NAA25 as a regulator of insulin signaling: Integration of FOXO1 imaging CRISPRi screen and Mendelian randomization.

Disclosure: W. He: None. N. Loganathan: None. K.W. Mak: None. E. McIlwraith: None. D.D. Belsham: None. Insulin signals through the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R) in hypothalamic neurons to control food intake and peripheral metabolism. A contributor to obesity, and subsequent comorbidities such type 2 diabetes and heart disease, is the development of cellular insulin resistance in hypothalamic neurons. However, the molecular changes to neuronal insulin signaling remain to be fully elucidated. MicroRNAs (miRNAs) inhibit translation of specific mRNAs; thus, this study aimed to understand the involvement of miRNAs in the regulation of insulin signaling and resistance in hypothalamic neurons. To profile miRNAs expressed in hypothalamic neurons, RNA from the whole hypothalami of 14-week-old CD1 male mice (n = 4), as well as the immortalized hypothalamic neuronal cell lines mHypoE-46 (n = 3) and mHypoA-59 (n = 3), each co-expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP), were assessed using the Affymetrix GeneChip miRNA 4.0 Array. Notably, miR-16 family members, including miR-16-5p, miR-15b-5p, and miR-322-5p, were among the most highly expressed miRNAs in each case. In the mHypoA-59 neurons, overexpression of miR-322-5p for 24 hours decreased the mRNA levels of Igf1r (-26.1%; p = 0.0007; n = 4) and the protein level of INSR-beta (-31.8%; p = 0.0023). These results suggest that the miR-16 family plays an inhibitory role in insulin signaling in hypothalamic neurons. To study the involvement of miRNAs in hyperinsulinemia-induced neuronal insulin resistance, the mHypoE-46 neurons were treated with 100 nM insulin for 24 hours to induce cellular insulin resistance, as characterized by decreased INSR-beta protein (-96.3%; p < 0.0001; n = 4) and a resulting decrease in insulin-induced phosphorylation of protein kinase B (AKT) (-67.6%; p = 0.01; n = 4). GeneChip miRNA array analysis showed that insulin overexposure disrupted the expression of 48 miRNAs (p < 0.05, n = 3), including the upregulation of miR-18a-3p, mir-322, miR-494-3p, and miR-671-3p. Upon RT-qPCR validation (n = 3), we established that prolonged (24 hours), but not acute (1-6 hours), insulin exposure upregulated miR-18a-3p (+62%; p = 0.0104), miR-671-3p (+111%; p = 0.0079), and miR-1983 (+97%; p = 0.0181). miR-671-3p, miR-494-3p, and miR-18a-3p have been shown to decrease phosphatase and tensin homolog (PTEN) levels, which can promote neuronal insulin resistance based on bioinformatic analysis. Overall, these results suggest hyperinsulinemia disrupts the expression of specific miRNAs in hypothalamic neurons to promote cellular insulin resistance. Knowledge derived from these studies will provide insight into hypothalamus-derived miRNAs that could be targeted for miRNA-based diagnostics and therapeutics for early central insulin resistance in humans.(Supported by the CRC, CIHR, NSERC, EndoSoc, and BBDC) Presentation: Saturday, June 17, 2023

Inflammatory processes can disrupt tissue homeostasis and promote metabolic disturbances, including insulin resistance. Pro-inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6), mediate this process. Since skeletal muscle is one of the major insulin-sensitive tissues, it is crucial to search for molecular links that promote insulin resistance during inflammation. Protein tyrosine phosphatases (PTPs) negatively regulate insulin signaling in multiple organs and models. To gain insight into the potential interactions of cytokines and PTPs in skeletal muscle, we characterized the effects and kinetics of cytokine stimulation on insulin signaling in murine C2C12 muscle cells. The protein level and activities of PTP non-receptor type 1 (PTPN1/PTP1B) and type 2 (PTPN2/TCPTP) were evaluated after cytokine stimulation and addressed by pharmacological inhibition or siRNA-mediated knockdown (KD). TNF, IL-1β and IL-6 elicited different kinetics and expression patterns of the respective PTPs and insulin signaling molecules, while surprisingly, insulin action was preserved. Furthermore, PTPN1 and PTPN2 had only minor effects on insulin signaling in C2C12 cells with siRNA-mediated compensatory PTP regulation. However, pan-PTP inhibition by sodium orthovanadate confirms that PTPs are negative regulators of insulin signaling. In summary, our data provide insights into the balance of the physiological and pathophysiological effects of cytokines and targeting individual PTPs to regulate insulin signaling in C2C12 cells. Additionally, our findings highlight the complex dynamics and interplay of cytokines, PTPs and metabolic pathways. This should be acknowledged when new therapeutic tools are developed to address inflammation-induced diseases such as type 2 diabetes or related comorbidities.

190-OR: Lrtm1, a Novel Regulator of Insulin Signaling and Metabolism

Regulation of insulin signaling by microRNAs in smooth muscle cells may contribute to diabetic vascular disease. The two smooth muscle enriched miRNAs miR-143 and miR-145 have been reported to target mediators of insulin signaling in non-smooth muscle cells. In this study, we aimed to determine the importance of this regulation in vascular smooth muscle cells, where expression of miR-143/145 is much higher than in other cell types.Smooth muscle cells deficient of the miR-143/145 cluster were used, as well as smooth muscle cells transfected with mimics/inhibitors for either miR-143 or miR-145.We found that deletion of miR-143/145 in smooth muscle results in a dramatic upregulation IRS-1 expression and insulin signaling, and an increased insulin-induced glucose uptake. Furthermore, specific modulation of either miR-145 or miR-143 expression regulated specific targets (IRS-1, ORP8 and the IGF-1 receptor) in the insulin signaling pathway. Consequently, transient inhibition or overexpression of either miR-143 or miR-145 was sufficient to regulate insulin signaling in smooth muscle cells.In conclusion, the results of this study support an important role for both miR-143 and miR-145 in the regulation of insulin signaling and glucose uptake in vascular smooth muscle cells.

Insulin resistance is a major aspect of type 2 diabetes (T2D), which results from impaired insulin signaling in target cells. Signaling to regulate forkhead box protein O1 (FOXO1) may be the most important mechanism for insulin to control transcription. Despite this, little is known about how insulin regulates FOXO1 and how FOXO1 may contribute to insulin resistance in adipocytes, which are the most critical cell type in the development of insulin resistance. We report a detailed mechanistic analysis of insulin control of FOXO1 in human adipocytes obtained from non-diabetic subjects and from patients with T2D. We show that FOXO1 is mainly phosphorylated through mTORC2-mediated phosphorylation of protein kinase B at Ser(473) and that this mechanism is unperturbed in T2D. We also demonstrate a cross-talk from the MAPK branch of insulin signaling to stimulate phosphorylation of FOXO1. The cellular abundance and consequently activity of FOXO1 are halved in T2D. Interestingly, inhibition of mTORC1 with rapamycin reduces the abundance of FOXO1 to the levels in T2D. This suggests that the reduction of the concentration of FOXO1 is a consequence of attenuation of mTORC1, which defines much of the diabetic state in human adipocytes. We integrate insulin control of FOXO1 in a network-wide mathematical model of insulin signaling dynamics based on compatible data from human adipocytes. The diabetic state is network-wide explained by attenuation of an mTORC1-to-insulin receptor substrate-1 (IRS1) feedback and reduced abundances of insulin receptor, GLUT4, AS160, ribosomal protein S6, and FOXO1. The model demonstrates that attenuation of the mTORC1-to-IRS1 feedback is a major mechanism of insulin resistance in the diabetic state.

The present study was aimed to investigate the protective role of metformin in type 2 diabetic nephropathic rats and elucidate regulation of unwanted insulin signaling in the kidney as the underlying mechanism. Diabetes mellitus was established in male Sprague Dawley rats by a single intraperitoneal injection of 40 mg/kg streptozotocin. A sustained level of blood glucose over 16.7 mM was defined as type 2 diabetes mellitus in this study. Rats were randomly classified into the normal group, diabetes mellitus group, diabetes mellitus plus low-dose and high-dose metformin groups (100 and 200 mg/kg orally). Rats received treatment for 12 weeks after which all rats were sacrificed and kidney samples were collected. The samples were dissected and prepared for the observation of structural changes of renal glomerular podocytes between the groups microscopically. The expressions of IRS-1, Akt, nephrin, desmin, phosphatidylinositol-3-hydroxy kinase (PI3K)-p85 and extra cellular matrix were probed by western blot. Compared to the control group, the downregulated nephrin and elevated levels of desmin and collagen IV in the kidney were significantly reversed by metformin treatment. Furthermore, metformin recovered the atypical activities of insulin/PI3K-Akt signaling to the normal ranges. Metformin evidently reduced the renal damage in diabetic rats, which could be due to modulating lipid metabolism and insulin resistance.

Type 2 diabetes is characterized by insulin resistance in the expanding adipose tissue of obesity. The insulin resistance manifests in human adipocytes as system-wide impairment of insulin signalling. An exception is the regulation of transcription factor FOXO1 (forkhead box protein O1), which is phosphorylated downstream of mTORC2 (mammalian/mechanistic target of rapamycin in complex with raptor) and is therefore not exhibiting impaired response to insulin. However, the abundance, and activity, of FOXO1 is reduced by half in adipocytes from patients with diabetes. To elucidate the effect of reduced FOXO1 activity, we here transduced human adipocytes with a dominant-negative construct of FOXO1 (DN-FOXO1). Inhibition of FOXO1 reduced the abundance of insulin receptor, glucose transporter-4, ribosomal protein S6, mTOR and raptor. Functionally, inhibition of FOXO1 induced an insulin-resistant state network-wide, a state that qualitatively and quantitatively mimicked adipocytes from patients with type 2 diabetes. In contrast, and in accordance with these effects of DN-FOXO1, overexpression of wild-type FOXO1 appeared to augment insulin signalling. We combined experimental data with mathematical modelling to show that the impaired insulin signalling in FOXO1-inhibited cells to a large extent can be explained by reduced mTORC1 activity - a mechanism that defines much of the diabetic state in human adipocytes. Our findings demonstrate that FOXO1 is critical for maintaining normal insulin signalling of human adipocytes.

Insulin signaling is a conserved pathway that orchestrates glucose and lipid metabolism, energy balance, and inflammation, and its dysregulation compromises the homeostasis of multiple systems. Insulin resistance is a shared hallmark of several metabolic diseases, including obesity, metabolic syndrome, and type 2 diabetes, and has been associated with cognitive decline during aging and dementia. Numerous mechanisms promoting the development of peripheral and central insulin resistance have been described, although most of them were not completely clarified. In the last decades, several studies have highlighted that biliverdin reductase-A (BVR-A), over its canonical role in the degradation of heme, acts as a regulator of insulin signaling. Evidence from human and animal studies show that BVR-A alterations are associated with the aberrant activation of insulin signaling, metabolic syndrome, liver steatosis, and visceral adipose tissue inflammation in obese and diabetic individuals. In addition, recent findings demonstrated that reduced BVR-A levels or impaired BVR-A activation contribute to the development of brain insulin resistance and metabolic alterations in Alzheimer’s disease. In this narrative review, we will provide an overview on the literature by focusing on the role of BVR-A in the regulation of insulin signaling and how BVR-A alterations impact on cell dysfunctions in both metabolic and neurodegenerative disorders.

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes.

Suppression of the expression of the heterotrimeric G-protein Galpha(i2) in vivo has been shown to provoke insulin resistance, whereas enhanced insulin signaling is observed when Galpha(i2) is overexpressed in vivo. The basis for Galpha(i2) regulation of insulin signaling was explored in transgenic mice with targeted expression of the GTPase-deficient, constitutively active Q205L Galpha(i2) in fat and skeletal muscle. Phosphorylation of insulin receptor and IRS-1 in response to insulin challenge in vivo was markedly amplified in fat and skeletal muscle expressing Q205L Galpha(i2). The expression and activity of the protein-tyrosine phosphatase 1B (PTP1B), but not protein-tyrosine phosphatases SHP-1, SHP-2, and LAR, were constitutively decreased in tissues expressing the Q205L Galpha(i2), providing a direct linkage between insulin signaling and Galpha(i2). The loss of PTP1B expression may explain, in part, the loss of PTP1B activity in the iQ205L transgenic mice. Activation of Galpha(i2) in mouse adipocytes with lysophosphatidic acid was shown to decrease PTP1B activity, whereas pertussis toxin inactivates Galpha(i2), blocks lysophosphatidic acid-stimulated inhibition of PTP1B activity, and blocks tonic suppression of PTP1B activity by Galpha(i2). Elevation of intracellular cAMP in fat cells is shown to increase PTP1B activity, whereas either depression of cAMP levels or direct activation of Galpha(i2) suppresses PTP1B. These data provide the first molecular basis for the interplay between Galpha(i2) and insulin signaling, i.e. activation of Galpha(i2) can suppress both the expression and activity of PTP1B in insulin-sensitive tissues.

Insulin resistance is associated with obesity, cardiovascular disease, non-alcoholic fatty liver disease, and type 2 diabetes. These complications are exacerbated by a high-calorie diet, which we used to model type 2 diabetes in Drosophila melanogaster Our studies focused on the fat body, an adipose- and liver-like tissue that stores fat and maintains circulating glucose. A gene regulatory network was constructed to predict potential regulators of insulin signaling in this tissue. Genomic characterization of fat bodies suggested a central role for the transcription factor Seven-up (Svp). Here, we describe a new role for Svp as a positive regulator of insulin signaling. Tissue-specific loss-of-function showed that Svp is required in the fat body to promote glucose clearance, lipid turnover, and insulin signaling. Svp appears to promote insulin signaling, at least in part, by inhibiting ecdysone signaling. Svp also impairs the immune response possibly via inhibition of antimicrobial peptide expression in the fat body. Taken together, these studies show that gene regulatory networks can help identify positive regulators of insulin signaling and metabolic homeostasis using the Drosophila fat body.

Insulin resistance is the hallmark of type 2 diabetes; however, the mechanism underlying the development of insulin resistance is still not completely understood. Previous reports showed that posttranslational modifications of IRS play a critical role in insulin signaling, especially the phosphorylation of IRS by distinct kinases. While it is known that increasing Sirtuin1 deacetylase activity improves insulin sensitivity in the liver, the identity of its counterpart, an acetyl-transferase, remains unknown. Our recent study shows that elevated endotoxin (LPS) levels in the liver of obese mice lead to the induction of the acetyl-transferase P300 through the IRE1-XBP1s pathway. Subsequently, induced P300 impairs insulin signaling by acetylating IRS1 and IRS2 in the insulin signaling pathway. Therefore, the P300 acetyl-transferase activity appears to be a promising therapeutic target for the treatment of diabetes.

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.

PTEN and SHIP2 regulates PI3K/Akt pathway through focal adhesion kinase

The overproduction of very low density lipoprotein (VLDL) is an important cause for initiation and development of atherosclerosis, which is highly associated with insulin signaling. The aim of this work is to verify whether the inhibition of VLDL overproduction is an underlying mechanism for a Laminaria japonica polysaccharide (LJP61A (where LJP is L. japonica)) to resist atherosclerosis. LJP61A (50 and 200 mg/kg/day) was orally administered to a high-fat diet (HFD)-fed LDL receptor deficient mice for 14 weeks. LJP61A significantly attenuated insulin resistance, hepatic steatosis, atherosclerosis, and dyslipidemia. Meanwhile, LJP61A ameliorated the HFD-induced impairment of hepatic insulin signaling and reduced VLDL overproduction via regulating the expression of genes involved in the assembly and secretion of VLDL. To study the possibility that the inhibition of mammalian target of rapamycin complex 1 and stimulation of Forkhead box protein O1 (Foxo1) nuclear exclusion is a result of LJP61A via regulating insulin signaling, LJP61A was administrated to HepG2 cells in the presence or absence of mTOR inhibitor and Foxo1 inhibitor. Results showed that LJP61A alleviated VLDL overproduction via regulating insulin receptor substrate mediated phosphatidylinositide 3-kinase AKT mammalian target of rapamycin complex 1 and phosphatidylinositide 3-kinase AKT-Foxo1 signaling pathways. These results suggested that LJP61A ameliorated HFD-induced insulin resistance to attenuate VLDL overproduction possibly via regulating insulin signaling, leading to the inhibition of atherosclerosis.