Introduction. S Kapur 1, F Picard 1, M Perreault 1, Y Deshaies 1 and A Marette 1 *

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1 (2000) 24, Suppl 4, S36±S40 ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $ Nitric oxide: a new player in the modulation of energy metabolism S Kapur 1, F Picard 1, M Perreault 1, Y Deshaies 1 and A Marette 1 * 1 Department of Physiology, Lipid Research Unit, Centre de Recherche sur le MeÂtabolisme E nergeâtique (CREME), Laval University Hospital Research Center, QueÂbec, Canada Nitric oxide (NO) is a key messenger molecule in several cell types. NO formation is catalyzed by a family of NO synthases (NOS) that use L-arginine as a substrate. Rat adipose tissue expresses the inducible, macrophage-type, nitric oxide (NO) synthase isoform (inos). Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) markedly increases the expression and activity of inos in both white and brown adipose tissues, as well as in skeletal muscle. inos induction can be reproduced in vitro by treatment of cultured white or brown adipocytes or L6 myocytes with LPS and in ammatory cytokines (TNFa, IFNg). The physiological role of NO in adipose tissues and skeletal muscle is still obscure. Recent evidence suggests that NO may be implicated in the regulation of energy metabolism. Using both pharmacological and genetic models of inos invalidation, we have recently begun to uncover a role for NO in the modulation of glucose transport and lipoprotein hydrolysis. These studies support the emerging concept that NO may ful ll the dual role of modulating energy metabolism in both physiological and pathological conditions as well as contributing to local immune defense during in ammatory processes. (2000) 24, Suppl 4, S36±S40 Keywords: nitric oxide synthase isoform; lipopolysaccharide; tumor necrosis factor-a; adipocytes; skeletal muscle; obesity; insulin resistance; in ammation; endotoxemia Introduction Skeletal muscle and adipose tissues are the principal sites of glucose and lipid metabolism in the postabsorptive state. Both glucose and lipid metabolism are greatly affected in conditions associated with increased levels of in ammatory cytokines such as endotoxemia, but also including metabolic diseases such as obesity and diabetes. The mechanisms by which these immune molecules modulate energy metabolism are still unclear. In this paper, we will review the increasing body of evidence supporting the concept that NO generated by the inducible NO synthase (NOS) inos is implicated in this modulation. NO synthases in skeletal muscle and adipose tissues There are at least three isotypes of NOS: the endothelial cell NOS (enos), the neuronal type NOS (nnos), and the macrophage-type inducible NOS (inos). When induced, this latter NOS isotype generates NO at a much higher rate and for longer periods of time than constitutive nnos and enos enzymes. 1 We have *Correspondence: A Marette, Department of Physiology and Lipid Research Unit, Laval University Hospital Research Center, 2705 Laurier Boulevard, RC 9502, Ste-Foy, Que bec, Canada G1V 4G2. andre.marette@crchul.ulaval.ca previously shown that, in the basal state, skeletal muscle expresses both nnos and enos isoforms. 2 In fact, nnos is mostly expressed in type IIb muscle bers, whereas enos detection in skeletal muscle is in large part explained by the presence of vascular endothelial cells in the tissue. In the rat, inos is not detected to any signi cant level in resting muscle but it is induced following in vivo injection of lipopolysaccharide (LPS) (Figure 1). The endotoxin-mediated inos induction results in marked NO production by the isolated soleus and extensor digitorum longus (EDL) muscles in vitro. Unlike skeletal muscle, neither enos nor nnos can be detected to signi cant levels in rat adipose tissue (Figure 2). On the other hand, inos protein can be detected in both white (Figure 2) and brown adipose tissues (data not shown) of rats, even in the basal state. Whether inos expression in adipose tissues of normal rats is localized to adipocytes or other cell types (eg resident macrophages) is still unknown. As in muscle, endotoxin challenge markedly increased cellular inos protein concentrations and inos activity in both white and brown adipose tissues (Figure 3). The effect is marked (10 ± 20-fold increase in inos activity) and can be detected within 2 h of LPS exposure in vivo. Mechanism of inos induction in endotoxemia We have recently investigated the mechanism of inos activation in adipose tissue and skeletal

2 S37 Figure 1 LPS-induced inos expression in skeletal muscle. Rats were injected i.p. with LPS or saline and sacri ced 8 h later. Soleus and EDL muscles were removed and used for determination of inos protein contents or NO production. inos in soleus was detected by immunoprecipitation followed by SDS ± PAGE and Western blot analysis of inos immunoreactivity. Molecular weight standards are shown on the left. NO production was measured by the assessment of plasma concentrations of NO 2 =NO 3 by the Griess reaction. The results are representative of three independent experiments. **P < 0.01 vs control values. Figure 2 Expression of NOS isoforms in white adipose tissue. Tissue extracts (50 mg) of epididymal white adipose tissue (WAT) from a control rat were subjected to SDS ± PAGE and immunoblot analysis using isoform-speci c antibodies against inos, enos and nnos. Extracts from L6 myocytes, endothelial cells and skeletal muscle were used for positive controls of inos, enos and nnos, respectively. Only inos (mol wt 130 kda) was detected in control WAT. muscle of LPS-treated rats. Systemic administration of the bacterial endotoxin LPS causes the release of several cytokines (such as tumor necrosis factor-a (TNFa), interleukin-1 and -6, and interferon-g (IFNg)) 3,4 and these in ammatory molecules may activate inos expression in a concerted manner. We have therefore used cell lines of skeletal muscle (L6), and white adipose tissue (3T3-L1) to investigate the individual and combined effects of two cytokines (TNFa and IFNg) and LPS on inos activation in vitro. As shown in Figure 4, incubation of muscle or adipose cells with TNFa, LPS or IFNg alone did not increase NO production, as measured by the accumulation of nitrite in the incubation medium. A small increase in NO production was detected when the two Figure 3 Effects of LPS on inos protein levels and inos activity in epididymal (EWAT), perirenal (PWAT) and interscapular brown (BAT) adipose tissues. Rats were treated or not with LPS for 2, 4 or 8 h and tissue inos protein levels and inos activity were determined. The immunoblot is representative of three independent experiments with tissues from different animals. Results for inos activity are represented as means s.e. of two to three individual experiments. **P < 0.01 vs control values. (Reproduced from Am J Physiol 1999; 276: E635, with permission from the American Physiological Society.) cytokines were combined. However, maximal activation of inos was observed when cells were incubated with all these immune molecules. Indeed, the combined effect of LPS, TNFa and IFNg was clearly synergistic as compared to the effect of only two of these factors. Similar ndings were obtained with a brown adipocyte cell line (T37i) (data not shown). Since neither LPS alone nor individual cytokines could increase inos-mediated NO production by the muscle or adipose cells, it appears that the induction of this enzyme in vivo is mediated by a complex network of interactions between in ammatory cytokines and endotoxin at the cellular level. Our data indicate that in myocytes and white adipocytes, IFNg is the most potent inducer of inos but that other cytokines and LPS must be present to maximally induce the enzyme. Interestingly, we found that TNFa is more potent than IFNg in increasing inos activity in cultured brown adipocytes (Penfornis and Marette, unpublished data). In future studies, it will be important to de ne more precisely the transduction pathways involved in inos activation by cytokines and LPS. In particular, the potential roles of interleukin-1 and -6 released from activated adipose cells in contributing to inos activation remain to be examined. In any case, our results clearly establish that LPS and cytokines can induce inos expression by a direct interaction with the adipocyte or myocyte. Possible role of inos in coupling energy metabolism to immune function inos is implicated in host defense and it is synthesized de novo in response to a variety of in ammatory stimuli (reviewed in Nathan 5 ). Induction of inos in

3 S38 Figure 4 LPS and cytokines synergistically increase inos activity in myocytes and adipocytes. L6 myocytes or 3T3-L1 adipocytes were treated or not with the cytokines TNFa (10 ng=ml) and=or IFNg (200 U=ml) and=or LPS (10 mg=ml). Incubation time was 24 h for muscle cells and 48 h for adipocytes. After the incubation, nitrite accumulation in the incubation medium was measured under identical conditions by the Griess reaction. Results are mean s.e. of three independent experiments, each performed in triplicate with different batches of cells. *P < 0.05 vs control values; P < 0.05 vs TNFa IFNg values. muscle and fat cells may therefore contribute to the immune defence in in ammatory settings. It is also noteworthy that white and brown adipose tissues are dispersed throughout the body and are widely distributed along and even within major organs. It is therefore likely to represent a major source of local NO generation in endotoxic shock. Another possible role of inos in skeletal muscle and adipose tissues is to mediate the profound changes in energy metabolism that are known to occur during endotoxic shock. Indeed, endotoxemia is characterized by marked perturbations in both glucose and lipid metabolism in animals and humans, 6,7 and insulin resistance is commonly observed in this condition (see Figure 5). 8,9 We have summarized below some of our recent ndings that strongly support the hypothesis that inos-generated NO production is mediating the perturbations of energy metabolism in endotoxemia and possibly other in ammatory disorders. Figure 5 Proposed role of inos as a modulator of energy metabolism in altered metabolic states such as obesity and in ammatory diseases. Increased levels of in ammatory cytokines in these conditions lead to inos induction in skeletal muscle and overexpression of inos in adipose tissues. inosgenerated NO overproduction in muscle causes insulin resistance for glucose uptake and inhibits LPL activity, leading to insulin resistance and hypertriglyceridemia. On the other hand, increased expression of inos in adipose tissues may increase basal rates of lipolysis and inhibit insulin action on lipolysis, leading to increase circulating FFA levels. Role of inos in endotoxin-mediated changes in glucose metabolism It is well documented that endotoxemia elicits profound changes in whole-body glucose homeostasis in both animals and humans. 7 Septic patients have an accelerated rate of glucose clearance in the basal state. 10 On the other hand, sepsis is also associated with a state of insulin resistance, as evidenced by diminished glucose tolerance, hyperinsulinemia and impaired insulin action on peripheral glucose disposal. 8±10 We have previously demonstrated that cytokines and LPS increase glucose uptake in muscle

4 cells by inducing the expression of inos and NO. 11 On the contrary, exaggerated NO production upon inos induction also causes insulin resistance by interfering with insulin action to promote glucose transport into the muscle cells. 2,11 This effect is direct since cytokine-induced inos overexpression and inos-mediated inhibition of insulin-stimulated glucose uptake could be reproduced in L6 myocytes in culture. 11 The inhibitory effects of NO on insulin action may be particularly relevant in in ammatory conditions in which muscle insulin resistance is associated with elevated levels of cytokines such as TNFa, IFNg and interleukins. Moreover, inos induction may also be involved in obesity-linked metabolic alterations since the obese state is also associated with increased TNFa secretion by adipocytes and muscle cells (see below). Very recently, we have further con rmed the role of inos in LPS-induced muscle insulin resistance using pharmacological and genetic models of inos invalidation. Thus we rst showed that LPS-induced muscle insulin resistance in rats could be blocked by pretreatment with the speci c inos inhibitor aminoguanidine (AGN) (Kapur and Marette, unpublished data). Moreover, LPS-induced insulin resistance is signi cantly blunted in mice de cient for the inos gene (Perreault and Marette, unpublished data). These results strongly support our hypothesis that inos is a key player in the development of insulin resistance in endotoxemia and other in ammatory diseases. Role of inos in endotoxin-mediated changes in lipid metabolism We have also recently explored the possible role of inos in modulating lipid metabolism in skeletal muscle and adipose tissues of endotoxin-challenged rats. It is well known that endotoxemia increased plasma triglycerides and that this is explained, at least in part, through a clearance of lipoproteinbound triglycerides by peripheral tissues (see Hardardottir et al 6 ). This led us to test whether inos may be involved in this endotoxin-mediated alteration in triglyceride hydrolysis. It was found that LPS administration increased triglyceride levels in rats but that pre-treatment with the inos inhibitor AGN completely blocked this effect. Interestingly, LPS-mediated inhibition of LPL activity in skeletal muscle was also prevented by AGN pre-treatment, whereas the reduction in adipose tissue LPL activity was not affected by the inos inhibitor (Picard, Kapur, Marette and Deshaies, unpublished data). Very recently, we have also found that LPS failed to affect plasma triglyceride levels and LPL activity in skeletal muscle of inos-de cient mice, further documenting the primary role of inos in LPS-induced hypertriglyceridemia (Picard, Perreault, Marette and Deshaies, unpublished data). These results provide convincing evidence that inos induction is responsible for the marked inhibition of LPL activity in skeletal muscle of endotoxic rats and further indicate that muscle, and not adipose tissue, is the primary site of decreased lipoprotein hydrolysis in this condition. Endotoxemia is also characterized by an increased level of circulating free fatty acids (FFA), which is the consequence of increased release by the adipose cells. 10,12 Whether inos-mediated NO production is responsible for this increased fat cell lipolysis is not known. There is some evidence that NO can regulate adipocyte lipolysis. Indeed, Gaudiot et al 13 recently reported that some NO-releasing compounds inhibit catecholamine-stimulated lipolysis in rat fat cells. The NO-dependent inhibition of b-adrenoceptor-stimulated lipolysis has been recently con rmed in isolated human fat cells. 14 On the other hand, in the absence of catecholamines, NO donors can also increase basal lipolysis. 13 Whether NO is mediating the known stimulatory effects of cytokines (such as TNFa) on lipolysis remains to be tested. Furthermore, NO may also increase FFA release by interfering with insulin's ability to blunt lipolysis since NO overproduction inhibits insulin action in other cell types (myocytes). More studies are needed to con rm the role of NO in the increased release of FFA into the circulation of septic subjects. Is inos involved in the obesitylinked metabolic syndrome? TNFa expression is increased in adipose tissue of genetic and nutritional rodent models of obesity as well as in obese individuals. 15 ± 17 There is also evidence that TNFa secretion is increased in muscle cells of type II diabetic subjects. 18 This increased TNFa secretion, especially in the presence of other in ammatory cytokines in vivo, may induce inos expression in muscle and fat cells. It is therefore possible that an exaggerated production of NO through inos induction may contribute to the development of insulin resistance and hypertriglyceridemia in obesitylinked diabetes (Figure 5). We have preliminary evidence that inos expression is induced in skeletal muscle of high-fat fed rodents (Perreault and Marette, unpublished observations). Future studies will be aimed at testing whether inos invalidation (pharmacologically or genetically) of obese animals could reduce or even prevent the development of insulin resistance, hypertriglyceridemia and associated cardiovascular complications. Conclusions The studies reviewed in the present paper strongly support the tenet that inos is a key player in the modulation of energy metabolism in altered metabolic states such as in endotoxemia and other in ammatory S39

5 S40 conditions. Changes in both glucose metabolism and lipoprotein hydrolysis appear to be mostly explained by inos induction in skeletal muscle. inos expression is also markedly increased in white and brown adipose tissues of septic rats and the increased NO production may be involved in the modulation of fat cell lipolysis. What remains unclear is whether the inos-mediated perturbations in energy metabolism are part of an adaptative phenomenon to in ammatory settings or simply an uncontrolled secondary effect of high NO output. However, the concerted and tissuespeci c effects of inos-produced NO on glucose and lipid metabolisms strongly support the emerging concept that inos is playing a critical role in modulating energy metabolism in in ammatory conditions. Future studies may also reveal an important role for inos in the pathogenesis of obesity-linked insulin resistance and hyperlipidemia. Acknowledgements This work was supported by grants from the Medical Research Council of Canada and by scholarships from the Medical Research Council and the Fonds de la Recherche en Sante du QueÂbec to A Marette. S Kapur was supported by a fellowship from the Canadian Diabetes Association. F Picard and M Perreault were supported by studentships from the Medical Research Council of Canada and by the `Association du diabeáte du QueÂbec'. References 1 Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmac Rev 1991; 43: 109 ± Kapur S, Bedard S, Marcotte B, Cote CH, Marette A. Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action. Diabetes 1997; 46: 1691 ± Nathan CF. Secretory products of macrophages. J Clin Invest 1987; 79: 319 ± Andersson J, Nagy S, Bjork L, Abrams J, Holm S, Andersson U. Bacterial toxin-induced cytokine production studied at the single-cell level. Immunol Rev 1992; 127: 69 ± Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest 1997; 100: 2417 ± Hardardottir I, Moser AH, Memon R, Grunfeld C, Feingold KR. Effects of TNF, IL-1, and the combination of both cytokines on cholesterol metabolism in Syrian hamsters. Lymphokine Cytokine Res 1994; 13: 161 ± Frayn KN. Hormonal control of metabolism in trauma and sepsis. Clin Endocrinol 1986; 24: 577 ± Westfall MV, Sayeed MM. Basal- and insulin-stimulated muscle glucose transport in endotoxin and bacteremia rats. Am J Physiol 1988; 254: R673 ± R Shangraw RE, Jahoor F, Miyoshi H, Neff WA, Stuart CA, Herndon DN, Wolfe RR. Differentiation between septic and postburn insulin resistance. Metab Clin Exp 1989; 38: 983 ± Wolfe RR. Substrate utilization=insulin resistance in sepsis= trauma. Baillieres Clin Endocrinol Metab 1997; 11: 645 ± BeÂdard S, Marcotte B, Marette A. Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase. Biochem J 1997; 325: 487 ± Spitzer JA, Fish RE. Lipolytic patterns in isolated adipocytes of continuously endotoxemic rats. Circ Shock 1986; 18: 21 ± Gaudiot N, Jaubert AM, Charbonnier E, Sabourault D, Lacasa D, Giudicelli Y, Ribiere C. Modulation of white adipose tissue lipolysis by nitric oxide. J Biol Chem 1998; 273: ± Andersson K, Gaudiot N, Ribiere C, Elizalde M, Giudicelli Y, Arner P. A nitric oxide-mediated mechanism regulates lipolysis in human adipose tissue in vivo. Br J Pharmac 1999; 126: 1639 ± Hotamisligil GS, Spiegelman BM. Tumor necrosis factor alpha: a key component of the obesity ± diabetes link. Diabetes 1994; 43: 1271 ± Hotamisligil, GS, Arner, P, Caro, JF, Atkinson, RL, Spiegelman, BM. Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance. J Clin Invest 1995; 95: 2409 ± Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R, Simsolo RB. The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase. J Clin Invest 1995; 95: 2111 ± Saghizadeh M, Ong JM, Garvey WT, Henry RR, and Kern PA. The expression of TNF alpha by human muscle. Relationship to insulin resistance. J Clin Invest 1996; 97: 1111 ± 1116.