Abstract-Atrial fibrillation (AF), the most common chronic arrhythmia, increases the risk of stroke and is an independent predictor of mortality. Available pharmacological treatments have limited efficacy. Once initiated, AF tends to self-perpetuate, owing in part to electrophysiological remodeling in the atria; however, the fundamental mechanisms underlying this process are still unclear. We have recently demonstrated that chronic human AF is associated with increased atrial oxidative stress and peroxynitrite formation; we have now tested the hypothesis that these events participate in both pacing-induced atrial electrophysiological remodeling and in the occurrence of AF following cardiac surgery. In chronically instrumented dogs, we found that rapid (400 min Ϫ1) atrial pacing was associated with attenuation of the atrial effective refractory period (ERP). Treatment with ascorbate, an antioxidant and peroxynitrite decomposition catalyst, did not directly modify the ERP, but attenuated the pacing-induced atrial ERP shortening following 24 to 48 hours of pacing. Biochemical studies revealed that pacing was associated with decreased tissue ascorbate levels and increased protein nitration (a biomarker of peroxynitrite formation). Oral ascorbate supplementation attenuated both of these changes. To evaluate the clinical significance of these observations, supplemental ascorbate was given to 43 patients before, and for 5 days following, cardiac bypass graft surgery. Patients receiving ascorbate had a 16.3% incidence of postoperative AF, compared with 34.9% in control subjects. In combination, these studies suggest that oxidative stress underlies early atrial electrophysiological remodeling and offer novel insight into the etiology and potential treatment of an enigmatic and difficult to control arrhythmia. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2001;89:e32-e38.) Key Words: atrial fibrillation Ⅲ antioxidant Ⅲ ascorbate Ⅲ oxidative stress Ⅲ cardiac surgery A trial fibrillation (AF) is self-perpetuating 1 because of the combined effects of rate-induced electrophysiological and structural remodeling. 2 The earliest observed change in AF is an abbreviation of the atrial effective refractory period (ERP). The mechanisms by which high-rate activity results in electrophysiological remodeling are poorly understood. AF is also a frequent postoperative complication of cardiac surgery, with a reported incidence of 20% to 50%, increasing the risk of stroke. 3 Before arrhythmia onset, patients who experience postoperative AF exhibit increased atrial ectopy, abbreviation of monophasic action potential duration, and increased heart rate. 4 Evidence from animal models of atrial fibrillation, 5-7 as well as our studies of patients with postoperative AF, 8 supports a prominent role for myocyte calcium overload in initiating the process of atrial electrophysiological remodeling. We have documented both significant electrophysiological remodeling 8,9 and biochemical evidence of oxidative stress...

A method for the determination of free, active insulin in the sera of insulin-treated diabetics is described. This involved radioimmunoassay after extraction of free insulin with polyethylene glycol. Recovery tests with cold insulin showed 73 per cent recovery of free insulin and no recovery of bound insulin.The fasting free insulin levels were slightly lower in the patients than in normal persons; exceptions were some patients with complications expected to cause insulin resistance at the peripheral tissue level. Free insulin levels did not correlate with total insulin levels, antibody titers, insulin requirements or conditions of insulin treatment.A very slight increase of insulin was observed after glucose loading in insulin-treated patients, but a marked increase of the free insulin level followed by an exaggerated increase in the total insulin level was observed in a patient with the insulin autoimmune syndrome.The diurnal changes of the free insulin suggested the dynamic states of this fraction and its usefulness for determining control of diabetes with insulin. DIABETES 22: 590-600, August, 1973.Since Berson and Yalow introduced the radioimmunologic method for the determination of plasma insulin, 1 their original method and modifications have been widely used and the method's reliability generally accepted. The plasma insulin level in patients previously treated with insulin, however, cannot be determined by the original method or most of its modifications, due to the presence of human anti-insulin antibody in the plasma which combines with the added radioinsulin and interferes with the reaction between radioinsulin, endogenous insulin and guinea pig antiinsulin antibody.By Grodsky and Forsham's method in which the plasma insulin is extracted with acid ethanol prior to the immunoassay, 2 the presence of human antibody causes no interference in the radioimmunoassay. However, the obtained insulin level represents total insulins, including antibody-bound inactive insulin. For the study of insulin dynamics in patients treated with insulin, the determination of free, active insulin is required. In this report, a simple method for the determination of free insulin in serum containing insulin antibodies is described. MATERIALS AND METHODSExtraction of free insulin. To 1 ml. of plasma chilled in an ice water bath, 1 ml. of cold 25 per cent (w/w) polyethylene glycol solution was added with an automatic syringe, and the mixture was immediately agitated with a vortical mixer for one minute and centrifuged at 3,000 r.p.m. for forty-five minutes in a refrigerated centrifuge. The supernatant was used for the radioimmunoassay.Extraction of total insulin. A 1 ml. quantity of plasma and 0.2 ml. of 1.0 N HC1 were mixed and, after one hour at room temperature, 1.4 ml. of 25 per cent polyethylene glycol solution was added. After mixing with a vortical mixer, 0.2 ml. of 1.0 N NaOH was added and the mixture was again agitated with a vortical mixer. After centrifugation at 3,000 r.p.m. for forty-five minutes, the supernatant was...

. Overexpression of human adiponectin in transgenic mice results in suppression of fat accumulation and prevention of premature death by high-calorie diet. Am J Physiol Endocrinol Metab 293: E210-E218, 2007. First published March 27, 2007 doi:10.1152/ajpendo.00645.2006.-Adiponectin, a physiologically active polypeptide secreted by adipocytes, shows insulin-sensitizing, anti-inflammatory, and antiatherogenic properties in rodents and humans. To assess the effects of chronic hyperadiponectinemia on metabolic phenotypes, we established three lines of transgenic mice expressing human adiponectin in the liver. When maintained on a high-fat/high-sucrose diet, mice of two lines that had persistent hyperadiponectinemia exhibited significantly decreased weight gain associated with less fat accumulation and smaller adipocytes in both visceral and subcutaneous adipose tissues. Macrophage infiltration in adipose tissue was markedly suppressed in the transgenic mice. Expression levels of adiponectin receptors were not altered in skeletal muscle or liver. Circulating levels of endogenous adiponectin were elevated, whereas fasting glucose, insulin, and leptin levels were reduced compared with control mice. In the hyperadiponectinemic mice daily food intake was not altered, but oxygen consumption was significantly greater, suggesting increased energy expenditure. Moreover, high-calorie diet-induced premature death was almost completely prevented in the hyperadiponectinemic mice in association with attenuated oxidative DNA damage. The transgenic mice also showed longer life span on a conventional low-fat chow. In conclusion, transgenic expression of human adiponectin blocked the excessive fat accumulation and reduced the morbidity and mortality in mice fed a high-calorie diet. These observations may provide new insights into the prevention and therapy of metabolic syndrome in humans.adipocyte; macrophage; life span; 8-hyroxy-2-deoxyguanosine RESTRICTION OF CALORIC INTAKE retards age-related changes and extends the maximum life span in mammals (22, 37). The longevity is associated with a reduction in steady-state oxidative damage to proteins, lipids, and DNA in animals subjected to restricted caloric intake (13). These decreases in oxidative damage may be attributable to a decrease in the mitochondrial free radical generation rate in various tissues (4). Conversely, obese rats fed a high-calorie diet ad libitum have a shorter life span than their lean counterparts fed a low-calorie diet (36). The excessive production of oxidative stress resulting from high caloric intake may play a role in the premature death.Metabolic syndrome associated with obesity is a growing cause of morbidity and mortality in humans (16, 18). Accumulating evidence has revealed that a reduction in the serum level of adiponectin, a bioactive peptide secreted by adipocytes, is involved in the development of metabolic syndrome and the acceleration of atherosclerosis. The serum level of adiponectin is decreased in patients with obesity (1), type 2 diabetes (17)...

Previously, we have shown that the adipocyte-specific nuclear form of sterol regulatory element-binding protein-1c (nSREBP-1c) transgenic mice spontaneously developed hepatic lesions that are similar to those of human nonalcoholic steatohepatitis (NASH) with a concomitant elevation of plasma TNF-α. In this study, we analyzed the role of TNF-α in the progression of nonalcoholic fatty liver disease (NAFLD). We established a Tnf knockout nSREBP-1c transgenic mouse line. Glucose tolerance and liver histology were examined at the age of 20 weeks. The gene expression and protein levels were assessed by quantitative RT-PCR and Western blot, respectively. The Tnf knockout improved glucose tolerance and significantly reduced the prevalence of hepatic steatosis (20% vs. 100%, p<0.0001) and fibrosis (15% vs. 65%, p=0.0057). The expressions of Acaca, Scd1, Mcp1, Tgfb1, Col1a1, and Timp1 were increased in the liver from the original nSREBP-1c transgenic mice. However, gene upregulation was reduced in the livers from the Tnf(-/-) nSREBP-1c transgenic mice. Furthermore, the hepatic levels of TIMP1 protein were increased in the original nSREBP-1c transgenic mice but not in Tnf(-/-) nSREBP-1c transgenic mice. To assess the direct effect of TNF-α on the expression of the genes, we cultured primary hepatocytes in the presence of TNF-α and found that TNF-α increased the expression of Mcp1, Tgfb1, and Timp1 in hepatocytes. These observations indicate that TNF-α plays a pivotal role in the development of NAFLD and progression to NASH through upregulating key molecules associated with lipid metabolism, inflammatory cytokines, and fibrosis in the liver.

ObjectiveMetformin is known to have a beneficial effect on body weight and body composition, although the precise mechanism has not been elucidated yet. The aim of this study is to investigate the effects of metformin on energy metabolism and anthropometric factors in both human subjects and rats.MethodsIn human studies, metformin (1500mg/day) was administered to 23 healthy subjects and 18 patients with type 2 diabetes for 2 weeks. Metabolic parameters and energy metabolism were measured during a meal tolerance test in the morning before and after the treatment of metformin. In animal studies, 13 weeks old SD rats were fed 25–26 g of standard chow only during 12-hours dark phase with either treated by metformin (2.5mg/ml in drinking water) or not for 2 weeks, and metabolic parameters, anthropometric factors and energy metabolism together with expressions related to fat oxidation and adaptive thermogenesis were measured either in fasting or post-prandial state at 15 weeks old.ResultsPost-prandial plasma lactate concentration was significantly increased after the metformin treatment in both healthy subjects and diabetic patients. Although energy expenditure (EE) did not change, baseline respiratory quotient (RQ) was significantly decreased and post-prandial RQ was significantly increased vice versa following the metformin treatment in both groups. By the administration of metformin to SD rats for 2 weeks, plasma levels of lactate and pyruvate were significantly increased in both fasting and post-prandial states. RQ during a fasting state was significantly decreased in metformin-treated rats compared to controls with no effect on EE. Metformin treatment brought about a significant reduction of visceral fat mass compared to controls accompanied by an up-regulation of fat oxidation-related enzyme in the liver, UCP-1 in the brown adipose tissue and UCP-3 in the skeletal muscle.ConclusionFrom the results obtained, beneficial effects of metformin on visceral fat reduction has been demonstrated probably through a mechanism for a potential shift of fuel resource into fat oxidation and an upregulation of adaptive thermogenesis independent of an anorexigenic effect of this drug.