Plasma levels of adiponectin are decreased in patients with nonalcoholic fatty liver disease (NAFLD), but the relationship among plasma adiponectin, insulin sensitivity, and histological features is unclear. In 174 NAFLD patients and 42 controls, we examined plasma adiponectin concentrations in relation to 1) lipid profile, indices of insulin resistance, and features of the metabolic syndrome (n = 174); 2) hepatic insulin resistance (clamp technique with tracer infusion) (10 patients); and 3) histological features at liver biopsy (n = 116). When the data from all subjects were combined, plasma adiponectin levels were positively associated with increased age, female gender, and plasma high-density lipoprotein levels, and negatively associated with waist circumference, body mass index, triglycerides, indices of insulin resistance, and aminotransferase levels, and also predicted the presence of the metabolic syndrome. In step-wise regression, increased age, female gender, waist circumference, triglyceride levels, and homeostasis model assessment independently associated with adiponectin (adjusted R(2), 0.329). In NAFLD, adiponectin was only associated with increased age, female gender, and triglycerides (adjusted R(2), 0.245). When the measured histological parameters were included in the model, plasma adiponectin levels were also inversely proportional to the percentage of hepatic fat content (adjusted R(2), 0.221), whereas necroinflammation and fibrosis did not fit in the model. Adiponectin was negatively correlated with insulin-suppressed endogenous glucose production during the clamp (P = 0.011). The results demonstrate that decreased levels of circulating adiponectin in NAFLD are related to hepatic insulin sensitivity and to the amount of hepatic fat content. Hypoadiponectinemia in NAFLD is part of a metabolic disturbance characterized by ectopic fat accumulation in the central compartment.

Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.

Supplementary key words endocannabinoids • validation • reference intervalsThe endocannabinoids (ECs) are bioactive lipid mediators derived from membrane phospholipids. Since the discovery of the fi rst lipid mediator of the endocannabinoid system (ECS), arachidonoyl-ethanolamide (AEA), also called anandamide ( 1 ), several molecules belonging to this family were identifi ed, the most important being 2-arachidonoyl-glycerol (2AG) and its isomer 1AG among MAGs ( 2, 3 ), palmitoyl-ethanolamide (PEA) and oleoylethanolamide (OEA) among the NAEs, to which AEA belongs ( 4 ). Both 2AG and AEA act on cannabinoid receptor type 1 (CB1) and type 2 (CB2), whereas PEA and OEA act by infl uencing AEA metabolism and binding the peroxisome proliferator-activated receptor ␣ ( 5, 6 ), thus defi ned endocannabinoid related compounds (ERC ).Abstract The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefi niteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/ MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoylethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by online purifi cation was chosen, allowing high-throughput and reliability. We estimated gender-specifi c reference intervals on 121 healthy normal weight subjects fulfi lling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas signifi cantly higher MAG levels were found in males compared with females. MAGs also signifi cantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of defi nitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research fi eld into the clinical fi eld. -Fanelli,F

There are no studies in vivo on the effects of insulin on androgens and sex hormone-binding globulin (SHBG) in men. We, therefore, investigated the effects of insulin suppression on testosterone and SHBG in two groups of eight nondiabetic adult obese men and six healthy normal weight men who underwent diazoxide treatment (100 mg, three times daily) for 7 days. Blood samples for hormone determination were obtained before the subjects had been selected for the study, immediately before diazoxide administration, and on the last day of treatment. A 24-h oral glucose tolerance test was also performed for glucose, insulin, and C-peptide determinations before and on the last day of treatment. Only one subject experienced significant side-effects, and no significant changes in mean body weight were found during the treatment. Diazoxide administration worsened glucose tolerance in several subjects and reduced fasting and glucose-stimulated insulin levels by approximately 50% in both control and obese subjects. No significant difference was present between historical and pretreatment hormone values in either group. Moreover, there were no differences in pretreatment gonadotropin and SHBG concentrations between the two groups, whereas testosterone (free and total) levels were lower in the obese than in the control subjects. After diazoxide administration, testosterone (free and total) decreased slightly, but significantly, whereas LH and SHBG significantly increased in both groups. Diazoxide treatment increased estradiol levels in controls, but not in obese men. In conclusion, these results indicate that in vivo, insulin is capable of stimulating testosterone production and, simultaneously, of inhibiting SHBG concentrations in both normal weight and obese men.

Objective: We measured blood levels of obestatin, total ghrelin, and the ghrelin/obestatin ratio and their relationship with anthropometric and metabolic parameters, adiponectin and insulin resistance, in overweight/obese and normal-weight women. Design: Outpatients Unit of Endocrinology of the S Orsola-Malpighi Hospital of Bologna, Italy. Methods: Fasting obestatin, ghrelin, adiponectin and lipid levels, fasting and glucose-stimulated oral glucose tolerance test insulin, and glucose levels were measured in 20 overweight/obese and 12 controls. The fasting ghrelin/obestatin ratio was calculated; the homeostasis model assessment-IR (HOMA-IR) and insulin sensitivity index (ISI composite ) were calculated as indices of insulin resistance. Results: Obese women had higher obestatin and lower ghrelin blood levels, and a lower ghrelin/obestatin ratio compared with controls. In all subjects, obestatin was significantly and positively correlated with total cholesterol and triglycerides, but not with ghrelin, anthropometric, and metabolic parameters. In the obese women, however, obestatin and ghrelin concentrations were positively correlated. By contrast, the ghrelin/obestatin ratio was significantly and negatively correlated with body mass index, waist, waist-to-hip ratio, fasting insulin, and HOMA-IR, and positively with ISI composite but not with adiponectin. None of these parameters were correlated with the ghrelin/obestatin ratio in the obese. Conclusions: Increased obestatin, decreased ghrelin levels, and a decreased ghrelin/obestatin ratio characterize obesity in women. This supports the hypothesis that the imbalance of ghrelin and obestatin may have a role in the pathophysiology of obesity. On the other hand, some relevant differences between our data on circulating levels of obestatin and the ghrelin/obestatin ratio in obese subjects and those reported in the few studies published so far imply that further research is needed.European Journal of Endocrinology 157 295-301

In a previous study we were the first to describe a negative correlation between circulating ghrelin concentrations and androgen levels in human plasma, suggesting an interaction between ghrelin and the endocrine regulation of reproductive physiology. We now investigated a potential direct regulatory influence of circulating androgens on plasma ghrelin levels. Fourteen obese women with polycystic ovary syndrome (PCOS) on a hypocaloric diet were randomly assigned to treatment groups (open-labeled design), receiving either placebo (no.=7) or the antiandrogen flutamide (no.=7) for 6 months. Anthropometry, visceral (VAT) and subcutaneous (SAT) adipose tissue (quantified by computerized tomography), plasma hormone levels, insulin sensitivity indexes (Quantitative Insulin-Sensitivity Check Index-QUICKI) and Homeostatic Model Assessment applied to the oral glucose tolerance test (HOMA(OGTT)) were evaluated at baseline and at the end of the study. Body weight decreased and insulin resistance indexes improved in both groups. A tendency toward a greater loss of VAT was observed in the flutamide group. Only in the flutamide group was a significant reduction of androgens levels observed. Plasma ghrelin levels significantly increased following treatment with flutamide, while ghrelin remained unchanged in the placebo group. We observed a negative correlation between changes of ghrelin levels and changes of androgen plasma concentration in the flutamide-treated group. In the same group a positive correlation was found between plasma ghrelin changes and insulin sensitivity as expressed by HOMA(OGTT). Analysis in a multiple regression model, however, showed that plasma ghrelin changes were mainly due to changes of androgen levels rather than improved insulin sensitivity. We, therefore, conclude that androgens are independent modulators of circulating ghrelin concentrations.

Ghrelin is a peripheral circulating hormone, mainly released from the stomach, which can stimulate food intake. We studied fed, fasted and fasted-refed prepuberal gilts in order to outline possible changes in gastric mucosal ghrelin cells and in plasma ghrelin profiles in reponse to food deprivation. Acyl-ghrelin-immunoreactive cells were numerous in oxyntic glands, less abundant in cardiac glands and least frequent in pyloric glands, with the addition of a minor population of labelled cells in the gastric pit mucosa. When fed and fasted animals were compared (72-h fast versus fed; n=4 each), no clear-cut differences were revealed in labelled cell numbers, nor in their staining intensity. An RIA for plasma porcine acyl-ghrelin (noctanoylated at Ser-3), not recognizing des-acyl-ghrelin, was validated. Plasma acyl-ghrelin progressively increased upon fasting (over 6, 12, 24 and 48 h); ghrelin levels significantly (P<0·05) higher than those prefast were reached at 72 h. After refeeding, plasma ghrelin was rapidly restored to basal values by 6 h. In the same animals, plasma insulin was significantly reduced throughout the fasting period (6-72 h), while rapidly increasing after refeeding. Non-esterified fatty acid levels increased during fasting (12-72 h) and rapidly returned to low values after refeeding. In conclusion, the present study demonstrates that starvation and refeeding influence ghrelin plasma level in prepuberal gilts. The absence of detectable changes in ghrelin cells, as seen in immunohistochemistry, could be due to a large intracellular storage of potentially releasable acyl-ghrelin.

No abstract