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Tadigoppula Narender - One of the best experts on this subject based on the ideXlab platform.
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4-Hydroxyisoleucine attenuates the inflammation-mediated insulin resistance by the activation of AMPK and suppression of SOCS-3 coimmunoprecipitation with both the IR-β subunit as well as IRS-1
Molecular and Cellular Biochemistry, 2016Co-Authors: Sudeep Gautam, Rohit Singh, Tadigoppula Narender, Nayab Ishrat, Pragya Yadav, Arvind K. SrivastavaAbstract:It is known that 4-Hydroxyisoleucine (4-HIL) from seeds of Trigonella foenum-graecum has beneficial effects on low-grade inflammation; therefore, the insulin signaling as well as the anti-inflammatory effects of 4-HIL in TNF-α-induced insulin resistance in C2C12 myotubes was studied with an aim to dissect out the mechanism(s) of the inflammation-mediated insulin resistance. TNF-α suppressed insulin-stimulated glucose transport rate and increased Ser-307 phosphorylation of insulin receptor substrate-1 (IRS-1). However, the treatment of 4-Hydroxyisoleucine enhanced insulin-stimulated glucose transport rate via the activation of AMP-activated protein kinase (AMPK) in a dose-dependent manner. 4-HIL also increases the tyrosine phosphorylation of both IR-β and IRS-1. Moreover, coimmunoprecipitation (Co-IP) of insulin receptor-β (IR-β) subunit with IRS-1 was found to be increased by 4-Hydroxyisoleucine. Concentration of SOCS-3 protein and coimmunoprecipitation of SOCS-3 protein with both the IR-β subunit as well as IRS-1 was found to be decreased by 4-HIL. We conclude that the 4-Hydroxyisoleucine reverses the insulin resistance by the activation of AMPK and suppression of SOCS-3 coimmunoprecipitation with both the IR-β subunit as well as IRS-1.
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4-Hydroxyisoleucine ameliorates fatty acid-induced insulin resistance and inflammatory response in skeletal muscle cells.
Molecular and cellular endocrinology, 2014Co-Authors: Chandan K. Maurya, Rohit Singh, Tadigoppula Narender, Natasha Jaiswal, K. Venkateswarlu, Akhilesh Kumar TamrakarAbstract:The 4-Hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum-graecum was investigated for its metabolic effects to ameliorate free fatty acid-induced insulin resistance in skeletal muscle cells. An incubation of L6 myotubes with palmitate inhibited insulin stimulated-glucose uptake and -translocation of glucose transporter 4 (GLUT4) to the cell surface. Addition of 4-HIL strongly prevented this inhibition. We then examined the insulin signaling pathway, where 4-HIL effectively inhibited the ability of palmitate to reduce insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1), protein kinase B (PKB/AKT), AKT substrate of 160 kD (AS160) and glycogen synthase kinase 3β (GSK-3β) in L6 myotubes. Moreover, 4-HIL presented strong inhibition on palmitate-induced production of reactive oxygen species (ROS) and associated inflammation, as the activation of NF-κB, JNK1/2, ERK1/2 and p38 MAPK was greatly reduced. 4-HIL also inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-stimulated IRS-1 tyrosine phosphorylation in the presence of palmitate, leading to enhanced insulin sensitivity. These findings suggested that 4-HIL could inhibit palmitate-induced, ROS-associated inflammation and restored insulin sensitivity through regulating IRS-1 function.
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4-Hydroxyisoleucine stimulates glucose uptake by increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-3-kinase-dependent pathway
European Journal of Nutrition, 2012Co-Authors: Natasha Jaiswal, Arvind K. Srivastava, Tadigoppula Narender, Chandan K. Maurya, K. Venkateswarlu, P. Sukanya, Akhilesh K. TamrakarAbstract:Purpose To determine the effect of 4-Hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum - graecum , on glucose uptake and the translocation of glucose transporter 4 (GLUT4) to plasma membrane in skeletal muscle cells and to investigate the underlying mechanisms of action. Methods Rat skeletal muscle cells (L6-GLUT4 myc ) were treated with 4-HIL, and the effect on glucose uptake was determined by measuring the incorporation of radio-labeled 2-deoxy-[^3H]- d -glucose (2-DG) into the cell. Translocation of GLUT4 myc to plasma membrane was measured by an antibody-coupled colorimetric assay. Results The prolonged exposure (16 h) of L6-GLUT4 myc myotubes to 4-HIL caused a substantial increase in the 2-DG uptake and GLUT4 translocation to the cell surface, without changing the total amount of GLUT4 and GLUT1. Cycloheximide treatment reversed the effect of 4-HIL on GLUT4 translocation to the basal level suggesting the requirement of new protein synthesis. The 4-HIL-induced increase in GLUT4 translocation was completely abolished by wortmannin, and 4-HIL significantly increased the basal phosphorylation of AKT (Ser-473), but did not change the mRNA expression of AKT, IRS-1, GLUT4, and GSK3β. Conclusion Results suggest that 4-HIL stimulates glucose uptake in L6-GLUT4 myc myotubes by enhancing translocation of GLUT4 to the cell surface in a PI-3-kinase/AKT-dependent mechanism.
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4-Hydroxyisoleucine stimulates glucose uptake by increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-3-kinase-dependent pathway.
European journal of nutrition, 2012Co-Authors: Natasha Jaiswal, Arvind K. Srivastava, Tadigoppula Narender, Chandan K. Maurya, K. Venkateswarlu, P. Sukanya, Akhilesh K. TamrakarAbstract:Purpose To determine the effect of 4-Hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum-graecum, on glucose uptake and the translocation of glucose transporter 4 (GLUT4) to plasma membrane in skeletal muscle cells and to investigate the underlying mechanisms of action.
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Antihyperglycaemic effect of an unusual amino acid (4-Hydroxyisoleucine) in C57BL/KsJ-db/db mice
Natural product research, 2010Co-Authors: Amar B. Singh, Akhilesh K. Tamarkar, Shweta, Tadigoppula Narender, Arvind K. SrivastavaAbstract:The present report confirms the anti-hyperglycaemic and anti-dyslipidemic properties of 4-Hydroxyisoleucine, an unusual amino acid isolated from Trigonella foenum-graecum seeds, for the first time in a well-characterised model of type II diabetes, i.e. db/db mice. 4-Hydroxyisoleucine, when given orally to these mice at 50 mg kg(-1) dose level, significantly (p < 0.05) declined their elevated blood glucose, plasma insulin, triglycerides, total cholesterol, low-density lipoprotein-cholesterol levels and raised their declined plasma high-density lipoprotein-cholesterol level. These results indicate that 4-Hydroxyisoleucine exhibits significant potential as an anti-diabetic agent by suppressing progression of type II diabetic states that is suggested by enhancement of insulin sensitivity and glucose uptake in peripheral tissue.
Arvind K. Srivastava - One of the best experts on this subject based on the ideXlab platform.
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4-Hydroxyisoleucine attenuates the inflammation-mediated insulin resistance by the activation of AMPK and suppression of SOCS-3 coimmunoprecipitation with both the IR-β subunit as well as IRS-1
Molecular and Cellular Biochemistry, 2016Co-Authors: Sudeep Gautam, Rohit Singh, Tadigoppula Narender, Nayab Ishrat, Pragya Yadav, Arvind K. SrivastavaAbstract:It is known that 4-Hydroxyisoleucine (4-HIL) from seeds of Trigonella foenum-graecum has beneficial effects on low-grade inflammation; therefore, the insulin signaling as well as the anti-inflammatory effects of 4-HIL in TNF-α-induced insulin resistance in C2C12 myotubes was studied with an aim to dissect out the mechanism(s) of the inflammation-mediated insulin resistance. TNF-α suppressed insulin-stimulated glucose transport rate and increased Ser-307 phosphorylation of insulin receptor substrate-1 (IRS-1). However, the treatment of 4-Hydroxyisoleucine enhanced insulin-stimulated glucose transport rate via the activation of AMP-activated protein kinase (AMPK) in a dose-dependent manner. 4-HIL also increases the tyrosine phosphorylation of both IR-β and IRS-1. Moreover, coimmunoprecipitation (Co-IP) of insulin receptor-β (IR-β) subunit with IRS-1 was found to be increased by 4-Hydroxyisoleucine. Concentration of SOCS-3 protein and coimmunoprecipitation of SOCS-3 protein with both the IR-β subunit as well as IRS-1 was found to be decreased by 4-HIL. We conclude that the 4-Hydroxyisoleucine reverses the insulin resistance by the activation of AMPK and suppression of SOCS-3 coimmunoprecipitation with both the IR-β subunit as well as IRS-1.
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In vitro anti-hyperglycemic activity of 4-Hydroxyisoleucine derivatives.
Phytomedicine : international journal of phytotherapy and phytopharmacology, 2014Co-Authors: Venkateswarlu Korthikunta, Jyotsana Pandey, Rohit Singh, Rohit Srivastava, Arvind K. Srivastava, Akhilesh K. Tamrakar, Narender TadigoppulaAbstract:Abstract The nonproteinogenic amino acid, 4-Hydroxyisoleucine (1) has been isolated in large quantities from the fenugreek (T. foenum-graecum) seeds. Few novel derivatives (3–11 and 13–18) were prepared from the naturally occurring 4-Hydroxyisoleucine (1) and screened for their in vitro glucose uptake stimulatory effect in L-6 skeletal muscle cells. The derivatives 6, 7, 8, 10 and 11 exhibited better glucose uptake stimulatory activity than parent compound, 4-Hydroxyisoleucine at 5 and 10 µM concentrations and compounds 7 and 11 enhanced translocation of insulin sensitive glucose transporters-4 in skeletal muscle cells.
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4-Hydroxyisoleucine improves insulin resistance by promoting mitochondrial biogenesis and act through AMPK and Akt dependent pathway.
Fitoterapia, 2014Co-Authors: Arun Kumar Rawat, Venkateswarlu Korthikunta, Akhilesh K. Tamrakar, Narender Tadigoppula, Sudeep Gautam, Savita Pal, Arvind K. SrivastavaAbstract:4-Hydroxyisoleucine (4-HIL) is an unusual amino acid isolated from fenugreek seeds (Trigonella foenum graecum L). Various studies have shown that it acts as an antidiabetic agent yet its mechanism of action is not clear. We therefore investigated the effect 4-HIL on the high fructose diet fed streptozotocin induced diabetic rats and L6 myotubes. 4-HIL (50 mg/kg) has improved blood lipid profile, glucose tolerance and insulin sensitivity in a diabetic rat model. It has increased the glucose uptake in L6 myotubes in AMPK-dependent manner and upregulated the expression of genes (PGC-1α, PGC-1β, CPT 1 and CPT 2), which have role in mitochondrial biogenesis and energy metabolism in the liver, skeletal muscles as well as in L6 myotubes. Interestingly, it also increased the AMPK and Akt expression along with their phosphorylated forms in the liver and muscle tissues of treated animals. Altogether we concluded that 4-HIL acts to improve insulin resistance by promoting mitochondrial biogenesis in high fructose diet fed STZ induced diabetic rats.
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4-Hydroxyisoleucine stimulates glucose uptake by increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-3-kinase-dependent pathway
European Journal of Nutrition, 2012Co-Authors: Natasha Jaiswal, Arvind K. Srivastava, Tadigoppula Narender, Chandan K. Maurya, K. Venkateswarlu, P. Sukanya, Akhilesh K. TamrakarAbstract:Purpose To determine the effect of 4-Hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum - graecum , on glucose uptake and the translocation of glucose transporter 4 (GLUT4) to plasma membrane in skeletal muscle cells and to investigate the underlying mechanisms of action. Methods Rat skeletal muscle cells (L6-GLUT4 myc ) were treated with 4-HIL, and the effect on glucose uptake was determined by measuring the incorporation of radio-labeled 2-deoxy-[^3H]- d -glucose (2-DG) into the cell. Translocation of GLUT4 myc to plasma membrane was measured by an antibody-coupled colorimetric assay. Results The prolonged exposure (16 h) of L6-GLUT4 myc myotubes to 4-HIL caused a substantial increase in the 2-DG uptake and GLUT4 translocation to the cell surface, without changing the total amount of GLUT4 and GLUT1. Cycloheximide treatment reversed the effect of 4-HIL on GLUT4 translocation to the basal level suggesting the requirement of new protein synthesis. The 4-HIL-induced increase in GLUT4 translocation was completely abolished by wortmannin, and 4-HIL significantly increased the basal phosphorylation of AKT (Ser-473), but did not change the mRNA expression of AKT, IRS-1, GLUT4, and GSK3β. Conclusion Results suggest that 4-HIL stimulates glucose uptake in L6-GLUT4 myc myotubes by enhancing translocation of GLUT4 to the cell surface in a PI-3-kinase/AKT-dependent mechanism.
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4-Hydroxyisoleucine stimulates glucose uptake by increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-3-kinase-dependent pathway.
European journal of nutrition, 2012Co-Authors: Natasha Jaiswal, Arvind K. Srivastava, Tadigoppula Narender, Chandan K. Maurya, K. Venkateswarlu, P. Sukanya, Akhilesh K. TamrakarAbstract:Purpose To determine the effect of 4-Hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum-graecum, on glucose uptake and the translocation of glucose transporter 4 (GLUT4) to plasma membrane in skeletal muscle cells and to investigate the underlying mechanisms of action.
Qin Cai - One of the best experts on this subject based on the ideXlab platform.
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4 Hydroxyisoleucine improves insulin resistance in hepg2 cells by decreasing tnf α and regulating the expression of insulin signal transduction proteins
Molecular Medicine Reports, 2015Co-Authors: Feng Gao, Mohammad Ishraq Zafar, Liumeng Jian, Qin Cai, Raja Adeel ShafqatAbstract:Previous studies have indicated that 4‑Hydroxyisoleucine (4‑HIL) improves insulin resistance, however, the underlying mechanisms remain to be elucidated. In the present study, the molecular mechanisms underlying how 4‑HIL improves insulin resistance in hepatocytes were examined. HepG2 cells were co‑cultured with insulin and a high glucose concentration to obtain insulin‑resistant (IR) HepG2 cells. Insulin sensitivity was determined by measuring the glucose uptake rate. The IR HepG2 cells were treated with different concentrations of 4‑HIL to determine its effect on IR Hep2 cells. The levels of tumor necrosis factor‑α (TNF‑α) were measured by an enzyme‑linked immunosorbent assay and protein levels of TNF‑α converting enzyme (TACE)/tissue inhibitor of metalloproteinase 3 (TIMP3), insulin receptor substrate (IRS)‑1, IRS‑2, phosphorylated (p)‑IRS‑1 (Ser307) and glucose transporter type 4 (GLUT4) were measured by western blot analysis. The results of the present study demonstrated that insulin‑induced glucose uptake was reduced in IR HepG2 cells; however, this reduction was reversed by 4‑HIL in a dose‑dependent manner. 4‑HIL achieved this effect by downregulating the expression of TNF‑α and TACE, and upregulating the expression of TIMP3 in IR HepG2 cells. In addition, 4‑HIL increased the expression of the insulin transduction regulators IRS‑1 and GLUT4, and decreased the expression of p‑IRS‑1 (Ser307), without affecting the expression of IRS‑2. The present study suggests that 4‑HIL improved insulin resistance in HepG2 cells by the following mechanisms: 4‑HIL reduced TNF‑α levels by affecting the protein expression of the TACE/TIMP3 system and 4‑HIL stimulated the expression of IRS‑1 and GLUT4, but inhibited the expression of p‑IRS‑1 (Ser307).
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4-Hydroxyisoleucine ameliorates an insulin resistant-like state in 3T3-L1 adipocytes by regulating TACE/TIMP3 expression
Drug design development and therapy, 2015Co-Authors: Feng Gao, Mohammad Ishraq Zafar, Raja Adeel Shafqat, Liumeng Jian, Qin CaiAbstract:Obesity-associated insulin resistance (IR) is highly correlated with soluble tumor necrosis factor-α (sTNF-α), which is released from transmembranous TNF-α by TNF-α converting enzyme (TACE). In vivo, TACE activity is suppressed by tissue inhibitor of metalloproteinase 3 (TIMP3). Agents that can interact with TACE/TIMP3 to improve obesity-related IR would be highly valuable. In the current study, we assessed whether (2S,3R,4S)-4-Hydroxyisoleucine (4-HIL) could modulate TACE/TIMP3 and ameliorate an obesity-induced IR-like state in 3T3-L1 adipocytes.3T3-L1 adipocytes were incubated in the presence of 25 mM glucose and 0.6 nM insulin to induce an IR-like state, and were then treated with different concentrations of 4-HIL or 10 µM pioglitazone (positive control). The glucose uptake rate was determined using the 2-deoxy-[(3)H]-D-glucose method, and the levels of sTNF-α in the cell supernatant were determined using ELISA. The protein expression of TACE, TIMP3, and insulin signaling-related molecules was measured using western blotting.Exposure to high glucose and insulin for 18 hours increased the levels of sTNF-α in the cell supernatant. The phosphorylation of insulin receptor substrate-1 (IRS-1) Ser(307) and Akt Ser(473) was increased, whereas the protein expression of IRS-1, Akt, and glucose transporter-4 was decreased. The insulin-induced glucose uptake was reduced by 67% in 3T3-L1 adipocytes, which indicated the presence of an IR-like state. The above indexes, which demonstrated the successful induction of an IR-like state, were reversed by 4-HIL in a dose-dependent manner by downregulating and upregulating the protein expression of TACE and TIMP3 proteins, respectively.4-HIL improved an obesity-associated IR-like state in 3T3-L1 adipocytes by targeting TACE/TIMP3 and the insulin signaling pathway.
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4 Hydroxyisoleucine ameliorates an insulin resistant like state in 3t3 l1 adipocytes by regulating tace timp3 expression
Drug Design Development and Therapy, 2015Co-Authors: Feng Gao, Mohammad Ishraq Zafar, Raja Adeel Shafqat, Liumeng Jian, Qin CaiAbstract:Obesity-associated insulin resistance (IR) is highly correlated with soluble tumor necrosis factor-α (sTNF-α), which is released from transmembranous TNF-α by TNF-α converting enzyme (TACE). In vivo, TACE activity is suppressed by tissue inhibitor of metalloproteinase 3 (TIMP3). Agents that can interact with TACE/TIMP3 to improve obesity-related IR would be highly valuable. In the current study, we assessed whether (2S,3R,4S)-4-Hydroxyisoleucine (4-HIL) could modulate TACE/TIMP3 and ameliorate an obesity-induced IR-like state in 3T3-L1 adipocytes.3T3-L1 adipocytes were incubated in the presence of 25 mM glucose and 0.6 nM insulin to induce an IR-like state, and were then treated with different concentrations of 4-HIL or 10 µM pioglitazone (positive control). The glucose uptake rate was determined using the 2-deoxy-[(3)H]-D-glucose method, and the levels of sTNF-α in the cell supernatant were determined using ELISA. The protein expression of TACE, TIMP3, and insulin signaling-related molecules was measured using western blotting.Exposure to high glucose and insulin for 18 hours increased the levels of sTNF-α in the cell supernatant. The phosphorylation of insulin receptor substrate-1 (IRS-1) Ser(307) and Akt Ser(473) was increased, whereas the protein expression of IRS-1, Akt, and glucose transporter-4 was decreased. The insulin-induced glucose uptake was reduced by 67% in 3T3-L1 adipocytes, which indicated the presence of an IR-like state. The above indexes, which demonstrated the successful induction of an IR-like state, were reversed by 4-HIL in a dose-dependent manner by downregulating and upregulating the protein expression of TACE and TIMP3 proteins, respectively.4-HIL improved an obesity-associated IR-like state in 3T3-L1 adipocytes by targeting TACE/TIMP3 and the insulin signaling pathway.
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4‑Hydroxyisoleucine improves insulin resistance in HepG2 cells by decreasing TNF‑α and regulating the expression of insulin signal transduction proteins
Molecular medicine reports, 2015Co-Authors: Feng Gao, Mohammad Ishraq Zafar, Liumeng Jian, Qin Cai, Raja Adeel ShafqatAbstract:Previous studies have indicated that 4‑Hydroxyisoleucine (4‑HIL) improves insulin resistance, however, the underlying mechanisms remain to be elucidated. In the present study, the molecular mechanisms underlying how 4‑HIL improves insulin resistance in hepatocytes were examined. HepG2 cells were co‑cultured with insulin and a high glucose concentration to obtain insulin‑resistant (IR) HepG2 cells. Insulin sensitivity was determined by measuring the glucose uptake rate. The IR HepG2 cells were treated with different concentrations of 4‑HIL to determine its effect on IR Hep2 cells. The levels of tumor necrosis factor‑α (TNF‑α) were measured by an enzyme‑linked immunosorbent assay and protein levels of TNF‑α converting enzyme (TACE)/tissue inhibitor of metalloproteinase 3 (TIMP3), insulin receptor substrate (IRS)‑1, IRS‑2, phosphorylated (p)‑IRS‑1 (Ser307) and glucose transporter type 4 (GLUT4) were measured by western blot analysis. The results of the present study demonstrated that insulin‑induced glucose uptake was reduced in IR HepG2 cells; however, this reduction was reversed by 4‑HIL in a dose‑dependent manner. 4‑HIL achieved this effect by downregulating the expression of TNF‑α and TACE, and upregulating the expression of TIMP3 in IR HepG2 cells. In addition, 4‑HIL increased the expression of the insulin transduction regulators IRS‑1 and GLUT4, and decreased the expression of p‑IRS‑1 (Ser307), without affecting the expression of IRS‑2. The present study suggests that 4‑HIL improved insulin resistance in HepG2 cells by the following mechanisms: 4‑HIL reduced TNF‑α levels by affecting the protein expression of the TACE/TIMP3 system and 4‑HIL stimulated the expression of IRS‑1 and GLUT4, but inhibited the expression of p‑IRS‑1 (Ser307).
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4-Hydroxyisoleucine improves hepatic insulin resistance by restoring glycogen synthesis in vitro.
International journal of clinical and experimental medicine, 2015Co-Authors: Qin Cai, Mohammad Ishraq Zafar, Liumeng Jian, Lun Cai, Feng GaoAbstract:Introduction/Aims: Hydroxyisoleucine (4-HIL), derived from fenugreek seeds, improves insulin resistance in 3T3-L1 adipocytes and L6 myotubes. However, the effects of 4-HIL on liver glycogen synthesis and hepatic insulin resistance have not been described. The aim of this study was to investigate the effects of 4-HIL on glycogen synthesis in a tumor necrosis factor-α (TNF-α)-induced insulin resistance model using HepG2 cells. Materials and methods: HepG2 cells were divided into eight groups: control; TNF-α; and 5, 10, or 20 μM 4-HIL without or with TNF-α. Glycogen and protein expression were evaluated using a glycogen assay kit and western blotting, respectively. Results: Glycogen levels did not differ between the 4-HIL groups and control (P>0.05), but were decreased significantly in the TNF-α group (P
Feng Gao - One of the best experts on this subject based on the ideXlab platform.
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4-Hydroxyisoleucine relieves inflammation through iRhom2-dependent pathway in co-cultured macrophages and adipocytes with LPS stimulation.
BMC complementary medicine and therapies, 2020Co-Authors: Cong Zhou, Feng Gao, Rui Chen, Jiaoyue ZhangAbstract:4-Hydroxyisoleucine (4-HIL) is an active ingredient extracted from Trigonella foenum-graecum L., a Chinese traditional herbal medicine, which exerts the efficacy of anti-obesity and anti-diabetes. We previously reported that 4-HIL potentiates anti-inflammatory and anti-insulin resistance effects through down-regulation of TNF-α and TNF-α converting enzyme (TACE) in 3 T3-L1 adipocytes and HepG2 cells. In the present study, we further investigate the effects and mechanisms of 4-HIL on obesity-induced inflammation in RAW264.7 macrophages and 3 T3-L1 adipocytes co-culture system. RAW264.7 macrophages and 3 T3-L1 adipocytes were co-cultured to mimic the microenvironment of adipose tissue. siRNA-iRhom2 transfection was performed to knockdown iRhom2 expression in RAW264.2 macrophages. The mRNA and protein expression of iRhom2 and TACE were measured by real-time quantitative PCR (RT-qPCR) and western blotting. The production of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), IL-6 and IL-10 were evaluated by ELISA. The ratio of M2/M1 was detected by flow cytometry. 4-HIL significantly repressed the mRNA and protein levels of iRhom2 and TACE in RAW264.7 macrophages after LPS stimulated. Meanwhile, the levels of pro-inflammatory cytokines, including TNF-α, MCP-1, and IL-6, were substantially suppressed by 4-HIL in the co-culture system. Moreover, the level of anti-inflammatory cytokine IL-10 was increased significantly by 4-HIL in the co-culture system after LPS stimulation. Additionally, the ratio of M2/M1 was also increased by 4-HIL in the co-culture system after LPS stimulation. Finally, these effects of 4-HIL were largely enhanced by siRNA-iRhom2 transfection. Taken together, our results indicated that obesity-induced inflammation was potently relieved by 4-HIL, most likely through the iRhom2-dependent pathway.
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4-Hydroxyisoleucine: A Potential New Treatment for Type 2 Diabetes Mellitus
BioDrugs : clinical immunotherapeutics biopharmaceuticals and gene therapy, 2016Co-Authors: Mohammad Ishraq Zafar, Feng GaoAbstract:4-Hydroxyisoleucine (4-HIL) is a compound found in Trigonella foenum-graecum (fenugreek) seeds, which have been used as part of traditional medicine to treat diabetes mellitus. The synthesis of 4-HIL on a large scale is possible using fermentation methods (artificial synthesis) involving the isolation of the l-isoleucine dioxygenase gene from Bacillus thuringiensis, which can yield a greater quantity of 4-HIL than that produced with conventional methods (82 % attained with fermentation methods vs. 0.6–39 % attained with conventional methods). In studies of rats and humans, T. foenum-graecum improved laboratory parameters associated with renal dysfunction and dyslipidemia, increased levels of antioxidants and hormones that are altered in patients with type 2 diabetes mellitus (T2DM), and decreased fasting blood glucose, 2-h postprandial plasma glucose, and glycated hemoglobin. Similarly, in in vitro and preclinical studies, 4-HIL decreased glucose levels, hepatic glucose production, glucose/insulin ratios, indicators of hepatic damage, triglycerides, and total cholesterol, and increased utilization of glucose and levels of high-density lipoprotein cholesterol. Studies in humans are needed to determine whether 4-HIL is safer and more effective than current medications for the treatment of T2DM.
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4 Hydroxyisoleucine improves insulin resistance in hepg2 cells by decreasing tnf α and regulating the expression of insulin signal transduction proteins
Molecular Medicine Reports, 2015Co-Authors: Feng Gao, Mohammad Ishraq Zafar, Liumeng Jian, Qin Cai, Raja Adeel ShafqatAbstract:Previous studies have indicated that 4‑Hydroxyisoleucine (4‑HIL) improves insulin resistance, however, the underlying mechanisms remain to be elucidated. In the present study, the molecular mechanisms underlying how 4‑HIL improves insulin resistance in hepatocytes were examined. HepG2 cells were co‑cultured with insulin and a high glucose concentration to obtain insulin‑resistant (IR) HepG2 cells. Insulin sensitivity was determined by measuring the glucose uptake rate. The IR HepG2 cells were treated with different concentrations of 4‑HIL to determine its effect on IR Hep2 cells. The levels of tumor necrosis factor‑α (TNF‑α) were measured by an enzyme‑linked immunosorbent assay and protein levels of TNF‑α converting enzyme (TACE)/tissue inhibitor of metalloproteinase 3 (TIMP3), insulin receptor substrate (IRS)‑1, IRS‑2, phosphorylated (p)‑IRS‑1 (Ser307) and glucose transporter type 4 (GLUT4) were measured by western blot analysis. The results of the present study demonstrated that insulin‑induced glucose uptake was reduced in IR HepG2 cells; however, this reduction was reversed by 4‑HIL in a dose‑dependent manner. 4‑HIL achieved this effect by downregulating the expression of TNF‑α and TACE, and upregulating the expression of TIMP3 in IR HepG2 cells. In addition, 4‑HIL increased the expression of the insulin transduction regulators IRS‑1 and GLUT4, and decreased the expression of p‑IRS‑1 (Ser307), without affecting the expression of IRS‑2. The present study suggests that 4‑HIL improved insulin resistance in HepG2 cells by the following mechanisms: 4‑HIL reduced TNF‑α levels by affecting the protein expression of the TACE/TIMP3 system and 4‑HIL stimulated the expression of IRS‑1 and GLUT4, but inhibited the expression of p‑IRS‑1 (Ser307).
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4-Hydroxyisoleucine ameliorates an insulin resistant-like state in 3T3-L1 adipocytes by regulating TACE/TIMP3 expression
Drug design development and therapy, 2015Co-Authors: Feng Gao, Mohammad Ishraq Zafar, Raja Adeel Shafqat, Liumeng Jian, Qin CaiAbstract:Obesity-associated insulin resistance (IR) is highly correlated with soluble tumor necrosis factor-α (sTNF-α), which is released from transmembranous TNF-α by TNF-α converting enzyme (TACE). In vivo, TACE activity is suppressed by tissue inhibitor of metalloproteinase 3 (TIMP3). Agents that can interact with TACE/TIMP3 to improve obesity-related IR would be highly valuable. In the current study, we assessed whether (2S,3R,4S)-4-Hydroxyisoleucine (4-HIL) could modulate TACE/TIMP3 and ameliorate an obesity-induced IR-like state in 3T3-L1 adipocytes.3T3-L1 adipocytes were incubated in the presence of 25 mM glucose and 0.6 nM insulin to induce an IR-like state, and were then treated with different concentrations of 4-HIL or 10 µM pioglitazone (positive control). The glucose uptake rate was determined using the 2-deoxy-[(3)H]-D-glucose method, and the levels of sTNF-α in the cell supernatant were determined using ELISA. The protein expression of TACE, TIMP3, and insulin signaling-related molecules was measured using western blotting.Exposure to high glucose and insulin for 18 hours increased the levels of sTNF-α in the cell supernatant. The phosphorylation of insulin receptor substrate-1 (IRS-1) Ser(307) and Akt Ser(473) was increased, whereas the protein expression of IRS-1, Akt, and glucose transporter-4 was decreased. The insulin-induced glucose uptake was reduced by 67% in 3T3-L1 adipocytes, which indicated the presence of an IR-like state. The above indexes, which demonstrated the successful induction of an IR-like state, were reversed by 4-HIL in a dose-dependent manner by downregulating and upregulating the protein expression of TACE and TIMP3 proteins, respectively.4-HIL improved an obesity-associated IR-like state in 3T3-L1 adipocytes by targeting TACE/TIMP3 and the insulin signaling pathway.
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4 Hydroxyisoleucine ameliorates an insulin resistant like state in 3t3 l1 adipocytes by regulating tace timp3 expression
Drug Design Development and Therapy, 2015Co-Authors: Feng Gao, Mohammad Ishraq Zafar, Raja Adeel Shafqat, Liumeng Jian, Qin CaiAbstract:Obesity-associated insulin resistance (IR) is highly correlated with soluble tumor necrosis factor-α (sTNF-α), which is released from transmembranous TNF-α by TNF-α converting enzyme (TACE). In vivo, TACE activity is suppressed by tissue inhibitor of metalloproteinase 3 (TIMP3). Agents that can interact with TACE/TIMP3 to improve obesity-related IR would be highly valuable. In the current study, we assessed whether (2S,3R,4S)-4-Hydroxyisoleucine (4-HIL) could modulate TACE/TIMP3 and ameliorate an obesity-induced IR-like state in 3T3-L1 adipocytes.3T3-L1 adipocytes were incubated in the presence of 25 mM glucose and 0.6 nM insulin to induce an IR-like state, and were then treated with different concentrations of 4-HIL or 10 µM pioglitazone (positive control). The glucose uptake rate was determined using the 2-deoxy-[(3)H]-D-glucose method, and the levels of sTNF-α in the cell supernatant were determined using ELISA. The protein expression of TACE, TIMP3, and insulin signaling-related molecules was measured using western blotting.Exposure to high glucose and insulin for 18 hours increased the levels of sTNF-α in the cell supernatant. The phosphorylation of insulin receptor substrate-1 (IRS-1) Ser(307) and Akt Ser(473) was increased, whereas the protein expression of IRS-1, Akt, and glucose transporter-4 was decreased. The insulin-induced glucose uptake was reduced by 67% in 3T3-L1 adipocytes, which indicated the presence of an IR-like state. The above indexes, which demonstrated the successful induction of an IR-like state, were reversed by 4-HIL in a dose-dependent manner by downregulating and upregulating the protein expression of TACE and TIMP3 proteins, respectively.4-HIL improved an obesity-associated IR-like state in 3T3-L1 adipocytes by targeting TACE/TIMP3 and the insulin signaling pathway.
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4 Hydroxyisoleucine an unusual amino acid as antidyslipidemic and antihyperglycemic agent
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: T Narender, Anju Puri, Tanvir Khaliq, Rashmi Saxena, Geetika Bhatia, Ramesh ChandraAbstract:Trigonella foenum-graecum, commonly known as fenugreek, is an annual herbaceous plant. From the seeds of T. foenum-graecum an unusual amino acid, 4-Hydroxyisoleucine 5, has been isolated, which significantly decreased the plasma triglyceride levels by 33% (P<0.002), total cholesterol (TC) by 22% (P<0.02), and free fatty acids by 14%, accompanied by an increase in HDL-C/TC ratio by 39% in the dyslipidemic hamster model.
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4-Hydroxyisoleucine an unusual amino acid as antidyslipidemic and antihyperglycemic agent
Bioorganic & medicinal chemistry letters, 2005Co-Authors: Tadigoppula Narender, Shweta, Anju Puri, Tanvir Khaliq, Rashmi Saxena, Geetika Bhatia, Ramesh ChandraAbstract:Trigonella foenum-graecum, commonly known as fenugreek, is an annual herbaceous plant. From the seeds of T. foenum-graecum an unusual amino acid, 4-Hydroxyisoleucine 5, has been isolated, which significantly decreased the plasma triglyceride levels by 33% (P
