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      4-Hydroxyisoleucine ameliorates an insulin resistant-like state in 3T3-L1 adipocytes by regulating TACE/ TIMP3 expression


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          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 (2 S,3 R,4 S)-4-hydroxyisoleucine (4-HIL) could modulate TACE/ TIMP3 and ameliorate an obesity-induced IR-like state in 3T3-L1 adipocytes.

          Materials and methods

          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-[ 3H]- 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.

          Most cited references24

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          Adipocytes as regulators of energy balance and glucose homeostasis.

          Adipocytes have been studied with increasing intensity as a result of the emergence of obesity as a serious public health problem and the realization that adipose tissue serves as an integrator of various physiological pathways. In particular, their role in calorie storage makes adipocytes well suited to the regulation of energy balance. Adipose tissue also serves as a crucial integrator of glucose homeostasis. Knowledge of adipocyte biology is therefore crucial for understanding the pathophysiological basis of obesity and metabolic diseases such as type 2 diabetes. Furthermore, the rational manipulation of adipose physiology is a promising avenue for therapy of these conditions.
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            Tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction.

            A number of studies have demonstrated that tumor necrosis factor-alpha (TNF-alpha) is associated with profound insulin resistance in adipocytes and may also play a critical role in the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. Reports on the mechanism of TNF-alpha action have been somewhat contradictory. GLUT4 down-regulation has been implicated as a possible cause of insulin resistance as has been the reduced kinase function of the insulin receptor. Here we examine the effects of tumor necrosis factor on the protein components thought to be involved in insulin-stimulated glucose transport in adipocytes, namely the insulin receptor, its major substrate IRS-1, and the insulin responsive glucose transporter GLUT4. Prolonged exposure (72-96 h) of 3T3-L1 adipocytes to TNF-alpha causes a substantial reduction (>80%) in IRS-1 and GLUT4 mRNA and protein as well as a lesser reduction (>50%) in the amount of the insulin receptor. Nevertheless, the remaining proteins appear to be biochemically indistinguishable from those in untreated adipocytes. Both the insulin receptor and IRS-1 are tyrosine-phosphorylated to the same extent in response to acute insulin stimulation following cellular TNF-alpha exposure. Furthermore, the ability of the insulin receptor to phosphorylate exogenous substrate in the test tube is also normal following its isolation from TNF-alpha-treated cells. These results are confirmed by the reduced but obvious level of insulin-dependent glucose transport and GLUT4 translocation observed in TNF-alpha-treated adipocytes. We conclude that the insulin resistance of glucose transport in 3T3-L1 adipocytes exposed to TNF-alpha for 72-96 h results from a reduced amount in requisite proteins involved in insulin action. These results are consistent with earlier studies indicating that TNF-alpha reduces the transcriptional activity of the GLUT4 gene in murine adipocytes, and reduced mRNA transcription of a number of relevant genes may be the general mechanism by which TNF-alpha causes insulin resistance in adipocytes.
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              The role of TNF alpha in adipocyte metabolism.

              Tumor necrosis factor-alpha (TNF alpha) is a multifunctional cytokine which exerts a myriad of biological actions in different tissues and species. Many of these actions can perturb the normal regulation of energy metabolism. In adipose tissue, in particular, TNF alpha has been demonstrated to regulate or interfere with adipocyte metabolism at numerous sites including transcriptional regulation, glucose and fatty acid metabolism and hormone receptor signaling. The implications of these perturbations in disease states and the current understanding of the molecular mechanisms utilised by TNF alpha are discussed herein.

                Author and article information

                Drug Des Devel Ther
                Drug Des Devel Ther
                Drug Design, Development and Therapy
                Drug Design, Development and Therapy
                Dove Medical Press
                20 October 2015
                : 9
                : 5727-5736
                [1 ]Department of Endocrinology, Union Hospital, Wuhan, Hubei, People’s Republic of China
                [2 ]Department of Medicine, Tongji Hospital, Wuhan, Hubei, People’s Republic of China
                [3 ]Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China
                [4 ]Chengdu First People’s Hospital, Sichuan, People’s Republic of China
                Author notes
                Correspondence: Furong Lu, Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan, Hubei 430022, People’s Republic of China,s Email furonglulove@ 123456163.com
                © 2015 Gao et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License

                The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.

                Original Research

                Pharmacology & Pharmaceutical medicine
                4-hydroxyisoleucine,obesity-related insulin resistance,soluble tnf-α,tace,timp3,insulin signaling,glucose uptake,fenugreek seed


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