4-hydroxyisoleucine and Insulin-Resistance

Topics: Animals; Glucose; Humans; In Vitro Techniques; Insulin Resistance; Isoleucine; Liver; Liver Function Tests; MAP Kinase Signaling System; Obesity; Plant Extracts; Trigonella

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.

Topics: Adenylate Kinase; Androstadienes; Animals; Cell Line; Deoxyglucose; Enzyme Activation; Glucose Transporter Type 4; Immunoprecipitation; Inflammation; Insulin Receptor Substrate Proteins; Insulin Resistance; Isoleucine; Mice; Muscle Fibers, Skeletal; Phosphorylation; Suppressor of Cytokine Signaling 3 Protein; Tumor Necrosis Factor-alpha; Wortmannin

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).

Topics: ADAM Proteins; ADAM17 Protein; Dose-Response Relationship, Drug; Gene Expression Regulation; Glucose; Glucose Transporter Type 4; Hep G2 Cells; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Isoleucine; Phosphorylation; Signal Transduction; Tissue Inhibitor of Metalloproteinase-3; Trigonella; Tumor Necrosis Factor-alpha

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.

Topics: 3T3-L1 Cells; ADAM Proteins; ADAM17 Protein; Adipocytes; Animals; Deoxyglucose; Dose-Response Relationship, Drug; Insulin; Insulin Resistance; Isoleucine; Mice; Pioglitazone; Signal Transduction; Thiazolidinediones; Time Factors; Tissue Inhibitor of Metalloproteinase-4; Tissue Inhibitor of Metalloproteinases

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.

Topics: Animals; Cell Line; Extracellular Signal-Regulated MAP Kinases; Fatty Acids; Glucose Transporter Type 4; GTPase-Activating Proteins; Inflammation; Insulin Receptor Substrate Proteins; Insulin Resistance; Isoleucine; MAP Kinase Signaling System; Muscle, Skeletal; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Reactive Oxygen Species

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.

Topics: AMP-Activated Protein Kinases; Animals; Blood Glucose; Cells, Cultured; Diabetes Mellitus, Experimental; Gene Expression Regulation; Insulin Resistance; Isoleucine; Male; Mitochondria; Muscle Fibers, Skeletal; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Signal Transduction

To investigate the effect of 4-hydroxyisoleucine (4-HIL), an active component of Trigonella Foenum-graecum L. on high glucose induced insulin resistance (IR) in 3T3-L1 adipocytes, and to explore underlying molecular mechanisms.. 3T3-L1 adipocytes were treated with 25 mmol/L glucose and 0.6 nmol/L insulin to induce IR. They were intervened by different concentrations of 4-HIL (at 5, 10, and 20 micromol/L). [3H]-Deoxy-D-glucose up-taking method was used to detect the glucose uptake. The mRNA expression of cellular tumor necrosis factor-alpha (TNF-alpha) was detected by polymerase chain reaction (PCR). The content of TNF-alpha in the culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Palmitic acid (PA) acted as the control.. After intervened by 25 mmol/L glucose and 0.6 nmol/L insulin for 18 h, the insulin-stimulated glucose transportation in 3T3-L1 adipocytes was inhibited by 63%. The mRNA expression of cellular TNF-alpha in adipocytes significantly increased, when compared with that in normal adipocytes (P < 0.05). The level of TNF-alpha secreted in the culture supernatant was increased by 70 pg/mL (P < 0.05). Similar changes occurred in the PA group. After exposure to 4-HIL (5, 10, or 20 micromol/L) for 24 h, the glucose transportation was increased by 35%, 50%, and 60%, respectively. PCR results showed that along with increasing 4-HIL concentrations, the mRNA expression of cellular TNF-alpha showed a decreasing trend, showing statistical difference when compared with the model group and the PA group (P < 0.05). Compared with the model group, the TNF-alpha level in the supernatant was respectively reduced by 10 pg/mL, 18 pg/mL, and 39 pg/mL after intervention (P < 0.05).. 4-HIL could remarkably improve high glucose-induced IR in 3T3-L1 adipocytes. Meanwhile, 4-HIL could inhibit the secretion of TNF-alpha.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Glucose; Insulin; Insulin Resistance; Isoleucine; Male; Mice; Trigonella; Tumor Necrosis Factor-alpha

Fenugreek (Trigonella foenum graecum L) is a plant traditionally used for the treatment of diabetes. It contains an unusual amino acid, 4-hydroxyisoleucine, demonstrated to have insulinotropic and antidiabetic properties in animal models. Here we examine the effect of 4-hydroxyisoleucine on liver function and blood glucose in two rat models of insulin resistance, fructose-fed rats and streptozotocin-induced diabetes type 2. In fructose-fed rats, levels of glucose and liver damage marker aspartate transaminase were markedly (84% and 93%, respectively) and significantly elevated compared with controls (p < 0.001 for both). Alanine transaminase was elevated slightly (18%), and all markers were restored to near control values after treatment with 4-hydroxyisoleucine at 50 mg/kg per day for 8 weeks, the effect being significant (p < 0.01) for all markers. This prolonged exposure to 4-hydroxyisoleucine was well tolerated in control animals and did not alter levels of glucose or liver damage markers significantly. In diabetic rats, treatment with 4-hydroxyisoleucine did not affect glucose or liver damage markers, but did improve HDL-cholesterol levels (31% increase, p < 0.05). These findings indicate 4-hydroxyisoleucine as a useful and well-tolerated treatment for insulin resistance, both directly as a hypoglycaemic and also as a protective agent for the liver.

Topics: Alanine Transaminase; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Fructose; Hypoglycemic Agents; Insulin Resistance; Isoleucine; Liver; Male; Plant Extracts; Rats; Rats, Wistar; Trigonella

ID-1101 (4-hydroxyisoleucine), an amino acid extracted from fenugreek seeds, exhibits an interesting glucose-dependent insulin-stimulating activity. The present study was undertaken to investigate a possible extrapancreatic effect of ID-1101 on insulin signaling and action besides its previously described insulinotropic action. Insulin-sensitizing effects of ID-1101 were investigated in rat in vivo by three different approaches: 1) using euglycemic hyperinsulinemic clamps in two different rat models of insulin resistance, i.e., Zucker fa/fa rats and rats fed a sucrose-lipid diet; 2) measuring liver and muscle phosphatidylinositol (PI) 3-kinase activity after an acute injection of ID-1101 in normal and insulin-resistant diabetic rats; and 3) after chronic treatment in two rat models of insulin resistance. Euglycemic hyperinsulinemic clamp experiments revealed that ID-1101 can improve insulin resistance through an increase of peripheral glucose utilization rate in sucrose-lipid-fed rats and by decreasing hepatic glucose production in Zucker fa/fa rats. Moreover, we demonstrated that a single injection of ID-1101 activates the PI 3-kinase activity in liver and muscle from normal rats but also in muscle from diabetic rats. Finally, chronic ID-1101 treatment significantly reduced insulinemia in type 2 diabetic rats and reduced the progression of hyperinsulinemia in insulin-resistant obese Zucker fa/fa rats. These findings clearly demonstrate that ID-1101 can reduce insulin resistance through activation of the early steps of insulin signaling in peripheral tissues and in liver. In summary, ID-1101, besides its insulinotropic effect, directly improves insulin sensitivity, making it a potentially very valuable therapeutic agent for diabetes treatment.

Topics: Animals; Diabetes Mellitus, Experimental; Diet; Enzyme Activation; Glucose; Glucose Clamp Technique; Hyperinsulinism; Insulin; Insulin Resistance; Isoleucine; Lipids; Liver; Male; Muscle, Skeletal; Niacinamide; Obesity; Phosphatidylinositol 3-Kinases; Rats; Rats, Sprague-Dawley; Rats, Zucker; Signal Transduction; Sucrose