Telomere length elongation after weight loss intervention in obese adults.

INTRODUCTION: Telomeres may be considered markers of biological aging, shorter telomere length is associated with some age-related diseases; in several studies short telomere length has also been associated to obesity in adults and adolescents. However the relationship between telomere complex functions and obesity is still not clear. Aim of the study was to assess telomere length (TL) in adults' obese subjects before and after weight loss obtained by placement of bioenteric intragastric balloon (BIB) for 6months. METHODS: We enrolled 42 obese subjects before and after BIB placement as weight loss intervention. Blood samples were collected in order to obtain DNA from leukocyte to measure TL by quantitative PCR. RESULTS: Data were analyzed only in 37 subjects with complete data; all presented important body weight loss (124.06±26.7 vs 105.40±23.14, p<0.001) and more interesting they presented a significant increase in TL (3.58±0.83 vs 5.61±3.29, p<0.001). Moreover we observed a significant positive correlation between TL elongation and weight loss (r=0.44, p=0.007) as well as an inverse correlation between TL at baseline and TL elongation (r=-0.35, p=0.03).The predictors of TL elongation were once again weight loss and short TL at baseline (respectively p=0.007 and p=0.003). CONCLUSIONS: Our study shows that weight loss is associated to telomere lengthening in a positive correlation: the greater weight loss the greater telomere lengthening; moreover telomere lengthening is more significant in those subjects with shortest telomeres at baseline.

WITHDRAWN: Telomeres and atherosclerosis.

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Increased appearance rate of 27-hydroxycholesterol in vivo in hypercholesterolemia: a possible compensatory mechanism.

BACKGROUND AND AIMS: The first step in the alternative pathway of bile acid biosynthesis is the 27-hydroxylation of cholesterol, which takes place both in liver and extrahepatic tissues. This pathway is believed to play a role in peripheral cholesterol degradation. Aim of this study was to investigate the impact of hyperlipidemia on 27-hydroxycholesterol appearance rate, and to assess the effects induced by treatment with statins. METHODS AND RESULTS: Seven patients with familial hypercholesterolemia and eight patients with familial combined hyperlipidemia underwent determination of 27-hydroxylation rates in vivo by i.v. infusion of deuterated 27-hydroxycholesterol. Isotope enrichment was assayed by gas chromatography-mass spectrometry, allowing to calculate 27-hydroxycholesterol appearance rates. Six normocholesterolemic subjects were regarded as controls. In some hypercholesterolemic patients the infusions were repeated during treatment with atorvastatin or rosuvastatin. Hydroxylation rates were higher in hypercholesterolemic patients (8.7 ± 2.5 mg/h; controls, 3.4 ± 2.0 mg/h; combined hyperlipidemia, 4.4 ± 1.6 mg/h; mean ± SD, P < 0.01 vs both). After statin treatment, both plasma cholesterol levels and hydroxylation rates dropped by nearly 50%. No difference was detectable between the two statins. A linear correlation was shown between plasma cholesterol and 27-hydroxylation rates. CONCLUSION: Hypercholesterolemia associates with increased 27-hydroxycholesterol appearance rates, which decrease during hypocholesterolemic treatment. The correlation with cholesterol levels supports the view that 27-hydroxylation may act as a compensatory mechanism in a condition of larger plasma cholesterol pool. A regulatory role for hepatic and extrahepatic nuclear receptors seems reasonable. These data prompt novel pharmacological approaches for the management of hypercholesterolemia and the prevention of atherosclerosis.

Risk for cardiovascular events in an Italian population of patients with type 2 diabetes.

BACKGROUND AND AIM: This study aims to analyse the risk of cardiovascular events in a local cohort of patients with type 2 diabetes, and to evaluate the prognostic accuracy of four algorithms used to estimate cardiovascular risk: the Framingham study, United Kingdom Prospective Diabetes Study (UKPDS), Riskard study and Progetto Cuore. METHOD AND RESULTS: We analysed clinical charts of the Diabetes Clinics of Modena for the period 1991-95. Patients in the age range of 35-65 with type 2 diabetes and no previous cardiovascular disease were eligible. The incidence of new cardiovascular disease was compared with estimated rates deriving from the different functions. A stratification was obtained in subgroups at different cardiovascular risk, allowing comparison between the algorithms. A total of 1532 patients were eligible; women presented a worse cardiovascular risk profile. An absolute 10-year rate of cardiovascular events of 14.9% was observed. Comparing patients with events with event-free subjects, we found significant differences in systolic blood pressure, age at visit, smoking, high-density lipoprotein (HDL)-cholesterol, duration of diabetes, glycosylated haemoglobin (HbA1c) and co-morbidities. Comparing the estimated risk rate according to the different functions, Italian algorithms were more consistent with observed data; however, Progetto Cuore and Riskard show underestimation of events when applied to females. CONCLUSIONS: Estimation of cardiovascular risk is dependent on the algorithm adopted and on the baseline risk of the reference cohort. Functions designed for a specific population, including risk variables peculiar for diabetes, should be adopted to increase the performance of such functions which is clearly unsatisfactory at present.

Genetic polymorphisms in non-alcoholic fatty liver disease: interleukin-6-174G/C polymorphism is associated with non-alcoholic steatohepatitis.

BACKGROUND: Environmental and genetic factors play a role in the pathogenesis and natural history of non-alcoholic fatty liver disease (NAFLD). METHODS: In 114 subjects with NAFLD we report the prevalence and correlation with clinical parameters of three polymorphisms: interleukin-6 (-174G/C), plasma cell differentiation antigen (K121Q) and microsomal transfer protein (-493G/T). In 59 biopsied patients with NAFLD the polymorphisms were also related to histological features. RESULTS: IL-6 -174C variant was more prevalent (p<0.01) in NAFLD compared to controls. In the NAFLD group, C carriers had higher HOMA-IR and fasting insulin than G carriers (p<0.05). The prevalence of IL-6/C variant was higher (83%) in biopsied than in not biopsied subjects (66%) (p<0.05). In biopsied subjects, C carriers had higher HOMA and fasting insulin (p<0.05) compared than those with G allele. The prevalence of IL-6 -174G/C polymorphism was significantly higher in NASH than in NAFLD (p=0.048). At logistic regression analysis IL-6 -174C was an independent predictor of both NAFLD (OR 4.116, C.I. 1.126-15.048) and NASH (OR 7.035, C.I. 1.167-42.394). Conversely, the distribution of PC-1 and MTP polymorphisms was not significantly different compared to the control group, nor associated with clinical or histological characteristics. CONCLUSIONS: Our data suggest that IL-6 -174C genetic polymorphisms, involved in inflammation and insulin resistance, are associated with NASH. These data may contribute to the understanding of the genetic susceptibility to NAFLD.

Nuclear receptors as potential molecular targets in cholesterol accumulation conditions: insights from evidence on hepatic cholesterol degradation and gallstone disease in humans.

The liver plays a central role in the regulation of cholesterol homeostasis. Hepatic cholesterol content is maintained by a complex interplay between input and output pathways; alterations in the balance among these processes may lead to accumulation of excess cholesterol in body compartments with potentially deleterious consequences at the level of blood vessels (atherosclerosis) and biliary tract (gallstone disease). Molecular biology has brought new insights into this field. Nuclear receptors have been shown to play a key role in the "sensing" of intracellular cholesterol levels and in the triggering of metabolic responses via the sterol regulatory element binding protein (SREBP) cascade. A nuclear receptor for bile acids, farnesoid X receptor (FXR), has been identified and the molecular pathways underlying feedback inhibition of bile acid synthesis, the main mechanism of irreversible degradation of cholesterol, have been clarified. Such regulation involves a number of additional coactivators/corepressors of the transcription of the limiting enzyme of bile acid synthesis, cholesterol 7alpha-hydroxylase. Finally, the main transporters of biliary lipids (bile acids, phospholipids and cholesterol) have been described; most of them undergo transcriptional control by nuclear receptors, allowing regulation of biliary lipid efflux in conditions of different intracellular availability. Despite a body of evidence coming from experimental models the intimate mechanisms of regulation have not been clearly defined and direct evidence in humans is rather limited. This review will focus on the role of nuclear receptors in the regulation of hepatic cholesterol degradation and biliary lipid secretion, and on the theoretical applications from a pharmacotherapeutic perspective.

Age-related changes in bile acid synthesis and hepatic nuclear receptor expression.

BACKGROUND: Recent data highlighted the role of nuclear receptors in the transcriptional regulation of the limiting enzyme of bile acid synthesis, cholesterol 7alpha-hydroxylase, in cellular and animal models. This study was designed to analyze the effects of age on cholesterol 7alpha-hydroxylase and related nuclear receptor expression in human livers. DESIGN: Surgical liver biopsies were obtained in 23 patients requiring operation on the gastrointestinal tract. mRNA levels of cholesterol 7alpha-hydroxylase and related nuclear receptors and co-activators were assayed by quantitative real-time RT-PCR. Serum levels of 7alpha-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis, were assayed by gas-liquid chromatography:mass spectrometry. RESULTS: Ageing was inversely correlated with serum 7alpha-hydroxy-4-cholesten-3-one and with cholesterol 7alpha-hydroxylase mRNA levels (r = -0.44 and r = -0.45 on a semi-log scale, respectively, P < 0.05). Among different nuclear factors, cholesterol 7alpha-hydroxylase mRNA best correlated with hepatocyte nuclear factor-4 (r = 0.55 on a log scale, P < 0.05); hepatocyte nuclear factor-4 levels were also inversely correlated with age (r = -0.64 on a semi-log scale, P < 0.05). Age was inversely correlated with serum insulin-like growth factor-I levels, which were directly correlated with hepatocyte nuclear factor-4 and cholesterol 7alpha-hydroxylase expression. No suppressive effect of short heterodimer partner expression on cholesterol 7alpha-hydroxylase was observed. CONCLUSIONS: Ageing associates with reduced bile acid synthesis, possibly related to decreased hepatic expression of hepatocyte nuclear factor-4 and consequently of cholesterol 7alpha-hydroxylase. Age-related modifications of the growth hormone/insulin-like growth factor axis might play a role. These findings may help to elucidate the pathophysiology of age-related modifications of cholesterol metabolism.

Decreased hepatic expression of PPAR-gamma coactivator-1 in cholesterol cholelithiasis.

BACKGROUND: Cholesterol cholelithiasis (gallstone disease) is a common disease in the Western world. The aim of the present study was to analyze the hepatic expression of a number of nuclear receptors involved in bile acid metabolism in human cholesterol gallstone disease. MATERIALS AND METHODS: Surgical liver biopsies were obtained from 11 patients with untreated cholesterol cholelithiasis and nine gallstone-free subjects; mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1) and related nuclear receptors and coactivators were assayed by quantitative real-time RT-PCR. RESULTS: No differences between the two groups were detected in mRNA levels of CYP7A1 and related nuclear receptors, with the exception of peroxysome proliferator-activated receptor-gamma coactivator 1 (PGC-1), which was significantly (P < 0.01) less expressed in gallstone subjects. Expression of PGC-1 was linearly correlated with farnesoid X receptor (FXR) in gallstone patients (r = 0.87 on a log scale, P < 0.01), but not in control subjects; in gallstone patients PGC-1 expression was also correlated with hepatocyte nuclear factor 4 (HNF-4) (r = 0.78, P < 0.01). CONCLUSION: These findings suggest that PGC-1 can play a role in the prevention of cholesterol gallstone disease in humans; this might take place via interaction with the bile acid receptor FXR, whose protective role in cholelithiasis has been suggested by recent evidence in animal models and other coactivators. The present data might help to understand the pathophysiology and possibly focus on new therapeutical targets in cholesterol gallstone disease.

Review article: diabetes, genetics and ethnicity.

The prevalence of insulin resistance and diabetes has increased in the past decades at an alarming rate in all Western countries and in those countries which are adopting a 'western life style'. This trend suggests the impact of environmental factors such as diet, obesity and physical activity on the pathogenesis of diabetes. However it is known that the prevalence and variation of prevalence, as consequence of environmental changes, it is different in various ethnic groups. Studies conducted in multiethnic populations suggest that some ethnic groups, such as Hispanics or Asian Indians, might have a particular predisposition, possibly on genetic basis, to develop insulin resistance and diabetes, when exposed to adverse conditions. According to the 'thrifty gene' hypothesis, a clustering of different genetic defects or polymorphisms, developed as genetic advantage in some populations, could predispose some ethnic groups to insulin resistance and diabetes in presence of an increased food supply. Multiple mutations, associated with small changes in insulin sensitivity, when combined, may induce a significant reduction in insulin sensitivity. This review deals with the possible relevance of genetic factors in the expression of insulin resistance and diabetes in relation to ethnicity.

Review article: the metabolic syndrome and non-alcoholic fatty liver disease.

Metabolic syndrome represents a common risk factor for premature cardiovascular disease and cancer whose core cluster includes diabetes, hypertension, dyslipidaemia and obesity. The liver is a target organ in metabolic syndrome patients in which it manifests itself with non-alcoholic fatty liver disease spanning steatosis through hepatocellular carcinoma via steatohepatitis and cirrhosis. Given that metabolic syndrome and non-alcoholic fatty liver disease affect the same insulin-resistant patients, not unexpectedly, there are amazing similarities between metabolic syndrome and non-alcoholic fatty liver disease in terms of prevalence, pathogenesis, clinical features and outcome. The available drug weaponry for metabolic syndrome includes aspirin, metformin, peroxisome proliferator-activated receptor agonists, statins, ACE (angiotensin I-converting enzyme) inhibitors and sartans, which are potentially or clinically useful also to the non-alcoholic fatty liver disease patient. Studies are needed to highlight the grey areas in this topic. Issues to be addressed include: diagnostic criteria for metabolic syndrome; nomenclature of non-alcoholic fatty liver disease; enlargement of the clinical spectrum and characterization of the prognosis of insulin resistance-related diseases; evaluation of the most specific clinical predictors of metabolic syndrome/non-alcoholic fatty liver disease and assessment of their variability over the time; characterization of the importance of new risk factors for metabolic syndrome with regard to the development and progression of non-alcoholic fatty liver disease.

Suppression of bile acid synthesis, but not of hepatic cholesterol 7alpha-hydroxylase expression, by obstructive cholestasis in humans.

Regulation of bile acid synthesis, a key determinant of cholesterol homeostasis, is still incompletely understood. To elucidate the feedback control exerted on bile acid biosynthesis in humans with obstructive cholestasis, 16 patients with bile duct obstruction were studied. In vivo 7alpha-hydroxylation, reflecting bile acid synthesis, was assayed in 13 of them by tritium release analysis. Serum 27-hydroxycholesterol was determined by gas chromatography-mass spectrometry. In a subgroup, hepatic cholesterol 7alpha-hydroxylase mRNA was assayed by real-time polymerase chain reaction (PCR), enzyme activity was determined by isotope incorporation, and microsomal cholesterol content was assayed by gas chromatography-mass spectrometry. Age-matched control subjects were studied in parallel. Hydroxylation rates were lower in cholestatic patients (108 +/- 33 mg of cholesterol per day, mean +/- SEM; controls: 297 +/- 40 mg/d; P <.01). The reduction was proportional to the severity of cholestasis, and synthetic rates were normalized in 4 subjects restudied after resolution of biliary obstruction. Consistent findings were obtained by analysis of serum 7alpha-hydroxycholesterol levels. On the other hand, hepatic cholesterol 7alpha-hydroxylase mRNA, microsomal enzyme activity, and cholesterol content tended to be increased in cholestasis. Finally, serum 27-hydroxycholesterol levels were slightly reduced in cholestatic subjects and were not related with the severity of the disease. Suppression of in vivo bile acid synthesis with no corresponding reduction in tissue 7alpha-hydroxylase expression and activity is consistent with nontranscriptional, posttranslational levels of regulation; these may play a role in the feedback control of bile acid synthesis in particular conditions. Alteration of the alternate biosynthetic pathway seems unlikely according to the present data.

Review article: effect of bile salt pool composition on hepatic and biliary functions.

The enterohepatic recirculation of bile salts exerts important regulatory effects on many hepatic, biliary and intestinal functions: such regulation is likely to depend, to a large extent, on the physical-chemical property of hydrophobicity of the recirculating pool. The present review summarizes the main experimental evidence carried out by our research group over the past two decades, in the attempt to investigate systematically the relationships between structural properties and biological effects of bile acids in humans. Hydrophobic bile acids (chenodeoxycholic acid, deoxycholic acid), but not hydrophilic acids (ursodeoxycholic acid), significantly suppressed hepatic activity of HMG-CoA reductase, the limiting step of cholesterol synthesis, and in vivo cholesterol 7alpha-hydroxylation, the limiting step of bile acid synthesis. The output of biliary cholesterol and phospholipid was also directly related to the hydrophobicity of the bile acid pool. Finally, treatment with chenodeoxycholic acid, but not with ursodeoxycholic acid, significantly decreased gall-bladder emptying rates. When turning to the in vitro model of HepG2 cells, hydrophobic bile acids were found to induce greater cytotoxic and pro-apoptotic effects. From this series of studies, we conclude that the regulatory effects of bile acids on the liver and biliary tract are largely dependent on the hydrophobic-hydrophilic balance of the recirculating bile acid pool.

Regulation of ob gene expression: evidence for epinephrine-induced suppression in human obesity.

Leptin acts as satiety factor and increases energy expenditure. Studies conducted on animals and in vitro on adipocytes culture have shown that infusion of catecholamines leads to a significant reduction of ob gene expression; it appears of interest to evaluate the in vivo effects of adrenergic activation on the expression of the ob gene in humans. We studied ob gene expression in adipose tissue samples from 13 obese subjects before and after epinephrine (25 ng/min x kg ideal body weight for 3 h) and 6 obese patients during saline infusion. Hormonal infusion led to a significant increase in epinephrine plasma levels (from 27 +/- 4 to 339 +/- 75 pg/mL; P < 0.001), plasma free fatty acids (from 0.73 +/- 0.05 to 0.98 +/- 0.07; P < 0.05), heart rate (13.5 +/- 3.1 beats/min; F = 2.9; P < 0.03), and systolic blood pressure (F = 2.7; P < 0.05), whereas diastolic blood pressure did not show significant variation. Plasma leptin levels decreased by the end of the infusion (from 63 +/- 13 to 49 +/- 11 ng/mL; P < 0.05), and ob messenger ribonucleic acid levels were significantly reduced (decrease amounting to 47 +/- 5% of basal values). Our study shows that adrenergic activation contributes to regulate ob messenger ribonucleic acid levels in humans. The interaction between epinephrine and leptin may operate during metabolic and psychological stress to regulate energy expenditure and food intake.