The fatty liver and insulin resistance

Obesity is not necessary to observe insulin resistance in humans since severe insulin resistance also characterizes patients lacking subcutaneous fat such as those with HAART (highly-active antiretroviral therapy) - associated lipodystrophy. Both the obese and the lipodystrophic patients have, however, an increase in the amount of fat hidden in the liver. Liver fat content can be non-invasively accurately quantified by proton magnetic resonance spectroscopy. It is closely correlated with fasting insulin and direct measures of hepatic insulin sensitivity while the amount of subcutaneous adipose tissue is not. The causes of interindividual variation in liver fat content independent of obesity are largely unknown but could involve differences in signals from adipose tissue such as in the amount of adiponectin produced and differences in fat intake. Adiponectin deficiency characterizes both lipodystrophic and obese insulin resistant individuals, and serum levels correlate with liver fat content. Liver fat content can be decreased by weight loss. In addition, treatment of both lipodystrophic and type 2 diabetic patients with PPARgamma agonists but not metformin decreases liver fat and increases adiponectin levels. Markers of liver fat such as serum alanine aminotransferase activity have been shown to predict type 2 diabetes in several studies independent of obesity. The fatty liver thus may help to explain why some but not all obese individuals are insulin resistant and why even lean individuals may be insulin resistant, and thereby at risk of developing type 2 diabetes and cardiovascular disease.     

Adiponectin is required for PPARγ-mediated improvement of endothelial function in diabetic mice

Rosiglitazone is a PPARγ agonist commonly used to treat diabetes. In addition to improving insulin sensitivity, rosiglitazone restores normal vascular function by a mechanism that remains poorly understood. Here we show that adiponectin is required to mediate the PPARγ effect on vascular endothelium of diabetic mice. In db/db and diet-induced obese mice, PPARγ activation by rosiglitazone restores endothelium-dependent relaxation of aortae, whereas diabetic mice lacking adiponectin or treated with an anti-adiponectin antibody do not respond. Rosiglitazone stimulates adiponectin release from fat explants, and subcutaneous fat transplantation from rosiglitazone-treated mice recapitulates vasodilatation in untreated db/db recipients. Mechanistically, adiponectin activates AMPK/eNOS and cAMP/PKA signaling pathways in aortae, which increase NO bioavailability and reduce oxidative stress. Taken together, these results demonstrate that adipocyte-derived adiponectin is required for PPARγ-mediated improvement of endothelial function in diabetes. Thus, the adipose tissue represents a promising target for treating diabetic vasculopathy.     

Regulation of gene expression by activation of the peroxisome proliferator-activated receptor gamma with rosiglitazone (BRL 49653) in human adipocytes.

To better define the mechanism of action of the thiazolidinediones, we incubated freshly isolated human adipocytes with rosiglitazone and investigated the changes in mRNA expression of genes encoding key proteins of adipose tissue functions. Rosiglitazone (10(-6) M, 4 h) increased p85alphaphosphatidylinositol 3-kinase (p85alphaPI-3K) and uncoupling protein-2 mRNA levels and decreased leptin expression. The mRNA levels of insulin receptor, IRS-1, Glut 4, lipoprotein lipase, hormone-sensitive lipase, acylation-stimulating protein, fatty acid transport protein-1, angiotensinogen, plasminogen activator inhibitor-1, and PPARgamma1 and gamma2 were not modified by rosiglitazone treatment. Activation of RXR, the partner of PPARgamma, in the presence of rosiglitazone, increased further p85alphaPI-3K and UCP2 mRNA levels and produced a significant augmentation of Glut 4 expression. Because p85alphaPI-3K is a major component of insulin action, the induction of its expression might explain, at least in part, the insulin-sensitizing effect of the thiazolidinediones.     

Thiazolidinedione response in familial lipodystrophy patients with LMNA mutations: a case series

Type 2 familial partial lipodystrophy (FPLD2) patients show impaired glucose and lipid metabolism resulting from lipodystrophic 'lipid pressure' and an intrinsic defect in skeletal muscle metabolism. Since mutated lamin A may interfere with peroxisome proliferator activator gamma (PPARγ) expression, we hypothesized that PPARγ stimulation improves fat distribution and metabolic abnormalities in these patients. 5 nondiabetic FPLD2 patients were treated with rosiglitazone over 12 months. We assessed body composition, body fat distribution, and skinfold thickness/subcutaneous tissue thickness. We also determined venous glucose, insulin, and free fatty acid (FFA) concentrations, and respiratory quotient (RQ) before and during oral glucose tolerance testing. Adipose tissue and muscle fasting and postprandial metabolism were studied by microdialysis. Within 12 months treatment, hip circumference increased from 93.6±2.78 cm to 96.2±2.3 cm (p<0.05). Rosiglitazone reduced fasting glucose levels and liver transaminases. Baseline and postprandial FFA concentrations were significantly lower after 12 months treatment. RQ and muscle interstitial pyruvate and lactate did not respond to treatment. We conclude that PPARγ stimulation with rosiglitazone modestly improves glucose metabolism in FPLD2 patients presumably through proximal adipose tissue expansion. The intrinsic muscular metabolic defect does not respond to rosiglitazone.     

Adipose-specific overexpression of GLUT4 reverses insulin resistance and diabetes in mice lacking GLUT4 selectively in muscle

Adipose tissue plays an important role in glucose homeostasis and affects insulin sensitivity in other tissues. In obesity and type 2 diabetes, glucose transporter 4 (GLUT4) is downregulated in adipose tissue, and glucose transport is also impaired in muscle. To determine whether overexpression of GLUT4 selectively in adipose tissue could prevent insulin resistance when glucose transport is impaired in muscle, we bred muscle GLUT4 knockout (MG4KO) mice to mice overexpressing GLUT4 in adipose tissue (AG4Tg). Overexpression of GLUT4 in fat not only normalized the fasting hyperglycemia and glucose intolerance in MG4KO mice, but it reduced these parameters to below normal levels. Glucose infusion rate during a euglycemic clamp study was reduced 46% in MG4KO compared with controls and was restored to control levels in AG4Tg-MG4KO. Similarly, insulin action to suppress hepatic glucose production was impaired in MG4KO mice and was restored to control levels in AG4Tg-MG4KO. 2-deoxyglucose uptake during the clamp was increased approximately twofold in white adipose tissue but remained reduced in skeletal muscle of AG4Tg-MG4KO mice. AG4Tg and AG4Tg-MG4KO mice have a slight increase in fat mass, a twofold elevation in serum free fatty acids, an approximately 50% increase in serum leptin, and a 50% decrease in serum adiponectin. In MG4KO mice, serum resistin is increased 34% and GLUT4 overexpression in fat reverses this. Overexpression of GLUT4 in fat also reverses the enhanced clearance of an oral lipid load in MG4KO mice. Thus overexpression of GLUT4 in fat reverses whole body insulin resistance in MG4KO mice without restoring glucose transport in muscle. This effect occurs even though AG4Tg-MG4KO mice have increased fat mass and low adiponectin and is associated with normalization of elevated resistin levels.     

Subcutaneous Abdominal Adipose Tissue Subcompartments: Potential Role in Rosiglitazone Effects

Abdominal visceral tissue (VAT) and subcutaneous adipose tissue (SAT), comprised of superficial‐SAT (sSAT) and deep‐SAT (dSAT), are metabolically distinct. The antidiabetic agents thiazolidinediones (TZDs), in addition to their insulin‐sensitizing effects, redistribute SAT suggesting that TZD action involves adipose tissue depot‐specific regulation. We investigated the expression of proteins key to adipocyte metabolism on differentiated first passage (P1) preadipocytes treated with rosiglitazone, to establish a role for the diverse depots of abdominal adipose tissue in the insulin‐sensitizing effects of TZDs. Adipocytes and preadipocytes were isolated from sSAT, dSAT, and VAT samples obtained from eight normal subjects. Preadipocytes (P1) left untreated (U) or treated with a classic differentiation cocktail (DI) including rosiglitazone (DIR) for 9 days were evaluated for strata‐specific differences in differentiation including peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) and lipoprotein lipase (LPL) expression, insulin sensitivity via adiponectin and glucose transport‐4 (GLUT4), glucocorticoid metabolism with 11β‐hydroxysteroid dehydrogenase type‐1 (11βHSD1), and alterations in the adipokine leptin. While depot‐specific differences were absent with the classic differentiation cocktail, with rosiglitazone sSAT had the most potent response followed by dSAT, whereas VAT was resistant to differentiation. With rosiglitazone, universal strata effects were observed for PPAR‐γ, LPL, and leptin, with VAT in all cases expressing significantly lower basal expression levels. Clear dSAT‐specific changes were observed with decreased intracellular GLUT4. Specific sSAT alterations included decreased 11βHSD1 whereas secreted adiponectin was potently upregulated in sSAT with respect to dSAT and VAT. Overall, the subcompartments of SAT, sSAT, and dSAT, appear to participate in the metabolic changes that arise with rosiglitazone administration.     

The effect of the HIV protease inhibitor ritonavir on proliferation, differentiation, lipogenesis, gene expression and apoptosis of human preadipocytes and adipocytes.

HIV patients in highly active antiretroviral therapy (HAART) develop lipodystrophy and insulin resistance. Protease inhibitors have been shown to alter adipocyte metabolism in murine cell lines. In this study, biological effects of the HIV protease inhibitor, ritonavir, were investigated on human SGBS preadipocytes and adipocytes. Ritonavir dose-dependently impaired preadipocyte proliferation and adipogenic differentiation. Gene expression analysis measured by real-time PCR, showed no effect of ritonavir (up to 20 microM) on expression of mRNA of PPARgamma2 and SREBP1c, but suppressed adiponectin mRNA while increasing IL-6 mRNA expression. In human adipocytes, ritonavir at therapeutic concentrations inhibited insulin-stimulated lipogenesis, reduced GLUT4 mRNA, fatty acid synthase and adiponectin expression, while increasing IL-6 mRNA expression. Finally, long-term treatment (72 and 120 h) of SGBS adipocytes but not preadipocytes with ritonavir induced apoptosis in up to 15% of the cells. All together, these data show effects of ritonavir on human preadipocytes and adipocytes aiming at reducing adipose tissue mass and increasing insulin resistance. These in vitro findings may partly explain the clinical findings in patients under HAART. Furthermore, SGBS cells may serve as a useful tool in further investigation of the mechanism of protease inhibitor action in human adipocytes.     

Increased angiotensinogen production in epicardial adipose tissue during cardiac surgery: possible role in a postoperative insulin resistance

Critical illness induces among other events production of proinflammatory cytokines that in turn interfere with insulin signaling cascade and induce insulin resistance on a postreceptor level. Recently, local renin-angiotensin system of adipose tissue has been suggested as a possible contributor to the development of insulin resistance in patients with obesity. The aim of our study was to determine local changes of the renin-angiotensin system of subcutaneous and epicardial adipose tissue during a major cardiac surgery, which may serve as a model of an acute stress potentially affecting endocrine function of adipose tissue. Ten patients undergoing elective cardiac surgery were included into the study. Blood samples and samples of subcutaneous and epicardial adipose tissue were collected at the beginning and at the end of the surgery. Blood glucose, serum insulin and adiponectin levels were measured and mRNA for angiotensinogen, angiotensin-converting enzyme and angiotensin II type 1 receptor were determined in adipose tissue samples using RT PCR. Cardiac surgery significantly increased both insulin and blood glucose levels suggesting the development of insulin resistance, while serum adiponectin levels did not change. Expression of angiotensinogen mRNA significantly increased in epicardial adipose tissue at the end of surgery relative to baseline but remained unchanged in subcutaneous adipose tissue. Fat expression of angiotensin-converting enzyme and type 1 receptor for angiotensin II were not affected by surgery. Our study suggests that increased angiotensinogen production in epicardial adipose tissue may contribute to the development of postoperative insulin resistance.     

Differential patterns of serum concentration and adipose tissue expression of chemerin in obesity: Adipose depot specificity and gender dimorphism

Chemerin, a recognized chemoattractant, is expressed in adipose tissue and plays a role in adipocytes differentiation and metabolism. Gender- and adipose tissue-specific differences in human chemerin expression have not been well characterized. Therefore, these differences were assessed in the present study. The body mass index (BMI) and the circulating levels of chemerin and other inflammatory, adiposity and insulin resistance markers were assessed in female and male adults of varying degree of obesity. Chemerin mRNA expression was also measured in paired subcutaneous and visceral adipose tissue samples obtained from a subset of the study subjects. Serum chemerin concentrations correlated positively with BMI and serum leptin levels and negatively with high density lipoprotein (HDL)-cholesterol levels. No correlation was found between serum chemerin concentrations and fasting glucose, total cholesterol, low density lipoprotein (LDL)-cholesterol, triglycerides, insulin, C-reactive protein or adiponectin. Similarly, no relation was observed with the homeostasis model assessment for insulin resistance (HOMA-IR) values. Gender- and adipose tissue-specific differences were observed in chemerin mRNA expression levels, with expression significantly higher in women than men and in subcutaneous than visceral adipose tissue. Interestingly, we found a significant negative correlation between circulating chemerin levels and chemerin mRNA expression in subcutaneous fat. Among the subjects studied, circulating chemerin levels were associated with obesity markers but not with markers of insulin resistance. At the tissue level, fat depot-specific differential regulation of chemerin mRNA expression might contribute to the distinctive roles of subcutaneous vs. visceral adipose tissue in human 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.     

Actions of PPARgamma agonism on adipose tissue remodeling, insulin sensitivity, and lipemia in absence of glucocorticoids

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists improve insulin sensitivity and lipemia partly through enhancing adipose tissue proliferation and capacity for lipid retention. The agonists also reduce local adipose glucocorticoid production, which may in turn contribute to their metabolic actions. This study assessed the effects of a PPARgamma agonist in the absence of glucocorticoids (adrenalectomy, ADX). Intact, ADX, and intact pair-fed (PF) rats were treated with the PPARgamma agonist rosiglitazone (RSG) for 2 wk. RSG increased inguinal (subcutaneous) white (50%) and brown adipose tissue (6-fold) weight but not that of retroperitoneal (visceral) white adipose tissue. ADX but not PF reduced fat accretion in both inguinal and retroperitoneal adipose depots but did not affect brown adipose mass. RSG no longer increased inguinal weight in ADX and PF rats but increased brown adipose mass, albeit less so than in intact rats. RSG increased cell proliferation in white (3-fold) and brown adipose tissue (6-fold), as assessed microscopically and by total DNA, an effect that was attenuated but not abrogated by ADX. RSG reduced the expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) in all adipose depots. RSG improved insulin sensitivity (reduction in fasting insulin and homeostasis model assessment of insulin resistance, both -50%) and triacylglycerolemia (-75%) regardless of the glucocorticoid status, these effects being fully additive to those of ADX and PF. In conclusion, RSG partially retained its ability to induce white and brown adipose cell proliferation and brown adipose fat accretion and further improved insulin sensitivity and lipemia in ADX rats, such effects being therefore independent from the PPARgamma-mediated modulation of glucocorticoids.     

Differences in adiponectin protein expression: effect of fat depots and type 2 diabetic status.

Adiponectin is an adipocyte-derived hormone associated with insulin sensitivity and atherosclerotic risk. As central rather than gluteofemoral fat is known to increase the risk of type 2 diabetes and cardiovascular disease, we investigated the mRNA and protein expression of adiponectin in human adipose tissue depots. RNA was extracted from 46 human adipose tissue samples from non-diabetic subjects aged 44.33 +/- 12.4 with a BMI of 28.3 +/- 6.0 (mean +/- SD). The samples were as follows: 21 abdominal subcutaneous, 13 omentum, 6 thigh; samples were also taken from diabetic subjects aged 66.6 +/- 7.5 with BMI 28.9 +/- 3.17; samples were: 6 abdominal subcutaneous; 3 thigh. Quantitative PCR and Western analysis was used to determine adiponectin content. Protein content studies determined that when compared with non-diabetic abdominal subcutaneous adipose tissue (Abd Sc AT) (values expressed as percentage relative to Abd Sc AT -100 %). Adiponectin protein content was significantly lower in non-diabetic omental AT (25 +/- 1.6 %; p < 0.0001, n = 6) and in Abd Sc AT from diabetic subjects (36 +/- 1.5 %; p < 0.0001, n = 4). In contrast, gluteal fat maintained high adiponectin protein content from non-diabetic patients compared with diabetic patients. An increase in BMI was associated with lower adiponectin protein content in obese ND Abd Sc AT (25 +/- 0.4 %; p < 0.0001). These findings were in agreement with the mRNA expression data. In summary, this study indicates that adiponectin protein content in non-diabetic subjects remains high in abdominal subcutaneous fat, including gluteal fat, explaining the high serum adiponectin levels in these subjects. Omental fat, however, expresses little adiponectin. Furthermore, abdominal and gluteal subcutaneous fat appears to express significantly less adiponectin once diabetic status is reached. In conclusion, the adipose tissue depot-specific expression of adiponectin may influence the pattern of serum adiponectin concentrations and subsequent disease risk.     

Central role of the adipocyte in the insulin-sensitising and cardiovascular risk modifying actions of the thiazolidinediones

Insulin resistance is a key metabolic defect in type 2 diabetes that is exacerbated by obesity, especially if the excess adiposity is located intra-abdominally/centrally. Insulin resistance underpins many metabolic abnormalities-collectively known as the insulin resistance syndrome-that accelerate the development of cardiovascular disease. Thiazolidinedione anti-diabetic agents improve glycaemic control by activating the nuclear receptor peroxisome proliferator activated receptor-gamma (PPARgamma). This receptor is highly expressed in adipose tissues. In insulin resistant fat depots, thiazolidinediones increase pre-adipocyte differentiation and oppose the actions of pro-inflammatory cytokines such as tumour necrosis factor-alpha. The metabolic consequences are enhanced insulin signalling, resulting in increased glucose uptake and lipid storage coupled with reduced release of free fatty acids (FFA) into the circulation. Metabolic effects of PPARgamma activation are depot specific-in people with type 2 diabetes central fat mass is reduced and subcutaneous depots are increased. Thiazolidinediones increase insulin sensitivity in liver and skeletal muscle as well as in fat, but they do not express high levels of PPARgamma, suggesting that improvement in insulin action is indirect. Reduced FFA availability from adipose tissues to liver and skeletal muscle is a pivotal component of the insulin-sensitising mechanism in these latter two tissues. Adipocytes secrete multiple proteins that may both regulate insulin signalling and impact on abnormalities of the insulin resistance syndrome--this may explain the link between central obesity and cardiovascular disease. Of these proteins, low plasma adiponectin is associated with insulin resistance and atherosclerosis--thiazolidinediones increase adipocyte adiponectin production. Like FFA, adiponectin is probably an important signalling molecule regulating insulin sensitivity in muscle and liver. Adipocyte production of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, and angiotensin II secretion are partially corrected by PPARgamma activation. The favourable modification of adipocyte-derived cardiovascular risk factors by thiazolidinediones suggests that these agents may reduce cardiovascular disease as well as provide durable glycaemic control in type 2 diabetes.