Exercise increases TBC1D1 phosphorylation in human skeletal muscle.

Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% Vo(2 max)). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser(711) (AMPK), TBC1D1 Ser(231) (AMPK), TBC1D1 Ser(660) (AMPK), TBC1D1 Ser(700) (AMPK), and TBC1D1 Thr(590) (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser(711), TBC1D1 Ser(231), and TBC1D1 Ser(660) but had no effect on TBC1D1 Ser(700). Exercise did not increase TBC1D1 Thr(590) phosphorylation or TBC1D1/AS160 PAS phosphorylation, consistent with the lack of Akt activation. These data demonstrate that a single bout of exercise regulates TBC1D1 and AS160 phosphorylation on multiple sites in human skeletal muscle.

The anti-diabetic AMPK activator AICAR reduces IL-6 and IL-8 in human adipose tissue and skeletal muscle cells.

Increased production of inflammatory cytokines is suggested to be of importance for initiation and progression of insulin resistance. Chronic treatment with the AMP analogue 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR) has been shown to improve insulin sensitivity and prevent the development of diabetes in rodents. We investigated the effects of AICAR on the expression and production of inflammatory cytokines from human adipose tissue and skeletal muscle cells as well as intact rat skeletal muscles in vitro. AICAR dose-dependently decreased interleukin-6 (IL-6) gene expression and secretion in human adipose tissue and in human skeletal muscle cells. In parallel, AICAR inhibited interleukin-8 (IL-8) secretion in human adipose tissue and skeletal muscle cells, as well as reduced IL-8 gene expression in skeletal muscle cells. In intact rat extensor digitorum longus (EDL) muscle fibres AICAR markedly decreased IL-6 and IL-8 gene expression. In conclusion, AICAR inhibits the production of IL-6 and IL-8 human adipose tissue and in skeletal muscle cells. We suggest that decreased cytokine production might play a role for the AICAR-induced increase in insulin sensitivity.

Adiponectin receptors in human adipose tissue: effects of obesity, weight loss, and fat depots.

OBJECTIVE: To investigate the presence and regulatory properties of the adiponectin receptors, AdipoR1 and AdipoR2, in human adipose tissue (AT) and in isolated human adipocytes. RESEARCH METHODS AND PROCEDURES: The effect of obesity, weight loss, and gender on expression of AdipoR1 and AdipoR2 was investigated in subcutaneous AT. The influence of fat distribution on these receptors was investigated in paired samples of subcutaneous and omental AT. Gene expression of these receptors was quantified by reverse transcriptase-polymerase chain reaction. RESULTS: AdipoR1 mRNA levels were approximately 10-fold higher than adipoR2 in both AT fragments and in isolated adipocytes. AdipoR1 expression was lower in AT from obese subjects (p < 0.05) compared with that from normal-weight subjects, and AdipoR1 displayed a negative correlation with BMI (r = -0.53, p < 0.01). In obese subjects, weight loss (approximately 12 kg) increased AdipoR1 expression by 80% in AT (p < 0.01). Concerning regional differences, AdipoR1 showed significantly lower expression in omental AT than in subcutaneous AT (p < 0.01). No gender difference was observed in the expression of these receptors. In human preadipocyte cultures, AdipoR1 expression was not induced during the differentiation process, whereas AdipoR2 was induced by 5-fold (p < 0.05). DISCUSSION: AdipoR1 is highly expressed in human AT, indicating that adiponectin may have biological effects in AT in an autocrine/paracrine manner. AdipoR1 expression in AT is reduced in obese subjects and is increased after weight loss. Thus, it can be suggested that adiponectin might have reduced biological effects in AT due to low levels of adiponectin receptors in obese subjects and in omental adipocytes, which may further aggravate the negative metabolic effect of low levels of adiponectin characterizing the obese state.

Monocyte chemoattractant protein-1 release is higher in visceral than subcutaneous human adipose tissue (AT): implication of macrophages resident in the AT.

UNLABELLED: Human adipose tissue (AT) produces several adipokines including monocyte chemoattractant protein (MCP)-1, involved in the pathogenesis of atherosclerosis. OBJECTIVE: Human AT cultures, isolated adipocytes, and stromal-vascular cells were used to investigate the relationship among AT-resident macrophages, MCP-1, and adiposity and the regulation of MCP-1. RESULTS: mRNA levels of specific macrophage markers (CD68 and CD14) are correlated with adiposity in sc AT and visceral AT (P < 0.05). MCP-1 production is higher in stromal-vascular cells vs. adipocytes (P < 0.01) and correlates with macrophage markers in both AT compartments (P < 0.05). MCP-1 release is higher in obese subjects (P < 0.05) and in VAT (P < 0.01), but after adjusting for AT-resident macrophages, the differences disappear. MCP-1 is stimulated by IL-1beta, TNF-alpha, IL-8, IL-4, and IL-6 + IL-6-soluble receptor and is decreased by dexamethasone, IL-10, metformin, and thiazolidinediones. DISCUSSION: MCP-1 is correlated with specific macrophage markers, adiposity, and AT localization, but the relationship seems to be related to the number of AT-resident macrophages. Despite this, MCP-1 may be involved in obesity-related health complications, and the decrease of MCP-1 by metformin and thiazolidinediones suggests that these antidiabetic compounds have antiinflammatory properties improving the low-grade inflammatory state observed in obesity.

Lower expression of adiponectin mRNA in visceral adipose tissue in lean and obese subjects.

Adiponectin is an adipocyte-specific protein suggested to play a role in mediating the metabolic effects of obesity. In the present study, we investigated adiponectin mRNA levels in both visceral and subcutaneous abdominal adipose tissue (AT) from lean and obese subjects. Investigations on both "fresh" fat biopsies and incubations of AT fragments were performed. Regional differences in the effects of the cytokine interleukin-1beta (IL-1beta) were investigated. Adiponectin gene expression was 33% lower in visceral AT than in subcutaneous AT of lean subjects (P < 0.05), and 28% lower in obese subjects, albeit non-significant (P = 0.3). In both lean and obese subjects adiponectin mRNA expression in incubated AT fragments was significantly lower in visceral AT than in subcutaneous AT (lean: P < 0.01; obese: P < 0.05). No difference was found in adiponectin mRNA levels in gluteal compared to abdominal subcutaneous AT. IL-1beta suppressed adiponectin mRNA levels substantially in both subcutaneous and visceral AT. In conclusion, adiponectin gene expression is lower in visceral AT than in subcutaneous abdominal AT, suggesting subcutaneous AT to be more important for circulating adiponectin levels.

AICAR stimulates adiponectin and inhibits cytokines in adipose tissue.

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) can be used as an experimental tool to activate 5'-AMP-activated protein kinase (AMPK) and has been shown to improve insulin sensitivity. In parallel adiponectin also seems to activate AMPK and to improve insulin sensitivity. We have investigated the effects of AICAR on the gene expression of adiponectin and on gene expression and release of cytokines in human adipose tissue in vitro. AICAR stimulated AMPK alpha1 activity 3-4-fold (p<0.001), and dose-dependently increased adiponectin mRNA levels with significant stimulation (2-4-fold) at AICAR concentrations of 0.5-2mM (p<0.05). The adipose tissue protein release of tumor necrosis factor-alpha (TNF- alpha) and interleukin-6 (IL-6) was decreased by AICAR (p<0.05). In conclusion, AICAR stimulated adipose tissue AMPK alpha1 activity and adiponectin gene expression, while attenuating the release of TNF-alpha and IL-6. Reduced concentrations of these cytokines and increased levels of adiponectin might play a role for the insulin sensitizing effects of AICAR.

Higher production of IL-8 in visceral vs. subcutaneous adipose tissue. Implication of nonadipose cells in adipose tissue.

IL-8 is released from human adipose tissue. Circulating IL-8 is increased in obese compared with lean subjects and is associated with measures of insulin resistance, development of atherosclerosis, and cardiovascular disease. We studied 1) the production and release of IL-8 in vitro from paired samples of subcutaneous (SAT) and visceral (VAT) adipose tissue and 2) the production of IL-8 from whole adipose tissue, isolated adipocytes, and nonfat cells of adipose tissue. IL-8 release from VAT was fourfold higher than from SAT (P < 0.05), and IL-8 mRNA was twofold higher in VAT compared with SAT (P < 0.01). Dexamethasone (50 nM) attenuated IL-8 production by 50% (P < 0.05), and IL-1beta (2 microg/l) increased IL-8 production up to 15-fold (P < 0.001). IL-8 release from whole SAT explants correlated with body mass index (BMI; r = 0.78; P < 0.001), as did IL-8 release from nonfat cells (r = 0.79; P < 0.001). However, no correlation was found between IL-8 release from the fraction of isolated adipocytes and BMI (r = 0.01). In conclusion, we demonstrated an increased release of IL-8 from VAT compared with SAT. Furthermore, our data suggest that the observed elevation in circulating levels of IL-8 in obese subjects is due primarily to the release of IL-8 from nonfat cells from adipose tissue. The high levels of IL-8 release from human adipose tissue and accumulation of this tissue in obese subjects may account for some of the increase in circulating IL-8 observed in obesity.

Increased expression of TNF-alpha, IL-6, and IL-8 in HALS: implications for reduced adiponectin expression and plasma levels.

Human immunodeficiency virus (HIV)-associated lipodystrophy syndrome (HALS) is a side effect of highly active antiretroviral therapy of HIV-infected patients; however, the mechanism of the lipodystrophy and insulin resistance seen in this syndrome remains elusive. Adiponectin, an adipocyte-specific protein, is thought to play an important role in regulating insulin sensitivity. We investigated circulating levels and gene expression of adiponectin in subcutaneous abdominal adipose tissue (AT) from 18 HIV-infected patients with HALS compared with 18 HIV-infected patients without HALS. Implications of cytokines for adiponectin levels were investigated by determining circulating levels of TNF-alpha, IL-6, and IL-8 as well as gene expression of these cytokines in AT. HALS patients exhibited 40% reduced plasma adiponectin levels (P < 0.05) compared with non-HALS subjects. Correspondingly, adiponectin mRNA levels in AT were reduced by >50% (P = 0.06). HALS patients were insulin resistant, and a positive correlation was found between plasma adiponectin and insulin sensitivity (r = 0.55, P < 0.01) and percent limb fat (r = 0.61, P < 0.01). AT mRNA of TNF-alpha, IL-6, and IL-8 was increased in AT of HALS subjects (P < 0.05), and both AT TNF-alpha mRNA and plasma TNF-alpha were negatively correlated to plasma adiponectin (P < 0.05). Finally, TNF-alpha was found in vitro to inhibit human AT adiponectin mRNA by 80% (P < 0.05). In conclusion, HALS patients have reduced levels of plasma adiponectin and adiponectin mRNA in AT. Increased cytokine mRNA in AT is hypothesized to exert an inhibitory effect on adiponectin gene expression and, consequently, to play a role in the reduced plasma adiponectin levels found in HALS patients.

Regulation of adiponectin by adipose tissue-derived cytokines: in vivo and in vitro investigations in humans.

Adiponectin is an adipose tissue-specific protein that is abundantly present in the circulation and suggested to be involved in insulin sensitivity and development of atherosclerosis. Because cytokines are suggested to regulate adiponectin, the aim of the present study was to investigate the interaction between adiponectin and three adipose tissue-derived cytokines (IL-6, IL-8, and TNF-alpha). The study was divided into three substudies as follows: 1) plasma adiponectin and mRNA levels in adipose tissue biopsies from obese subjects [mean body mass index (BMI): 39.7 kg/m2, n = 6] before and after weight loss; 2) plasma adiponectin in obese men (mean BMI: 38.7 kg/m2, n = 19) compared with lean men (mean BMI: 23.4 kg/m2, n = 10) before and after weight loss; and 3) in vitro direct effects of IL-6, IL-8, and TNF-alpha on adiponectin mRNA levels in adipose tissue cultures. The results were that 1) weight loss resulted in a 51% (P < 0.05) increase in plasma adiponectin and a 45% (P < 0.05) increase in adipose tissue mRNA levels; 2) plasma adiponectin was 53% (P < 0.01) higher in lean compared with obese men, and plasma adiponectin was inversely correlated with adiposity, insulin sensitivity, and IL-6; and 3) TNF-alpha (P < 0.01) and IL-6 plus its soluble receptor (P < 0.05) decreased adiponectin mRNA levels in vitro. The inverse relationship between plasma adiponectin and cytokines in vivo and the cytokine-induced reduction in adiponectin mRNA in vitro suggests that endogenous cytokines may inhibit adiponectin. This could be of importance for the association between cytokines (e.g., IL-6) and insulin resistance and atherosclerosis.