we identified to interfere with INSR splicing and cancer cell behavior. We hypothesize that decreasing the expression of the A variant will lead to decreased tumor growth and increased susceptibility to current treatment regimens that will be particularly beneficial to AA men that express high levels of the INR-A isoform. Citation Format: Safiya Khurshid, Matias Montes, Frank Rigo, Dawn S Chandler. Insulin receptor splicing regulation as a potential target for improved prostate cancer disparity outcomes [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D118.

Vitamin D actions in neurons require the PI3K pathway for both enhancing insulin signaling and rapid depolarizing effects

Despite correlations between low vitamin D levels and diabetes incidence/severity, supplementation with vitamin D has not been widely effective in improving glucose parameters. This may be due to a lack of knowledge regarding how low vitamin D levels physiologically affect glucose homeostasis. We have previously shown that the brain may be a critical area for vitamin d-mediated action on peripheral glucose levels. However, the mechanisms for how vitamin D acts in the brain are unknown. We utilized a multimodal approach to determine the mechanisms by which vitamin D may act in the brain. We first performed an unbiased search (RNA-sequencing) for pathways affected by vitamin D. Vitamin D (125-dihydroxyvitamin D3; 1,25D3) delivered directly into the third ventricle of obese animals differentially regulated multiple pathways, including the insulin signaling pathway. The insulin signaling pathway includes PI3K, which is important in the brain for glucose regulation. Since others have shown that vitamin D acts through the PI3K pathway in non-neuronal cells (muscle and bone), we hypothesized that vitamin D may act in neurons through a PI3K-dependent pathway. In a hypothalamic cell-culture model (GT1-7 cells), we demonstrate that 1,25D3 increased phosphorylation of Akt in the presence of insulin. However, this was blocked with pre-treatment of wortmannin, a PI3K inhibitor. 1,25D3 increased gene transcription of several genes within the PI3K pathway, including Irs2 and p85, without affecting expression of InsR or Akt. Since we had previously shown that 1,25D3 has significant effects on neuronal function, we also tested if the PI3K pathway could mediate rapid actions of vitamin D. We found that 1,25D3 increased the firing frequency of neurons through a PI3K-dependent mechanism. Collectively, these data support that vitamin D enhances insulin signaling and neuronal excitability through PI3K dependent processes which involve both transcriptional and membrane-initiated signaling events.

SAT-723 Effects of Organohalogenated Endocrine Disrupting Chemicals on Cell Proliferation and Gene Expression in GH3 Somatolactotropes

Endocrine Disrupting Chemicals (EDCs) are substances that have been increasingly implicated in many serious pathologies, such as tumor formation, metabolic, growth and reproductive disorders. The economic and health burden of exposure to these compounds has an annual predicted cost in excess of €150 billion, across the EU regions alone. Of the growing list of compounds that act as EDCs, the organohalogenated compounds (OHCs), polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) have been associated with an increased risk of pituitary disease. We have previously reported that feline patients with hypersomatotropism (acromegaly) are exposed to elevated levels of PBDEs and PCBs in their environment. However, the mechanisms by which these compounds might directly influence somatotroph function have yet to be established. In this study, we use the GH3 rat somatolactotrope cell line to investigate how two PCB congeners - 138 and 153 - influence cell proliferation (using a Crystal Violet assay) and somatotrope gene expression (using a multiplex RT-qPCR approach to examine expression of Esr1, Esr2, Sstr1, Sstr2, Sstr3, Sstr4, Sstr5, Insr, Tshr, Pou1f1, Ghrhr2, Gh). GH3 cells were treated with Phenol Red-free media in the absence or presence of either PCB138 or 153 (-10 to -6 M), or in combination (-10 to -6M) for up to 72h. Treatment with either PCB alone, or in combination, caused significant concentration-dependent, biphasic changes in cell proliferation at each time point, but with a different profile of response on each day (significantly increased at high pM/low nM concentrations); there was no evidence of toxicity at maximum concentrations (-6M). Gene expression changes were determined in GH3 cells treated in the absence or presence of either -8M or -6M PCB138 or 153 for 24h. Differential effects of these compounds were seen on the expression of Sstr3, Sstr4, Sstr5 and Insr; all other gene transcripts were unaffected. These findings reveal that GH3 cells exposed to physiologically relevant concentrations of PCB138 and 153, alone or in combination, show concentration-dependent increases in cell proliferation; furthermore, the expression of genes associated with therapeutic targets for the treatment of acromegaly (i.e. SSTRs) are differentially affected by exposure to PCB138 and 153. Our data indicate a potential mechanism for EDCs in the onset of acromegaly, that require further, in vivo, investigations.

Anti‐adipogenic Effects of Anthocyanin‐enriched Riceberry Rice Extract (Oryza sativa L.) in 3T3‐L1 Preadipocytes

Riceberry rice containing anthocyanins is a new pigmented variety of rice from Thailand with various biological activities such as antioxidant, anti‐inflammatory, anti‐hyperglycemia, and anti‐cancer. The present study aimed to investigate the effects of Riceberry rice extract (RBE) on adipogenic differentiation in mice 3T3‐L1 cells. RBE was obtained using water followed by freeze dryer lyophilization. Then, total phenolic compounds, total anthocyanin contents, and major anthocyanins in RBE were analyzed. The 3T3‐L1 preadipocytes were cultured in differentiation media and supplemented with RBE 1, 10, 20 and 50 μg/ml concentrations. After 8 days of incubation, cell viability, mature adipocyte number, triglyceride levels, and glucose uptake were determined. The mRNA expression of transcription factors and adipogenic genes including PPARγ, C/EBPα, FasN, ACC, aP2, AdipoQ, Leptin, Resistin, InsR, and Glut4 were also examined. The results revealed that RBE contained total phenolic compounds (63.33±2.4 mg gallic acid equivalent/g extract) and total anthocyanin (10.13±0.14 mg cyanidin‐3‐glucoside/g extract). The anthocyanins detected were cyanidin‐3‐glucoside and peonidin‐3‐glucoside. In 3T3‐L1 cells, RBE significantly reduced the number of mature adipocytes and triglyceride accumulation in a concentration‐dependent manner. In addition, the treatment of 3T3‐L1 cells with the various RBE concentrations did not cause cytotoxicity. Furthermore, the mRNA expression of major transcription factors, PPARγ and C/EBPα were significantly downregulated by RBE (20 and 50 μg/ml) in both early phase (day 3) and late phase (day 8) of adipogenesis. At 50 μg/ml RBE, there was a significant reduction in mRNA expression levels of all adipogenic genes. Interestingly, RBE (10, 20 and 50 μg/ml) significantly reduced glucose uptake into adipocytes via downregulated InsR and Glut4 mRNA expression. These findings suggest that RBE could potentially be used as an anti‐obesity agent by inhibiting adipogenesis and reducing glucose uptake in adipocytes.Support or Funding InformationDutsadi Phiphat Scholarship from Chulalongkorn University, LSU‐SVM CORP

Inhibitory effect of genistein and PTP1B on grasshopper Oedaleus asiaticus development

Protein tyrosine kinase (PTKs) and protein tyrosine phosphatase (PTPs) collectively regulate the insect insulin-signaling pathway (ISP), cell growth, metabolism, proliferation, differentiation, cell communication and monitoring of immune responses. We studied the inhibitory response of the grasshopper Oedaleus asiaticus Bey-Bienko to treat with the flavonoid genistein (PTK inhibitor), the enzyme PTP1B-IN-1 (PTP1B inhibitor) and control (dimethyl sulfoxide (DMSO)). Mean survival rates were not significantly different amongst the three treatments, but growth rate, body mass, and overall biological performance were reduced significantly following treatment with genistein. On comparison, treatment with PTP1B-IN-1 was not significantly different from the control. Relative gene expression of INSR, IRS, PI3K, PDK, and Akt was substantially lower (P < 0.05) following treatment with genistein compared to PTP1B-IN-1 and control. Similarly, gene expression and protein phosphorylation levels of the PTK5, PTP1B → FOXO cascade, indicators of stress response in the ISP, were also reduced in response to genistein treatments. The activity of the protective enzymes (POD and CAT) was upregulated by FOXO in response to PTP1B-IN-1. Our results demonstrated that genistein negatively regulates the ISP pathway, with a consequent effect on growth and development of O. asiaticus, as higher energy was required for detoxification. Given the negative effect of genistein on O. asiaticus physiology and development, the compound could be used in a biopesticide formulation for insect pest control in grasslands and other crop plants as a potential application to grasshopper biological control.

Could perturbed fetal development of the ovary contribute to the development of polycystic ovary syndrome in later life?

Polycystic ovary syndrome (PCOS) affects around 10% of young women, with adverse consequences on fertility and cardiometabolic outcomes. PCOS appears to result from a genetic predisposition interacting with developmental events during fetal or perinatal life. We hypothesised that PCOS candidate genes might be expressed in the fetal ovary when the stroma develops; mechanistically linking the genetics, fetal origins and adult ovarian phenotype of PCOS. In bovine fetal ovaries (n = 37) of 18 PCOS candidate genes only SUMO1P1 was not expressed. Three patterns of expression were observed: early gestation (FBN3, GATA4, HMGA2, TOX3, DENND1A, LHCGR and FSHB), late gestation (INSR, FSHR, and LHCGR) and throughout gestation (THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX and KRR1). A splice variant of FSHB exon 3 was also detected early in the bovine ovaries, but exon 2 was not detected. Three other genes, likely to be related to the PCOS aetiology (AMH, AR and TGFB1I1), were also expressed late in gestation. Significantly within each of the three gene groups, the mRNA levels of many genes were highly correlated with each other, despite, in some instances, being expressed in different cell types. TGFβ is a well-known stimulator of stromal cell replication and collagen synthesis and TGFβ treatment of cultured fetal ovarian stromal cells inhibited the expression of INSR, AR, C8H9orf3 and RAD50 and stimulated the expression of TGFB1I1. In human ovaries (n = 15, < 150 days gestation) many of the same genes as in bovine (FBN3, GATA4, HMGA2, FSHR, DENND1A and LHCGR but not TOX3 or FSHB) were expressed and correlated with each other. With so many relationships between PCOS candidate genes during development of the fetal ovary, including TGFβ and androgen signalling, we suggest that future studies should determine if perturbations of these genes in the fetal ovary can lead to PCOS in later life.

Sites and sources of sympathoexcitation in obese male rats: role of brain insulin.

In males, obesity increases sympathetic nerve activity (SNA), but the mechanisms are unclear. Here, we investigate insulin, via an action in the arcuate nucleus (ArcN), and downstream neuropathways, including melanocortin receptor 3/4 (MC3/4R) in the hypothalamic paraventricular nucleus (PVN) and dorsal medial hypothalamus (DMH). We studied conscious and α-chloralose-anesthetized Sprague-Dawley rats fed a high-fat diet, which causes obesity prone (OP) rats to accrue excess fat and obesity-resistant (OR) rats to maintain fat content, similar to rats fed a standard control (CON) diet. Nonspecific blockade of the ArcN with muscimol and specific blockade of ArcN insulin receptors (InsR) decreased lumbar SNA (LSNA), heart rate (HR), and mean arterial pressure (MAP) in OP, but not OR or CON, rats, indicating that insulin supports LSNA in obese males. In conscious rats, intracerebroventricular infusion of insulin increased MAP only in OP rats and also improved HR baroreflex function from subnormal to supranormal. The brain sensitization to insulin may elucidate how insulin can drive central SNA pathways when transport of insulin across the blood-brain barrier may be impaired. Blockade of PVN, but not DMH, MC3/4R with SHU9119 decreased LSNA, HR, and, MAP in OP, but not OR or CON, rats. Interestingly, nanoinjection of the MC3/4R agonist melanotan II (MTII) into the PVN increased LSNA only in OP rats, similar to PVN MTII-induced increases in LSNA in CON rats after blockade of sympathoinhibitory neuropeptide Y Y1 receptors. ArcN InsR expression was not increased in OP rats. Collectively, these data indicate that obesity increases SNA, in part via increased InsR signaling and downstream PVN MC3/4R.

Gestational diabetes and maternal obesity are associated with sex-specific changes in miRNA and target gene expression in the fetus.

Background/ObjectivePregnancies complicated by gestational diabetes (GDM) or maternal obesity have been linked to the development of diabetes, obesity and fatty liver disease later in life with sex-specific manifestations. Alterations in miRNA expression in offspring exposed to GDM and maternal obesity and effects on hepatic development are unknown. Here we describe how exposure to maternal obesity in utero leads to sex-specific changes in miRNA and target gene expression in human fetal liver.MethodsCandidate miRNA expression was measured in 2nd trimester amniotic fluid (AF) from women with GDM. Targets of differentially expressed miRNAs were determined and pathway enrichment of target genes was performed. MiRNA and target gene expression were measured in a separate cohort of 2nd trimester primary human fetal hepatocytes (PHFH) exposed to maternal obesity via QPCR and western blot. All studies were IRB approved.ResultsGDM exposed AF had significant increases in miRNAs 199a-3p, 503-5p, and 1268a (fold change (FC) ≥1.5, p<0.05). Female offspring specific analysis showed enrichment in miRNAs 378a-3p, 885-5p, and 7-1-3p (p<0.05). MiRNA gene targets were enriched in hepatic pathways. Key genes regulating de novo lipogenesis were upregulated in obesity exposed PHFH, especially in males. Significantly altered miRNAs in GDM AF were measured in obese exposed PHFH, with consistent increases in miRNAs 885-5p, 199-3p, 503-5p, 1268a and 7-1-3p (FC ≥1.5, p<0.05). Female PHFH exposed to maternal obesity had increased expression of miR-885-5p, miR-199-3p, miR-503-5p, miR-1268s and miR-7-1-3p, (p<0.05), corresponding to decreased target genes expression for ABCA1, PAK4 and INSR. In male PHFHs, no miRNA changes were measured but there was increased expression of ABCA1, PAK4, and INSR (p<0.05).ConclusionOur data suggest sex-specific changes in miRNA and gene expression in PHFH may be one mechanism contributing to the sexual dimorphism of metabolic disease in offspring exposed to GDM and maternal obesity in utero.

Differential Effects of Insulin and IGF1 Receptors on ERK and AKT Subcellular Distribution in Breast Cancer Cells.

Insulin and insulin-like growth factor-1 (IGF1) have important roles in breast cancer development. The recent identification of nuclear insulin (INSR) and IGF1 (IGF1R) receptors provides a novel paradigm in the area of signal transduction. The fact that INSR and IGF1R can function as transcription factors, capable of binding DNA and controlling transcription, adds a new layer of biological complexity by conferring upon cell-surface receptors the ability to regulate genomic events. The present study was designed to assess the hypothesis that insulin and IGF1 pathways elicit differential effects on subcellular distribution and activation of ERK1/2 and AKT. To this end, MCF7 breast cancer-derived cell lines with specific INSR or IGF1R disruption were employed. In addition, small interfering RNA technology was used to specifically down-regulate INSR or IGF1R expression in T47D breast cancer cells. DNA affinity chromatography assays were conducted to address the specific binding of ERK1/2 and AKT to the IGF1R promoter region. We demonstrate that both INSR and IGF1R exhibit a nuclear localization in breast cancer-derived cells. In addition, the insulin and IGF1 pathways have different effects on the subcellular distribution (and, particularly, the nuclear presence) of ERK1/2 and AKT molecules. Both cytoplasmic mediators are capable of binding and transactivating the IGF1R promoter. In conclusion, our data are consistent with the notion that, in addition to their classical roles as targets for insulin-like molecules, both ERK1/2 and AKT are involved in transcriptional control of the IGF1R gene. This previously unrecognized regulatory loop may provide mechanistic advantages to breast cancer cells. Given the potential role of INSR and IGF1R as therapeutic targets in oncology, it will be of clinical relevance to address the future use of nuclear receptors and their downstream cytoplasmic mediators as biomarkers for INSR/IGF1R targeted therapy.