GLUT4 Activity Research Articles

An antifibrotic compound that ameliorates hyperglycaemia and fat accumulation in cell and HFD mouse models.

Appropriate management of blood glucose levels and the prevention of complications are important in the treatment of diabetes. We have previously reported on a compound named HPH-15 that is not only antifibrotic but also AMP-activated protein kinase (AMPK)-activating. In this study, we evaluated whether HPH-15 is useful as a therapeutic medication for diabetes. We examined the effects of HPH-15 on AMPK activation, glucose uptake, fat accumulation and lactic acid production in L6-GLUT4, HepG2 and 3T3-L1 cells, as a model of muscle, liver and fat tissue, respectively. Additionally, we investigated the glucose-lowering, fat-accumulation-suppressing, antifibrotic and AMPK-activating effect of HPH-15 in mice fed a high-fat diet (HFD). HPH-15 at a concentration of 10 µmol/l increased AMPK activation, glucose uptake and membrane translocation of GLUT4 in each cell model to the same extent as metformin at 2 mmol/l. The production of lactic acid (which causes lactic acidosis) in HPH-15-treated cells was equal to or less than that observed in metformin-treated cells. In HFD-fed mice, HPH-15 lowered blood glucose from 11.1±0.3 mmol/l to 8.2±0.4 mmol/l (10 mg/kg) and 7.9±0.4 mmol/l (100 mg/kg) and improved insulin resistance. The HPH-15 (10 mg/kg) group showed the same level of AMPK activation as the metformin (300 mg/kg) group in all organs. The HPH-15-treated HFD-fed mice also showed suppression of fat accumulation and fibrosis in the liver and fat tissue; these effects were more significant than those obtained with metformin. Mice treated with high doses of HPH-15 also exhibited a 44% reduction in subcutaneous fat. HPH-15 activated AMPK at lower concentrations than metformin in vitro and in vivo and improved blood glucose levels and insulin resistance in vivo. In addition, HPH-15 was more effective than metformin at ameliorating fatty liver and adipocyte hypertrophy in HFD-fed mice. HPH-15 could be effective in preventing fatty liver, a common complication in diabetic individuals. Additionally, in contrast to metformin, high doses of HPH-15 reduced subcutaneous fat in HFD-fed mice. Presumably, HPH-15 has a stronger inhibitory effect on fat accumulation and fibrosis than metformin, accounting for the reduction of subcutaneous fat. Therefore, HPH-15 is potentially a glucose-lowering medication that can lower blood glucose, inhibit fat accumulation and ameliorate liver fibrosis.

Study of the Effects of Deuterium-Depleted Water on the Expression of GLUT4 and Insulin Resistance in the Muscle Cell Line C2C12.

Deuterium-depleted water (DDW) is used in the treatment of many diseases, including cancer and diabetes. To detect the effect of DDW on gene expression and activation of the insulin-responsive transporter GLUT4 as a mechanism for improving the pathology of diabetes, we investigated the GLUT4 expression and glucose uptake at various concentrations of DDW using the myoblast cell line C2C12 differentiated into myotubes. GLUT4 gene expression significantly increased under deuterium depletion, reaching a maximum value at a deuterium concentration of approximately 50 ppm, which was approximately nine times that of natural water with a deuterium concentration of 150 ppm. GLUT4 protein also showed an increase at similar DDW concentrations. The membrane translocation of GLUT4 by insulin stimulation reached a maximum value at a deuterium concentration of approximately 50-75 ppm, which was approximately 2.2 times that in natural water. Accordingly, glucose uptake also increased by up to 2.2 times at a deuterium concentration of approximately 50 ppm. Drug-induced insulin resistance was attenuated, and the glucose uptake was four times higher in the presence of 10 ng/mL TNF-α and three times higher in the presence of 1 μg/mL resistin at a deuterium concentration of approximately 50 ppm relative to natural water. These results suggest that DDW promotes GLUT4 expression and insulin-stimulated activation in muscle cells and reduces insulin resistance, making it an effective treatment for diabetes.

Exercise alleviates diabetic complications by inhibiting oxidative stress-mediated signaling cascade and mitochondrial metabolic stress in GK diabetic rat tissues.

Type 2 diabetes, obesity (referred to as "diabesity"), and metabolic syndrome associated with increased insulin resistance and/or decreased insulin sensitivity have been implicated with increased oxidative stress and inflammation, mitochondrial dysfunction, and alterations in energy metabolism. The precise molecular mechanisms of these complications, however, remain to be clarified. Owing to the limitations and off-target side effects of antidiabetic drugs, exercise-induced control of hyperglycemia and increased insulin sensitivity is a preferred strategy to manage "diabesity" associated complications. In this study, we have investigated the effects of moderate exercise (1h/day, 5days a week for 60days) on mitochondrial, metabolic, and oxidative stress-related changes in the liver and kidney of type 2 diabetic Goto-Kakizaki (GK) rats. Our previous study, using the same exercise regimen, demonstrated improved energy metabolism and mitochondrial function in the pancreas of GK diabetic rats. Our current study demonstrates exercise-induced inhibition of ROS production and NADPH oxidase enzyme activity, as well as lipid peroxidation and protein carbonylation in the liver and kidney of GK rats. Interestingly, glutathione (GSH) content and GSH-peroxidase and GSH reductase enzymes as well as superoxide dismutase (SOD) activities were profoundly altered in diabetic rat tissues. Exercise helped in restoring the altered GSH metabolism and antioxidant homeostasis. An increase in cytosolic glycolytic enzyme, hexokinase, and a decrease in mitochondrial Kreb's cycle enzyme was observed in GK diabetic rat tissues. Exercise helped restore the altered energy metabolism. A significant decrease in the activities of mitochondrial complexes and ATP content was also observed in the GK rats and exercise regulated the activities of the respiratory complexes and improved energy utilization. Activation of cytochrome P450s, CYP 2E1, and CYP 3A4 was observed in the tissues of GK rats, which recovered after exercise. Altered expression of redox-responsive proteins and translocation of transcription factor NFκB-p65, accompanied by activation of AMP-activated protein kinase (AMPK), SIRT-1, Glut-4, and PPAR-γ suggests the induction of antioxidant defense responses and increased energy metabolism in GK diabetic rats after exercise.

Autocrine FGF1 signaling promotes glucose uptake in adipocytes

Fibroblast growth factor 1 (FGF1) is an autocrine growth factor released from adipose tissue during over-nutrition or fasting to feeding transition. While local actions underlie the majority of FGF1's anti-diabetic functions, the molecular mechanisms downstream of adipose FGF receptor signaling are unclear. We investigated the effects of FGF1 on glucose uptake and its underlying mechanism in murine 3T3-L1 adipocytes and in ex vivo adipose explants from mice. FGF1 increased glucose uptake in 3T3-L1 adipocytes and epididymal WAT (eWAT) and inguinal WAT (iWAT). Conversely, glucose uptake was reduced in eWAT and iWAT of FGF1 knockout mice. We show that FGF1 acutely increased adipocyte glucose uptake via activation of the insulin-sensitive glucose transporter GLUT4, involving dynamic crosstalk between the MEK1/2 and Akt signaling proteins. Prolonged exposure to FGF1 stimulated adipocyte glucose uptake by MEK1/2-dependent transcription of the basal glucose transporter GLUT1. We have thus identified an alternative pathway to stimulate glucose uptake in adipocytes, independent from insulin, which could open new avenues for treating patients with type 2 diabetes.

An Overview of Phytotherapy Used in the Management of Type II Diabetes.

Diabetes mellitus is related to unconstrained high blood sugar and linked with long-term impairment, dysfunction and failure of several organs. Since 1980, the global frequency of diabetes has almost doubled in the adult population. In very rare cases due to poor prevention and management programs, diabetes causes worsening of health and reduced lifespan of the world population, thus impacting on the world's economy. Supplements, however, help in the improvement of nutritional deficiencies. Phytotherapeutics has the advantage of being economical and easy to access with marginal side effects. So, it is a preferred candidate for the management of diabetes. Currently, a multitude of pharmaceuticals are used which are obtained from natural sources having medicinal properties. The mechanistic approaches are based on the regulation of insulin signaling pathways, translocation of GLUT-4 receptors and/or activation of PPAR γ. These natural compounds include numerous flavonoids which help in preventing glucose absorption by preventing the absorption of α-amylase and α-glucosidase. But to validate the efficacy and safety profile of these compounds, detailed validatory clinical studies are required. This review majorly focuses on the mechanistic approaches of various naturally derived compounds relevant for the condition of Diabetes Mellitus.

Resveratrol in Treating Diabetes and Its Cardiovascular Complications: A Review of Its Mechanisms of Action.

Diabetes mellitus (DM) is one of the most prevalent chronic diseases worldwide. High morbidity and mortality caused by DM are closely linked to its complications in multiple organs/tissues, including cardiovascular complications, diabetic nephropathy, and diabetic neuropathy. Resveratrol is a plant-derived polyphenolic compound with pleiotropic protective effects, ranging from antioxidant and anti-inflammatory to hypoglycemic effects. Recent studies strongly suggest that the consumption of resveratrol offers protection against diabetes and its cardiovascular complications. The protective effects of resveratrol involve the regulation of multiple signaling pathways, including inhibition of oxidative stress and inflammation, enhancement of insulin sensitivity, induction of autophagy, regulation of lipid metabolism, promotion of GLUT4 expression, and translocation, and activation of SIRT1/AMPK signaling axis. The cardiovascular protective effects of resveratrol have been recently reviewed in the literature, but the role of resveratrol in preventing diabetes mellitus and its cardiovascular complications has not been systematically reviewed. Therefore, in this review, we summarize the pharmacological effects and mechanisms of action of resveratrol based on in vitro and in vivo studies, highlighting the therapeutic potential of resveratrol in the prevention and treatment of diabetes and its cardiovascular complications.

Effects of Moderate-Intensity Physical Training on Skeletal Muscle Substrate Transporters and Metabolic Parameters of Ovariectomized Rats.

A deficit of estrogen is associated with energy substrate imbalance, raising the risk of metabolic diseases. Physical training (PT) is a potent metabolic regulator through oxidation and storage of substrates transported by GLUT4 and FAT CD36 in skeletal muscle. However, little is known about the effects of PT on these carriers in an estrogen-deficit scenario. Thus, the aim of this study was to determine the influence of 12 weeks of PT on metabolic variables and GLUT4 and FAT CD36 expression in the skeletal muscle of animals energetically impaired by ovariectomy (OVX). The trained animals swam 30 min/day, 5 days/week, at 80% of the critical load intensity. Spontaneous physical activity was measured biweekly. After training, FAT CD36 and GLUT4 expressions were quantified by immunofluorescence in the soleus, as well as muscular glycogen and triglyceride of the soleus, gluteus maximus and gastrocnemius. OVX significantly reduced FAT CD36, GLUT4 and spontaneous physical activity (p < 0.01), while PT significantly increased FAT CD36, GLUT4 and spontaneous physical activity (p < 0.01). PT increased soleus glycogen, and OVX decreased muscular triglyceride of gluteus maximus. Therefore, OVX can cause energy disarray through reduction in GLUT4 and FAT CD36 and their muscle substrates and PT prevented these metabolic consequences, masking ovarian estrogen’s absence.

Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity.

The leptin receptor (LepR) acts as a signaling nexus for the regulation of glucose uptake and obesity, among other metabolic responses. The functional role of LepR under leptin-deficient conditions remains unclear. This study reports that epiregulin (EREG) governed glucose uptake in vitro and in vivo in Lepob mice by activating LepR under leptin-deficient conditions. Single and long-term treatment with EREG effectively rescued glucose intolerance in comparative insulin and EREG tolerance tests in Lepob mice. The immunoprecipitation study revealed binding between EREG and LepR in adipose tissue of Lepob mice. EREG/LepR regulated glucose uptake without changes in obesity in Lepob mice via mechanisms, including ERK activation and translocation of GLUT4 to the cell surface. EREG-dependent glucose uptake was abolished in Leprdb mice which supports a key role of LepR in this process. In contrast, inhibition of the canonical epidermal growth factor receptor (EGFR) pathway implicated in other EREG responses, increased glucose uptake. Our data provide a basis for understanding glycemic responses of EREG that are dependent on LepR unlike functions mediated by EGFR, including leptin secretion, thermogenesis, pain, growth, and other responses. The computational analysis identified a conserved amino acid sequence, supporting an evolutionary role of EREG as an alternative LepR ligand.

Polyphenol-rich Indian ginger cultivars ameliorate GLUT4 activity in C2C12 cells, inhibit diabetes-related enzymes and LPS-induced inflammation: An in vitro study.

Diabetes is a chronic metabolic disorder that results in distorted insulin signaling and microvascular complications. Current antidiabetic drugs possess harmful long term side effects, necessitating the need for alternate or compliment therapy with lesser issues. Medicinal plants such as ginger have been reported to possess several beneficial activities including antidiabetic activity. The antidiabetic efficacy of microwave-assisted polyphenolic extracts of Indian ginger cultivars from Odisha (MPO) and Tamil Nadu (MPT) is reported here. MPT and MPO showed insulin stimulated glucose uptake of 1.74±0.25 and 1.47±0.15 fold at 6.25µg/ml of concentrations in C2C12 cells respectively when compared to control. MPT possessed α-amylase, α-glucosidase inhibitory and anti-glycation properties. It also showed DPPH radical scavenging activity (7.69±0.001%), inhibited LPS-induced nitric oxide production (1.06±0.004 fold) than the latter and increased the GLUT4 protein expression by 1.4 fold. Major active compounds such as shogaol and gingerol derivatives, curcumene, zingiberone were identified through GC-ESI/MS analysis and D-pinitol (cyclitol) was identified through HPLC analysis in this variety. This is the first paper to report the presence of an antidiabetic compound, D-pinitol, in the ginger variety. Polyphenol rich, biologically potent ginger extracts can be a good food and nutraceutical supplement to address diabetes and related complications. PRACTICAL APPLICATIONS: Ginger is a native spice of South Asian Countries including India. Ginger extracts possess several medicinal properties such as anti-inflammatory, antidiabetic and antioxidant activities. It is used to treat nausea, vomiting and commonly used as a food flavouring agent and dietary food supplement. Our study shows the antidiabetic, anti-glycation and antioxidant efficacy of polyphenol rich Indian ginger cultivars grown in different geographical regions. Variations in the biological activities between the MPT and MPO ginger variety was observed. Different environmental conditions and their corresponding metabolite accumulation can be correlated with the better activity shown by MPT variety. It showed an increased GLUT4 expression even at a lower dose of 6.25µg/ml. Ginger cultivar, especially MPT variety can be used as an adjuvant therapy for treating diabetes. Therefore, our study indicates that polyphenols rich ginger cultivar has major application in functional food product development.

Friedelin exhibits antidiabetic effect in diabetic rats via modulation of glucose metabolism in liver and muscle

Ethnopharmacological relevanceDemand for plant-based medications and therapeutics is increasing worldwide as of its potential effects and no toxic. Traditionally, so many medicinal plants are used to treat diabetes. Subsequently, investigation on medicinal plants was enduring to discover potential antidiabetic drugs. A. tetracantha is used traditionally to cure diabetes mellitus, cough, dropsy, chronic diarrhea, rheumatism, phthisis and smallpox. Scientifically, A. tetracantha has been reported as an antidiabetic agent. Friedelin, the isolated compound has been reported as hypolipidemic, antioxidant, scavenging of free radicals, antiulcer, anti-inflammatory, analgesic and antipyretic agent. Aim of the studyTo scrutinize the mechanism of antidiabetic activity of friedelin isolated from the leaves of A. tetracantha. Materials and methodsA. tetracantha leaves powder (5 kg) was soaked in hexane (15 L) to obtain hexane extract. Using column chromatography, the hexane extract was fractionated using a combination of solvents like hexane and ethyl acetate. 25 fractions were obtained and the fractions 13 and 14 yielded the compound, friedelin. Friedelin at the doses of 20 and 40 mg/kg was used to treated STZ -induced diabetic rats for 28 days. Later 28 days of treatment, the bodyweight changes, levels of blood glucose, insulin, SGOT, SGPT, SALP, liver glycogen and total protein were assessed. ResultsFriedelin significantly brought these altered levels to near normal. Moreover, friedelin also enhanced the translocation as well as activation of GLUT2 and GLUT4 through PI3K/p-Akt signaling cascade in skeletal muscles and liver on diabetic rats. ConclusionThis finding proved that friedelin has an anti-diabetic effect through insulin-dependent signaling cascade mechanism, thus it may lead to establishing a drug to treat type 2 diabetes mellitus.

Downregulation of the Glo1 Gene Is Associated with Reduced Adiposity and Ectopic Fat Accumulation in Spontaneously Hypertensive Rats.

Methylglyoxal (MG), a potent precursor of advanced glycation end-products (AGE), is increased in metabolic disorders such as diabetes and obesity. MG and other dicarbonyl metabolites are detoxified by the glyoxalase system in which glyoxalase 1, coded by the Glo1 gene, serves as the rate-limiting enzyme. In this study, we analyzed the effects of Glo1 downregulation on glucose and lipid metabolism parameters in spontaneously hypertensive rats (SHR) by targeting the Glo1 gene (SHR-Glo1+/− heterozygotes). Compared to SHR wild-type animals, SHR-Glo1+/− rats showed significantly reduced Glo1 expression and lower GLO1 activity in tissues associated with increased MG levels. In contrast to SHR controls, SHR-Glo1+/− rats exhibited lower relative weight of epididymal fat, reduced ectopic fat accumulation in the liver and heart, and decreased serum triglycerides. In addition, compared to controls, SHR-Glo1+/− rats showed reduced serum insulin and increased basal and insulin stimulated incorporation of glucose into white adipose tissue lipids (lipogenesis). Reduced ectopic fat accumulation in the heart was associated with significantly increased pAMPK/AMPK ratio and GLUT4 activity. These results provide evidence that Glo1 downregulation in SHR is associated with reduced adiposity and ectopic fat accumulation, most likely mediated by AMPK activation in the heart.

Kinetic Trans-omic Analysis Reveals Key Regulatory Mechanisms for Insulin-Regulated Glucose Metabolism in Adipocytes.

SummaryInsulin regulates glucose metabolism through thousands of regulatory mechanisms; however, which regulatory mechanisms are keys to control glucose metabolism remains unknown. Here, we performed kinetic trans-omic analysis by integrating isotope-tracing glucose flux and phosphoproteomic data from insulin-stimulated adipocytes and built a kinetic mathematical model to identify key allosteric regulatory and phosphorylation events for enzymes. We identified nine reactions regulated by allosteric effectors and one by enzyme phosphorylation and determined the regulatory mechanisms for three of these reactions. Insulin stimulated glycolysis by promoting Glut4 activity by enhancing phosphorylation of AS160 at S595, stimulated fatty acid synthesis by promoting Acly activity through allosteric activation by glucose 6-phosphate or fructose 6-phosphate, and stimulated glutamate synthesis by alleviating allosteric inhibition of Gls by glutamate. Most of glycolytic reactions were regulated by amounts of substrates and products. Thus, phosphorylation or allosteric modulator-based regulation of only a few key enzymes was sufficient to change insulin-induced metabolism.

Fibroblast growth factor-21 potentiates glucose transport in skeletal muscle fibers.

Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that is considered a myokine playing a role in a variety of endocrine functions, including regulation of glucose transport and lipid metabolism. Although FGF21 has been associated with glucose metabolism in skeletal muscle cells, its cellular mechanism in adult skeletal muscle fibers glucose uptake is poorly understood. In the present study, we found that FGF21 induced a dose-response effect, increasing glucose uptake in skeletal muscle fibers from flexor digitorum brevis muscle of mice, evaluated using the fluorescent glucose analog 2-NBDG (300 µM) in single living fibers. This effect was prevented by the use of either Cytochalasin B (5 µM) or Indinavir (100 µM), both antagonists of GLUT4 activity. The use of PI3K inhibitors such as Wortmannin (100 nM) and LY294002 (50 µM) completely prevented the FGF21-dependent glucose uptake. In fibers electroporated with the construct encoding GLUT4myc-eGFP chimera and stimulated with FGF21 (100 ng/mL), a strong sarcolemmal GLUT4 label was detected. This effect promoted by FGF21 was demonstrated to be dependent on atypical PKC-ζ, by using selective PKC inhibitors. FGF21 at low concentrations potentiated the effect of insulin on glucose uptake but at high concentrations, completely inhibited the uptake in the presence of insulin. These results suggest that FGF21 regulates glucose uptake by a mechanism mediated by GLUT4 and dependent on atypical PKC-ζ- in skeletal muscle.

Discovery of novel N-substituted thiazolidinediones (TZDs) as HDAC8 inhibitors: in-silico studies, synthesis, and biological evaluation.

Epigenetics plays a fundamental role in cancer progression, and developing agents that regulate epigenetics is crucial for cancer management. Among Class I and Class II HDACs, HDAC8 is one of the essential epigenetic players in cancer progression. Therefore, we designed, synthesized, purified, and structurally characterized novel compounds containing N-substituted TZD (P1-P25). Cell viability assay of all compounds on leukemic cell lines (CEM, K562, and KCL22) showed the cytotoxic potential of P8, P9, P10, P12, P19, and P25. In-vitro screening of different HDACs isoforms revealed that P19 was the most potent and selective inhibitor for HDAC8 (IC50 - 9.3μM). Thermal shift analysis (TSA) confirmed the binding of P19 to HDAC8. In-vitro screening of all compounds on the transport activity of GLUT1, GLUT4, and GLUT5 indicated that P19 inhibited GLUT1 (IC50 - 28.2μM). P10 and P19 induced apoptotic cell death in CEM cells (55.19% and 60.97% respectively) and P19 was less cytotoxic on normal WBCs (CC50 - 104.2μM) and human fibroblasts (HS27) (CC50 - 105.0μM). Thus, among this novel series of TZD derivatives, compound P19 was most promising HDAC8 inhibitor and cytotoxic on leukemic cells. Thus, P19 could serve as a lead for further development of optimized molecules with enhanced selectivity and potency.

Insulin‐Resistant Pathways Are Associated With Disease Activity in Rheumatoid Arthritis and Are Subject to Disease Modification Through Metabolic Reprogramming: A Potential Novel Therapeutic Approach

To investigate a role for insulin-resistant pathways in inflammation and therapeutic targeting for disease modification in rheumatoid arthritis (RA). RA disease activity and cardiovascular risk factors, including insulin resistance and body mass index (BMI), were assessed in an Irish RA cohort. Glucose transporter 1 (GLUT-1) and GLUT-4 activity in RA and osteoarthritis (OA) synovial tissue was examined using immunohistochemistry. Spontaneous release of proinflammatory mediators from ex vivo RA synovial explants and primary synovial fibroblast (SF) cell culture supernatants was quantified by enzyme-linked immunosorbent assay. Phosphorylated AMP-activated protein kinase (p-AMPK) and GLUT-1 protein expression was analyzed by Western blotting. Cellular glycolytic and oxidative phosphorylation was assessed using extracellular flux analysis. Insulin resistance was independently associated with both BMI (unstandardized coefficient B 0.113 [95% confidence interval (95% CI) 0.059-0.167]; P < 0.001) (n = 61) and swollen joint count in 28 joints (SJC28) (B 0.114 [95% CI 0.032-0.197]; P = 0.008) (n = 61). Increased GLUT-1 expression in RA synovium (n = 26) versus OA synovium (n = 16) was demonstrated (P = 0.0003), with increased expression in the lining, sublining, and vascular regions. In contrast, decreased GLUT-4 expression in the RA lining layer (n = 21) versus the OA lining layer (n = 8) was observed (P = 0.0358). Decreased GLUT-1 protein expression was observed in parallel with increased p-AMPK protein expression in SFs in the presence of metformin (n = 4). Metformin increased glycolytic activity and decreased oxidative phosphorylation in RASFs (n = 7) (P < 0.05 for both). Metformin or aminoimidazole carboxamide ribonucleotide presence decreased spontaneous production of interleukin-6 (IL-6), IL-8, and monocyte chemotactic protein 1 in RA synovial explants and SFs (n = 5-7). Insulin resistance is significantly associated with BMI and synovitis in RA, suggesting distinct interplay between glucose availability and inflammation in RA. Furthermore, the effect of metformin on proinflammatory mechanisms suggests a role for AMPK-modifying compounds in the treatment of RA.

NF-κB inhibitor QNZ protects human chondrocyte degeneration by promoting glucose uptake through Glut4 activation.

Glucose is not only an energy source but also raw material for proteoglycan biosynthesis of chondrocyte. The aim of the present study is to explore the role of QNZ in the progress of chondrocytes glucose uptake and investigate whether it will improve the chondrocytes degeneration through Glut4 activation. We isolated human chondrocytes from the cartilage by the patients who underwent total knee arthroplasty operations. Chondrocytes were pretreated with insulin or QNZ for 24 h. The uptake of glucose with stimulation, as well as the expression of Glut4, collagen II, aggrecan, MMP13, TNF-α, PCNA, and the p16 levels were determined by Western blot, quantitative reverse-transcription polymerase chain reaction (qPCR), or immunofluorescence. Both insulin and QNZ stimuli to chondrocytes contributed to the expression of Glut4 and glucose uptake compared to the normal cells. Additionally, collagen II and aggrecan expression was detected to a significant increase, along with the reduced levels of MMP13 and TNF-α after exposed to QNZ. Moreover, QNZ protected chondrocytes degeneration via promoting proliferation and delaying aging. After blocking Glut4, the glucose uptake significantly reduced in QNZ treatment, as well as the expression of collagen II and aggrecan. However, no significant changes were noticed in the MMP13 and TNF-α levels. The present study demonstrates that inhibition of NF-κB activation by QNZ would improve the glucose uptake through Glut4 activation, which plays an important role in the protection of chondrocytes degeneration.

Antioxidant-Conjugated Peptide Attenuated Metabolic Reprogramming in Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disorder instigated by pulmonary vascular cell proliferation. Activation of Akt was previously reported to promote vascular remodeling. Also, the irreversible nitration of Y350 residue in Akt results in its activation. NitroAkt was increased in PAH patients and the SU5416/Hypoxia (SU/Hx) PAH model. This study investigated whether the prevention of Akt nitration in PAH by Akt targeted nitroxide-conjugated peptide (NP) could reverse vascular remodeling and metabolic reprogramming. Treatment of the SU/Hx model with NP significantly decreased nitration of Akt in lungs, attenuated right ventricle (RV) hypertrophy, and reduced RV systolic pressure. In the PAH model, Akt-nitration induces glycolysis by activation of the glucose transporter Glut4 and lactate dehydrogenase-A (LDHA). Decreased G6PD and increased GSK3β in SU/Hx additionally shunted intracellular glucose via glycolysis. The increased glycolytic rate upregulated anaplerosis due to activation of pyruvate carboxylase in a nitroAkt-dependent manner. NP treatment resolved glycolytic switch and activated collateral pentose phosphate and glycogenesis pathways. Prevention of Akt-nitration significantly controlled pyruvate in oxidative phosphorylation by decreasing lactate and increasing pyruvate dehydrogenases activities. Histopathological studies showed significantly reduced pulmonary vascular proliferation. Based on our current observation, preventing Akt-nitration by using an Akt-targeted nitroxide-conjugated peptide could be a useful treatment option for controlling vascular proliferation in PAH.

Klotho induces insulin resistance possibly through interference with GLUT4 translocation and activation of Akt, GSK3β, and PFKfβ3 in 3T3-L1 adipocyte cells.

Klotho is considered as an anti-aging factor inducing insulin resistance and involved in type 2 diabetes. However, mechanisms by which klotho induces insulin resistance remain to be understood. Thus, in this study, we aimed to evaluate possible interference points of klotho with insulin signaling pathways in 3T3-L1 adipocyte cells by focusing on phosphorylation levels of Akt, GSK3β, PFK-fβ3, and GLUT4 translocation. Differentiation of 3T3-L1 cells to the adipocyte-like cells were performed using specific differentiation kit and confirmed by mRNA expression assay of PPARγ using qRT-PCR, and Sudan black staining of lipid droplets. Then cells were co-treated with klotho and insulin. Expression and translocation of GLUT4 mRNA were evaluated using qRT-PCR and Alexa flour 488 conjugated GLUT4 antibody, respectively. P-Akt/Akt, p-GSK3β/GSK3β, and p-PFKfβ3/PFKfβ3 ratios were determined in insulin and klotho/insulin treated cells using western blot. Our result indicated that GLUT4 expression were decreased to 0.72 ± 0.16 fold in insulin treated cells, however it was calculated 1.12 ± 0.25 fold in klotho/insulin treated cells. In addition, klotho prevented GLUT4 membrane translocation by 27.2% in comparison with insulin-treated cells (P < 0.05). Interestingly, in insulin/klotho co-treated cells, phospho-levels of Akt, GSK3β, and PFKfβ3 proteins was decreased to 2.34 ± 0.14, 2.29 ± 0.63, and 1.95 ± 0.37 fold in comparison with the insulin cells, (P < 0.05). In conclusion, our study indicated that klotho induces insulin resistance in adipocytes possibly through prevention of GLUT4 translocation, and interfere with phosphorylation of Akt, GSK3β, and PFKf3β intracellular signaling mediators by insulin.

Hypoglycemic and hypolipidemic effects of total saponins from Stauntonia chinensis in diabetic db/db mice

Stauntonia chinensis DC. has been utilised as a traditional herbal medicine for its anti‐hyperglycemic characteristic, which has been associated with triterpene saponins. The goal of the current evaluation was to examine hypoglycemic activity and affiliated mechanism of total saponins from S. chinensis. The chemical composition was analysed by HPLC‐ESI‐MS/MS. The fasting blood glucose, oral glucose tolerance test, insulin tolerance test, insulin and glycogen levels of type 2 diabetic db/db mice administered total saponins were quantified to determine the hypoglycemic effects. The serum lipid profiles were assessed to determine the hypolipidemic effects. Western blotting was used to quantify the protein levels of insulin receptor substrates (IRS)‐1/PI3K/AKT, AMPK/ACC and GLUT4. Twenty triterpene saponins were identified from the total saponins, which exhibited hypoglycemic activities and modulated hyperlipidemia that was associated with type 2 diabetes. The hypoglycemic effects were partly due to the activation of GLUT4, which is regulated by IRS‐1/PI3K/AKT. The activation of the AMPK/ACC signalling pathway may be responsible for the hypolipidemic activity. This study revealed that total saponins from S. chinensis have significant hypoglycemic and hypolipidemic activity in diabetic db/db mice, indicating that these may be utilised in the development of saponins based on S. chinensis for the treatment of type 2 diabetes.

Identification of baicalin from Bofutsushosan and Daisaikoto as a potent inducer of glucose uptake and modulator of insulin signaling-associated pathways

Type 2 diabetes mellitus (T2DM) is a chronic disease characterized by hyperglycemia that can lead to long-term complications including heart diseases, stroke, retinopathy, and renal failure. Treatment strategies include stimulating glucose uptake and controlling blood glucose level. Bofutsushosan (BOF) and Daisaikoto (DAI) are two herb-based kampo medicines that have been demonstrated to improve metabolism-associated disorders including obesity, hyperlipidemia, and nonalcoholic fatty liver. Given their bioactivities against metabolic syndromes, we explored in this study the effect of BOF and DAI extracts on glucose absorption and used them as source to identify phytochemical stimulator of glucose absorption. Glucose uptake and mechanistic studies were evaluated in differentiated C2C12 skeletal muscle cells, and HPLC analysis was used to determine the molecular bioactive constituents. Our results indicated that the ethanolic extracts of BOF and DAI (BOFEE and DAIEE, respectively) enhanced the glucose uptake ratio in the differentiated C2C12 cells, and further analysis identified the flavone baicalin as a major constituent capable of efficiently stimulating glucose absorption. Mechanistic studies revealed that the effect from baicalin involved the activation of IRS-1 and GLUT-4, and implicated the AMPK, PI3K/Akt, and MAPK/ERK signaling cascades. Due to its potency, we suggest that baicalin merit further evaluation as a potential candidate anti-hyperglycemic agent for the treatment and management of T2DM.