Obesity is the main risk factor for the development of type 2 diabetes. Activation of the central endocannabinoid system increases food intake and promotes weight gain. Blockade of the cannabinoid type 1 (CB-1) receptor reduces body weight in animals by central and peripheral actions; the role of the peripheral endocannabinoid system in human obesity is now being extensively investigated. We measured circulating endocannabinoid concentrations and studied the expression of CB-1 and the main degrading enzyme, fatty acid amide hydrolase (FAAH), in adipose tissue of lean (n ‫؍‬ 20) and obese (n ‫؍‬ 20) women and after a 5% weight loss in a second group of women (n ‫؍‬ 17). Circulating levels of anandamide and 1/2-arachidonoylglycerol were increased by 35 and 52% in obese compared with lean women (P < 0.05). Adipose tissue mRNA levels were reduced by ؊34% for CB-1 and ؊59% for FAAH in obese subjects (P < 0.05). A strong negative correlation was found between FAAH expression in adipose tissue and circulating endocannabinoids. Circulating endocannabinoids and CB-1 or FAAH expression were not affected by 5% weight loss. The expression of CB-1 and FAAH was increased in mature human adipocytes compared with in preadipocytes and was found in several human tissues. Our findings support the presence of a peripheral endocannabinoid system that is upregulated in human obesity. Diabetes 54: 2838 -2843, 2005 O besity is one of the main risk factors for the development of type 2 diabetes, and weight loss may be a successful means of reducing the number of patients affected by type 2 diabetes (1-4). Exogenous cannabinoids and endocannabinoids increase food intake and promote weight gain in animals by activating central endocannabinoid receptors (5-8). This phenomenon has been exploited in the treatment of cachexia using tetrahydrocannabinol (9). Endocannabinoids are derived from membrane phospholipids (anandamide [AEA]) or triglycerides (2-arachidonoylglycerol [2-AG]) (10). Endocannabinoids bind to the G-proteincoupled cannabinoid (CB) type 1 and type 2 receptors. In animals, CB-1 is expressed in the brain, gastrointestinal organs, and adipose tissue, whereas CB-2 is predominantly expressed on peripheral immune cells (11). Intracellular degradation by the enzyme fatty acid amide hydrolase (FAAH) limits endocannabinoid action (10).In genetic animal models of obesity, brain endocannabinoid levels are increased and CB-1 is downregulated (12,13). CB-1 gene-deficient mice are lean and resistant to diet-induced obesity (14). Similarly, pharmacological CB-1 blockade with SR141716 (rimonabant) reduces food intake and body weight (8,12,15). Central and peripheral mechanisms may contribute to this weight loss (16). Indeed, CB-1 activation in isolated mouse adipocytes increases the activity of the lipogenic enzyme lipoprotein lipase (16). Moreover, CB-1 blockade increases adiponectin gene expression in adipose tissue and elevates circulating adiponectin levels in the obese Zucker rat (17). Recently, the activation of CB-1 receptors in the...

Background-Angiotensin type 1 receptor (AT 1 R) blockers (ARB) have been shown to reduce the incidence of type 2 diabetes mellitus by an unknown molecular mechanism. The peroxisome proliferator-activated receptor-␥ (PPAR␥) is the central regulator of insulin and glucose metabolism improving insulin sensitivity. We investigated the regulation of PPAR␥ function by ARBs. Methods and Results-The ARBs irbesartan and telmisartan (10 mol/L) potently enhanced PPAR␥-dependent 3T3-L1 adipocyte differentiation associated with a significant increase in mRNA expression of the adipogenic marker gene adipose protein 2 (aP2), as measured by quantitative real-time polymerase chain reaction (irbesartan: 3.3Ϯ0.1-fold induction; telmisartan: 3.1Ϯ0.3-fold induction; both PϽ0.01). Telmisartan showed a more pronounced induction of aP2 expression in lower, pharmacologically relevant concentrations compared with the other ARBs. The ARB losartan enhanced aP2 expression only at high concentrations (losartan 100 mol/L: 3.6Ϯ0.3-fold induction; PϽ0.01), whereas eprosartan up to 100 mol/L had no significant effects. In transcription reporter assays, irbesartan and telmisartan (10 mol/L) markedly induced transcriptional activity of PPAR␥ by 3.4Ϯ0.9-fold and 2.6Ϯ0.6-fold (PϽ0.05), respectively, compared with 5.2Ϯ1.1-fold stimulation by the PPAR␥ ligand pioglitazone (10 mol/L). Irbesartan and telmisartan also induced PPAR␥ activity in an AT 1 R-deficient cell model (PC12W), demonstrating that these ARBs stimulate PPAR␥ activity independent of their AT 1 R blocking actions. Conclusions-The present study demonstrates that a specific subset of ARBs induces PPAR␥ activity, thereby promoting PPAR␥-dependent differentiation in adipocytes. 3 The underlying mechanism of the insulin-sensitizing/antidiabetic effect of ARBs is widely unknown.The nuclear hormone receptor peroxisome proliferator-activated receptor-␥ (PPAR␥) plays an important role in the regulation of insulin sensitivity. 4 Activated by its ligands such as prostaglandins or synthetic insulin-sensitizing thiazolidinediones/glitazones, PPAR␥ functions as a transcriptional regulator of multiple genes involved in glucose and lipid metabolism, thereby ameliorating type 2 diabetes. 4 To elucidate the underlying mechanisms of the antidiabetic effect of ARBs, we investigated the effects of different ARBs on PPAR␥ function in 3T3-L1 cells, an established cell model to study PPAR␥ function. Methods Cell CultureMouse 3T3-L1 preadipocytes were cultured and differentiated as previously described by using a standard differentiation mixture Semiquantitative RT-PCR and Quantitative Real-Time PCRReal-time polymerase chain reaction (PCR) was performed as previously described with an ABI 7000 sequence detection system for real-time PCR. 6 Mouse 18S ribosomal RNA for real-time PCR and hypoxanthine guanine phosphoribosyl transferase or ␤-actin for semiquantitative reverse transcription (RT)-PCR were chosen as endogenous controls (housekeeping genes). Transfection and Luciferase AssayTransient transfection and...

Abstract-The renin-angiotensin-aldosterone system has been causally implicated in obesity-associated hypertension. We studied the influence of obesity and weight reduction on the circulating and adipose tissue renin-angiotensin-aldosterone system in menopausal women. Blood samples were analyzed for angiotensinogen, renin, aldosterone, angiotensinconverting enzyme activity, and angiotensin II. In adipose tissue biopsy samples, we analyzed angiotensinogen, renin, renin-receptor, angiotensin-converting enzyme, and angiotensin II type-1 receptor gene expression. Obese women (nϭ19) had higher circulating angiotensinogen, renin, aldosterone, and angiotensin-converting enzyme than lean women (nϭ19), and lower angiotensinogen gene expression in adipose tissue. Seventeen women successfully participated in a weight reduction protocol over 13 weeks to reduce daily caloric intake by 600 kcal. Body weight was reduced by Ϫ5%, as were angiotensinogen levels by Ϫ27%, renin by Ϫ43%, aldosterone by Ϫ31%, angiotensin-converting enzyme activity by Ϫ12%, and angiotensinogen expression by Ϫ20% in adipose tissue (all PϽ0.05). The plasma angiotensinogen decrease was highly correlated with the waist circumference decline (rϭ0.74; PϽ0.001). Weight and renin-angiotensin-aldosterone system reductions were accompanied by a Ϫ7-mm Hg reduced systolic ambulatory blood pressure. These data suggest that a 5% reduction in body weight can lead to a meaningfully reduced renin-angiotensin-aldosterone system in plasma and adipose tissue, which may contribute to the reduced blood pressure. The renin-angiotensin-aldosterone system (RAAS) has been implicated by several authors. 3 In humans, increased circulating angiotensinogen (AGT), renin, aldosterone, and angiotensin-converting enzyme (ACE) activity were reported in obese subjects. 4 -10 Furthermore, increased RAAS gene expression was described in adipose tissue, especially in rodent models of obesity. 3,[11][12][13][14][15] The link between adipose tissue AGT gene expression and blood pressure was recently documented in 2 mouse models. Targeted AGT expression in adipocytes of wild-type and AGT knockout mice increased circulating AGT levels and blood pressure. 16 Targeted expression of 11␤-hydroxysteroid dehydrogenase-1 in adipocytes increased blood pressure, plasma AGT, and adipose tissue AGT gene expression in mice with a wild-type genetic background. 17,18 The relationship between blood pressure and the RAAS in obese humans comes mostly from observational and not from intervention studies. The influence of weight loss on RAAS activity, especially on AGT plasma levels and the adipose tissue RAAS, has not been explored. MethodsThe institutional review board approved both studies; all volunteers gave informed written consent. Thirty-eight white menopausal women participated in the cross-sectional study, 30 menopausal women started the weight reduction protocol, and 17 achieved the 5% body weight reduction goal. None had diabetes mellitus, liver disease, congestive heart failure, coronary heart diseas...

Studies in mice suggest that adipocytes serve as glucose sensors and regulate systemic glucose metabolism through release of serum retinol-binding protein 4 (RBP4). This model has not been validated in humans. RBP4 was highly expressed in isolated mature human adipocytes and secreted by differentiating human adipocytes. In contrast to the animal data, RBP4 mRNA was downregulated in subcutaneous adipose tissue of obese women, and circulating RBP4 concentrations were similar in normal weight, overweight, and obese women (n ‫؍‬ 74). RBP4 was positively correlated with GLUT4 expression in adipose tissue, independent of any obesity-associated variable. Five percent weight loss slightly decreased adipose RBP4 expression but did not influence circulating RBP4. In another set of experiments, we stratified patients (n ‫؍‬ 14) by low or high basal fasting interstitial glucose concentrations, as determined by the microdialysis technique. Venous glucose concentrations were similar throughout oral glucose tolerance testing, and basal RBP4 expression in adipose tissue and serum RBP4 concentrations were similar in the groups with higher and lower interstitial glucose levels. Our findings point to profound differences between rodents and humans in the regulation of adipose or circulating RBP4 and challenge the notion that glucose uptake by adipocytes has a dominant role in the regulation of RBP4. Diabetes 55:

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Low plasma levels of the anti-inflammatory factor adiponectin characterize obesity and insulin resistance. To elucidate the relationship between plasma levels of adiponectin, adiponectin gene expression in adipose tissue, and markers of inflammation, we obtained blood samples, anthropometric measures, and subcutaneous adipose tissue samples from 65 postmenopausal healthy women. Adiponectin plasma levels and adipose-tissue gene expression were significantly lower in obese subjects and inversely correlated with obesity-associated variables, including high-sensitive C-reactive protein (hs-CRP) and interleukin-6 (IL-6). Despite adjustment for obesity-associated variables, plasma levels of adiponectin were significantly correlated to adiponectin gene expression (partial r ‫؍‬ 0.38, P < 0.05). Furthermore, the inverse correlation between plasma levels of hs-CRP and plasma adiponectin remained significant despite correction for obesity-associated variables (partial r ‫؍‬ ؊0.32, P < 0.05), whereas the inverse correlation between adiponectin plasma levels or adiponectin gene expression in adipose tissue with plasma IL-6 were largely dependent on the clustering of obesity-associated variables. In conclusion, our data suggest a transcriptional mechanism leading to decreased adiponectin plasma levels in obese women and demonstrate that low levels of adiponectin are associated with higher levels of hs-CRP and IL-6, two inflammatory mediators and markers of increased cardiovascular risk. Diabetes 52:942-947, 2003

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Recent studies suggest that angiotensin II (Ang II) plays a role in the adipogenesis of murine preadipocytes. Here, we examined the role of Ang II for the differentiation of primary cultured human preadipocytes. Preadipocytes were isolated from human adipose tissue and stimulated to differentiate. Quantitation of gene expression during adipogenesis was performed for renin-angiotensin system (RAS) genes. The influence of the RAS on adipogenic differentiation was investigated by addition of either angiotensinogen (AGT), Ang II, or angiotensin receptor antagonists to the differentiation medium. We also examined the influence of adipocytes on adipogenesis by co-culture experiments. Expression of the RAS genes AGT, renin, angiotensin-converting enzyme, and Ang II type 1 receptor increased during adipogenesis. Stimulation of the Ang II type 1 receptor by Ang II reduced adipose conversion, whereas blockade of this receptor markedly enhanced adipogenesis. Adipocytes were able to inhibit preadipocyte differentiation in the co-culture, and this effect was abolished by blockade of the Ang II type 1 receptor. This finding points to a functional role of the RAS in the differentiation of human adipose tissue. Because AGT secretion and Ang II generation are characteristic features of adipogenesis, we postulate a paracrine negative-feedback loop that inhibits further recruitment of preadipocytes by maturing adipocytes. Diabetes 51: 1699 -1707, 2002 O besity is well recognized as a major risk factor for the development of type 2 diabetes. Paradoxically, loss of adipose tissue (1) or impaired adipogenic differentiation (2) have been proposed as important mechanisms underlying the development of insulin resistance and type 2 diabetes (lipotoxicity hypothesis [3]). Adipogenesis results from a precise interplay of transcription factors, enabling preadipocytes to accumulate lipids and respond to insulin (4). Interestingly, mature adipocytes secrete a host of vasoactive substances that influence adipogenesis (5), including the adipogenic vasodilators nitric oxide (6) and prostacyclin (7) and the antiadipogenic vasoconstrictor endothelin-1 (8), suggesting a paracrine role of vasoactive molecules in the regulation of adipocyte growth and differentiation.Angiotensin II (Ang II), another vasoactive molecule, has also recently been implicated in the modulation of adipogenesis (9). Thus, mature adipocytes express all components of the renin-angiotensin system (RAS), including angiotensinogen (AGT), the sole precursor of Ang II, as well as the angiotensin peptide forming enzymes renin, ACE, and chymase. Adipocytes also express the type 1 (AT 1 ) and type 2 (AT 2 ) angiotensin receptor subtypes (10). AGT expression and secretion increases during adipogenesis (11)(12)(13)(14), whereas suppression of the differentiation-specific element binding protein, the activator of differentiation-dependent AGT gene expression, suppressed AGT expression and adipogenesis in 3T3-L1 mouse clonal preadipocytes (15). Although previous studies in rodents poi...