Visfatin Promotes Monocyte Adhesion by Upregulating ICAM-1 and VCAM-1 Expression in Endothelial Cells via Activation of p38-PI3K-Akt Signaling and Subsequent ROS Production and IKK/NF-κB Activation.

BACKGROUND/AIMS: Visfatin is known to act as a mediator in several metabolic disorders, such as obesity, diabetes, and cardiovascular diseases. This study aimed to investigate the effect of visfatin on the adhesion of THP-1 monocytes to human vascular endothelial cells and the underlying mechanism. METHODS: Monocytes adhesion to endothelial cells was determined by using fluorescence-labeled monocytes. ICAM-1 and VCAM-1 expression in endothelial cells were measured by western blotting. Production of reactive oxygen species (ROS) was measured by using a fluorescent dye. The amounts of nuclear factor-kappa B (NF-κB) and phosphorylation of inhibitory factor of NF-κB (IκB) were determined by using western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined by using immunofluorescence. RESULTS: Here we showed that visfatin significantly caused the upregulation of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells, as well as enhanced monocyte adhesion to endothelial cells. Moreover, we found that inhibition of PI3K, Akt, and p38 MAPK activation significantly prevented visfatin-enhanced expression of ICAM-1 and VCAM-1 and monocyte adhesion to endothelial cells. Visfatin enhanced ROS production and IKK/NF-кB activation and then led to upregulation of ICAM-1 and VCAM-1 and enhanced monocyte adhesion to endothelial cells. These effects were also p38/PI3K/Akt-dependent. CONCLUSION: These results demonstrated that visfatin promoted monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression via the activation of p38/PI3K/Akt signaling and downstream ROS production and IKK/NF-кB activation.

Exercise reduces body fat and improves insulin sensitivity and pancreatic ß-cell function in overweight and obese male Taiwanese adolescents.

BACKGROUND: Improvements in insulin resistance and pancreatic ß-cell function have been shown following exercise in adults with obesity; however, few adolescent-based studies have been conducted. This study examined the impact of exercise training on body fat and insulin sensitivity and secretion in overweight and obese adolescents. METHODS: The effects of a 12-week exercise program on the parameters of adiposity and glucose homeostasis were investigated in 47 overweight and obese male adolescents. RESULTS: After the exercise training program, body weight, body mass index, waist circumference, and body fat were significantly decreased (P < 0.001). Improvements in insulin sensitivity (HOMA-IR: 1.40 vs. 0.86, P < 0.001) and the disposition index (5.84 vs. 12.77, P < 0.001) were also observed. Compared to baseline, oral glucose tolerance tests showed reduced glucose and insulin levels at all time points following the exercise training (all P < 0.001). Subgroup analysis of overweight and obese adolescents with abnormal glucose tolerance revealed that there was no difference in plasma glucose levels as compared to the lean group. CONCLUSIONS: A 12-week exercise training is effective in reducing body fat and improving insulin sensitivity and secretion. In addition, the benefits of the exercise intervention were even experienced by those with impaired glucose tolerance.

Using proteomics to discover novel biomarkers for fatty liver development and response to CB1R antagonist treatment in an obese mouse model.

Over activity of cannabinoid receptor type 1 (CB1R) plays a key role in increasing the incidence of obesity-induced non-alcoholic fatty liver disease. Tissue proteome analysis has been applied to investigate the bioinformatics regarding the mode of action and therapeutic mechanism. The aim of this study was to explore the potential pathways altered with CB1R in obesity-induced fatty liver. Male C57BL/6 mice were fed either a standard chow diet (STD) or a high-fat diet (HFD) with or without 1-week treatment of CB1R inverse agonist AM251 at 5 mg/kg. Then, liver tissues were harvested for 2DE analysis and protein profiles were identified by using MALDI-MS. Results showed that eight of significantly altered protein spots at the level of changes > twofold were overlapped among the three groups, naming major urinary protein 1, ATP synthase subunit ß, glucosamine-fructose-6-phosphate aminotransferase 1, zine finger protein 2, s-adenosylmethionine synthase isoform type-1, isocitrate dehydrogenase subunit α, epoxide hydrolase 2 and 60S acidic ribosomal protein P0. These identified proteins were involved in glucose/lipid metabolic process, xenobiotic metabolic system, and ATP synthesized process in mitochondria. Based on the findings, we speculated that CB1R blockade might exert its anti-metabolic disorder effect via improvement of mitochondrial function in hepatic steatosis in HFD condition.

Major urinary protein 1 interacts with cannabinoid receptor type 1 in fatty acid-induced hepatic insulin resistance in a mouse hepatocyte model.

Hepatic insulin resistance (HIR) is a metabolic abnormality characterized by increased gluconeogenesis which usually contributes from an elevation of free fatty acids. Cannabinoid receptor type 1 (CB1R) and major urinary protein 1 (MUP1) are thought to play pivotal roles in mitochondrial dysfunction, liver steatosis and insulin resistance. The aim of this study was to explore the role of MUP1 in CB1R-mediated HIR through the dysregulation of mitochondrial function in AML12 mouse hepatocytes challenged with high concentration of free fatty acids (HFFA). Firstly we observed that treatment of AM251, a selective CB1R antagonist, obviously reversed the HFFA-induced reduction of MUP1 protein expression both in vivo and in vitro. Additionally, our results revealed that AM251 also reverted HFFA-mediated decrease of the mRNA level of mitochondrial biogenesis-related factors, mtDNA amount, ATP production, mitochondrial respiratory complexes-I and -III, and mitochondrial membrane potential, thus consequently might correlate with a parallel reduction of ROS production. Meanwhile, AM251 attenuated HFFA-induced impairment of insulin signaling phosphorylation and elevation of phosphoenolpyrvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), two key enzymes of gluconeogenesis. Silence of MUP1 gene abolished the inhibitory effect of AM251 on HFFA-mediated elevation of PEPCK and G6Pase expression, whereas the suppression of insulin signaling and mRNA level of mitochondrial biogenesis-related factors were only partially recovered. Altogether, these findings suggest that the anti-HIR effect of AM251 via improvement of mitochondrial functions might occur in a MUP1-dependent manner.

Endothelin-1 exacerbates development of hypertension and atherosclerosis in modest insulin resistant syndrome.

Endothelin-1 (ET-1) is known as potent vasoconstrictor, by virtue of its mitogenic effects, and may deteriorate the process of hypertension and atherosclerosis by aggravating hyperplasia and migration in VSMCs. Our previous study demonstrated that insulin infusion caused sequential induction of hyperinsulinemia, hyperendothelinemia, insulin resistance, and then hypertension in rats. However, the underlying mechanism of ET-1 interfere insulin signaling in VSMCs remains unclear. To characterize insulin signaling during modest insulin resistant syndrome, we established and monitored rats by feeding high fructose-diet (HFD) until high blood pressure and modest insulin resistance occurred. To explore the role of ET-1/ETAR during insulin resistance, ETAR expression, ET-1 binding, and insulin signaling were investigated in the HFD-fed rats and cultured A-10 VSMCs. Results showed that high blood pressure, tunica medial wall thickening, plasma ET-1 and insulin, and accompanied with modest insulin resistance without overweight and hyperglycemia occurred in early-stage HFD-fed rats. In the endothelium-denuded aorta from HFD-fed rats, ETAR expression, but not ETBR, and ET-1 binding in aorta were increased. Moreover, decreasing of insulin-induced Akt phosphorylation and increasing of insulin-induced ERK phosphorylation were observed in aorta during modest insulin resistance. Interestingly, in ET-1 pretreated VSMCs, the increment of insulin-induced Akt phosphorylation was decreased whereas the increment of insulin-induced ERK phosphorylation was increased. In addition, insulin potentiated ET-1-induced VSMCs migration and proliferation due to increasing ET-1 binding. ETAR antagonist reversed effects of ET-1 on insulin-induced signaling and VSMCs migration and proliferation. In summary, modest insulin resistance syndrome accompanied with hyperinsulinemia leading to the potentiation on ET-1-induced actions in aortic VSMCs. ET-1 via ETAR pathway suppressed insulin-induced AKT activation, whereas remained insulin-induced ERK activation. ET-1 and insulin synergistically potentiated migration and proliferation mainly through ETAR/ERK dependent pathway, which is dominant in VSMCs during modest insulin resistance syndrome. Therefore, ET-1 and ETAR are potential targets responsible for the observed synergism effect in the hypertensive atherosclerotic process through enhancement of ET-1 binding, ET-1 binding, ETAR expression, and ET-1-induced mitogenic actions in aortic VSMCs.

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor.

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ETAR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT1R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ETAR expression, but not ETBR, in aorta and increased ET-1 binding, mainly to ETAR in A-10 VSMCs. Moreover, Ang II-enhanced ETAR expression was blunted and ET-1 binding was reduced by AT1R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ETAR expression and ET-1/ETAR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.

Effect of growth hormone on dawn phenomenon in patients with type 2 diabetes.

We aimed to investigate the involvement of growth hormone in dawn phenomenon and insulin sensitivity in patients with type 2 diabetes mellitus (T2DM). On six occasions separated by intervals of at least 3 days, subjects received early evening (16:00 hours) or late night (23:00 hours) pretreatment with subcutaneous injection of normal saline, human growth hormone, or octreotide. Modified euglycemic insulin clamp test was done 16 hours later and variable glucose infusion (M values) was determined. Plasma glucose, serum insulin, insulin-like growth factor-1, non-esterified fatty acids, and metabolic clearance rate of insulin (MCRI) were measured. Early evening application of growth hormone decreased MCRI 16 hours later, suggesting reduction in insulin sensitivity. Exogenous growth hormone injection reduced insulin sensitivity in T2DM patients. Results provide direct evidence for the role of growth hormone in regulating the insulin sensitivity in insulin-resistant patients.

Endothelin-1 induces lipolysis through activation of the GC/cGMP/Ca2+/ERK/CaMKIII pathway in 3T3-L1 adipocytes.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide produced and secreted mainly by endothelial cells. Recent studies indicate that ET-1 can regulate lipid metabolism, which may increase the risk of insulin resistance. Our previous studies revealed that ET-1 induced lipolysis in adipocytes, but the underlying mechanisms were unclear. 3T3-L1 adipocytes were used to investigate the effect of ET-1 on lipolysis and the underlying mechanisms. Glycerol levels in the incubation medium and hormone-sensitive lipase (HSL) phosphorylation were used as indices for lipolysis. ET-1 significantly increased HSL phosphorylation and lipolysis, which were completely inhibited by ERK inhibitor (PD98059) and guanylyl cyclase (GC) inhibitor (LY83583). LY83583 reduced ET-1-induced ERK phosphorylation. A Ca2+-free medium and PLC inhibitor caused significant decreases in ET-1-induced lipolysis as well as ERK and HSL phosphorylation, and IP3 receptor activator (D-IP3) increased lipolysis. ET-1 increased cGMP production, which was not affected by depletion of extracellular Ca2+. On the other hand, LY83583 diminished the ET-1-induced Ca2+ influx. Transient receptor potential vanilloid-1 (TRPV-1) antagonist and shRNA partially inhibited ET-1-induced lipolysis. ET-1-induced lipolysis was completely suppressed by CaMKIII inhibitor (NH-125). These results indicate that ET-1 stimulates extracellular Ca2+ entry and activates the intracellular PLC/IP3/Ca2+ pathway through a cGMP-dependent pathway. The increased cytosolic Ca2+ that results from ET-1 treatment stimulates ERK and HSL phosphorylation, which subsequently induces lipolysis. ET-1 induces HSL phosphorylation and lipolysis via the GC/cGMP/Ca2+/ERK/CaMKIII signaling pathway in 3T3-L1 adipocytes.

Resistin induces monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression in endothelial cells via p38MAPK-dependent pathway.

Resistin, firstly reported as an adipocyte-specific hormone, is suggested to be an important link between obesity and diabetes. Recent studies have suggested an association between resistin and atherogenic processes. The adhesion of circulating monocytes to endothelial cells is a critical step in the early stages of atherosclerosis. The purpose of the present study was to investigate the effect of resistin on the adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Our results showed that resistin caused a significant increase in monocyte adhesion. In exploring the underlying mechanisms of resistin action, we found that resistin-induced monocyte adhesion was blocked by inhibition of p38MAPK activation using SB203580 and SB202190. Furthermore, resistin increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by HUVECs and these effects were also p38MAPK-dependent. Resistin-induced monocyte adhesion was also blocked by monoclonal antibodies against ICAM-1 and VCAM-1. Taken together, these results show that resistin increases both the expression of ICAM-1 and VCAM-1 by endothelial cells and monocyte adhesion to HUVECs via p38MAPK-dependent pathways.

Clinical observations, molecular genetic analysis, and treatment of sitosterolemia in infants and children.

The clinical observation and treatment of young children with sitosterolemia has rarely been reported. We report clinical, biochemical, and molecular genetic observations and treatment outcomes for five Chinese children from four separate families presenting with sitosterolemia in whom we identified two new (Y329X, G269R) and three known (R446X, N437K, R389H) mutations in the ABCG5 gene. The R389H mutation was found in 50% of alleles. Three of these five patients received cholestyramine therapy with a very good response. However, all patients discontinued this therapy because of poor compliance. Finally, all patients were on ezetimibe therapy and had satisfactory total serum cholesterol levels, though their plant sterol levels were still higher than normal. Another noteworthy finding is that a female infant had a serum cholesterol level of 654 mg/dl at 7 months of age, despite being breast fed (with very tiny amounts of plant sterols) since birth and undergoing 4 months of ezetimibe administration. Although she failed to respond to ezetimibe during this period, she did show improvement when the therapy was started again at 2 years of age. It is possible that another 23-month-old female patient also responded more slowly to ezetimibe treatment than older patients.

Whether to report diabetes as the underlying cause-of-death? a survey of internists of different sub-specialties.

BACKGROUND: Cause-specific mortality is a commonly used endpoint of clinical trials or prospective studies. However, it is sometimes difficult for physician to determine the underlying-cause-of-death (UCD), especially for diabetic patients coexisted with cardiovascular diseases (CVD). The aim of this survey was to examine whether internists with different specialties have different opinions on the reporting of diabetes as the UCD. METHODS: A total of 549 physicians completed the questionnaire in Taiwan, which comprised seven hypothetical case scenarios, each indicating a different level of contribution of diabetes in initiating the chain of events leading to death. RESULTS: As a whole, endocrinologists were more likely than cardiologists and nephrologists to report diabetes as the UCD. The differences were more prominent when the diabetic patient had a coexisting CVD. In scenario 3 (a diabetic patient with hypertension who died from acute myocardial infarction), the percentage was 56% in endocrinologists, which was significantly higher than in cardiologists (42%) and nephrologists (41%). In scenario 4 (a diabetic patient with hypertension who died from cerebrovascular infarction), the percentage was 45% in endocrinologists, and only 31% in cardiologists and 36% in nephrologists. CONCLUSIONS: Internists of different sub-specialties do have different opinions on the reporting of diabetes as the UCD, especially when the diabetic patient has a coexisting CVD.

The Shipai cohort for cardiovascular metabolic risk factors and outcome study - Design and preliminary results.

BACKGROUND: The aim of this study was to identify genotypic and phenotypic cardiovascular metabolic risk factors, and to establish risk models of diseases, including diabetes mellitus, cardiovascular disease, stroke, kidney dysfunction and psychiatric disorders, in Taiwanese adults. METHODS: In 2009, a community-based cohort study was initiated in the Shipai area of the Shilin and Beitou districts in Taipei. Residents were randomly sampled by age (young adults: 35-44 years and middle-aged adults: 45-55 years) and urbanization (rural and urban). Residents who agreed to participate were scheduled to receive examinations (physical and blood) and answer questionnaires. A ten-year follow-up is anticipated. Metabolic syndrome (MetS) was defined based on the Adult Treatment Panel III guidelines, and individuals with only one or two of the five MetS components was identified for prevention target. RESULTS: The response rate of the 9000 invited residents was 10.1%. After screening, 906 participants were enrolled. While 31.0% (281) had no MetS components, 29.1% (264) had only one, and 22.0% (199) had two. MetS with at least three components was diagnosed in 17.9% (162) of the cohort. Concerning gender difference, 25.4% of men and 13.2% of women had MetS (p < 0.001). The percentage of MetS was higher in middle-aged participants than in young adults (20.5% versus 13.4%, p = 0.008). Forty-six percent of participants had central obesity. After adjusting for gender, age, and urbanization, the central obesity odds ratio for MetS was 23.7, with a 95% confidence internal of 13.1-42.7. CONCLUSION: Our preliminary results revealed a high MetS percentage among young and middle-aged adults in Taiwan, with central obesity being a particularly urgent prevention target. The research design and operational protocol of this cohort study may stimulate more research in the future.

Effect of reversing dark-light cycles on normal diurnal variation and related metabolic disturbance in rats.

Diurnal variation of glucose tolerance and insulin action was studied in male Sprague-Dawley rats with a normal or reversed light-dark cycle. A series of experiments conducted was at 12 AM and 12 PM in the two groups. All measurements were separated by a recovery period of at least 3 days and preceded by a 16-hour fast. Glucose tolerance and insulin action were measured by both an oral glucose tolerance test and intraperitoneal insulin tolerance test. Normal light-dark cycle rats had significantly (P < 0.05) greater insulin sensitivity at 12 PM than at 12 AM, whereas reversed light-dark cycle rats had the opposite results (P < 0.05). Rats in the normal light-dark cycle group had a significantly higher growth hormone concentration at 12 AM than at 12 PM, whereas rats in the reversed group had the opposite results. Measurement of insulin-stimulated glucose uptake of isolated adipocytes preincubated with or without 100 ng/ml growth hormone at 37 degrees C for 5 hours revealed that approximately 30% of insulin-stimulated glucose uptake was suppressed when adipocytes were treated with growth hormone. These results indicate that male rats exhibit significant diurnal variation of glucose tolerance and insulin sensitivity, and suggest that the concomitant diurnal variation of growth hormone may have a superimposed and amplifying effects on this variation.

Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting.

AIM: To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. METHODS: By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. RESULTS: A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). CONCLUSION: In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.

VISFATIN PROMOTES FOAM CELL FORMATION BY DYSREGULATING CD36, SRA, ABCA1, AND ABCG1 EXPRESSION IN RAW264.7 MACROPHAGES.

BACKGROUND: Visfatin is produced in and secreted from adipocytes. Increased circulating visfatin level is observed in obese subjects. Previous studies demonstrated that visfatin was involved in obesity-related cardiovascular diseases. AIMS: This study aims to explore the regulatory effects of adipokine visfatin on foam cell formation, a key step in the development of atherosclerosis. METHODS: Effect of visfatin on protein and mRNA expression of scavenger receptor and ATP binding cassette transporter in RAW264.7 macrophages were measured by western blotting and real-time RT-PCR. To confirm the influence of visfatin-regulated scavenger receptor and ATP binding cassette transporter to foam cell formation, the visfatin-caused changes of ox-LDL uptake, cholesterol efflux, and foam cell formation were determined. RESULTS: Visfatin significantly increased the expression of CD36 and scavenger receptor A (SRA), decreased the expression of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and had no effect on the expression of SR-B1. Visfatin increased oxidized-LDL (ox-LDL) uptake and decreased cholesterol efflux, which increased foam cell formation. The PI3K inhibitor LY294002 blocked the effect of visfatin on the protein and mRNA expression levels of CD36, SRA, and ABCG1 and ox-LDL uptake and cholesterol efflux. The ERK inhibitor PD98059 also prevented visfatin-induced ABCA1 instability and subsequently decreased cholesterol efflux. CONCLUSIONS: Visfatin upregulated CD36 and SRA expression and downregulated ABCA1 and ABCG1 expression, subsequently increased ox-LDL uptake and decreased cholesterol efflux, and finally promoted foam cell formation via the PI3K- and ERK-dependent pathways.

Effects of bariatric weight loss surgery on glucose metabolism, inflammatory cytokines, and serum tartrate-resistant acid phosphatase 5a in obese Chinese adults.

BACKGROUND: We determined effects of bariatric weight loss surgery on serum tartrate-resistant acid phosphatase 5a (TRACP 5a), inflammatory cytokines and glucose homeostasis in severely obese Chinese adults. METHODS: Severely obese adults undergoing bariatric surgery were recruited. Anthropometry, insulin resistance (IR), inflammatory markers and serum TRACP 5a were measured at baseline and 3, 6 and 12months postoperatively. RESULTS: Data of 93 patients, including 69 non-diabetic (non-DM group) and 24 diabetic (DM group), were analyzed. Anthropometry decreased significantly at 3months postoperatively in both groups; low-density lipoprotein cholesterol decreased obviously at 3, 6 and 12months in non-DM group, while improving significantly at 6 and 12months in DM group. Homeostasis model assessment for IR (HOMA-IR) improved significantly at 3, 6 and 12months in non-DM group and 12months in DM group. In DM group, C-reactive protein (CRP) decreased significantly at 3months postoperatively and inflammatory markers interleukin-6 (IL-6) and TRACP 5a improved at 6months postoperatively; in non-DM group, serum TRACP 5a decreased obviously at 12months postoperatively without significant changes in CRP and IL-6. CONCLUSION: Weight reduction by bariatric surgery decreases anthropometry, IR, lipids and inflammatory markers in severely obese Chinese adults.

Cannabinoid receptor type 1 mediates high-fat diet-induced insulin resistance by increasing forkhead box O1 activity in a mouse model of obesity.

Hepatic glucose production is promoted by forkhead box O1 (FoxO1) under conditions of insulin resistance. The overactivity of cannabinoid receptor type 1 (CB1R) partly causes increased liver fat deposits and metabolic dysfunction in obese rodents by decreasing mitochondrial function. The aim of the present study was to investigate the role of FoxO1 in CB1R-mediated insulin resistance through the dysregulation of mitochondrial function in the livers of mice with high-fat diet (HFD)-induced obesity. For this purpose, male C57BL/6 mice were randomly assigned to groups and either fed a standard diet (STD), a HFD, or a HFD with 1-week treatment of the CB1R inverse agonist, AM251, at 1 or 5 mg/kg. For in vitro experiments, AML12 hepatocytes were incubated with FoxO1 siRNA prior to challenge with arachidonyl-2'-chloroethylamide (ACEA) or a high concentration of free fatty acids (HFFA). Plasma parameters were analyzed using colorimetric methods. Liver histopathology and hepatic status markers were examined. The HFD-fed mice exhibited an increase in CB1R levels in the liver. Moreover, in response to increased hepatic oxidative stress, the HFD-fed mice also displayed hepatic mitochondrial dysfunction, as indicated by the decreased mRNA levels of carnitine palmitoyltransferase-1 (CPT-1), mitochondrial transcription factor A (TFAM), nuclear respiratory factor-1 (NRF-1) and citrate synthase. On the contrary, these effects in the HFD-fed mice were reversed by treatment with 5 mg/kg AM251. The administration of AM251 suppressed the induction of FoxO1, phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) expression in the livers of the mice fed a HFD by enhancing the phosphorylation of insulin signaling cascades thus, further lowering the high level of the homeostatic model assessment of insulin resistance (HOMA­IR) index. In our in vitro experiments, transfection with FoxO1 siRNA prevented the HFFA- and ACEA-induced decrease in the gene expression of mitochondrial biogenesis-related factors, and abrogated the HFFA- and ACEA-induced increase in PEPCK and G6Pase expression. Taken together, our findings suggest that the anti-insulin resistance effect of AM251, which leads to an improvement of mitochondrial function in hepatic steatosis, is mediated through FoxO1.

Chronic endothelin-1 infusion causes adipocyte hyperplasia in rats.

OBJECTIVE: The aim of this study was to investigate the regulatory mechanism of endothelin-1 (ET-1), an endothelium-derived vasoconstrictor, on adipogenesis in vitro and in vivo. METHODS: 3T3-L1 preadipocytes were used to explore the mechanisms mediating ET-1 actions on preadipocyte proliferation and adipocyte differentiation. To investigate the in vivo effect of ET-1, male Sprague-Dawley rats were infused with ET-1 or saline for 4 weeks via intraperitoneally implanted osmotic pumps, and the fat pad weight and adipocyte size of adipose tissues were measured. RESULTS: ET-1 stimulated preadipocyte proliferation and increased the cell number at the mitotic clonal expansion stage of adipocyte differentiation via the endothelin A receptor (ETAR) and activation of the protein kinase C (PKC) pathway. ET-1, via ETAR, inhibited adipocyte differentiation partially through an ERK-dependent pathway. Furthermore, no significant difference in the body weight and fat pad weight was observed in either ET-1- or saline-infused rats. Compared with saline-infused rats, the adipocyte cell number was significantly increased but the adipocyte size was significantly decreased in ET-1-infused rats. CONCLUSIONS: Chronic ET-1 infusion increased the number of small adipocytes without the change of white adipose tissue mass in rats, which were associated with ET-1-stimulated preadipocyte proliferation, but not ET-1-suppressed adipocyte differentiation.

The Ankle Brachial Index Exhibits Better Association of Cardiovascular Prognosis Than Non-High-Density Lipoprotein Cholesterol in Type 2 Diabetes.

OBJECTIVE: The association between ankle brachial index (ABI) and outcomes in diabetic subjects is controversial. The purpose of this study was to evaluate whether the ABI is more strongly associated with cardiovascular outcomes comparing with non-high-density lipoprotein cholesterol (non-HDL-c). RESEARCH DESIGN AND METHODS: A total of 452 type 2 diabetic subjects followed up for a mean of 5.8 years were grouped by ABI (<0.9 versus ≥0.9) and non-HDL-c (<100mg/dL versus ≥100mg/dL). Primary outcomes were composite events including all-cause mortality, hospitalization for coronary artery disease, stroke, revascularization, amputation and diabetic foot, and the secondary end point was all-cause mortality. RESULTS: Intergroup differences in percentage of men, duration of diabetes, hemoglobin A1c, total cholesterol, low-density lipoprotein cholesterol, triglycerides and estimated glomerular filtration rate were significant. A total of 64 composite events and 17 deaths were recorded. A higher number of composite events occurred in the group with abnormal ABI but optimal non-HDL-c than in those with suboptimal non-HDL-c but normal ABI (29% versus 11%, P < 0.05). A similar trend was observed in all-cause mortality (11% versus 1%, P < 0.05). The ABI was the dominant risk factor for both end points after adjusting other factors (for composite events, hazard ratio = 0.02, 95% CI: 0.00-0.10, P < 0.001 and for all-cause mortality, hazard ratio = 0.01, 95% CI: 0.00-0.28, P = 0.006). CONCLUSIONS: The ABI was more strongly associated with outcomes in diabetes than non-HDL-c. The ABI should be routinely screened in diabetes even without symptom.

Angiotensin II enhances insulin sensitivity in vitro and in vivo.

The renin-angiotensin system plays a critical role in the pathogenesis of obesity, obesity-associated hypertension, and insulin resistance. However, the biological actions of angiotensin II (AII) on insulin sensitivity remain controversial. Because angiotensinogen and AII receptors are expressed on adipose tissue, we investigated the effect of AII on the insulin sensitivity of isolated rat adipocytes. The results of a receptor binding assay showed the maximal AII binding capacity of adipocytes to be 8.3 +/- 0.9 fmol/7 x 10(6) cells and the dissociation constant to be 2.72 +/- 0.11 nM. Substantial expression of both type 1 and 2 AII (AT1 and AT2) receptors was detected by RT-PCR. AII had no effect on basal glucose uptake, but significantly potentiated insulin-stimulated glucose uptake; this effect was abolished by the AT1 antagonist, losartan. In addition, AII did not alter the insulin binding capacity of adipocytes, but increased insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit, Akt phosphorylation, and translocation of glucose transporter 4 to the plasma membrane. AII potentiated insulin-stimulated glucose uptake through the AT1 receptor and by alteration of the intracellular signaling of insulin. Intraperitoneal injection of Sprague Dawley rats with AII increased insulin sensitivity in vivo. In conclusion, we have shown that AII enhances insulin sensitivity both in vitro and in vivo, suggesting that dysregulation of the insulin-sensitizing effect of AII may be involved in the development of insulin resistance.