Inhibition Of Insulin Signaling Research Articles

Short-Term Statin Therapy Induces Hepatic Insulin Resistance Through HNF4α/PAQR9/PPM1α Axis Regulated AKT Phosphorylation.

Statins, the first-line medication for dyslipidemia, are linked to an increased risk of type 2 diabetes. But exactly how statins cause diabetes is yet unknown. In this study, a developed short-term statin therapy on hyperlipidemia mice show that hepatic insulin resistance is a cause of statin-induced diabetes. Statin medication raises the expression of progesterone and adiponectin receptor 9 (PAQR9) in liver, which inhibits insulin signaling through degradation of protein phosphatase, Mg2+/Mn2+ dependent 1 (PPM1α) to activate ERK pathway. STIP1 homology and U-box containing protein 1 (STUB1) is found to mediate ubiquitination of PPM1α promoted by PAQR9. On the other hand, decreased activity of hepatocyte nuclear factor 4 alpha (HNF4α) seems to be the cause of PAQR9 expression under statin therapy. The interventions on PAQR9, including deletion of PAQR9, caloric restriction and HNF4α activation, are all effective treatments for statin-induced diabetes, while liver specific over-expression of PPM1α is another possible tactic. The results reveal the importance of HNF4α-PAQR9-STUB1-PPM1α axis in controlling the statin-induced hepatic insulin resistance, offering a fresh insight into the molecular mechanisms underlying statin therapy.

Insulin and TLR4 Antagonist Improve Motor Impairments in a Rat Model of Parkinson’s Disease

Background: Insulin resistance is an important pathological hallmark of Parkinson’s disease (PD). Proinflammatory cytokines during neuroinflammation decrease insulin sensitivity by suppressing insulin signaling elements. Toll-like receptor 4 (TLR4), the main receptor involved in neuroinflammation, is also associated with the pathogenesis of PD. Objectives: The present study evaluated the effect of insulin, an insulin receptor antagonist, and a TLR4 inhibitor on behavioral deficits and insulin resistance induced by 6-hydroxydopamine (6-OHDA). Methods: Male Wistar rats were divided into nine groups: (1) sham (normal saline [NS] in the medial forebrain bundle [MFB]); (2) 6-OHDA (20 µg in the MFB); (3) 6-OHDA + NS; (4) 6-OHDA + dimethyl sulfoxide (DMSO); (5) 6-OHDA + insulin (2.5 IU/day, intracerebroventricular ([ICV]); (6) 6-OHDA + insulin (5 IU/day, intranasal [IN]); (7) 6-OHDA + insulin receptor antagonist (S961; 6.5 nM/kg, ICV); (8) 6-OHDA + TLR4 antibody (TAK242; 0.01 µg/rat, ICV); (9) 6-OHDA + insulin + TLR4 antibody. All treatments were administered for seven consecutive days. Motor performance was evaluated using apomorphine-induced rotation and cylinder tests. Gene expression and protein levels of α-synuclein, TLR4, insulin receptor substrate (IRS) 1, IRS2, and glycogen synthase kinase 3β (GSK3β) were measured by real-time PCR and western blotting, respectively, in the striatum. Results: Insulin and insulin combined with TAK242 improved motor deficits induced by 6-OHDA. Administration of the insulin receptor antagonist had no effect on motor deficits. The increased expression of α-synuclein and TLR4 following 6-OHDA was attenuated by insulin and TAK242. GSK3β levels, both mRNA and protein, were significantly increased by 6-OHDA and attenuated with insulin and TAK242. Conclusions: The findings suggest that 6-OHDA induces neurodegeneration via activation of TLR4 and GSK3β, indicating insulin resistance, and that insulin can improve these impairments. Moreover, TLR4 inhibition prevents insulin signaling dysfunction and improves behavioral and molecular impairments, highlighting the critical role of TLR4 in the development of insulin resistance in PD pathology.

The Impact of FBN1-α5β1 Axis in Fibro/Adipogenic Progenitor Cells (FAPCD9-) on Intramuscular Fat Content in Pigs.

Intramuscular fat (IMF) plays a crucial role in enhancing meat quality, enriching meat flavor, and overall improving palatability. In this study, Single-cell RNA sequencing was employed to analyze the longissimus dorsi (LD) obtained from Guangdong small-ear spotted pigs (GDSS, with high IMF) and Yorkshire pigs (YK, with low IMF). GDSS had significantly more Fibro/Adipogenic Progenitor (FAPs), in which the CD9 negative FAPs (FAPCD9-) having adipogenic potential, as demonstrated by in vitro assays using cells originated from mouse muscle. On the other hand, Yorkshire had more fibro-inflammatory progenitors (FIPs, marked with FAPCD9+), presenting higher expression of the FBN1-Integrin α5β1. FBN1-Integrin α5β1 could inhibit insulin signaling in FAPCD9-, suppressing adipogenic differentiation. Our results demonstrated that fat-type pigs possess a greater number of FAPCD9-, which are the exclusive cells in muscle capable of differentiating into adipocytes. Moreover, lean-type pigs exhibit higher expression of FBN1-Integrin α5β1 axis, which inhibits adipocyte differentiation. These results appropriately explain the observed higher IMF content in fat-type pigs.

Queen bee gut microbiota extends honeybee lifespan by inhibiting insulin signaling.

Queen and worker bees share the same genetic background but have vastly different lifespans. The gut microbiome regulates host health, suggesting that differences in lifespan between queen and worker bees could be related to gut bacteria. Herein, we used an innovative method to transplant gut microbiota from adult queen or worker bees to microbiota-free bees. The transplantation of queen gut microbiota to microbiota-free bees extended their lifespan. Insulin/insulin-like growth factor signaling, a highly conserved metabolic pathway related to lifespan, displayed identical expression profiles in natural queen bees and microbiota-free bees transplanted with queen microbiota. This finding significantly expands our understanding of the relationships between intestinal bacteria, host health, and the biology of aging.

Antioxidant Effects of Gude Bean (Cajanus cajan) to Homa-IR and IRS-1 of High Fat and High Fructose Diet Rats

This study aimed to determine the effect of antioxidants in Gude bean (Cajanus cajan) treatment on HOMA-IR level and IRS-1 gene expression on the rats induced with high fat and high fructose diet. This study used 25 male white rats (Rattus norvegicus) in two control groups and three treatment groups. The measurement of HOMA-IR was done before and after treatment using the blood plasma while IRS-1 measurement was after treatment using the muscle tissue. The results showed that rat blood glucose level after being induced high fat and high fructose diet reached average value higher (p<0.05) compared to the Normal group. The Gude bean treatment resulted the decrease of HOMA-IR level (p<0.05) and the increase of IRS-1 (p<0.05). Gude bean treatment can inhibit insulin signal interference by ROS synthesis inhabitation by decreasing HOMA-IR level and increase IRS-1 gene expression in rats induced high fat and fructose diet.

GPER/PKA-Dependent Enhancement of Hormone-Sensitive Lipase Phosphorylation in 3T3-L1 Adipocytes by Piceatannol.

We previously reported that piceatannol (PIC) had an anti-obesity effect only in ovariectomized (OVX) postmenopausal obesity mice. PIC was found to induce the phosphorylation of hormone-sensitive lipase (pHSL) in OVX mice. To elucidate the mechanism by which PIC activates HSL, we investigated the effect of PIC using 3T3-L1 adipocytes. PIC induced HSL phosphorylation at Ser563 in 3T3-L1 cells, as in vivo experiments showed. pHSL (Ser563) is believed to be activated through the β-adrenergic receptor (β-AR) and protein kinase A (PKA) pathways; however, the addition of a selective inhibitor of β-AR did not inhibit the effect of PIC. The addition of a PKA inhibitor with PIC blocked pHSL (Ser563), suggesting that the effects are mediated by PKA in a different pathway than β-AR. The addition of G15, a selective inhibitor of the G protein-coupled estrogen receptor (GPER), reduced the activation of HSL by PIC. Furthermore, PIC inhibited insulin signaling and did not induce pHSL (Ser565), which represents its inactive form. These results suggest that PIC acts as a phytoestrogen and phosphorylates HSL through a novel pathway that activates GPER and its downstream PKA, which may be one of the inhibitory actions of PIC on fat accumulation in estrogen deficiency.

Attenuating DNA hypermethylation enhances insulin signaling and reduces apoptosis in an in vitro model of DLB

AbstractBackgroundDementia with Lewy bodies (DLB) is the second most prevalent degenerative dementia after Alzheimer’s disease (AD). The cause of DLB is known to be associated with abnormal protein aggregations called Lewy bodies within affected neurons. Studies have shown that Lewy bodies are mainly composed of α‐synuclein. Normal α‐synuclein is a water‐soluble monomer. However, when triggered by certain factors, the folding of the protein changes, causing it to aggregate and accumulate inside neurons. Previously, we have demonstrated that amyloid β (Aβ) can promote α‐synuclein aggregation and neurotoxicity, which provides a molecular mechanism that reasonably explained the pathogenesis of DLB. A major risk factor for DLB is known to be aging, but its role in disease progression, especially with regard to epigenetic regulation, is still unclear.MethodIn this study, we investigated whether DNA hypermethylation, a common phenomenon in cellular aging, might be a factor in the progression of DLB. Through the in vitro experimental mode of DLB, we analyzed the content of α‐synuclein aggregations, the difference in cell viability and the activity changes of methylation‐related pathways in mouse HT‐22 neuronal cells.ResultWe found that the DLB neuronal cells display a significant tendency to undergo DNA hypermethylation, which inhibits stemness and reduces the content of neurotrophic factors. This makes it more difficult for neuronal cells to survive under stress. In addition, DLB neuronal cells also showed significant insulin resistance, which interferes with autophagic clearance, upregulates the senescence‐related pathways, and exacerbates the aggregation and accumulation of α‐synuclein.ConclusionDLB cells significantly increase the hypermethylation and thus inhibit insulin signaling, making the affected cells more vulnerable to neurotoxicity. Accordingly, the administration of epigenetic regulators by targeting neuronal DNA hypermethylation may lead to develop novel therapeutic strategies against DLB.

Retraction Note: SOCS3 inhibits insulin signaling in porcine primary adipocytes.

Retraction Note: SOCS3 inhibits insulin signaling in porcine primary adipocytes.

High-fructose consumption suppresses insulin signaling pathway accompanied by activation of macrophage and apoptotic markers in rat testis

Dietary high-fructose may cause metabolic disturbances; however, its effect on the reproductive system is little understood. The insulin signaling pathway is critical in testicular development, maintenance of microcirculation and spermatogenesis. Therefore, in this study, we aimed to investigate the impact of dietary high-fructose on insulin signaling pathway as well as macrophage and apoptotic markers in testicular tissue of rats. Fructose was administered to male Wistar rats as a 20% solution in drinking water for fifteen-week. Gene expression of ir-β, irs-1, irs-2, pi3k, akt, mtor, and enos in the testicular samples was determined by real-time PCR. Protein expression of IR, IRS-1, IRS-2, PI3K, Akt, phospho-Akt (p-Akt), mTOR, eNOS, phospho-eNOS (p-eNOS), and GLUT5 was established by analysis of Western Blot. Testicular expression of occludin, CD163, CD68, caspase-8, and caspase-3 was analyzed by using immunohistochemical assay. Testicular level of fructose was measured by colorimetric method. Dietary high-fructose decreased mRNA expressions of irs-1, irs-2, pi3k, and mtor in the testicular tissue of rats. Also, this dietary intervention impaired protein expressions of IR, IRS-1, IRS-2, PI3K, p-Akt, mTOR, eNOS, and p-eNOS as well as p-Akt/Akt and p-eNOS/eNOS ratios in the testis of rats. However, a high-fructose diet increased the expression of CD163, CD68, caspase-8 and caspase-3, but decreased that of occludin, in the testicular tissue of rats. The high-fructose consumption in rats suppresses testicular insulin signaling but activates macrophages-related factors and apoptotic markers. These changes induced by dietary fructose could be related to male reproductive dysfunction.

SAT053 Development Of A Novel Peptide-based Insulin Sensitizer Through Disruption Of Inositol Polyphosphate 5-Phosphatase K (SKIP) - p21-activated Kinase 1 (Pak1) Interactions

Abstract Disclosure: A.M. Kimbrough: None. J. Dev Gaddameedi: None. A.M. Lipchik: None. Type 2 diabetes (T2D) is a prominent, chronic metabolic disease that affects approximately 462 million people worldwide. The driving force behind T2D is insulin resistance (IR), defined as an impaired biological response to insulin, and leads to a multitude of more severe conditions including heart disease, stroke, and neuropathy. Most therapies fail to improve insulin sensitivity, and instead drive physiological compensation. It is imperative to develop new therapies to correct the underlying pathology by targeting mechanisms of IR. Inositol polyphosphate 5-phosphatase K (INPP5K/SKIP) is a potential target to treat IR. SKIP resides in the endoplasmic reticulum (ER) in resting cells, and translocates to the plasma membrane (PM) following growth factor stimulation. SKIP is recruited to the PM through binding to p21 activated kinase 1 (Pak1) and inhibits growth factor signaling through dephosphorylation of phosphoinositol-(3,4,5)-triphosphate. In obesity and IR, SKIP is overexpressed leading to PM localization and suppression of insulin signaling. Here, we developed a novel insulin sensitizer utilizing a peptide derived from Pak1 to inhibit SKIP-Pak1 interactions and prevent PM localization. We found that the Pak1 derived peptide displayed alpha helical properties and thermostabilized SKIP. In controlled in vitro assays, the Pak1 peptide was successfully taken up in target tissues. Additionally, treatment with the Pak1 peptides was sufficient to restore insulin sensitivity in skeletal muscle, adipose, and liver models of IR. Together these results demonstrate the use of a peptide-based insulin sensitizer as a novel therapeutic for T2D that requires further investigation. Presentation: Saturday, June 17, 2023

Insulin Inhibits Autophagy by Inhibiting the Binding of FoXO1 to the Promoter Region of GABARAPL1.

Hyperinsulinemia and insulin resistance in T2D have a potent suppressive effect on hepatic autophagy, however, the underlying mechanisms remain unclear. To explore the effect of insulin on hepatic autophagy and its possible signaling pathways, HL-7702 cells were treated with insulin with or without insulin signaling inhibitors. The interaction between insulin and the promoter region of GABARAPL1 was assessed through luciferase assay and EMSA. There were significant dose-dependent decreases in the number of intracellular autophagosomes and the protein levels of GABARAPL1 and beclin1 in insulin-treated HL-7702 cells. Insulin signaling inhibitors reversed the inhibitory effect of insulin on rapamycin-induced autophagy and autophagy-related gene upregulation. Insulin blocks the binding of FoxO1 to putative insulin response elements in GABARAPL1 gene promoter, leading to the repressed transcription of GABARAPL1 gene and the suppression of hepatic autophagy. Our study identified GABARAPL1 as a novel target of insulin in suppressing hepatic autophagy.

Regulated translocation of neutral sphingomyelinase-2 to the plasma membrane drives insulin resistance in steatotic hepatocytes

Obesity-associated diabetes is linked to the accumulation of ceramide in various organs, including the liver. The exact mechanisms by which ceramide contributes to diabetic pathology are unclear, but one proposed scenario is that ceramide accumulation may inhibit insulin signaling pathways. It is unknown however whether the excess ceramide is generated proximal to the insulin receptor, that is, at the plasma membrane (PM), where it could affect the insulin signaling pathway directly, or the onset of insulin resistance is due to ceramide-induced mitochondrial dysfunction and/or lipotoxicity. Using hepatic cell lines and primary cultures, gain- and loss- of function approach, and state-of-the art lipid imaging, this study shows that PM-associated neutral sphingomyelinase 2 (nSMase2) regulates ceramide homeostasis in fat-loaded hepatocytes and drives the onset of insulin resistance. Our results provide evidence of a regulated translocation of nSMase2 to the PM which leads to local generation of ceramide and insulin resistance in cells treated with palmitic acid (PAL), a type of fat commonly found in diabetogenic diets. Oleic acid, which also causes accumulation of lipid droplets, does not induce nSMase2 translocation and insulin resistance. Experiments using the acyl-biotin exchange method to quantify protein palmitoylation show that cellular PAL abundance regulates the rate of nSMase2 palmitoylation. Furthermore, while inhibition of nSMase2 with GW4869 prevents PAL-induced insulin resistance, the overexpression of wild type nSMase2 but not palmitoylation-defective mutant protein potentiates the suppressive effect of PAL on insulin signaling. Overall, this study identifies nSMase2 as a novel component of the mechanism of insulin resistance onset in fat-loaded hepatocytes, that is, cell-autonomous and driven by PAL.

Acrolein induces mitochondrial dysfunction and insulin resistance in muscle and adipose tissues in vitro and in vivo.

Type 2 diabetes mellitus (DM) is a common chronic condition characterized by persistent hyperglycemia and is associated with insulin resistance (IR) in critical glucose-consuming tissues, including skeletal muscle and adipose tissue. Oxidative stress and mitochondrial dysfunction are known to play key roles in IR. Acrolein is a reactive aldehyde found in the diet and environment that is generated as a fatty acid product through the glucose autooxidation process under hyperglycemic conditions. Our previous studies have shown that acrolein impairs insulin sensitivity in normal and diabetic mice, and this effect can be reversed by scavenging acrolein. This study demonstrated that acrolein increased oxidative stress and inhibited mitochondrial respiration in differentiated C2C12 myotubes and differentiated 3T3-L1 adipocytes. As a result, insulin signaling pathways were inhibited, leading to reduced glucose uptake. Treatment with acrolein scavengers, N-acetylcysteine, or carnosine ameliorated mitochondrial dysfunction and inhibited insulin signaling. Additionally, an increase in acrolein expression correlated with mitochondrial dysfunction in the muscle and adipose tissues of diabetic mice. These findings suggest that acrolein-induced mitochondrial dysfunction contributes to IR, and scavenging acrolein is a potential therapeutic approach for treating IR.

VAPB-mediated ER-targeting stabilizes IRS-1 signalosomes to regulate insulin/IGF signaling

The scaffold protein IRS-1 is an essential node in insulin/IGF signaling. It has long been recognized that the stability of IRS-1 is dependent on its endomembrane targeting. However, how IRS-1 targets the intracellular membrane, and what type of intracellular membrane is actually targeted, remains poorly understood. Here, we found that the phase separation-mediated IRS-1 puncta attached to endoplasmic reticulum (ER). VAPB, an ER-anchored protein that mediates tethers between ER and membranes of other organelles, was identified as a direct interacting partner of IRS-1. VAPB mainly binds active IRS-1 because IGF-1 enhanced the VAPB-IRS-1 association and replacing of the nine tyrosine residues of YXXM motifs disrupted the VAPB-IRS-1 association. We further delineated that the Y745 and Y746 residues in the FFAT-like motif of IRS-1 mediated the association with VAPB. Notably, VAPB targeted IRS-1 to the ER and subsequently maintained its stability. Consistently, ablation of VAPB in mice led to downregulation of IRS-1, suppression of insulin signaling, and glucose intolerance. The amyotrophic lateral sclerosis (ALS)-derived VAPB P56S mutant also impaired IRS-1 stability by interfering with the ER-tethering of IRS-1. Our findings thus revealed a previously unappreciated condensate-membrane contact (CMC), by which VAPB stabilizes the membraneless IRS-1 signalosome through targeting it to ER membrane.

PKCα Isoform Inhibits Insulin Signaling and Aggravates Neuronal Insulin Resistance.

Overexpression of PKCα has been linked to inhibit insulin signaling disrupting IRS-1 and Akt phosphorylations in skeletal muscle. PKCα inhibits IRS-1 and Akt phosphorylations, but not required for insulin-stimulated glucose transport in skeletal muscles. Inhibition of PKCα increased whereas in some studies decreased GLUT-4 levels at the plasma membrane in skeletal muscles and adipocytes. Controversial studies have reported opposite expression pattern of PKCα expression in insulin-resistant skeletal muscles. These findings indicate that the role of PKCα on insulin signaling is controversial and could be tissue specific. Evidently, studies are required to decipher the role of PKCα in regulating insulin signaling and preferably in other cellular systems. Utilizing neuronal cells, like Neuro-2a, SHSY-5Y and insulin-resistant diabetic mice brain tissues; we have demonstrated that PKCα inhibits insulin signaling, through IRS-Akt pathway in PP2A-dependent mechanism by an AS160-independent route involving 14-3-3ζ. Inhibition and silencing of PKCα improves insulin sensitivity by increasing GLUT-4 translocation to the plasma membrane and glucose uptake. PKCα regulates GSK3 isoforms in an opposite manner in insulin-sensitive and in insulin-resistant condition. Higher activity of PKCα aggravates insulin-resistant neuronal diabetic condition through GSK3β but not GSK3α. Our results mechanistically explored the contribution of PKCα in regulating neuronal insulin resistance and diabetes, which opens up new avenues in dealing with metabolic disorders and neurodegenerative disorders.

Azadirachtin exposure inhibit ovary development of Spodoptera litura (Lepidoptera: Noctuidae) by altering lipids metabolism event and inhibiting insulin signaling pathways

Lipids are main energy source for insects reproduction, which are becoming emerging target for pest management. Azadirachtin (AZA) is a multi-targeted and promising botanical insecticide, but its reproduction toxicity mechanism related to lipids metabolism is poorly understood. Here, we applied lipidomic and transcriptomic to provide a comprehensive resource for describing the effect of AZA on lipids remodeling in ovary of Spodoptera litura. The results showed that AZA exposure obviously altered the contents of 130 lipids subclasses (76 upregulated and 54 downregulated). In detail, AZA exposure changed the length and saturation degrees of fatty acyl chain of most glycerolipid, phospholipid and sphingolipid as well as the expression of genes related to biosynthesis of unsaturated fatty acids and fatty acids elongation. Besides, following the abnormal lipids metabolism, western blot analysis suggested that AZA induce insulin resistance-like phenotypes by inhibiting insulin receptor substrates (IRS) /PI3K/AKT pathway, which might be responsible for the ovary abnormalities of S. litura. Collectively, our study provided insights into the lipids metabolism event in S. litura underlying AZA exposure, these key metabolites and genes identified in this study would also provide important reference for pest control in future.

291-OR: CREG1 Regulates Hepatic Insulin Sensitivity and Insulin Receptor Trafficking

CREG1 is a small glycoprotein in the endosomal-lysosomal system involved in endocytic trafficking. It contains 3 N-linked glycosylation sites and is highly expressed in the liver. To elucidate its physiological functions, we generated Creg1fl/fl mice using CRISPR technology. Whole-body and hepatocyte-specific deletion of Creg1 was achieved by crossing Creg1fl/fl with UBC-cre-ERT2 and Albumin-cre mice, respectively. Both knockout mice developed elevated glycemia, glucose intolerance, and resistance to the blood glucose-lowering effect of insulin. Loss of hepatic CREG1 also aggravated high-fat diet-induced insulin resistance and hyperinsulinemia. This is likely caused by impaired insulin receptor (IR) trafficking because IR expression on the hepatocyte surface was significant reduced in hepatocyte specific Creg1 knockout mice. In in vitro studies using cultured hepatocytes, CREG1 overexpression increased IR recycling to the cell surface and enhanced insulin signaling, whereas its knockdown had the opposite effect. These findings indicate a cell autonomous role of CREG1 in hepatocyte IR trafficking and insulin signaling. Furthermore, SNP database survey identified 3 missense mutations of CREG1 at the N216 glycosylation site (N216I, N216K, and N216Y). When overexpressed in human hepatocytes, the N216I mutant inhibited insulin signaling in a dominant negative fashion. Taken together, these results suggest CREG1 regulates hepatic insulin sensitivity and signaling likely through its effect on IR trafficking. CREG1 glycosylation at N216 may be required for its function in these processes. Disclosure Y.Qi: None. J.Liu: None. J.C.Chao: None. S.A.Rahimi: None. L.Y.Lee: Speaker's Bureau; Abbott. S.Li: None. Funding Rutgers Biomedical and Health Sciences

Paeoniflorin mitigates high glucose-induced lifespan reduction by inhibiting insulin signaling in Caenorhabditis elegans.

In organisms, high glucose can cause several aspects of toxicity, including the lifespan reduction. Paeoniflorin is the major component of Paeoniaceae plants. Nevertheless, the possible effect of paeoniflorin to suppress high glucose toxicity in reducing lifespan and underlying mechanism are largely unclear. Thus, in this study, we examined the possible effect of paeoniflorin in suppressing high glucose (50mM)-induced lifespan reduction and the underlying mechanism in Caenorhabditis elegans. Administration with 16-64mg/L paeoniflorin could prolong the lifespan in glucose treated nematodes. Accompanied with this beneficial effect, in glucose treated nematodes, expressions of daf-2 encoding insulin receptor and its downstream kinase genes (age-1, akt-1, and akt-2) were decreased and expression of daf-16 encoding FOXO transcriptional factor was increased by 16-64mg/L paeoniflorin administration. Meanwhile, the effect of paeoniflorin in extending lifespan in glucose treated nematodes was enhanced by RNAi of daf-2, age-1, akt-1, and akt-2 and inhibited by RNAi of daf-16. In glucose treated nematodes followed by paeoniflorin administration, the increased lifespan caused by daf-2 RNAi could be suppressed by RNAi of daf-16, suggesting that DAF-2 acted upstream of DAF-16 to regulate pharmacological effect of paeoniflorin. Moreover, in glucose treated nematodes followed by paeoniflorin administration, expression of sod-3 encoding mitochondrial Mn-SOD was inhibited by daf-16 RNAi, and the effect of paeoniflorin in extending lifespan in glucose treated nematodes could be suppressed by sod-3 RNAi. Molecular docking analysis indicated the binding potential of paeoniflorin with DAF-2, AGE-1, AKT-1, and AKT-2. Therefore, our results demonstrated the beneficial effect of paeoniflorin administration in inhibiting glucose-induced lifespan reduction by suppressing signaling cascade of DAF-2-AGE-1-AKT-1/2-DAF-16-SOD-3 in insulin signaling pathway.

Effects of early life overnutrition and hyperandrogenism on spatial learning and memory in a rat model of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a complex disorder characterized by endocrine and metabolic abnormalities such as obesity and insulin resistance. PCOS is also associated with psychiatric disorders and cognitive impairment. The animal model of PCOS was induced by treating rats with 5α-dihydrotestosterone (5α-DHT) and additionally modified to induce adiposity by litter size reduction (LSR). Spatial learning and memory were assessed using the Barnes Maze test, and striatal markers of synaptic plasticity were analyzed. Striatal insulin signaling was estimated by the levels of insulin receptor substrate 1 (IRS1), its inhibitory phosphorylation at Ser307, and glycogen synthase kinase-3α/β (GSK3α/β) activity. Both LSR and DHT treatment significantly decreased striatal protein levels of IRS1, followed by increased GSK3α/β activity in small litters. Results of the behavioral study showed that LSR had a negative effect on learning rate and memory retention, whereas DHT treatment did not induce impairment in memory formation. While protein levels of synaptophysin, GAP43, and postsynaptic density protein 95 (PSD-95) were not altered by the treatments, DHT treatment induced an increase in phosphorylation of PSD-95 at Ser295 in both normal and small litters. This study revealed that LSR and DHT treatment suppressed insulin signaling by downregulating IRS1 in the striatum. However, DHT treatment did not have an adverse effect on learning and memory, probably due to compensatory elevation in pPSD-95-Ser295, which had a positive effect on synaptic strength. This implies that hyperandrogenemia in this setting does not represent a threat to spatial learning and memory, opposite to the effect of overnutrition-related adiposity.

Pit 1 transporter (SLC20A1) as a key factor in the NPP1-mediated inhibition of insulin signaling in human podocytes.

Podocytes are crucially involved in blood filtration in the glomerulus. Their proper function relies on efficient insulin responsiveness. The insulin resistance of podocytes, defined as a reduction of cell sensitivity to this hormone, is the earliest pathomechanism of microalbuminuria that is observed in metabolic syndrome and diabetic nephropathy. In many tissues, this alteration is mediated by the phosphate homeostasis-controlling enzyme nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). By binding to the insulin receptor (IR), NPP1 inhibits downstream cellular signaling. Our previous research found that hyperglycemic conditions affect another protein that is involved in phosphate balance, type III sodium-dependent phosphate transporter 1 (Pit 1). In the present study, we evaluated the insulin resistance of podocytes after 24 h of incubation under hyperinsulinemic conditions. Thereafter, insulin signaling was inhibited. The formation of NPP1/IR complexes was observed at that time. A novel finding in the present study was our observation of an interaction between NPP1 and Pit 1 after the 24 h stimulation of podocytes with insulin. After downregulation of the SLC20A1 gene, which encodes Pit 1, we established insulin resistance in podocytes that were cultured under native conditions, manifested as a lack of intracellular insulin signaling and the inhibition of glucose uptake via the glucose transporter type 4. These findings suggest that Pit 1 might be a major factor that participates in the NPP1-mediated inhibition of insulin signaling.