Fails to Affect Insulin-Stimulated Glucose Metabolism of Isolated Rat ...

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 7 Printed in U.S.A.

Acute and Chronic Exposure to Tumor Necrosis Factor-a Fails to Affect Insulin-Stimulated Glucose Metabolism of Isolated Rat Soleus Muscle* ¨ RNSINN, SUSANNE NESCHEN, OSWALD WAGNER, CLEMENS FU ¨ USL MICHAEL RODEN, MARCEL BISSCHOP, AND WERNER WALDHA Department of Medicine III (C.F., S.N., M.R., M.B., W.W.), Division of Endocrinology & Metabolism, and Department of Medical & Chemical Laboratory Diagnostics (O.W.), University of Vienna, Vienna, Austria A-1090 ABSTRACT To better understand the effects of tumor necrosis factor-a (TNFa) on insulin sensitivity, direct interaction of the peptide with freshly isolated rat soleus muscle strips was investigated. Muscles were exposed to TNFa at concentrations ranging from 0.01–5 nmol/liter. Rates of insulin-stimulated (5 or 100 nmol/liter) glucose metabolism were determined after periods of TNFa preexposure of 30 min, 6 h, and 24 h. Independent of exposure time, TNFa failed to exert any significant effect on rates of 3H-2-deoxy-glucose transport (stimulation by 100 nmol/liter insulin after preincubation without vs. with 5 nmol/liter TNFa, cpm/mgzh: 30 min, 779 6 29 vs. 725 6 29; 6 h, 652 6

56 vs. 617 6 60; 24 h, 911 6 47 vs. 936 6 31) or glucose incorporation into glycogen (mmol/gzh: 30 min, 5.19 6 0.22 vs. 5.25 6 0.41; 6 h, 2.08 6 0.10 vs. 2.09 6 0.17; 24 h, 2.51 6 0.21 vs. 2.41 6 0.26). In parallel, TNFa neither affected insulin-stimulated rates of glucose oxidation (CO2 production) and anaerobic glycolysis (lactate release), nor muscle glycogen content. In conclusion, these findings do not support the hypothesis of muscle insulin desensitization by TNFa via autocrine or paracrine mechanisms. The obtained data favor the concept that TNFa-dependent muscle insulin resistance in vivo depends on indirect effects rather than direct interaction of the peptide with skeletal muscle. (Endocrinology 138: 2674 –2679, 1997)

I

peripheral insulin resistance in obese Zucker rats. In contrast to its effects in Zucker rats, TNFa neutralization over a period of 4 weeks did not affect insulin sensitivity in obese NIDDM patients (10). Whether blunting of whole body insulin sensitivity by exogenous and endogenous TNFa in vivo is due to its direct interaction with insulin target tissues or rather mediated via indirect mechanisms is, however, not completely understood. Because up to 80% of insulin-stimulated glucose uptake is into muscle (2, 11), any major changes in whole body glucose clearance in response to infusion or neutralization of TNFa (4, 7, 8) have to involve considerable changes in muscle insulin sensitivity (7). Although a decrease in insulin action has been described in cultured L6 rat muscle cells preexposed to TNFa for 10 min to 12 h (12), no insulin resistance was observed in the same cell line after TNFa treatment for 4–8 days (13) or in cardiomyocytes after short-term exposure to the peptide (14). Hence, it is still unclear, whether TNFa is to influence glucose handling by direct interaction with native skeletal muscle. This study, therefore, was designed to elucidate if TNFa is to directly affect insulin-stimulated glucose metabolism in freshly isolated rat soleus muscle strips. Because time dependency of TNFa action in vitro has been described in other experimental settings (15–17), insulin-stimulated rates of glucose metabolism were determined after muscle preexposure to TNFa for 30 min, 6 h, and 24 h.

NSULIN RESISTANCE is a widespread feature of common metabolic diseases including obesity and type 2 diabetes (1, 2). The biochemical mechanisms responsible for deranged insulin sensitivity in the obese are not well understood, and recently the hypothesis has been raised that tumor necrosis factor-a (TNFa) may play a key role in the etiology of obesity-associated insulin resistance (for review see Ref. 3). This hypothesis is based on the observations that expression and release of TNFa is increased in adipose tissue from obese rodents and humans (4 – 6) and that TNFa carries a distinct potential to induce insulin resistance as indicated by blunted insulin effects on whole body glucose uptake and hepatic glucose output in TNFa-infused rats in vivo (7, 8). It is of note that during TNFa infusion, glucose uptake by skeletal muscle and skin revealed to be primarily responsible for decreased insulin-mediated whole body glucose utilization (7, 8). Furthermore, neutralization of endogenous TNFa in genetically obese insulin resistant Zucker rats for 3 days led to a 2- to 3-fold increase in insulin-stimulated peripheral glucose utilization in vivo without any change in the rate of hepatic glucose output (4). In parallel, insulin-induced autophosphorylation of the insulin receptor as well as phosphorylation of insulin receptor substrate-1 were restored to near control values in muscle and fat, but not liver (9), which suggests that endogenous TNFa contributes considerably to Received December 30, 1996. Address all correspondence and requests for reprints to: Clemens Fu¨rnsinn, Ph.D., Department of Medicine III, Division of Endocrinology and Metabolism, Wa¨hringer Gu¨rtel 18 –20, A-1090 Vienna, Austria. Email: [email protected]. * This work was supported by the Austrian Science Fund, Grant No. P11403-MED, and by the Diabetes Fund of the Netherlands (to M.B.).

Materials and Methods Rats and muscle preparation Male Sprague-Dawley rats were purchased from the breeding facilities of the University of Vienna (Himberg, Austria) and kept at an

2674

TNFa AND MUSCLE GLUCOSE METABOLISM artificial 12-h light, 12-h dark cycle at constant room temperature. Conventional laboratory diet and tap water were provided ad libitum until the evening before killing, when only food was withdrawn. Five-weekold rats at fasted body weights of approximately 140 g were killed by cervical dislocation between 0830 h and 0930 h. Immediately after killing, two longitudinal soleus muscle strips per leg were prepared, weighed (approximately 25 mg), and tied under tension on stainless steel clips as previously described (18).

TNFa Unless stated otherwise, human recombinant TNFa was from Sigma Chemical Co. (St. Louis, MO). Biological activity of the peptide was validated by TNFa-dependent accumulation of plasminogen activator inhibitor-1 in the supernatant of cultured human umbilical vein endothelial cells (ng/ml after 24 h: control, 56 6 11, vs. 1.4 nmol/liter TNFa, 275 6 23; P , 0.01; n 5 3 each).

Muscle incubation procedures Phase 1 (30 min, 6 h, or 24 h). Medium 199 (Sigma) supplemented with 5 mmol/liter HEPES, 25,000 U/liter penicillin G, 25 mg/liter streptomycin, and 0.25% (wt/vol) BSA was used as incubation medium (M199; pH 7.35). In the presence or absence of TNF-a, muscle strips were incubated for 30 min, 6 h, and 24 h, respectively. For short-term incubation (30 min), 25-ml Erlenmeyer flasks coated with Blue Slick solution (Serva, Heidelberg, Germany) and provided with 3 ml M199 were employed (1 strip/flask), whereas for long-term incubation (6 h and 24 h, respectively) muscles were put into coated 50-ml flasks provided with 20 ml M199 (6 strips/flask) so as not to touch the inner surface of the flask. Flasks were placed into a shaking water bath (37 C; 130 cycles/ min) and an atmosphere of 95% O2:5% CO2 was continuously provided within the flasks. During 24-h incubation, M199 was renewed every 5–7 h. Phase 2 (1 h). After phase 1, muscles were immediately transferred into a set of 25-ml flasks provided with 3 ml of M199 (1 strip/flask). In phase 2, M199 contained identical concentrations of TNFa as used in phase 1 and trace amounts of d-[U-14C] glucose or, alternatively, 2-deoxy-d[2,6-3H] glucose plus [U-14C] sucrose (all from Amersham, Amersham, UK) to determine rates of glucose transport, glucose incorporation into glycogen, and glucose oxidation in the absence or presence of insulin (Actrapid, Novo, Bagsvaerd, Denmark). After 60-min muscle strips were quickly removed, blotted, and frozen in liquid N2. Later, muscle strips were lysed in 1 mol/liter KOH at 70 C and the lysate used for further analytical procedures as described below.

Experimental design Before the effects of TNFa were investigated, a series of experiments was performed to confirm preservation of the stimulatory action of insulin on glucose metabolism in rat soleus muscle strips after long-term preincubation. To this end, muscle strips were preincubated for 30 min, 6 h, or 24 h in the absence of any exogenous peptide (phase 1) and selected parameters of glucose metabolism were then measured in the absence or presence of insulin (1, 10, and 100 nmol/liter; phase 2). To examine the dose-dependent effects of short- and long-term exposure of isolated muscle to TNFa on insulin-stimulated glucose metabolism, muscle strips were preincubated for 30 min, 6 h, or 24 h in the absence of insulin and in the presence of 10, 100, or 1000 pmol/liter TNFa (phase 1). Subsequently, selected parameters of glucose metabolism were measured in the presence of a maximally stimulating concentration of insulin (100 nmol/liter; phase 2). To investigate the effects of a concentration of 5 nmol/liter TNFa on glucose metabolism in the presence of submaximally and maximally insulin-stimulated glucose metabolism, muscle strips were preincubated for 30 min, 6 h, or 24 h in the absence vs. presence of 5 nmol/liter TNFa and in the absence of insulin (phase 1). Selected parameters of glucose metabolism were then measured in the presence of 5 nmol/liter and 100 nmol/liter insulin, respectively (phase 2). To control for potential influence of peptide source and solubility, the short-term effects of TNFa (5 nmol/liter) on insulin-stimulated (100 nmol/liter) glucose metabolism were examined employing TNFa from

2675

another source (GIBCO, Gaitherburg, MD) or, alternatively, in the presence of 1% (vol/vol) dimethyl sulfoxide.

Analytical procedures Net uptake rate of 2-deoxy-d-[2,6-3H] glucose, a glucose analogue that does not enter glycolysis and accumulates within the cell, was determined employing [14C] sucrose as an extracellular space marker by methods described previously (19). Glycogen synthesis is given as the net rate of conversion of [14C] glucose to [14C] glycogen as determined by methods described previously (18). Glucose oxidation, i.e. CO2 production, was calculated from conversion of [14C] glucose into 14CO2. To this end, the flasks were sealed during the last 45 min of muscle incubation, after which the muscle strips were quickly removed and the flasks were immediately resealed with a stopper carrying a hang-in container provided with 200 ml CO2-trapping solution (phenethylamine: methanol, 1:1). Using a syringe, 200 ml of 3 mmol/liter perchloric acid were injected into the incubation buffer within the flasks to quantitatively release CO2 from the medium. After incubation for at least 1 h at room temperature, the trapping solution was brought into scintillation fluid, which was vigorously shaken and counted for 14C-content. Anaerobic glycolysis, i.e. rate of lactate release, was calculated from M199 lactate concentration measured enzymatically by the lactate dehydrogenase method (20). For determination of muscle glycogen content, glycogen in the muscle lysate was completely degraded to glucose with amyloglucosidase (21). Glucose was then measured enzymatically by a commercial kit from Human (Taunusstein, Germany).

Statistics All data are presented as means 6 sem and a P , 0.05 was considered significant. For comparison of two groups, P values were calculated by two-tailed paired Student’s t test. Multiple comparisons with a control were performed after logarithmic transformation of data according to the method of Dunnett (22), where the effect of individual rat was controlled.

Results

Results depicted in Fig. 1 demonstrate that soleus muscle strips remained viable and well-responsive to insulin stimulation during incubation periods up to 24 h. Nevertheless, increased rates of glycolysis and glucose oxidation as well as decreased rates of glucose incorporation into glycogen were observed after prolonged incubation and were associated with lower muscle glycogen content. Exposure to 10, 100, and 1000 pmol/liter TNFa failed to affect insulin-stimulated muscle glucose metabolism. Rates of insulin-stimulated glucose transport, glycogen synthesis, glycolysis, and glycogen accumulation were neither influenced by short-term nor by long-term treatment with TNFa (Fig. 2). Table 1 demonstrates glucose metabolic rates prevailing in soleus muscle strips, which were exposed to 5 nmol/liter TNFa, a peptide concentration employed previously to describe insulin desensitization by TNFa of isolated nonmuscle tissue (14 –16, 23, 24). Both short- and long-term actions of TNFa on muscle glucose metabolism were not only tested with respect to insulin responsiveness (i.e. under maximal stimulation with 100 nmol/liter insulin), but also with respect to insulin sensitivity (i.e. under partial stimulation with 5 nmol/liter insulin). Under these experimental conditions, no influence on insulin sensitivity or insulin responsiveness of isolated rat soleus muscle was revealed, although muscles were exposed for up to 25 h to 5 nmol/liter TNFa. Furthermore, no significant effects of short-term TNFa exposure on insulin-stimulated glucose metabolism were ob-

2676

TNFa AND MUSCLE GLUCOSE METABOLISM

FIG. 1. Effects of prolonged incubation on muscle glucose metabolism in vitro. Isolated rat soleus muscle strips were preincubated for 30 min, 6 h, and 24 h, respectively, before rates of 3H-2-deoxy-glucose transport (A), glucose incorporation into glycogen (B), CO2 production (C), and lactate release (D) were determined in the presence of the indicated concentrations of insulin. Muscle glycogen content was measured after incubation (E). Means 6 SEM; n 5 6 each; a: P , 0.05, b: P , 0.01 vs. absence of insulin.

Endo • 1997 Vol 138 • No 7

FIG. 2. Effects of 10, 100, and 1000 pmol/liter TNFa on insulin-stimulated muscle glucose metabolism. Isolated rat soleus muscle strips were preexposed to the indicated concentrations of TNFa for 30 min, 6 h, or 24 h before rates of 3H-2-deoxy-glucose transport (A; n 5 6 each), glucose incorporation into glycogen (B; n 5 5–12 each), CO2 production (C; n 5 3–12 each), and lactate release (D; n 5 6 each) were determined in the presence of 100 nmol/liter insulin. Muscle glycogen content was measured after incubation (E; n 5 5–12 each). Means 6 SEM; no significance for presence vs. absence of TNFa.


2677

TABLE 1. Effects of 5 nmol/liter TNFa on insulin-stimulated glucose metabolism of isolated rat soleus muscle strips Insulin, (nmol/liter) TNFa, (nmol/liter)

5 0

5 5

100 0

100 5

n

Glucose transport (cpm/mg•h)

30 min 6h 24 h

753 6 49 626 6 51 842 6 25

663 6 32 637 6 51 814 6 34

779 6 29 652 6 56 911 6 47

725 6 29 617 6 60 936 6 31

6 6 6

Glycogen synthesis (mmol glucose U/g•h)

30 min 6h 24 h

3.29 6 0.21 1.78 6 0.16 1.89 6 0.22

3.34 6 0.24 1.83 6 0.23 2.08 6 0.09

5.19 6 0.22 2.08 6 0.10 2.51 6 0.21

5.25 6 0.41 2.09 6 0.17 2.41 6 0.26

12 6 6

CO2 production (mmol glucose U/g•h)

30 min 6h 24 h

0.39 6 0.03 0.83 6 0.06 2.59 6 0.24

0.42 6 0.04 0.85 6 0.05 2.81 6 0.22

0.46 6 0.04 0.99 6 0.07 2.26 6 0.27

0.42 6 0.03 0.98 6 0.06 2.28 6 0.10

12 6 6

Lactate release (mmol/g•h)

30 min 6h 24 h

15.9 6 0.6 17.4 6 0.6 15.4 6 0.6

14.3 6 0.9 18.0 6 1.6 15.7 6 1.2

17.0 6 1.0 20.0 6 1.3 16.1 6 1.0

17.0 6 0.8 19.4 6 0.9 17.0 6 1.1

6 6 6

Glycogen content (mmol glucosyl U/•g)

30 min 6h 24 h

10.4 6 0.5 11.6 6 0.9 8.7 6 0.8

10.4 6 0.5 12.5 6 1.5 8.9 6 0.6

12.3 6 0.4 11.5 6 1.0 9.1 6 0.7

12.4 6 0.6 11.3 6 1.0 7.5 6 0.3

12 6 6

Muscle strips were preexposed to TNFa for 30 min, 6 h, or 24 h before rates glucose metabolism were determined in the presence of submaximally or maximally stimulating concentrations of insulin. Muscle glycogen content was measured after incubation. Means 6 SEM; no significance for presence vs. absence of TNFa. TABLE 2. Effects of TNFa on insulin-stimulated glucose metabolism of isolated rat soleus muscle strips using peptide from an alternative source, or in the presence of detergent. TNFa, source

GIBCO

TNFa, nmol/liter DMSO, % (vol/vol)

0 0

Glucose transport (cpm/mg•h) Glycogen synthesis (mmol glucose U/g•h) CO2 production (mmol glucose U/g•h) Lactate release (mmol/g•h) Glycogen content (mmol glucosyl U/g)

Sigma 5 0

0 1

5 1

897 6 51

886 6 32

818 6 35

873 6 58

6.63 6 0.30

5.80 6 0.34

5.17 6 0.31

5.98 6 0.26

0.47 6 0.06

0.42 6 0.06

0.54 6 0.05

0.53 6 0.02

12.8 6 1.0

12.7 6 0.8

16.2 6 0.6

15.7 6 0.4

14.5 6 1.2

14.8 6 0.4

11.4 6 0.7

12.5 6 0.5

Muscle strips were preexposed to TNFa in the presence or absence of detergent for 30 min before rates of glucose metabolism were determined in the presence of 100 nmol/liter insulin. Muscle glycogen content was measured after incubation. Means 6 SEM; n 5 6 each; no significance for presence vs. absence of TNFa.

served, when the effects of TNFa from an alternative source or in the presence of detergent were determined (Table 2). Discussion

Infusion of TNFa in rats increases peripheral glucose uptake in the basal state but elicits a distinct loss in hepatic and peripheral insulin sensitivity in vivo, which includes decreased insulin-stimulated glucose uptake in rectus abdominus, gastrocnemius, and red as well as white quadriceps muscle (7, 8). Furthermore, experimental evidence has been provided that endogenous TNFa may be instrumental in the development of obesity-associated insulin resistance and derangement of glucose homeostasis in rats (3, 4). Because the circulating plasma levels of TNFa found in association with obesity are regarded too low to allow for endocrine effects on muscle tissue, it has been hypothesized that peptide release from fat cells located in the vicinity of muscle fibers may mediate TNFa action via paracrine mechanisms (3, 4, 25). Speculations also include potential autocrine mechanisms in spite of the observation that TNFa is expressed at much lower rates in muscle than in adipose tissue (25).

In this study, TNFa at concentrations ranging from 0.01–5 nmol/liter neither affected insulin-stimulated rates of glucose transport, glycogen synthesis, CO2 production, and lactate release, nor did TNFa exert any influence on glycogen content of isolated rat soleus muscle strips in vitro. Such failure of TNFa to directly affect insulin-stimulated soleus muscle glucose metabolism in vitro was substantiated under various experimental circumstances including TNFa pretreatment for 30 min, 6 h, and 24 h followed by both submaximal and maximal insulin stimulation. Although TNFa release from isolated muscle strips in vitro can not be excluded, it seems unlikely that autocrine mechanisms may have triggered maximal TNFa-stimulation in the control experiments and hence may have masked any effect of exogenous TNFa added to the incubation medium. Thus, in spite of such potential autocrine stimulation, others have found that plasma concentrations around 100 pmol/l TNFa were sufficient to induce distinct muscle insulin resistance in vivo (7) and that TNFa-induced insulin desensitization was welldescribable for various other isolated tissues (12, 16, 23, 24, 26 –28). Poor quality of employed TNFa was excluded as the

2678


cause of negative results by evaluation of its capability to release plasminogen activator inhibitor-1 from cultured human ubilical vein endothelial cells. The obtained data thus do not support the idea of TNFa to induce insulin resistance via direct interaction with skeletal muscle, which is known to express receptors for TNFa (29). Such lack of direct interaction with skeletal muscle glucose metabolism is in line with described effects on protein catabolism, which is markedly stimulated by TNFa in vivo (30, 31), whereas the peptide fails to directly affect protein breakdown of isolated muscle tissue in vitro (30, 32). In agreement with a previous report (33), the applied method for incubation of freshly isolated rat soleus muscle strips displayed blunted rates of glycogen synthesis, increased glycolysis, and decreased glycogen content upon prolonged incubation in the absence of plasma, hormones, and innervation when compared with short-term incubation. Such time-dependent increase in the rate of carbohydrate catabolism may be caused by depletion of im lipid stores resulting in an enhanced requirement for glucose as a fuel substrate, or may reflect a general increase in the metabolic rate due to the artificial environment. Nevertheless, muscle tissue remained viable even after incubation for 24 h as indicated by its preserved ability to respond to insulin, which dose-dependently triggered increases in the rates of glucose transport, glycogen synthesis, and glycolysis. The employed rat soleus muscle preparation therefore appears adequate for the investigation of short- and long-term TNFa action on insulin-stimulated glucose metabolism. Peptide concentrations and exposure periods applied in our experiments are in the range of that used to document TNFa-dependent insulin desensitization in isolated tissues including adipocytes, hepatoma cells, and the cultured rat skeletal muscle cell line L6 (12, 16, 23, 24, 26 –28). Most studies describing TNFa-dependent insulin desensitization in vitro did, however, not determine insulin-stimulated rates of glucose metabolism, but rather focused on processes involved in intracellular insulin signal transduction (16, 17, 23, 24, 26, 27), whereby phosphorylation of insulin receptor substrate-1 serine residues (23, 26) and activation of phosphotyrosine phosphatases (17) have been suggested to mediate TNFainduced insulin resistance. In a limited number of in vitro-studies, the stimulatory effect of insulin on glucose metabolism was determined and glucose transport has been found blunted in isolated adipocytes exposed to TNFa (15, 27). In the cultured rat skeletal muscle cell line L6, no insulin resistance was induced by 4 – 8 d of exposure to TNFa (13), whereas a distinct decrease in insulin-stimulated glucose transport was observed after TNFa-pretreatment for 1 h or 12 h (12). The latter finding was associated with reduced glucose incorporation into glycogen and glycogen synthase activity (12) suggesting that major differences exist in the interaction of TNFa with insulin in cultured L6 muscle cells vs. freshly isolated soleus muscle. Such discrepancies can not be explained by different source of TNFa, since peptide both from GIBCO (used for L6 cells; 12) and Sigma (used in this study) did not affect soleus muscle glucose handling in vitro. Difference in responses to TNFa thus are likely to reflect different type of tissue employed with freshly isolated native muscle tissue relating

Endo • 1997 Vol 138 • No 7

closer to the physiological situation than a cultured muscle cell line. Even using freshly isolated muscle, however, direct conclusions to the physiological situation are subject to limitations principally applying in vitro studies, which include that full muscle sensitivity to the effects of insulin and/or TNFa may depend on innervation and normal blood perfusion. Although muscles dominated by white glycolytic fast-twitch fibers as well as muscles dominated by red oxidative slow-twitch fibers are affected by TNFa-dependent insulin desensitization in vivo (7, 8), deviations in metabolic response to TNFa in vitro may to some extent depend on muscle fiber type. In the case of soleus muscle fiber type is mainly slow-twitch, although presence of a small amount fast-twitch fibers can not be excluded. In that context it is of note that TNFa has also been described to inhibit insulindependent activation of phosphatidylinositol 3-kinase in adipocytes, but not in cardiomyocytes (14), which carry a high oxidative capacity and hence resemble red rather than white skeletal muscle fibers. In conclusion, this study demonstrates failure of TNFa to affect insulin-stimulated glucose metabolism by direct interaction with rat soleus muscle in vitro and hence does not provide evidence for TNFa-dependent muscle insulin desensitization via autocrine or paracrine mechanisms as hypothesized by others (3, 4, 25). Our findings rather favour the concept that TNFa-dependent muscle insulin resistance in vivo is mediated indirectly via interaction with other tissues and may involve counterregulatory hormone release (7, 34) or lipid metabolism (9, 35). Acknowledgments We appreciate the help of the staff at the Biomedical Research Center, University of Vienna, who took care of the rats.

References 1. Olefsky JM, Kolterman OG, Scarlett JA 1982 Insulin action and resistance in obesity and noninsulin-dependent type II diabetes mellitus. Am J Physiol 243:E15–E30 2. DeFronzo RA 1988 The triumvirate b-cell, muscle, liver: a collusion responsible for NIDDM. Diabetes 37:667– 687 3. Hotamisligil GS, Spiegelman BM 1994 Tumor necrosis factor a: a key component of the obesity-diabetes link. Diabetes 43:1271–1278 4. Hotamisligil GS, Shargill NS, Spiegelman BM 1993 Adipose expression of tumor necrosis factor-a: direct role in obesity-linked insulin resistance. Science 259:87–91 5. Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R, Simsolo RB 1995 The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase. J Clin Invest 95:2111–2119 6. Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM 1995 Increased adipose tissue expression of tumor necrosis factor-a in human obesity and insulin resistance. J Clin Invest 95:2409 –2415 7. Lang CH, Dobrescu C, Bagby GJ 1992 Tumor necrosis factor impairs insulin action on peripheral glucose disposal and hepatic glucose output. Endocrinology 130:43–52 8. Sierra P, Ling PR, Istfan NW, Bistrian BR 1995 Fish oil feeding improves muscle glucose uptake in tumor necrosis factor-treated rats. Metabolism 44:1365–1370 9. Hotamisligil GS, Budavari A, Murray D, Spiegelman BM 1994 Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role for tumor necrosis factor-a. J Clin Invest:1543–1549 10. Ofei F, Hurel S, Newkirk J, Sopwith M, Taylor R 1996 Effects of an engineered human anti-TNF-a antibody (CDP571) on insulin sensitivity and glycemic control in patients with NIDDM. Diabetes 45:881– 885 11. Baron AD, Brechtel G, Wallace P, Edelman SV 1988 Rates and tissue sites of non-insulin and insulin-mediated glucose uptake in humans. Am J Physiol 255:E769 –E774

TNFa AND MUSCLE GLUCOSE METABOLISM 12. Begum N, Ragolia L 1996 Effect of tumor necrosis factor-a on insulin action in cultured rat skeletal muscle cells. Endocrinology 137:2441–2446 13. Ranganathan S, Davidson MB 1996 Effect of tumor necrosis factor-a on basal and insulin-stimulated glucose transport in cultured muscle and fat cells. Metabolism 45:1089 –1094 14. Liu L, Hauner H, Eckel J 1996 TNFa-induzierte Insulinresistenz in humanen Adipozyten. Diabetes und Stoffwechsel 5:66 (Abstract) 15. Hauner H, Petruschke T, Russ M, Ro¨hrig K, Eckel J 1995 Effects of tumor necrosis factor alpha (TNFa) on glucose transport and lipid metabolism of newly-differentiated human fat cells in cell culture. Diabetologia 38:764 –771 16. Guo D, Donner DB 1995 Tumor necrosis factor promotes phosphorylation and binding of insulin receptor substrate 1 to phosphatidylinositol 3-kinase in 3T3–L1 adipocytes. J Biol Chem 271:615– 618 17. Kroder G, Bossenmaier B, Kellerer M, Capp E, Stoyanov B, Mu¨hlho¨fer A, Berti L, Horikoshi H, Ullrich A, Ha¨ring H 1996 Tumor necrosis factor-a- and hyperglycemia-induced insulin resistance. J Clin Invest 97:1471–1477 18. Crettaz M, Prentki M, Zanietti D, Jeanrenaud B 1980 Insulin resistance in soleus muscle from obese Zucker rats. Involvement of several defective sites. Biochem J 186:525–534 19. Fu¨rnsinn C, Ebner K, Waldha¨usl W 1995 Failure of GLP-1(7–36)amide to affect glycogenesis in rat skeletal muscle. Diabetologia 38:864 – 867 20. Engel RC, Jones JB 1978 Causes and elimination of erratic blanks in enzymatic metabolic assays involving the use of NAD1 in alkaline hydrazine buffers: improved conditions for the assay of L-glutamate, L-lactate and other metabolites. Anal Biochem 88:475– 484 21. Dimitriadis GD, Leighton B, Vlachonikolis IG, Parry-Billings M, Challiss RAJ, West D, Newsholme EA 1988 Effects of hyperthyroidism on the sensitivity of glycolysis and glycogen synthesis to insulin in the soleus muscle of the rat. Biochem J 253:87–92 22. Dunnett CW 1964 New tables for multiple comparisons with a control. Biometrics 290:482– 491 23. Kanety H, Feinstein R, Papa MZ, Hemi R, Karasik A 1995 Tumor necrosis factor a-induced phosphorylation of insulin receptor substrate-1 (IRS-1). Possible mechanism for suppression of insulin-stimulated tyrosine phosphorylation of IRS-1. J Biol Chem 270:23780 –23784

2679

24. Feinstein R, Kanety H, Papa MZ, Lunenfeld B, Karasik A 1993 Tumor necrosis factor-a suppresses insulin-induced tyrosine phosphorylation of insulin receptor and its substrates. J Biol Chem 268:26055–26058 25. Saghizadeh M, Ong JM, Garvey WT, Henry RR, Kern PA 1996 The expression of TNFa by human muscle. Relationship to insulin resistance. J Clin Invest 97:1111–1116 26. Hotamisligil GS, Peraldi P, Budavari A, Ellis R, White MF, Spiegelman BM 1996 IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-a- and obesity-induced insulin resistance. Science 271:665– 668 27. Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM 1994 Tumor necrosis factor a inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 91:4854 – 4858 28. Begum N, Ragolia L, Srinivasan M 1996 Effect of tumor necrosis factor-a on insulin-stimulated mitogen activated protein kinase cascade in cultured rat skeletal muscle cells. Eur J Biochem 238:214 –220 29. Hofmann C, Lorenz K, Braithwaite SS, Colca JR, Palazuk BJ, Hotamisligil GS, Spiegelman BM 1994 Altered gene expression for tumor necrosis factor-a and its receptors during drug and dietary modulation of insulin resistance. Endocrinology 134:264 –270 30. Goodman MN 1991 Tumor necrosis factor induces skeletal muscle protein breakdown in rats. Am J Physiol 260:E727–E730 31. Charters Y, Grimble RF 1989 Effect of recombinant human tumor necrosis factor a on protein synthesis in liver, skeletal muscle and skin of rats. Biochem J 258:493– 497 32. Rofe AM, Conyers RA, Bais R, Gamble JR, Vadas MA 1987 The effects of recombinant tumor necrosis factor (cachectin) on metabolism in isolated rat adipocyte, hepatocyte and muscle preparations. Biochem J 247:789 –792 33. Stace PB, Marchington DR, Kerbey AL, Randle PJ 1990 Long term culture of rat soleus muscle in vitro. Its effects on glucose utilization and insulin sensitivity. FEBS Lett 273:91–94 34. Evans DA, Jacobs DO, Wilmore DW 1989 Tumor necrosis factor enhances glucose uptake by peripheral tissues. Am J Physiol 257:R1182–R1189 35. Sakurai Y, Zhang X, Wolfe RR 1993 Short-term effects of tumor necrosis factor on energy and substrate metabolism in dogs. J Clin Invest 91:2437–2445