Insulin Receptor Phosphorylation Research Articles

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

Patients with obesity and metabolic syndrome, as well as individuals with pre-type 2 diabetes, exhibit hyperinsulinemia prior to development of hyperglycemia due to underlying skeletal muscle insulin resistance. To mimic the effect of chronic hyperstimulation of the insulin receptor (IR), we overexpressed the IR specifically in skeletal muscle (IRMOE). This led to skeletal muscle post-receptor desensitization, with increased IR and IRS-1(Tyr) phosphorylation but decreased phosphorylation of Akt Ser473 and Thr308. To identify the regulators involved in this post-receptor insulin resistance, we performed RNA sequencing of IRMOE muscle. Among the most upregulated genes was Lrtm1, a poorly characterized leucine rich repeat transmembrane molecule while highly expressed in the heart and skeletal muscle. Overexpression of Lrtm1 in C2C12 myoblasts led to increased phosphorylation of early insulin signaling molecules (the insulin receptor, IRS-1, and Shc), but reduced phosphorylation of the downstream molecules Akt (at both Ser473 and Thr308) and Erk Thr202/204 following insulin stimulation. Mice with knockout of Lrtm1 (Lrtm1KO) showed significant increases in percent lean mass and decreases in percent fat mass, as well as a slight reduction in overall body weight. In addition, Lrtm1KO mice were more active than controls in both metabolic cages and an open field behavioral tests. Lrtm1KO mice also exhibited improved glucose tolerance and insulin sensitivity, especially in males, even on normal chow diet. Most importantly, Lrtm1KO mice were protected from high fat diet induced obesity and metabolic syndrome, with the knockouts showing 18% less body weight gain, 30% lower fat mass, significantly improved glucose tolerance and improved insulin sensitivity. Thus, Lrtm1 is a novel regulator of post-receptor insulin signaling and glucose metabolism that may provide a new target for therapy insulin resistant prediabetes and type 2 diabetes. Disclosure G. Wang: Employee; Agilent. Y. Yu: None. W. Chen: None. W. Cai: None. X. Liu: None. C. Kahn: None.

In Vitro Biological Characterization of Recombinant Insulin Aspart from Biogenomics and Originator Insulin Aspart.

Bioassays are used to identify the pharmacological activity of new or chemically unknown compounds, as well as their undesirable effect, including toxicity. Biological assays are also required to ensure the quality, safety, and efficacy of recombinant biologics to confirm its biosimilarity to its originator. In the present study, analytical similarity between the biosimilar and its innovator is established by in vitro bioassays. The objective of this study was to show the comparative in vitro characterization of the recombinant insulin aspart from BioGenomics with its originator insulin aspart, using relevant biological assays. In vitro assays such as receptor binding, receptor autophosphorylation, glucose uptake, and mitogenic potential were analyzed for biological characterization of BioGenomics recombinant insulin aspart (BGL-ASP) manufactured by BioGenomics Limited and NovoRapid® as the reference medicinal product (RMP) manufactured by NovoNordisk. Insulin receptor binding was studied by a state-of-the-art method, surface plasmon resonance (SPR) for biomolecular interactions. The receptor autophosphorylation assay measures the phosphorylated insulin receptor in cell lysates. The glucose uptake assay measures the uptake of glucose by 3T3-L1 cells in the presence of insulin. Lipogenesis was studied in treated 3T3-L1 cells by detecting the accumulation of lipid droplets in the cells. Mitogenic effect was studied by cell proliferation assay using MCF-7 cells. A rabbit bioidentity test was performed by measuring the sudden decrease in blood glucose in the presence of insulin. The binding studies showed thatthe affinity of BGL-ASP was highly comparable to NovoRapid®. Insulin receptor autophosphorylation, glucose uptake, and lipogenesis demonstrated high similarity to the RMP. The mitogenic assay for BGL-ASP did not show any proliferative effect and was comparable to the RMP. The in vivo bioidentity test showed that the BGL-ASP is highly similar to the innovator, NovoRapid®. The biological characterization studies of BGL-ASP demonstrated high binding and functional similarity to NovoRapid®.

Physicochemical and Functional Characterization of Medicinal Product Rinsulin® R

Introduction. Currently, biosimilars have found quite widespread use in the treatment of a number of chronic and life-threatening diseases. Thanks to them, there is a significant decrease of the economic pressure of biological drugs on the health care system and wide access of patients to effective and safe medicines is ensured. One of the most important stages of proving biosimilarity is the physicochemical and functional characterization of proteins. This set of studies is generally accepted, as sensitive as possible and allows us to give a conclusion about the compliance of the biosimilar with the original drug.Aim. Conducting physicochemical and functional characterization of medicinal product Rinsulin® R (GP40051) in comparison with the original drug Humulin® Regular.Materials and methods. Primary structure was analyzed by high-performance liquid chromatography with mass spectrometry detection and matrix assisted laser desorption/ionization. The identity of the higher protein structures was proved by the methods of circular dichroism, capillary isoelectric focusing, spectrometry and dynamic light scattering. The comparability of the impurity profiles of the preparations was evaluated using the methods of exclusive chromatography and reverse-phase high-performance liquid chromatography. Functional characterization included a metabolic cell test "glucose uptake" and insulin receptor–binding assay (kinetics of binding to type A and B receptors, phosphorylation of insulin receptor).Results and discussion. In the course of this research, the identity of the physicochemical and functional characteristics of GP40051 was shown. A complete overlap of primary sequence, high-order structures and impurity profiles was demonstrated between the comparison drug GP40051 and the reference drug Humulin® Regular. Functional studies have shown that GP40051 and Humulin® Regulars have the same activity.Conclusion. The results of the quality comparability study demonstrated similarity of Rinsulin® R to the reference medicinal product Humulin® Regular, providing the scientific basis for conducting a specifically designed clinical programme, and supported registration in Russian Federation.

Butyrate acts as a G-protein-coupled receptor ligand that prevents high glucose-induced amyloidogenesis in N2a cells through the protein kinase B/glycogen synthase kinase-3β pathway.

Butyrate acts as a G-protein-coupled receptor ligand that prevents high glucose-induced amyloidogenesis in N2a cells through the protein kinase B/glycogen synthase kinase-3β pathway.

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-α (TNF-α). Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-α, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

"Multiomics" Analyses Combined with Systems Pharmacology Reveal the Renoprotection of Mangiferin Monosodium Salt in Rats with Diabetic Nephropathy: Focus on Improvements in Renal Ferroptosis, Renal Inflammation, and Podocyte Insulin Resistance.

We explored the protection of mangiferin monosodium salt (MGM) on kidney injury in rats with streptozotocin (STZ)-induced diabetic nephropathy (DN) by "multiomics" analysis combined with systems pharmacology, with a specific focus on ferroptosis, inflammation, and podocyte insulin resistance (IR) signaling events in kidneys. MGM treatment afforded renoprotective effects on rats with STZ-induced DN by alleviating systemic IR-induced renal inflammation and podocyte IR. These mechanisms were correlated mainly with the MGM treatment-induced inhibition of the mitogen-activated protein kinase/nuclear factor-kappa B axis and activation of the phosphorylated insulin receptor substrate 1(Tyr608)/phosphorylated phosphatidylinositol 3-kinase/phosphorylated protein kinase B axis in the kidneys of DN rats. MGM had an ameliorative function in renal ferroptosis in rats with STZ-induced DN by upregulating mevalonate-mediated antioxidant capacities (glutathione peroxidase 4 and ferroptosis suppressor protein 1/coenzyme Q10 axis) and weakening acyl-CoA synthetase long-chain family member 4-mediated proferroptotic generation of lipid drivers in kidneys. MGM may be a promising alternative strategy for the treatment of DN.

Aspalathin alleviates skeletal muscle insulin resistance and mitochondrial dysfunction.

Natural compounds may bear promising therapeutic benefits against metabolic diseases such as type 2 diabetes mellitus (T2DM), which are characterized by a state of insulin resistance and mitochondrial dysfunction. Here, we examined the cellular mechanisms by which aspalathin, a dihydrochalcone C-glucoside unique to rooibos, may ameliorate palmitate-induced insulin resistance and mitochondrial dysfunction in cultured C2C12 myotubules. This current study demonstrated that aspalathin remains effective in improving glucose uptake in insulin-resistant skeletal muscle cells, supported by the upregulation of insulin-dependent signaling that involves the activation of insulin receptor (IR) and direct phosphorylation of protein kinase B (AKT). Interestingly, aspalathin also improved mitochondrial respiration and function, which was evident by an increased expression of carnitine palmitoyltransferase 1 (Cpt1), fatty acid transport protein 1 (Fatp1), sirtuin 1 (Sirt1), nuclear respiratory factor 1 (Nrf1), and transcription factor A, mitochondrial (Tfam). Importantly, our results showed that aspalathin treatment was effective in ameliorating the devastating outcomes of insulin resistance and mitochondrial dysfunction that are linked with an undesired pro-inflammatory response, by reducing the levels of well-known pro-inflammatory markers such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and protein kinase C-theta (PKC-theta). Thus, beyond improving glucose uptake and insulin signaling, the current study brings a new perspective in the therapeutic benefits of aspalathin in improving mitochondrial respiration and blocking inflammation to attenuate the detrimental effect of palmitate in skeletal muscle cells.

In silico screening and identification of leads against multi-targets of type II diabetes in Plectranthus vettiveroides

Generally, phytochemicals have activity on multi-targets and such molecules have been used as effective drugs against the diseases caused by multi-factorial causation like diabetes mellitus (DM). Plectranthus vettiveroides (Jacob) N.P. Singh & B.D. Sharma has been used as a polychrest against various diseases including type II diabetes in tradition system of medicine. The present investigation aimed to validate anti-diabetic property of the root derived essential oil of P. vettiveroides and identification of leads with activity on six targets of DM through in silico method. A total of 78 phytochemicals determined through Gas chromatography –mass spectrometry (GC-MS) analysis of the essential oil were docked with each of the six selected targets namely 11β-hydroxysteroid dehydrogenase type 1, dipeptidyl peptidase IV, protein-tyrosine phosphatase 1B, human pancreatic alpha-amylase, human lysosomal acid-alpha-glucosidase, and phosphorylated insulin receptor tyrosine kinase. The structures of the phytochemicals were retrieved from PubChem and the targets were retrieved from Protein Data Bank (PDB). Docking was carried out in AutoDockVina and the molecules with free energy of binding ≤-6 kcal/mol were considered as hit molecule. Out of 78 phytochemicals screened, 39 have activity on all the tested targets. Based on the docking interaction, drug-likeness and absorption, distribution, metabolism, excretion and toxicity (ADMET) property the best lead molecule against each target were determined. The overall results revealed that basically majority of the hits are terpinoides and not induced any toxicity. Therefore, the oil as such can be recommended as medicament against DM, since the individual, synergistic and cumulative effect of phytochemicals may concomitantly inhibit multi-targets of DM and act as an effective drug.

아이스플랜트 추출물의 3T3-L1 지방세포 내 TNF-α 유도 염증 및 인슐린 저항성 개선 효과

Obesity is classified as a low-grade chronic inflammatory disease. Obesity-induced inflammation is associated with the development of insulin resistance (IR). In this study, we investigated the effects of Mesembryanthemum crystallinum extract (MCE) on tumor necrosis factor-alpha (TNF-α)-induced inflammation and IR in 3T3-L1 adipocytes, as well as underlying mechanisms. Lipolysis was monitored by oil red O staining. Glucose uptake was measured by 2-deoxyglucose (2-DG). The production of cytokines and adiponectin was evaluated using enzyme-linked immunosorbent assay (ELISA). Protein expression was measured using western blotting. IR was induced in differentiated 3T3-L1 adipocytes by treatment with TNF-α. MCE treatment decreased lipolysis while increasing adiponectin production. MCE inhibited the production of pro-inflammatory cytokines such as interleukn (IL)-6 and monocyte chemoattractant protein (MCP)-1, as well as the Nuclear factor-κB (NF-κB) translocation. Additionally, MCE increased glucose uptake, prevented the protein expression of phosphorylated insulin receptor supstrate (IRS) and increased the protein expression of Glucose Transporter Type 4 (GLUT4) in a dose-dependent manner. These results demonstrate the potential of MCE in improving chronic inflammation and insulin sensitivity in obesity. Therefore, MCE can be used as a novel candidate for developing functional foods.

The CX3CL1 intracellular domain exhibits neuroprotection via insulin receptor/insulin-like growth factor receptor signaling

CX3CL1, also known as fractalkine, is best known for its signaling activity through interactions with its cognate receptor CX3CR1. However, its intrinsic function that is independent of interaction with CX3CR1 remains to be fully understood. We demonstrate that the intracellular domain of CX3CL1 (CX3CL1-ICD), generated upon sequential cleavages by α-/β-secretase and γ-secretase, initiates a back signaling activity, which mediates direct signal transmission to gene expression in the nucleus. To study this, we fused a synthetic peptide derived from CX3CL1-ICD, named Tet34, with a 13-amino acid tetanus sequence at the N terminus to facilitate translocation into neuronal cells. We show that treatment of mouse neuroblastoma Neuro-2A cells with Tet34, but not its scrambled control (Tet34s), induced cell proliferation, as manifested by changes in protein levels of transcription factors and progrowth molecules cyclin D1, PCNA, Sox5, and Cdk2. Further biochemical assays reveal elevation of phosphorylated insulin receptor β subunit, insulin-like growth factor-1 receptor β subunit, and insulin receptor substrates as well as activation of proliferation-linked kinase AKT. In addition, transgenic mice overexpressing membrane-anchored C-terminal CX3CL1 also exhibited activation of insulin/insulin-like growth factor-1 receptor signaling. Remarkably, we found that this Tet34 peptide, but not Tet34s, protected against endoplasmic reticulum stress and cellular apoptosis when Neuro-2A cells were challenged with toxic oligomers of β-amyloid peptide or hydrogen peroxide. Taken together, our results suggest that CX3CL1-ICD may have translational potential for neuroprotection in Alzheimer's disease and for disorders resulting from insulin resistance.

Magnesium increases insulin-dependent glucose uptake in adipocytes.

BackgroundType 2 diabetes (T2D) is characterized by a decreased insulin sensitivity. Magnesium (Mg2+) deficiency is common in people with T2D. However, the molecular consequences of low Mg2+ levels on insulin sensitivity and glucose handling have not been determined in adipocytes. The aim of this study is to determine the role of Mg2+ in the insulin-dependent glucose uptake.MethodsFirst, the association of low plasma Mg2+ with markers of insulin resistance was assessed in a cohort of 395 people with T2D. Secondly, the molecular role of Mg2+ in insulin-dependent glucose uptake was studied by incubating 3T3-L1 adipocytes with 0 or 1 mmol/L Mg2+ for 24 hours followed by insulin stimulation. Radioactive-glucose labelling, enzymatic assays, immunocytochemistry and live microscopy imaging were used to analyze the insulin receptor phosphoinositide 3-kinases/Akt pathway. Energy metabolism was assessed by the Seahorse Extracellular Flux Analyzer.ResultsIn people with T2D, plasma Mg2+ concentration was inversely associated with markers of insulin resistance; i.e., the lower Mg2+, the more insulin resistant. In Mg2+-deficient adipocytes, insulin-dependent glucose uptake was decreased by approximately 50% compared to control Mg2+condition. Insulin receptor phosphorylation Tyr1150/1151 and PIP3 mass were not decreased in Mg2+-deficient adipocytes. Live imaging microscopy of adipocytes transduced with an Akt sensor (FoxO1-Clover) demonstrated that FoxO1 translocation from the nucleus to the cytosol was reduced, indicting less Akt activation in Mg2+-deficient adipocytes. Immunocytochemistry using a Lectin membrane marker and at the membrane located Myc epitope-tagged glucose transporter 4 (GLUT4) demonstrated that GLUT4 translocation was diminished in insulin-stimulated Mg2+-deficient adipocytes compared to control conditions. Energy metabolism in Mg2+ deficient adipocytes was characterized by decreased glycolysis, upon insulin stimulation.ConclusionsMg2+ increases insulin-dependent glucose uptake in adipocytes and suggests that Mg2+ deficiency may contribute to insulin resistance in people with T2D.

Biochemical assessment of uric acid and magnesium in prediabetic patients in Peshawar-KP: A case-control study.

Background: Background: There is a lack of information regarding serum uric acid (SUA) and magnesium levels among the pre-diabetics population. This study aimed to assess the biochemical assessment of both uric acid and magnesium in prediabetics of the Peshawar-KP region.
 Methodology: In this case-control study, patients having normal glucose levels were considered as control, and prediabetics were cases. Fasting blood glucose (FPG) levels between 100-125 mg/dl or HbA1c levels between 5.7 to 6.4% were considered pre-diabetic. SUA was assessed by the Uricase method, serum Mg+ by the Calmagite calorimetric method, and fasting glucose was assessed by the GOD-POD enzymatic method.
 Results: According to our findings, mean values of serum magnesium were 2.12 ± 0.60 mg/dl (p=0.026) and 1.10 ± 0.82 mg/dl (p=0.001) in male and female of control group whereas, 1.60 ± 0.44 mg/dl (p=0.004) and 0.90 ± 0.22 mg/dl (p=0.002) in male and female of cases group respectively. Correspondingly, mean value of SUA was recorded as 4.14 ± 0.66 mg/dl (p=0.008) and 3.12 ± 0.38 mg/dl (p=0.004) in male and female of control group whereas 8.10 ± 1.00 mg/dl (p=0.002) and 6.22 ± 0.44 mg/dl (p=0.003) in male and female of cases and subjects group respectively. It is noticeable that mean serum magnesium and SUA values were higher in the male of both groups compared to females.
 Conclusion: The study concluded that lower serum magnesium levels in prediabetics might result from the closure of K+ channels, defective phosphorylation of insulin receptors, or diminished glucokinase activity. Higher uric acid levels in prediabetics might result from inflammation, oxidative stress, and hypertrophy of the tubules.

Combination of chronic stress with fructose diet increases AMP-activated protein kinase phosphorylation and affects agouti-related protein and proopiomelanocortin expression in the hypothalamus of male Wistar rats.

Appetite regulation in the hypothalamus is dependent on hormonal signals from the periphery, such as insulin and leptin, and can be modulated by both sugar-rich diet and stress. Our aim was to explore the effects of 9-week feeding with 20% fructose solution combined with 4-week chronic unpredictable stress, on appetite-regulating neuropeptides and modulatory role of leptin and insulin signalling in the hypothalamus of male Wistar rats. Energy intake, body mass and adiposity, as well as circulatory leptin and insulin concentrations were assessed. Hypothalamic insulin signalling was analysed at the level of glucose transporters, as well as at the protein level and phosphorylation of insulin receptor, insulin receptor supstrate-1, Akt and ERK. Phosphorylation of AMP-activated protein kinase (AMPK), level of protein tyrosine phosphatase 1B (PTP1B) and expression of leptin receptor (ObRb) and suppressor of cytokine signalling 3 (SOCS3) were also analysed, together with the expression of orexigenic agouti-related protein (AgRP) and anorexigenic proopiomelanocortin (POMC) neuropeptides. The results revealed that stress decreased body mass and adiposity, blood leptin level and expression of ObRb, SOCS3 and POMC, while combination with fructose diet led to marked increase of AgRP, associated with AMPK phosphorylation despite increased plasma insulin. Reduced Akt, enhanced ERK activity and elevated PTP1B were also observed in the hypothalamus of these animals. In conclusion, our results showed that joint effects of fructose diet and stress are more deleterious than the separate ones, since inappropriate appetite control in the hypothalamus may provide a setting for the disturbed energy homeostasis in the long run.

Malting barley carbon dots-mediated oxidative stress promotes insulin resistance in mice via NF-κB pathway and MAPK cascade

BackgroundFood-borne carbon dots (CDs) are widely generated during food processing and are inevitably ingested by humans causing toxicity. However, the toxic effects of food-borne CDs on the blood glucose metabolism are unknown.ResultsIn this study, we brewed beer via a representative strategy and extracted the melting-barley CDs (MBCDs) to explore the toxic effects on blood glucose in mice. We found the accumulation of fluorescent labeled MBCDs in various organs and oral administration of MBCDs can cause visceral toxicity, manifested as liver damage. Mice were orally administered MBCDs (5 and 25 mg/kg) for 16 weeks, and increased levels of fasting blood glucose were observed in both MBCDs-treated groups. Transcriptomic analyses revealed that MBCDs activate oxidative stress, inflammatory responses, the MAPK cascade, and PI3K/Akt signaling in mice livers. Mechanistically, MBCDs exposure-induced reactive oxygen species (ROS) overproduction activates the nuclear factor-κB (NF-κB) signaling pathway and MAPK cascade, thereby promoting phosphorylated insulin receptor substrate (IRS)-1 at Ser307 and inducing insulin resistance (IR). Meanwhile, the IR promoted gluconeogenesis, which enhanced MBCDs-induced hyperglycemia of mice. Importantly, inhibition of the ROS significantly attenuated the MBCDs-induced inflammatory response and MAPK cascade, thereby alleviating IR and hyperglycemia in mice.ConclusionIn summary, this study revealed that MBCDs promote ROS overproduction and thus induced IR, resulting in imbalance of glucose homeostasis in mice. More importantly, this study was further assessed to reveal an imperative emphasis on the reevaluation of dietary and environmental CDs exposure, and has important implications for T2DM prevention research.Graphical

Quercetin ameliorated insulin resistance via regulating METTL3-mediated N6-methyladenosine modification of PRKD2 mRNA in skeletal muscle and C2C12 myocyte cell line

Background and aimsN6-Methyladenosine (m6A) modification is involved in many pathological processes, including insulin resistance (IR). Quercetin (Que), a bioactive compound with strong antioxidant activity, has potential therapeutic effects on IR-related metabolic diseases. The aim of this study is to investigate the roles of m6A and Que in hyperinsulinemia. Methods and resultsMale C57Bl/6 mice received a high-fat diet (HFD) for 8 weeks to establish an IR model. Que treatment reduced the body weight, blood glucose, plasma triglycerides (TG) and serum insulin, ameliorated IR, and decreased oxidative stress in HFD-fed mice. Cellular IR model was established in C2C12 cells by palmitic acid (PA) stimulation, and a noncytotoxic dose of Que was found to promote glucose uptake and inhibit oxidative stress. Moreover, methyltransferase-like 3 (METTL3) and serine–threonine kinase protein kinase D2 (PRKD2) was downregulated in skeletal muscle of HFD-fed mouse and in PA-induced C2C12 cells. The online bioinformatic tool SRAMP revealed that there were multiple m6A modification sites in the PRKD2 mRNA sequence. Downregulation of METTL3 enhanced PRKD2 expression by reducing m6A level and promoting mRNA stability in PRKD2 mRNA transcript. Que decreased m6A, METTL3, and phosphorylated insulin receptor substrate 1 (p-IRS1) levels, increased the protein expression of PRKD2, glucose transporter type 4 (GLUT4) and p-AKT, promoted glucose uptake, and reduced oxidative stress in PA-induced C2C12 cells. Moreover, METTL3 overexpression or PRKD2 silence reversed the inhibitory effects of Que on the levels of MDA and p-IRS1 and the promotive effects on glucose uptake, superoxide dismutase (SOD), GSH and GLUT4 and p-AKT levels. ConclusionQue promoted glucose uptake, repressed oxidative stress and improved IR through METTL3-mediated m6A of PRKD2 mRNA.

N-Acetylcysteine Enhances the Recovery of Ischemic Limb in Type-2 Diabetic Mice

Critical limb ischemia (CLI) is a severe complication of diabetes mellitus that occurs without effective therapy. Excessive reactive oxygen species (ROS) production and oxidative stress play critical roles in the development of diabetic cardiovascular complications. N-acetylcysteine (NAC) reduces ischemia-induced ROS production. The present study aimed to investigate the effect of NAC on the recovery of ischemic limb in an experimental model of type-2 diabetes. TALLYHO/JngJ diabetic and SWR/J non-diabetic mice were used for developing a CLI model. For NAC treatment, mice received NAC (1 mg/mL) in their drinking water for 24 h before initiating CLI, and continuously for the duration of the experiment. Blood flow, mechanical function, histology, expression of antioxidant enzymes including superoxide dismutase (SOD)-1, SOD-3, glutathione peroxidase (Gpx)-1, catalase, and phosphorylated insulin receptor substrate (IRS)-1, Akt, and eNOS in ischemic limb were evaluated in vivo or ex vivo. Body weight, blood glucose, plasma advanced glycation end-products (AGEs), plasma insulin, insulin resistance index, and plasma TNF-a were also evaluated during the experiment. NAC treatment effectively attenuated ROS production with preserved expressions of SOD-1, Gpx-1, catalase, phosphorylated Akt, and eNOS, and enhanced the recovery of blood flow and function of the diabetic ischemic limb. NAC treatment also significantly decreased the levels of phosphorylated IRS-1 (Ser307) expression and plasma TNF-α in diabetic mice without significant changes in blood glucose and AGEs levels. In conclusion, NAC treatment enhanced the recovery of blood flow and mechanical function in ischemic limbs in T2D mice in association with improved tissue redox/inflammatory status and insulin resistance.

Angiotensin II Inhibits Insulin Receptor Signaling in Adipose Cells

Angiotensin II (Ang II) is a critical regulator of insulin signaling in the cardiovascular system and metabolic tissues. However, in adipose cells, the regulatory role of Ang II on insulin actions remains to be elucidated. The effect of Ang II on insulin-induced insulin receptor (IR) phosphorylation, Akt activation, and glucose uptake was examined in 3T3-L1 adipocytes. In these cells, Ang II specifically inhibited insulin-stimulated IR and insulin receptor substrate-1 (IRS-1) tyrosine-phosphorylation, Akt activation, and glucose uptake in a time-dependent manner. These inhibitory actions were associated with increased phosphorylation of the IR at serine residues. Interestingly, Ang II-induced serine-phosphorylation of IRS was not detected, suggesting that Ang II-induced desensitization begins from IR regulation itself. PKC inhibition by BIM I restored the inhibitory effect of Ang II on insulin actions. We also found that Ang II promoted activation of several PKC isoforms, including PKCα/βI/βII/δ, and its association with the IR, particularly PKCβII, showed the highest interaction. Finally, we also found a similar regulatory effect of Ang II in isolated adipocytes, where insulin-induced Akt phosphorylation was inhibited by Ang II, an effect that was prevented by PKC inhibitors. These results suggest that Ang II may lead to insulin resistance through PKC activation in adipocytes.

Upregulation of the serine palmitoyltransferase subunit SPTLC2 by endoplasmic reticulum stress inhibits the hepatic insulin response

Endoplasmic reticulum (ER) stress is induced by various conditions, such as inflammation and the presence of excess nutrients. Abnormal accumulation of unfolded proteins leads to the activation of a collective signaling cascade, termed the unfolded protein response (UPR). ER stress is reported to perturb hepatic insulin response metabolism while promoting insulin resistance. Here, we report that ER stress regulates the de novo biosynthesis of sphingolipids via the activation of serine palmitoyltransferase (SPT), a rate-limiting enzyme involved in the de novo biosynthesis of ceramides. We found that the expression levels of Sptlc1 and Sptlc2, the major SPT subunits, were upregulated and that the cellular concentrations of ceramide and dihydroceramide were elevated by acute ER stress inducers in primary hepatocytes and HepG2 cells. Sptlc2 was upregulated and ceramide levels were elevated by tunicamycin in the livers of C57BL/6J wild-type mice. Analysis of the Sptlc2 promoter demonstrated that the transcriptional activation of Sptlc2 was mediated by the spliced form of X-box binding protein 1 (sXBP1). Liver-specific Sptlc2 transgenic mice exhibited increased ceramide levels in the liver and elevated fasting glucose levels. The insulin response was reduced by the inhibition of the phosphorylation of insulin receptor β (IRβ). Collectively, these results demonstrate that ER stress induces activation of the de novo biosynthesis of ceramide and contributes to the progression of hepatic insulin resistance via the reduced phosphorylation of IRβ in hepatocytes.

Pomegranate peel polyphenols alleviate insulin resistance through the promotion of insulin signaling pathway in skeletal muscle of metabolic syndrome rats

Insulin resistance (IR) has been considered to be an important causative factor of metabolic syndrome (MetS). The present study investigated whether pomegranate peel polyphenols (PPPs) could prevent the development of MetS by improving IR in rats. Male Sprague-Dawley (SD) rats were fed high fat diet (HFD) to induce MetS and supplemented with different dosages of PPPs for 12 weeks. The results showed that HFD-induced insulin resistant rats had disordered metabolism of blood glucose, blood lipid, and terrible muscle fiber morphology when compared with normal diet-fed rats, but PPPs treatment at a dosage of 300 mg/kg·day significantly reversed these negative effects. Moreover, in skeletal muscle tissue of insulin resistant rats, PPPs treatments significantly increased the protein expressions of insulin receptor (InsR) and phosphorylated insulin receptor substrate 1 (IRS-1), stimulated peroxisome proliferator activated receptor gamma (PPARγ) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT/PKB) signaling pathway, and aggrandized the protein levels of phosphorylated glycogen synthase kinase-3β (GSK-3β) and glucose transporter 4 (GLUT4). Our results suggest that PPPs possess of the beneficial effects on alleviating IR by enhancing insulin sensitivity and regulating glucose metabolism.

Upregulation of GLUT4 and PI3K, and downregulation of GSK3 mediate the anti-hyperglycemic effects of proanthocyanidins.

Diabetes mellitus is the most common chronic metabolic disorder worldwide. The present study was designed to investigate the potential role of cinnamon bark extract oligomeric proanthocyanidins (OPCs) in controlling streptozotocin (STZ)-induced hyperglycemia and to clarify the underlying molecular mechanisms underlying its effects. For this purpose, 60 male rats were equally divided into six groups as follows: The normal control group; OPC control group (non-diabetic rats treated with OPC at 300 mg/kg orally for 21 days); the untreated diabetic control group; the wortmannin control group [diabetic rats treated with wortmannin at 1 mg/kg, intraperitoneal (i.p.) on the final day of the experiment]; the OPC diabetic group (diabetic rats treated with OPC at 300 mg/kg orally for 21 days); and the OPC diabetic + wortmannin co-treated group (diabetic rats treated with OPC at 300 mg/kg/day for 21 consecutive days and then 24 h after the final OPC dose treated with a single wortmannin injection at 1 mg/kg, i.p.). The results indicated that OPC ameliorated the diabetic state, as evidenced by a significant decrease in serum glucose levels, and a significant increase in the levels of insulin, amylin, insulin receptor phosphorylation, glycogen and glucose transporter-4 translocation; it also improved the lipid profile in STZ-diabetic rats. On the whole, the findings of the present study provide biochemical evidence that OPC treatment is effective as an anti-diabetic and anti-hyperlipidemic agent by enhancing glucose uptake through the activation of insulin receptor kinase activity and the PI3K/Akt pathway.