Regulation of adiponectin and leptin gene expression in white and brown adipose tissues: influence of beta3-adrenergic agonists,retinoic acid,leptin and fasting

Circulating adiponectin levels fall whereas leptin levels rise with obesity, suggesting that regulation of these two adipocyte-derived hormones may be simultaneously influenced by common obesity-related factors. We examined adiponectin mRNA levels in WAT and in some instances, brown adipose tissue (BAT) following fasting and refeeding, acute and chronic administration of a beta(3)-adrenergic agonist, acute treatment with retinoic acid (RA) and a glucocorticoid, and following chronic infusion of leptin and compared the expression of adiponectin to that of leptin in each circumstance. Serum concentrations of adiponectin were also reported for most of the treatments. Fasting diminished and refeeding reversed both adiponectin and leptin gene expression. Peripheral injection of the beta(3)-adrenergic agonist, CL316,243, suppressed both leptin and adiponectin expression in WAT. A small but significant reduction in adiponectin expression in BAT was also observed following this treatment. Although CL316,23 lowered serum leptin levels markedly, it did not affect serum adiponectin levels. A chronic 7-day infustion of CL316,243 resulted in an elevation of adiponectin expression in WAT and serum concentrations in contrast to suppressions in both mRNA and serum levels of leptin by a similar treatment as previously reported. Chronic administration of leptin did not alter adiponectin synthesis in WAT compared to controls, but prevented the reduction in adiponectin synthesis associated with pair feeding. Food restriction through pair feeding also diminished adiponectin expression in BAT. Collectively, although leptin and adiponectin are inversely correlated with obesity, leptin does not appear to participate directly in adiponectin synthesis. The short-term regulation of the two adipokine expression in WAT is somewhat similar, perhaps subjective to common control of energy balance. The long-term regulation of adiponectin expression in WAT appears to be the opposite of that of leptin and may be more sensitive to changes in adiposity or insulin sensitivity.     

Leptin and its receptor are controlled by 17beta-estradiol in peripheral tissues of ovariectomized rats

It has been widely shown that there is a complex interaction between sex steroids and leptin effects on body weight. In this sense, the absence of female sex steroids is linked to a significant increase in body weight, which seems to be related to an impairment of the central actions of leptin. The present study was designed to elucidate the effects of two different treatments with 17beta-estradiol on leptin receptor and serum leptin levels in ovariectomized rats, a model of postmenopausal condition. Our results have shown that plasma leptin levels in ovariectomized rats were lower than in estradiol-treated animals, thereby supporting a positive effect of this steroid. Recent information has extended leptin actions to peripheral tissues, mainly to insulin-dependent tissues, this effect being related to metabolic actions. To better understand the peripheral effects of leptin and their possible regulation by estradiol treatment, we have analyzed leptin receptor expression in the skeletal muscle and the adipose tissue. Our results showed a tissue-specific regulation of this protein: Ob-Rb expression in the adipose tissue decreased when the time of treatment or the dose of estradiol administered increased, suggesting less sensitivity to leptin in this tissue, whereas in the skeletal muscle the changes in this protein followed the same profile as the plasma leptin levels. We think that this specific regulation could ensure a different response of each tissue toward the same serum leptin level. Further studies to clarify this situation are ongoing.     

Endurance training partially reverses dietary-induced leptin resistance in rodent skeletal muscle

Leptin acutely stimulates skeletal muscle fatty acid (FA) metabolism in lean rodents and humans. This stimulatory effect is eliminated following the feeding of high-fat diets in rodents as well as in obese humans. The mechanism(s) responsible for the development of skeletal muscle leptin resistance is unknown; however, a role for increased suppressor of cytokine signaling-3 (SOCS3) inhibition of the leptin receptor has been demonstrated in other rodent tissues. Furthermore, whether exercise intervention is an effective strategy to prevent or attenuate the development of skeletal muscle leptin resistance has not been investigated. Toward this end, 48 Sprague-Dawley rats (175-190 g; approximately 2-3 mo of age) were fed control or high-fat (60% kcal) diets for 4 wk and either remained sedentary or were treadmill trained. In control diet-fed animals that remained sedentary (CS) or were endurance trained (CT), leptin stimulated FA oxidation (CS +32 +/- 15%, CT +30 +/- 17%; P < 0.05), suppressed triacylglycerol (TAG) esterification (CS -17 +/- 7%, CT -24 +/- 8%; P < 0.05), and reduced the esterification-to-oxidation ratio (CS -19 +/- 13%, CT -29 +/- 10%; P < 0.001) in soleus muscle. High-fat feeding induced leptin resistance in the soleus of sedentary rats (FS), whereas endurance exercise training (FT) restored the ability of leptin to suppress TAG esterification (-19 +/- 9%, P = 0.038). Training did not completely restore the ability of leptin to stimulate FA oxidation. High-fat diets stimulated SOCS3 mRNA expression irrespective of training status (FS +451 +/- 120%, P = 0.024; FT +381 +/- 141%, P = 0.023). Thus the development of skeletal muscle leptin resistance appears to involve an increase in SOCS3 mRNA expression. Endurance training was generally effective in preventing the development of leptin resistance, although this did not appear to require a decrease in SOCS3 expression. Future studies should examine changes in the actual protein content of SOCS3 in muscle and establish whether aerobic exercise is also effective in treating leptin resistance in humans.     

Intracerebroventricular leptin infusion improves glucose homeostasis in lean type 2 diabetic MKR mice via hepatic vagal and non-vagal mechanisms

MKR mice, lacking insulin-like growth factor 1 receptor (IGF-1R) signaling in skeletal muscle, are lean yet hyperlipidemic, hyperinsulinemic, and hyperglycemic, with severe insulin resistance and elevated hepatic and skeletal muscle levels of triglycerides. We have previously shown that chronic peripheral administration of the adipokine leptin improves hepatic insulin sensitivity in these mice independently of its effects on food intake. As central leptin signaling has been implicated in the control of peripheral glucose homeostasis, here we examined the ability of central intracerebroventricular leptin administration to affect energy balance and peripheral glucose homeostasis in non-obese diabetic male MKR mice. Central leptin significantly reduced food intake, body weight gain and adiposity, as well as serum glucose, insulin, leptin, free fatty acid and triglyceride levels relative to ACSF treated controls. These reductions were accompanied by increased fat oxidation as measured by indirect calorimetry, as well as increased oxygen consumption. Central leptin also improved glucose tolerance and hepatic insulin sensitivity determined using the euglycemic-hyperinsulinemic clamps relative to pair fed vehicle treated controls, as well as increasing the rate of glucose disappearance. Hepatic vagotomy only partially reversed the ability of central leptin to improve glucose tolerance. These results demonstrate that central leptin dramatically improves insulin sensitivity independently of its effects on food intake, in a lean mouse model of type 2 diabetes. The findings also suggest that: 1) both hepatic vagal and non-vagal pathways contribute to this improvement, and 2) central leptin alters glucose disposal in skeletal muscle in this model.     

In vivo effect of Trigonella foenum graecum on the expression of pyruvate kinase, phosphoenolpyruvate carboxykinase, and distribution of glucose transporter (GLUT4) in alloxan-diabetic rats

Plasma glucose levels are maintained by a precise balance between glucose production and its use. Liver pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK), 2 key enzymes of glycolysis and gluconeogenesis, respectively, play a crucial role in this glucose homeostasis along with skeletal muscle glucose transporter (GLUT4). In the diabetic state, this balance is disturbed owing to the absence of insulin, the principal factor controlling this regulation. In the present study, alloxan-diabetic animals having high glucose levels of more than 300 mmol/L have been taken and the administration of Trigonella seed powder (TSP) to the diabetic animals was assessed for its effect on the expression of PK and PEPCK in liver and GLUT4 distribution in skeletal muscle of alloxan-diabetic rats. TSP treatment to the diabetic animals resulted in a marked decrease in the plasma glucose levels. Trigonella treatment partially restored the altered expression of PK and PEPCK. TSP treatment also corrected the alterations in the distribution of GLUT4 in the skeletal muscle.     

Chronic leptin administration decreases fatty acid uptake and fatty acid transporters in rat skeletal muscle.

Chronic leptin administration reduces triacylglycerol content in skeletal muscle. We hypothesized that chronic leptin treatment, within physiologic limits, would reduce the fatty acid uptake capacity of red and white skeletal muscle due to a reduction in transport protein expression (fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm)) at the plasma membrane. Female Sprague-Dawley rats were infused for 2 weeks with leptin (0.5 mg/kg/day) using subcutaneously implanted miniosmotic pumps. Control and pair-fed animals received saline-filled implants. Leptin levels were significantly elevated (approximately 4-fold; p < 0.001) in treated animals, whereas pair-fed treated animals had reduced serum leptin levels (approximately -2-fold; p < 0.01) relative to controls. Palmitate transport rates into giant sarcolemmal vesicles were reduced following leptin treatment in both red (-45%) and white (-84%) skeletal muscle compared with control and pair-fed animals (p < 0.05). Leptin treatment reduced FAT mRNA (red, -70%, p < 0.001; white, -48%, p < 0.01) and FAT/CD36 protein expression (red, -32%; p < 0.05) in whole muscle homogenates, whereas FABPpm mRNA and protein expression were unaltered. However, in leptin-treated animals plasma membrane fractions of both FAT/CD36 and FABPpm protein expression were significantly reduced in red (-28 and -34%, respectively) and white (-44 and -56%, respectively) muscles (p < 0.05). Across all experimental treatments and muscles, palmitate uptake by giant sarcolemmal vesicles was highly correlated with the plasma membrane FAT/CD36 protein (r = 0.88, p < 0.01) and plasma membrane FABPpm protein (r = 0.94, p < 0.01). These studies provide the first evidence that protein-mediated long chain fatty acid transport is subject to long term regulation by leptin.     

Changes in UCP mRNA expression levels in brown adipose tissue and skeletal muscle after feeding a high-energy diet and relationships with leptin, glucose and PPARgamma

Brown adipose tissue and skeletal muscle are known to be important sites for nonshivering thermogenesis. In this context, it is accepted that uncoupling proteins (UCPs) are involved in such process, but little is known about the physiological regulation of these proteins as affected by the intake of a high-energy (cafeteria) diet inducing fat deposition. In this study, the UCP messenger RNA (mRNA) expression in interscapular brown adipose tissue (iBAT) and skeletal muscle was assessed to evaluate the influence of a dietary manipulation on energy homeostasis regulation. We report a statistically significant increase in mRNA levels of iBAT UCP1 and UCP3 and a statistical marginal rise in skeletal muscle UCP3 mRNA expression after feeding a high-energy diet, whereas no changes in UCP2 expression were found in either tissue. Furthermore, significant positive associations between iBAT UCP1 and UCP3 mRNA levels with serum leptin were found. Although the expression of the beta(3) adrenoceptor (beta(3)AR) was about 50% in the lean controls compared with the obese group in iBAT, no statistically significant changes were observed concerning peroxisome proliferator-activated receptor gamma2 (PPARgamma2) mRNA levels in muscle or iBAT. We conclude that feeding a diet inducing weight and fat gain produces different outcomes on iBAT and skeletal muscle UCP mRNA expression, revealing a tissue-dependent response for the three UCPs. Results suggest that the regulation of UCP expression in both tissues under these specific dietary conditions may be related to leptin circulating levels.     

Injury of skeletal muscle and specific cytokines induce the expression of gap junction channels in mouse dendritic cells

Dendritic cells (DCs) in culture express at least connexin43, a protein subunit of gap junctions, and form gap junction channels, which could be important for T-cells activation. Here, we evaluated whether DCs express connexins in vivo and also to identify components of their microenvironment that regulate the functional expression of gap junctions. In vivo studies were performed in lymph nodes of mice under control conditions or after skeletal muscle damage. In double immunolabeling studies, connexin45 was frequently detected in DEC205(+) DCs in lymph nodes of control animals, whereas connexin43 was rarely found in DCs. However, connexin43 was upregulated in DCs after skeletal muscle damage. Upregulation of connexin43 gene expression by tissue damage was also confirmed in mice carrying a beta-galactosidase reporter gene in a connexin43 allele. The effect of several cytokines on the expression of functional gap junctions between cultured DCs was also tested. Under control conditions, cultured DCs did not communicate via gap junctions. However, after treatment with keratinocyte-conditioned medium or cytokine mixtures containing at least TNF-alpha and IL-1beta, they became transiently coupled through a pathway sensitive to octanol, a gap junction blocker. Cellular coupling induced by effective cytokine mixtures was prevented by IL-6. Single cytokines (TNF-alpha, IL-1beta, IFN-gamma, or IL-6) or other mixtures than the described above did not induce coupling via gap junctions. Increased levels of connexin43 and connexin45 protein and mRNA accompanied the appearance of cellular coupling. These studies provide demonstration of connexin expression and regulation by specific danger signals in DCs.     

Waste management - cytokines, growth factors and cachexia

Muscle damage with a lack of regeneration, manifests itself in several life-threatening diseases, including cancer cachexia, congestive heart failure, AIDS and sepsis. Often misdiagnosed as a condition simply of weight loss, cachexia is actually a highly complex metabolic disorder involving features of anorexia, anaemia, lipolysis and insulin resistance. A significant loss of lean body mass arises from such conditions, resulting in wasting of skeletal muscle. Unlike starvation, the weight loss seen in chronic illnesses arises equally from loss of muscle and of fat. The cachectic state is particularly problematic in cancer, typifying poor prognosis and often lowering responses to chemotherapy and radiation treatment. More than half of cancer patients suffer from cachexia, and strikingly, nearly one-third of cancer deaths are related to cachexia rather than the tumour burden. In considering this disorder, we are faced with a conundrum; how is it possible for uncontrolled growth to prevail in the tumour, in the face of unrestrained tissue loss in our muscles? Consistently, the catabolic state has been associated with a shift in the homeostatic balance between muscle synthesis and degradation mediated by the actions of growth factors and cytokines. Indeed, tumour necrosis factor-alpha (TNF-alpha) levels are raised in several animal models of cachectic muscle wasting, whereas the insulin-like growth factor (IGF) system acts potently to regulate muscle development, hypertrophy and maintenance. This concept of skeletal muscle homeostasis, often viewed as the net balance between two separate processes of protein synthesis and degradation has however changed. More recently, the view is that these two biochemical processes are not occurring independently of each other but in fact are finely co-ordinated by a web of intricate signalling networks. This review, therefore, aims to discuss data currently available regarding the mechanisms of degeneration and regeneration with specific emphasis on the potential and controversial cross-talk which may exist between anabolic growth factors (e.g. IGF-I) and catabolic cytokines (e.g. TNF-alpha). Also importantly, the potential impact at a cellular level of exercise, diet and age will be addressed. Finally, the ability to 'hi-jack' signalling pathways traditionally believed to be for growth and survival or death will be reviewed. It is anticipated that such a review will highlight significant gaps in our knowledge of the cachectic state as well as provide caution with regards to therapeutics suggesting total block on inflammatory processes such as that associated with TNF-alpha action.     

Absence of Functional Leptin Receptor Isoforms in the POUND (Leprdb/lb) Mouse Is Associated with Muscle Atrophy and Altered Myoblast Proliferation and Differentiation

Objective

Leptin receptors are abundant in human skeletal muscle, but the role of leptin in muscle growth, development and aging is not well understood. Here we utilized a novel mouse model lacking all functional leptin receptor isoforms (POUND mouse, Lepr^db/lb) to determine the role of leptin in skeletal muscle.

Methods and Findings

Skeletal muscle mass and fiber diameters were examined in POUND mice, and primary myoblast cultures were used to determine the effects of altered leptin signaling on myoblast proliferation and differentiation. ELISA assays, integrated pathway analysis of mRNA microarrays, and reverse phase protein analysis were performed to identify signaling pathways impacted by leptin receptor deficiency. Results show that skeletal muscle mass and fiber diameter are reduced 30–40% in POUND mice relative to wild-type controls. Primary myoblast cultures demonstrate decreased proliferation and decreased expression of both MyoD and myogenin in POUND mice compared to normal mice. Leptin treatment increased proliferation in primary myoblasts from muscles of both adult (12 months) and aged (24 months) wild-type mice, and leptin increased expression of MyoD and myogenin in aged primary myoblasts. ELISA assays and protein arrays revealed altered expression of molecules associated with the IGF-1/Akt and MAPK/MEK signaling pathways in muscle from the hindlimbs of mice lacking functional leptin receptors.

Conclusion

These data support the hypothesis that the adipokine leptin is a key factor important for the regulation of skeletal muscle mass, and that leptin can act directly on its receptors in peripheral tissues to regulate cell proliferation and differentiation.     

Effects of leptin replacement on hypothalamic-pituitary growth hormone axis function and circulating ghrelin levels in ob/ob mice

Leptin-deficient obese mice (ob/ob) have decreased circulating growth hormone (GH) and pituitary GH and ghrelin receptor (GHS-R) mRNA levels, whereas hypothalamic GH-releasing hormone (GHRH) and somatostatin (SST) expression do not differ from lean controls. Given the fact that GH is suppressed in diet-induced obesity (a state of hyperleptinemia), it remains to be determined whether the absence of leptin contributes to changes in the GH axis of ob/ob mice. Therefore, to study the impact of leptin replacement on the hypothalamic-pituitary GH axis of ob/ob mice, leptin was infused for 7 days (sc), resulting in circulating leptin levels that were similar to wild-type controls (approximately 1 ng/ml). Leptin treatment reduced food intake, body weight, and circulating insulin while elevating circulating n-octanoyl ghrelin concentrations. Leptin treatment did not alter hypothalamic GHRH, SST, or GHS-R mRNA levels compared with vehicle-treated controls. However, leptin significantly increased pituitary GH and GHRH-R expression and tended to enhance circulating GH levels, but this latter effect did not reach statistical significance. In vitro, leptin (1 ng/ml, 24 h) did not affect pituitary GH, GHRH-R, or GHS-R mRNA but did enhance GH release. The in vivo effects of leptin on circulating hormone and pituitary mRNA levels were not replicated by pair feeding ob/ob mice to match the food intake of leptin-treated mice. However, leptin did prevent the fall in hypothalamic GHRH mRNA and circulating IGF-I levels observed in pair-fed mice. These results demonstrate that leptin replacement has positive effects on multiple levels of GH axis function in ob/ob mice.     

Fatty acid oxidation and triacylglycerol hydrolysis are enhanced after chronic leptin treatment in rats

Leptin acutely increases fatty acid (FA) oxidation and triacylglycerol (TG) hydrolysis and decreases TG esterification in oxidative rodent muscle. However, the effects of chronic leptin administration on FA metabolism in skeletal muscle have not been examined. We hypothesized that chronic leptin treatment would enhance TG hydrolysis as well as the capacity to oxidize FA in soleus (SOL) muscle. Female Sprague-Dawley rats were infused for 2 wk with leptin (LEPT; 0.5 mg x kg(-1) x day(-1)) by use of subcutaneously implanted miniosmotic pumps. Control (AD-S) and pair-fed (PF-S) animals received saline-filled implants. Subsequently, FA metabolism was monitored for 45 min in isolated, resting, and contracting (20 tetani/min) SOL muscles by means of pulse-chase procedures. Food intake (-33 +/- 2%, P < 0.01) and body mass (-12.5 +/- 4%, P = 0.01) were reduced in both LEPT and PF-S animals. Leptin levels were elevated (+418 +/- 7%, P < 0.001) in treated animals but reduced in PF-S animals (-73 +/- 8%, P < 0.05) relative to controls. At rest, TG hydrolysis was increased in leptin-treated rats (1.8 +/- 2.2, AD-S vs. 23.5 +/- 8.1 nmol/g wet wt, LEPT; P < 0.001). In contracting SOL muscles, TG hydrolysis (1.5 +/- 0.6, AD-S vs. 3.6 +/- 1.0 micromol/g wet wt, LEPT; P = 0.02) and palmitate oxidation (18.3 +/- 6.7, AD-S vs. 45.7 +/- 9.9 nmol/g wet wt, LEPT; P < 0.05) were both significantly increased by leptin treatment. Chronic leptin treatment had no effect on TG esterification either at rest or during contraction. Markers of overall (citrate synthase) and FA (hydroxyacyl-CoA dehydrogenase) oxidative capacity were unchanged with leptin treatment. Protein expression of hormone-sensitive lipase (HSL) was also unaltered following leptin treatment. Thus leptin-induced increases in lipolysis are likely due to HSL activation (i.e., phosphorylation). Increased FA oxidation secondary to chronic leptin treatment is not due to an enhanced oxidative capacity and may be a result of enhanced flux into the mitochondrion (i.e., carnitine palmitoyltransferase I regulation) or electron transport uncoupling (i.e., uncoupling protein-3 expression).     

Decreased splenic mRNA expression levels of TNF-α and IL-6 in diet-induced obese animals

Obesity could be considered as a systemic low-grade inflammatory condition affecting inflammation markers. Adipose tissue synthesizes cytokines whose degree of elevation may depend on the obesity status. Recently, new information is collected on the cross-talking between immune system and adipose tissue in obesity. We report hereby that tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) gene expression in spleen of diet-induced obese animals were markedly decreased (more than 50%) as a consequence of the high fat feeding during five weeks. Interestingly, a very significant negative correlation was found between splenic TNF-alpha mRNA levels and total fat pads (r = -0.806, p = 0.000). These findings support the hypothesis that TNF-alpha gene expression may follow different trends in obese animals adipocytes and splenocytes.     

Resistance exercise decreases skeletal muscle tumor necrosis factor alpha in frail elderly humans.

Skeletal muscle protein and function decline with advancing age but the underlying pathophysiology is poorly understood. To test the hypothesis that the catabolic cytokine tumor necrosis factor alpha (TNF-alpha) contributes to this process, we studied the effects of aging and resistance exercise on TNF-alpha expression in human muscle. Using in situ hybridization, TNF-alpha message was localized to myocytes in sections of skeletal muscle from elderly humans. Both TNF-alpha mRNA and protein levels were elevated in skeletal muscle from frail elderly (81+/-1 year) as compared to healthy young (23+/-1 year) men and women. To determine whether resistance exercise affects TNF-alpha expression, frail elderly men and women were randomly assigned to a training group or to a nonexercising control group. Muscle biopsies were performed before and after 3 months. Muscle TNF-alpha mRNA and protein levels decreased in the exercise group but did not change in the control group. Muscle protein synthesis rate in the exercise group was inversely related to levels of TNF-alpha protein. These data suggest that TNF-alpha contributes to age-associated muscle wasting and that resistance exercise may attenuate this process by suppressing skeletal muscle TNF-alpha expression.     

High-fat diet with acyl-ghrelin treatment leads to weight gain with low inflammation, high oxidative capacity and normal triglycerides in rat muscle

Obesity is associated with muscle lipid accumulation. Experimental models suggest that inflammatory cytokines, low mitochondrial oxidative capacity and paradoxically high insulin signaling activation favor this alteration. The gastric orexigenic hormone acylated ghrelin (A-Ghr) has antiinflammatory effects in vitro and it lowers muscle triglycerides while modulating mitochondrial oxidative capacity in lean rodents. We tested the hypothesis that A-Ghr treatment in high-fat feeding results in a model of weight gain characterized by low muscle inflammation and triglycerides with high muscle mitochondrial oxidative capacity. A-Ghr at a non-orexigenic dose (HFG: twice-daily 200-µg s.c.) or saline (HF) were administered for 4 days to rats fed a high-fat diet for one month. Compared to lean control (C) HF had higher body weight and plasma free fatty acids (FFA), and HFG partially prevented FFA elevation (P<0.05). HFG also had the lowest muscle inflammation (nuclear NFkB, tissue TNF-alpha) with mitochondrial enzyme activities higher than C (P<0.05 vs C, P = NS vs HF). Under these conditions HFG prevented the HF-associated muscle triglyceride accumulation (P<0.05). The above effects were independent of changes in redox state (total-oxidized glutathione, glutathione peroxidase activity) and were not associated with changes in phosphorylation of AKT and selected AKT targets. Ghrelin administration following high-fat feeding results in a novel model of weight gain with low inflammation, high mitochondrial enzyme activities and normalized triglycerides in skeletal muscle. These effects are independent of changes in tissue redox state and insulin signaling, and they suggest a potential positive metabolic impact of ghrelin in fat-induced obesity.     

Association of resistin with visceral fat and muscle insulin resistance

Maturing Sprague-Dawley (S-D) rats develop obesity and skeletal muscle insulin resistance. To investigate the relationship between fat mass and insulin responses, we performed surgical removal of the epididymal and retroperitoneal depots of visceral adipose tissue (VF) or sham surgery (SHAM) in male rats aged 4 months. At sacrifice, 30 days later, the mass of visceral fat was 48% lower (p<0.05) in VF- compared to SHAM, while subcutaneous fat was essentially unchanged. VF- animals displayed increased insulin responses in isolated strips of skeletal muscle. Insulin-stimulated glucose transport was increased 28% in soleus muscle (p<0.05), with a trend toward a 31% increase in extensor digitorum longus muscle (p=0.058). Glucose tolerance was not significantly affected by surgical fat removal. In VF- animals, serum resistin was reduced 26% (p<0.05) and serum adiponectin was reduced 30% (p<0.05), with trends for reductions in IL-4 (58% reduction, p=0.084) and IL-6 (56% reduction, p=0.123). TNF-alpha, leptin and free fatty acids (NEFAs) were unchanged. We conclude that in maturing S-D rats, increased visceral adiposity leads to an increase in systemic release in resistin and possibly interleukins. Elevation of circulating cytokines may play a role in the development of muscle insulin resistance.     

High-dose leptin activates human leukocytes via receptor expression on monocytes

Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 +/- 5% for monocytes, 12 +/- 4% for neutrophils, and 5 +/- 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-alpha and IL-6 mRNA and a dose-dependent production of TNF-alpha and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3-5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-alpha (5-fold), IL-6 (19-fold), and IFN-gamma (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.     

Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin, hyperphagia, increased tissue lipid content, and TNF-alpha

Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-alpha in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-alpha has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-alpha.     

Insulin-like growth factor-1 and muscle wasting in chronic heart failure

Chronic heart failure is a clinical syndrome of cardiac origin, which affects various organ systems. It is associated with metabolic abnormalities leading to a catabolic syndrome in advanced stages of the disease. As in several other chronic diseases, skeletal muscle dysfunction and structural muscle abnormalities result in progressive muscle wasting and cachexia. These changes are accompanied by increased expression of proinflammatory cytokines, increased rate of apoptosis and activation of the proteolytic ubiquitin-proteasome pathway. Further, reduced expression of the local anabolic insulin-like growth factor-1 has been demonstrated in skeletal muscle of animals and patients with chronic heart failure. This suppression occurs in the presence of normal serum levels of insulin-like growth factor-1. In addition to catabolic effects of proinflammatory cytokines, these recent findings are consistent with reduced anabolism involving altered local insulin-like growth factor-1 levels in progressive muscle atrophy in chronic heart failure. This article describes local effects of insulin-like growth factor-1 on skeletal muscle function and morphology, its role in stem cell recruitment and muscle regeneration as well as its regulation in circumstances of muscle inflammation and wasting.     

Immunization of mice with T cell-dependent antigens promotes IL-6 and TNF-alpha production in muscle cells

IL-6 and TNF-alpha are proinflammatory cytokines involved in various inflammatory or non-inflammatory disorders characterized by muscle wasting. While infiltrating leukocytes are known to be the major source of these cytokines, it is unclear whether muscle cells also contribute to local inflammation. In this study, we first showed that cultured muscle cells and naive mouse muscle tissue were capable of producing IL-6 and TNF-alpha. We demonstrated an increased expression of IL-6 and TNF-alpha on muscle sections of C57BL/6J mice immunized with acetylcholine receptor (AChR) in the complete Freund's adjuvant (CFA) or with CFA only. In the presence of IL-6 or TNF-alpha, cultured AChR-expressing mouse (G-8) and human (TE671) skeletal muscle cells showed significantly decreased alpha-bungarotoxin-binding sites as measured by cellular ELISA. Moreover, IL-6- or TNF-alpha-treated muscle cells displayed a considerable increase in apoptotic cell ratios. Collectively, this study suggests a direct role for these two cytokines in muscle cell destruction and a possibility of muscle cell damage via an autocrine fashion.