Impaired regulation of the TNF-alpha converting enzyme/tissue inhibitor of metalloproteinase 3 proteolytic system in skeletal muscle of obese type 2 diabetic patients: a new mechanism of insulin resistance in humans - PubMed (original) (raw)

. 2009 Oct;52(10):2169-81.

doi: 10.1007/s00125-009-1451-3. Epub 2009 Jul 25.

S Kamath, A O Chavez, V E Centonze, M Veerasamy, A Barrentine, J J Wewer, D K Coletta, C Jenkinson, R M Jhingan, D Smokler, S Reyna, N Musi, R Khokka, M Federici, D Tripathy, R A DeFronzo, F Folli

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Impaired regulation of the TNF-alpha converting enzyme/tissue inhibitor of metalloproteinase 3 proteolytic system in skeletal muscle of obese type 2 diabetic patients: a new mechanism of insulin resistance in humans

A Monroy et al. Diabetologia. 2009 Oct.

Abstract

Aims/hypothesis: TNF-alpha levels are increased in obesity and type 2 diabetes. The regulation of TNF-alpha converting enzyme (TACE) and its inhibitor, tissue inhibitor of metalloproteinase 3 (TIMP3), in human type 2 diabetes is unknown.

Methods: We examined TACE/TIMP3 regulation: (1) in lean and obese normal glucose tolerant (NGT) individuals and in type 2 diabetes patients; (2) following 6 h of lipid/saline infusion in NGT individuals; and (3) in cultured human myotubes from lean NGT individuals incubated with palmitate. Insulin sensitivity was assessed by a euglycaemic clamp and TACE/TIMP3 was evaluated by confocal microscopy, RT-PCR, western blotting and an in vitro activity assay. Circulating TNF-alpha, TNF-alpha-receptor 1 (TNFR1), TNF-alpha-receptor 2 (TNFR2), IL-6 receptor (IL-6R), vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) levels were evaluated.

Results: TIMP3 levels were reduced and TACE enzymatic activity was increased in type 2 diabetes skeletal muscle. TACE expression, and TACE, TNF-alpha, TNFR1 and IL-6R levels were increased in type 2 diabetes, and positively correlated with insulin resistance. A 6 h lipid infusion into NGT individuals decreased insulin-stimulated glucose metabolism by 25% with increased TACE, decreased expression of the gene encoding TIMP3 and increased IL-6R release. Palmitate induced a dramatic reduction of TIMP3 and increased the TACE/TIMP3 ratio in cultured myotubes.

Conclusions/interpretation: TACE activity was increased in skeletal muscle of obese type 2 diabetes patients and in lipid-induced insulin resistance. We propose that dysregulation of membrane proteolysis by TACE/TIMP3 of TNF-alpha and IL-6R is an important factor for the development of skeletal muscle insulin resistance in obese type 2 diabetes patients by a novel autocrine/paracrine mechanism.

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Figures

Fig. 1

Fig. 1

Subcellular distribution of TACE and TIMP3 in skeletal muscle of NGT individuals. a Subcellular fractionation method outline. b, c Immunoblot analysis of human skeletal muscle subcellular fractions employing anti-TACE (b) and anti-TIMP3 (c) antibodies. *p<0.05 (_n_=3–4 for b and c). Data are means ± SEM. ER, endoplasmic reticulum

Fig. 2

Fig. 2

mRNA for TACE, and TIMP3, pro-TNF-α and TNF-α protein abundance in human skeletal muscle. a Expression of TACE was measured by quantitative real-time RT-PCR and normalised for 18S ribosomal RNA in vastus lateralis (skeletal) muscle. Relative quantification was calculated by the 2−ΔΔCt where ΔCt is Ct−18S Ct. Each reaction was done in duplicate. b Western blot analysis of TACE protein in vastus lateralis muscle. Protein was extracted from total homogenate, solubilised and separated on SDS-PAGE, transferred to nitrocellulose membranes and incubated with rabbit polyclonal anti-TACE antibody. Abundance of TIMP3 (c), pro-TNF-α (d), TNF-α (e) and β-actin (f) in vastus lateralis muscle. Data are means ± SEM. *p<0.01 vs lean, **p<0.01 vs lean, †p<0.05 vs type 2 diabetes (_n_=10–12 per group). T2DM, type 2 diabetes mellitus

Fig. 3

Fig. 3

TACE activity in skeletal muscle in vitro and circulating TACE substrates in lean and obese individuals and type 2 diabetes patients. a, b TACE peptide cleavage was measured by a fluorimetric assay with different quantities of skeletal muscle protein: a relative fluorescence units (RFUs; black circles, 1 µg; white circles, 5 µg; black inverted triangles, 10 µg; white triangles, 15 µg; black squares, 30 µg); b AUCs. c TACE peptide cleavage (RFUs) at 10 µg muscle homogenate protein from lean NGT (black circles) and obese NGT individuals (white circles) and type 2 diabetes (T2DM) patients (black inverted triangles). d AUCs for TACE activity in vitro in lean NGT, obese NGT and type 2 diabetes patients. e, f Correlations between log10 TACE activity and NEFA (e, _r_=0.460, _r_2=0.211, _p_=0.05) and log10 TACE activity and log10 M (f, _r_=0.471, _r_2=0.222, _p_=0.036). g, h Serum concentration (measured with ELISA) of sTNFR1 (g) and sIL-6R (h) in lean NGT and obese NGT individuals and type 2 diabetic patients. Data are means ± SEM. *p<0.05, **p<0.01 (_n_=20 for TACE activity and 8–10 for sTNFR1 and sIL-6R)

Fig. 4

Fig. 4

a Insulin-stimulated glucose metabolism (M value) was measured with the euglycaemic insulin clamp during saline and lipid infusion. b–f mRNA expression for TACE (b), TIMP3 (c), IR (d), IRS-1 (e) and AKT (f) was measured in human vastus lateralis (skeletal) muscle before and after a 6 h lipid or saline infusion in lean NGT individuals. g Serum concentrations of sIL-6R before and after a 2–6 h lipid infusion, and after a 2 h euglycaemic insulin 80 mU m−2 min−1 clamp. Data are means ± SEM. *p<0.05, **p<0.01, ***p<0.001, all compared with basal (_n_=10–12 individuals per group). LBM, lean body mass

Fig. 5

Fig. 5

Expression in human myotubes of TACE (a), TIMP3 (b), TACE/TIMP3 ratio (c) and β-actin (d). *p<0.05, 200 µmol/l NEFA vs BSA control (BSA-Ctrl); **p<0.05, 400 µmol/l NEFA vs BSA. †p<0.05, 200 µmol/l NEFA vs 400 µmol/l NEFA. _n_=4–6 per group. Data are means ± SEM

Fig. 6

Fig. 6

a TACE levels in human myotubes infected with adenovirus encoding TACE or GFP for control. b–d Increased TNF-α shedding in the presence of increased abundance of both pro-TNF-α and TNF-α. e, f AKT and phospho-AKT (Ser 473) levels in myotubes in the presence (+) and absence (−) of insulin (10 nmol/l). g, h AMPK and phospho-AMPK levels in the presence (+) and absence (−) of insulin. i Phospho-ACC levels in human myotubes in the presence (+) or absence (−) of insulin. *p<0.05 vs GFP and absence of insulin; **p< 0.01 vs GFP. _n_=4–6 per group. Data are means ± SEM. j β-Actin as a protein loading control for pro-TNF-α and TNF-α levels. _n_=4 per group. Data are means ± SEM. Values are expressed as a percentage of control

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