Indole supplementation ameliorates MCD-induced NASH in mice.

Indole is a microbiota metabolite that functions to protect against obesity-associated non-alcoholic fatty liver disease. The present study examined the extent to which indole supplementation alleviates the severity of non-alcoholic steatohepatitis (NASH), which is the advanced form of non-alcoholic fatty liver disease. In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregations of macrophages in the liver compared with control diet-fed mice. Upon indole supplementation, the severity of MCD-induced hepatic steatosis and inflammation, as well as liver fibrosis, was significantly decreased compared with that of MCD-fed and control-treated mice. In vitro, indole treatment caused significant decreases in lipopolysaccharide-induced proinflammatory responses in hepatocytes incubated with either basal or MCD-mimicking media. However, indole treatment only significantly decreased lipopolysaccharide-induced proinflammatory responses in bone marrow-derived macrophages incubated with basal, but not MCD-mimicking media. These differential effects suggest that, relative to the responses of macrophages to indole, the responses of hepatocytes to indole appeared to make a greater contribution to indole alleviation of NASH, in particular liver inflammation. While indole supplementation decreased liver expression of desmin in MCD-fed mice, treatment of LX2 cells (a line of hepatic stellate cells) with indole also decreased the expression of various markers of hepatic stellate cell fibrogenic activation. Lastly, indole supplementation decreased intestinal inflammation in MCD-fed mice, suggesting that decreased intestinal inflammation also was involved in indole alleviation of NASH. Collectively, these results demonstrate that indole supplementation alleviates MCD-induced NASH, which is attributable to, in large part, indole suppression of hepatocyte proinflammatory responses and hepatic stellate cell fibrogenic activation, as well as intestinal proinflammatory responses.

Adipocyte inducible 6-phosphofructo-2-kinase suppresses adipose tissue inflammation and promotes macrophage anti-inflammatory activation.

Obesity-associated inflammation in white adipose tissue (WAT) is a causal factor of systemic insulin resistance. To better understand how adipocytes regulate WAT inflammation, the present study generated chimeric mice in which inducible 6-phosphofructo-2-kinase was low, normal, or high in WAT while the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (Pfkfb3) was normal in hematopoietic cells, and analyzed changes in high-fat diet (HFD)-induced WAT inflammation and systemic insulin resistance in the mice. Indicated by proinflammatory signaling and cytokine expression, the severity of HFD-induced WAT inflammation in WT â†’ Pfkfb3+/- mice, whose Pfkfb3 was disrupted in WAT adipocytes but not hematopoietic cells, was comparable with that in WT â†’ WT mice, whose Pfkfb3 was normal in all cells. In contrast, the severity of HFD-induced WAT inflammation in WT â†’ Adi-Tg mice, whose Pfkfb3 was over-expressed in WAT adipocytes but not hematopoietic cells, remained much lower than that in WT â†’ WT mice. Additionally, HFD-induced insulin resistance was correlated with the status of WAT inflammation and comparable between WT â†’ Pfkfb3+/- mice and WT â†’ WT mice, but was significantly lower in WT â†’ Adi-Tg mice than in WT â†’ WT mice. In vitro, palmitoleate decreased macrophage phosphorylation states of Jnk p46 and Nfkb p65 and potentiated the effect of interleukin 4 on suppressing macrophage proinflammatory activation. Taken together, these results suggest that the Pfkfb3 in adipocytes functions to suppress WAT inflammation. Moreover, the role played by adipocyte Pfkfb3 is attributable to, at least in part, palmitoleate promotion of macrophage anti-inflammatory activation.

Adipose tissue inflammation and systemic insulin resistance in mice with diet-induced obesity is possibly associated with disruption of PFKFB3 in hematopoietic cells.

Obesity-associated inflammation in white adipose tissue (WAT) is a causal factor of systemic insulin resistance; however, precisely how immune cells regulate WAT inflammation in relation to systemic insulin resistance remains to be elucidated. The present study examined a role for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in hematopoietic cells in regulating WAT inflammation and systemic insulin sensitivity. Male C57BL/6J mice were fed a high-fat diet (HFD) or low-fat diet (LFD) for 12 weeks and examined for WAT inducible 6-phosphofructo-2-kinase (iPFK2) content, while additional HFD-fed mice were treated with rosiglitazone and examined for PFKFB3 mRNAs in WAT stromal vascular cells (SVC). Also, chimeric mice in which PFKFB3 was disrupted only in hematopoietic cells and control chimeric mice were also fed an HFD and examined for HFD-induced WAT inflammation and systemic insulin resistance. In vitro, adipocytes were co-cultured with bone marrow-derived macrophages and examined for adipocyte proinflammatory responses and insulin signaling. Compared with their respective levels in controls, WAT iPFK2 amount in HFD-fed mice and WAT SVC PFKFB3 mRNAs in rosiglitazone-treated mice were significantly increased. When the inflammatory responses were analyzed, peritoneal macrophages from PFKFB3-disrputed mice revealed increased proinflammatory activation and decreased anti-inflammatory activation compared with control macrophages. At the whole animal level, hematopoietic cell-specific PFKFB3 disruption enhanced the effects of HFD feeding on promoting WAT inflammation, impairing WAT insulin signaling, and increasing systemic insulin resistance. In vitro, adipocytes co-cultured with PFKFB3-disrupted macrophages revealed increased proinflammatory responses and decreased insulin signaling compared with adipocytes co-cultured with control macrophages. These results suggest that PFKFB3 disruption in hematopoietic cells only exacerbates HFD-induced WAT inflammation and systemic insulin resistance.

Comparison of two regimens for patients with thyroid-associated ophthalmopathy receiving intravenous methyl prednisolone: A single center prospective randomized trial.

Intravenous (i.v.) glucocorticoid is recommended for active moderate-to-severe thyroid-associated ophthalmopathy (TAO). However, the details of the treatment schedule are still debatable. The present prospective randomized trial was performed to compare clinical outcomes and serum cytokines between the two regimens. A cohort of 90 patients with active moderate-to-severe TAO was randomized to receive i.v. methyl prednisolone on a weekly protocol or daily scheme. The response rate was evaluated at the 12-week follow-up visit. Serum interleukin (IL)-2, IL-6 and IL-17 levels were measured in 160 patients with TAO, 60 patients with isolated Graves' disease (GD) and 60 normal control (NC) at baseline, as well as patients with active moderate-to-severe TAO at the 12th week after treatment. The daily scheme had a higher response rate than the weekly protocol without a significant difference (77.8 vs. 63.6%, P>0.05). No major adverse events were recorded under either regimen. Overall, minor events were more common on the daily scheme (11.36 vs. 4.35%, P<0.05)than on the weekly protocol, whereas the deterioration of eye symptoms (two patients) was only reported on the weekly protocol. At baseline, the IL-17 level in the TAO group was higher than that in the isolated GD and NC groups (P<0.05). In addition, the IL-17 level in the active TAO group was higher than that in the inactive TAO group (P<0.05). Furthermore, the IL-17 level had significantly decreased under the two regimens at the 12-week visit (P<0.05). In conclusion, for patients with active moderate-to-severe TAO, daily i.v. glucocorticoid therapy has a relative higher response rate than the weekly protocol with a few more minor adverse events. These two regimens have their own merits with regard to adverse effects. IL-17 has the potential to be a biomarker for evaluating TAO activity and treatment effects.

Adoptive transfer of Pfkfb3-disrupted hematopoietic cells to wild-type mice exacerbates diet-induced hepatic steatosis and inflammation.

BACKGROUND AND OBJECTIVES: Hepatic steatosis and inflammation are key characteristics of non-alcoholic fatty liver disease (NAFLD). However, whether and how hepatic steatosis and liver inflammation are differentially regulated remains to be elucidated. Considering that disruption of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3/iPfk2) dissociates fat deposition and inflammation, the present study examined a role for Pfkfb3/iPfk2 in hematopoietic cells in regulating hepatic steatosis and inflammation in mice. METHODS: Pfkfb3-disrupted (Pfkfb3 +/-) mice and wild-type (WT) littermates were fed a high-fat diet (HFD) and examined for NAFLD phenotype. Also, bone marrow cells isolated from Pfkfb3 +/- mice and WT mice were differentiated into macrophages for analysis of macrophage activation status and for bone marrow transplantation (BMT) to generate chimeric (WT/BMT- Pfkfb3 +/-) mice in which Pfkfb3 was disrupted only in hematopoietic cells and control chimeric (WT/BMT-WT) mice. The latter were also fed an HFD and examined for NAFLD phenotype. In vitro, hepatocytes were co-cultured with bone marrow-derived macrophages and examined for hepatocyte fat deposition and proinflammatory responses. RESULTS: After the feeding period, HFD-fed Pfkfb3 +/- mice displayed increased severity of liver inflammation in the absence of hepatic steatosis compared with HFD-fed WT mice. When inflammatory activation was analyzed, Pfkfb3 +/- macrophages revealed increased proinflammatory activation and decreased anti-proinflammatory activation. When NAFLD phenotype was analyzed in the chimeric mice, WT/BMT-Pfkfb3 +/- mice displayed increases in the severity of HFD-induced hepatic steatosis and inflammation compared with WT/BMT-WT mice. At the cellular level, hepatocytes co-cultured with Pfkfb3 +/- macrophages revealed increased fat deposition and proinflammatory responses compared with hepatocytes co-cultured with WT macrophages. CONCLUSIONS: Pfkfb3 disruption only in hematopoietic cells exacerbates HFD-induced hepatic steatosis and inflammation whereas the Pfkfb3/iPfk2 in nonhematopoietic cells appeared to be needed for HFD feeding to induce hepatic steatosis. As such, the Pfkfb3/iPfk2 plays a unique role in regulating NAFLD pathophysiology.

Mice lacking adenosine 2A receptor reveal increased severity of MCD-induced NASH.

Adenosine 2A receptor (A2AR) exerts a protective role in obesity-related non-alcoholic fatty liver disease. Here, we examined whether A2AR protects against non-alcoholic steatohepatitis (NASH). In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregation of macrophages in the liver compared with mice fed a chow diet. MCD feeding also significantly increased the numbers of A2AR-positive macrophages/Kupffer cells in liver sections although decreasing A2AR amount in liver lysates compared with chow diet feeding. Next, MCD-induced NASH phenotype was examined in A2AR-disrupted mice and control mice. Upon MCD feeding, A2AR-disruptd mice and control mice displayed comparable decreases in body weight and fat mass. However, MCD-fed A2AR-disrupted mice revealed greater liver weight and increased severity of hepatic steatosis compared with MCD-fed control mice. Moreover, A2AR-disupted mice displayed increased severity of MCD-induced liver inflammation, indicated by massive aggregation of macrophages and increased phosphorylation states of Jun-N terminal kinase (JNK) p46 and nuclear factor kappa B (NFκB) p65 and mRNA levels of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6. In vitro, incubation with MCD-mimicking media increased lipopolysaccharide (LPS)-induced phosphorylation states of JNK p46 and/or NFκB p65 and cytokine mRNAs in control macrophages and RAW264.7 cells, but not primary hepatocytes. Additionally, MCD-mimicking media significantly increased lipopolysaccharide-induced phosphorylation states of p38 and NFκB p65 in A2AR-deficient macrophages, but insignificantly decreased lipopolysaccharide-induced phosphorylation states of JNK p46 and NFκB p65 in A2AR-deficient hepatocytes. Collectively, these results suggest that A2AR disruption exacerbates MCD-induced NASH, which is attributable to, in large part, increased inflammatory responses in macrophages.

A Randomized Trial of Insulin Glargine plus Oral Hypoglycemic Agents versus Continuous Subcutaneous Insulin Infusion to Treat Newly Diagnosed Type 2 Diabetes.

AIMS: Basal insulin plus oral hypoglycemic agents (OHAs) has not been investigated for early intensive antihyperglycemic treatment in people with newly diagnosed type 2 diabetes. This study is aimed at comparing the short-term (over a period of 12 days) effects of basal insulin glargine plus OHAs and continuous subcutaneous insulin infusion (CSII) on glycemic control and beta-cell function in this setting. METHODS: An open-label parallel-group study. Newly diagnosed hospitalized patients with type 2 diabetes and fasting plasma glucose (FPG) ≥11.1 mmol/L or glycated hemoglobin (HbA1c) ≥9% (75 mmol/mol) were randomized to CSII or insulin glargine in combination with metformin and gliclazide. The primary outcome measure was the mean amplitude of glycemic excursions (MAGE), and secondary endpoints included time to reach glycemic control target (FPG < 7 mmol/L and 2-hour postprandial plasma glucose < 10 mmol/L), markers of ß-cell function, and hypoglycemia. RESULTS: Subjects in the CSII (n = 35) and basal insulin plus OHA (n = 33) groups had a similar significant reduction from baseline to end of treatment in glycated albumin (-6.44 ± 3.23% and- 6.42 ± 3.56%, P = 0.970). Groups A and B have comparable time to glycemic control (3.6 ± 1.2 days and 4.0 ± 1.4 days), MAGE (3.40 ± 1.40 mmol/L vs. 3.16 ± 1.38 mmol/L; p = 0.484), and 24-hour mean blood glucose (7.49 ± 0.96 mmol/L vs. 7.02 ± 1.03 mmol/L). Changes in the C-peptide reactivity index, the secretory unit of islet in transplantation index, and insulin secretion-sensitivity index-2 indicated a greater ß-cell function improvement with basal insulin plus OHAs versus CSII. CONCLUSIONS: Short-term insulin glargine plus OHAs may be an alternative to CSII for initial intensive therapy in people with newly diagnosed type 2 diabetes.

Utility of Neck Circumference for Identifying Metabolic Syndrome by Different Definitions in Chinese Subjects over 50 Years Old: A Community-Based Study.

AIMS: Whether neck circumference (NC) could be used as a valuable tool for identifying metabolic syndrome (MS) by different criteria in Chinese is still unclear. METHODS: We conducted a cross-sectional survey from October 2010 to January 2011 in Shipai community, Guangzhou, Guangdong Province, China. A total of 1473 subjects aged over 50 years were investigated. We measured height, weight, NC, waist circumference, blood pressure, blood glucose, and lipids in all subjects. MS was identified by criteria of the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III), Chinese Diabetes Society (CDS), and International Diabetes Federation (IDF). RESULTS: Mean NC was 38.0 ± 2.7 cm in men and 34.2 ± 2.5 cm in women. By using receiver operating characteristic curves, the area under the curve (AUC) of NC for identifying MS (IDF) was 0.823 in men and 0.777 in women, while for identifying MS (CDS), it was 0.788 in men and 0.762 in women. The AUC of NC for diagnosing MS (ATP III) was 0.776 in men and 0.752 in women. The optimal cut points of NC for MS were 38.5 cm by three definitions in men, while those were 34.2 cm, 33.4 cm, and 34.0 cm in women by IDF, ATP III, and CDS definitions, respectively. No significant difference was observed between the AUC of NC and BMI for diagnosing MS by using different criteria (all p > 0.05). CONCLUSIONS: NC is associated with MS by different definitions in Chinese subjects over 50 years old. It may be a useful tool to identify MS in a community population.

Protective effects of Ginkgo biloba extract (EGb761) and its constituents quercetin and ginkgolide B against beta-amyloid peptide-induced toxicity in SH-SY5Y cells.

Ginkgo biloba extract EGb761 has been shown to protect against beta-amyloid peptide (Abeta)-induced neurotoxicity but the specific mechanisms remain unclear. In the present study, effects of EGb761 and two of its constituents, quercetin and ginkgolide B, on the cytotoxic action of Abeta (1-42) were tested with human neuroblastoma SH-SY5Y cells. We found that EGb761 was able to block Abeta (1-42)-induced cell apoptosis, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and activation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling pathways. Both quercetin and ginkgolide B may be involved in the inhibitory effects of EGb761 on JNK, ERK1/2 and Akt signaling pathways. Ginkgolide B also helped to improve mitochondrial functions but quercetin failed to show this effect. Additional experiments suggest that, protective effects of EGb761 against Abeta toxicity may be associated with its antioxidant and platelet activating factor (PAF) antagonist activities. Quercetin but not ginkgolide B is one of the constituents responsible for the antioxidant action of EGb761. Both quercetin and ginkgolide B may be involved in the PAF antagonist activity of EGb761. Overall, actions of individual EGb761 components provide further insights into direct mechanisms underlying the neuroprotective effects of EGb761.

Dosage effects of EGb761 on hydrogen peroxide-induced cell death in SH-SY5Y cells.

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, is one of the most popular herbal supplements, taken for its multivalent properties. In this study, dosage effects of EGb761 on hydrogen peroxide (H(2)O(2))-induced apoptosis of human neuroblastoma SH-SY5Y cells were investigated. It was found that H(2)O(2)-induced apoptotic cell death in SH-SY5Y cells, which was revealed in DNA fragmentation, mitochondrial membrane potential depolarization, and activation of Akt, c-Jun N-terminal kinases (JNK) and caspase 3. Low doses of EGb761 (50-100 microg/ml) inhibited H(2)O(2)-induced cell apoptosis via inactivation of Akt, JNK and caspase 3 while high doses of EGb761 (250-500 microg/ml) enhanced H(2)O(2) toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments revealed that H(2)O(2) decreased intracellular GSH content, which was also inhibited by low concentrations of EGb761 but enhanced after high concentrations of EGb761 treatment. This further suggests to us that dosage effects of EGb761 on apoptotic signaling proteins may be correlated with regulation of cell redox state. Therefore, treatment dosage may be one of the vital factors that determine the specific action of EGb761 on oxidative stress-induced cell apoptosis. To understand the mechanisms of dosage effects of EGb761 may have important clinical implications.