A2A adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A2AARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A2AAR stimulation in a rat model of arthritis. We investigated A2AAR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A2AARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A2AAR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A2AAR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A2AAR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A2AAR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A2AAR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A2AARs in lymphocytes from RA patients. The effectiveness of A2AAR stimulation in a rat model of arthritis supported the role of A2AAR agonists as potential pharmacological treatment for RA.

Hydrogen (H2) acts as a therapeutic antioxidant. However, there are few reports on H2 function in other capacities in diabetes mellitus (DM). Therefore, in this study, we investigated the role of H2 in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ)-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H2 promoted 2-[14C]-deoxy-d-glucose (2-DG) uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-activated protein kinase (AMPK), although it did not stimulate the translocation of Glut2 in Hep G2 cells. H2 significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p.) and oral (p.o.) administration. However, long-term p.o. administration of H2 had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H2 exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.

As the linker between the A chain and B chain of proinsulin, C-peptide displays high variability in length and amino acid composition, and has been considered as an inert byproduct of insulin synthesis and processing for many years. Recent studies have suggested that C-peptide can act as a bioactive hormone, exerting various biological effects on the pathophysiology and treatment of diabetes. In this study, we analyzed the coevolution of insulin molecules among vertebrates, aiming at exploring the evolutionary characteristics of insulin molecule, especially the C-peptide. We also calculated the correlations of evolutionary rates between the insulin and the insulin receptor (IR) sequences as well as the domain-domain pairs of the ligand and receptor by the mirrortree method. The results revealed distinctive features of C-peptide in insulin intramolecular coevolution and correlated residue substitutions, which partly supported the idea that C-peptide can act as a bioactive hormone, with significant sequence features, as well as a linker assisting the formation of mature insulin during synthesis. Interestingly, the evolution of C-peptide exerted the highest correlation with that of the insulin receptor and its ligand binding domain (LBD), implying a potential relationship with the insulin signaling pathway.

Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague–Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK) and downstream components of phosphatidylinositol 3-kinase (PI3K) signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC). Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKC)ζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB)/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr308) and (Ser473) and AS160 phosphorylation. Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1) mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of components of the AMPK and downstream to PIK3 (aPKC, Akt), and improve GLUT4 trafficking in skeletal muscle. These data help explain the mechanism how exercise regulates glucose homeostasis in diabetic rats.

Background

We tested the concordance of the two diagnostic criteria for diabetes using fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) by the Japan Diabetes Society (JDS) and American Diabetes Association (ADA).

Methods

We used data from 7,328 subjects without known diabetes who participated in a voluntary health checkup program at least twice between 1998 and 2006, at intervals ≤2 years. For repeat participants who attended the screening over two times, data from the first and second checkups were used for this study. At the first visit, diabetes was diagnosed both at FPG ≥7.0 mmol/L and HbA1c ≥6.5% using the JDS criteria. In addition, diabetes was diagnosed using two ADA criteria; ADA-FPG diabetes for persistent fasting hyperglycemia (FPG ≥7.0 mmol/L) or ADA-HbA1c diabetes for hyper-glycated hemoglominemia (HbA1c ≥6.5%), both at the first and second checkups. Subsequently, the concordance of diagnosis between the JDS and the ADA criteria was evaluated.

Results

At the first checkup, 153 (2.1%) persons were diagnosed with diabetes by the JDS criteria. They had higher levels of risk factors for diabetes than non-diabetic subjects. Using the first and second checkups, 174 (2.4%) and 175 (2.4%) were diagnosed with diabetes by the ADA-FPG criteria, respectively. Among 153 subjects diagnosed with diabetes by the JDS criteria, 125 (81.7%) and 129 (84.3%) had ADA-FPG and ADA-HbA1c diabetes, respectively. The kappa coefficients of the JDS criteria with ADA-FPG and ADA-HbA1c criteria were 0.759 and 0.782 (P<0.001), respectively. In the subgroup analysis stratified by sex, the concordance was well preserved at the kappa coefficients around 0.8 (between 0.725 and 0.836).

Conclusion

The JDS diagnostic criteria for diabetes have a substantial and acceptable concordance with the ADA criteria. The JDS criteria may be a practical method for diagnosing diabetes that maintains compatibility with the ADA criteria.

Aims/Hypothesis

Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats.

Methods

Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast. The purified protein was administered at 10–30 mg/kg i.p. b.i.d. to diabetic db/db mice for 2 weeks. Furthermore, high molecular weight human and globular mouse adiponectin batches were administered at 5–15 mg/kg i.p. b.i.d. to diabetic sand rats for 12 days.

Results

Surprisingly, none of our batches had any effect on blood glucose, HbA1c, plasma lipids or body weight in diabetic db/db mice or sand rats. In vitro biological, biochemical and biophysical data suggest that the protein was correctly folded and biologically active.

Conclusions/Interpretation

Recombinant adiponectin is ineffective at lowering blood glucose in diabetic db/db mice or sand rats.

Background

StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs.

Methodology/Principal Findings

Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (−26.4% and −41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and 3H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7.

Conclusions/Significance

Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.

Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.

Background

Unexplained intrauterine growth restriction (IUGR) may be a consequence of placental insufficiency; however, its etiology is not fully understood. We surmised that defective placentation in IUGR dysregulates cellular bioenergic homeostasis, leading to increased autophagy in the villous trophoblast. The aims of this work were (1) to compare the differences in autophagy, p53 expression, and apoptosis between placentas of women with normal or IUGR pregnancies; (2) to study the effects of hypoxia and the role of p53 in regulating trophoblast autophagy; and (3) to investigate the relationship between autophagy and apoptosis in hypoxic trophoblasts.

Methodology/Principal Findings

Compared with normal pregnant women, women with IUGR had higher placental levels of autophagy-related proteins LC3B-II, beclin-1, and damage-regulated autophagy modulator (DRAM), with increased p53 and caspase-cleaved cytokeratin 18 (M30). Furthermore, cytotrophoblasts cultured under hypoxia (2% oxygen) in the presence or absence of nutlin-3 (a p53 activity stimulator) had higher levels of LC3B-II, DRAM, and M30 proteins and increased Bax mRNA expression compared with controls cultured under standard conditions. In contrast, administration of pifithrin-α (a p53 activity inhibitor) during hypoxia resulted in protein levels that were similar to those of the control groups. Moreover, cytotrophoblasts transfected with LC3B, beclin-1, or DRAM siRNA had higher levels of M30 compared with the controls under hypoxia. However, transfection with Bcl-2 or Bax siRNA did not cause any significant change in the levels of LC3B-II in hypoxic cytotrophoblasts.

Conclusions/Significance

Together, these results suggest that there is a crosstalk between autophagy and apoptosis in IUGR and that p53 plays a pivotal and complex role in regulating trophoblast cell turnover in response to hypoxic stress.

Background

This study aimed to explore the association of MTNR1B genetic variants with gestational plasma glucose homeostasis in pregnant Chinese women.

Methods

A total of 1,985 pregnant Han Chinese women were recruited and evaluated for gestational glucose tolerance status with a two-step approach. The four MTNR1B variants rs10830963, rs1387153, rs1447352, and rs2166706 which had been reported to associate with glucose levels in general non-pregnant populations, were genotyped in these women. Using an additive model adjusted for age and body mass index (BMI), association of these variants with gestational fasting and postprandial plasma glucose (FPG and PPG) levels were analyzed by multiple linear regression; relative risk of developing gestational glucose intolerance was calculated by logistic regression. Hardy-Weinberg Equilibrium was tested by Chi-square and linkage disequilibrium (LD) between these variants was estimated by measures of D′ and r2.

Results

In the pregnant Chinese women, the MTNR1B variant rs10830963, rs1387153, rs2166706 and rs1447352 were shown to be associated with the increased 1 hour PPG level (p = 8.04×10−10, 5.49×10−6, 1.89×10−5 and 0.02, respectively). The alleles were also shown to be associated with gestational glucose intolerance with odds ratios (OR) of 1.64 (p = 8.03×10−11), 1.43 (p = 1.94×10−6), 1.38 (p = 1.63×10−5) and 1.24 (p = 0.007), respectively. MTNR1B rs1387153, rs2166706 were shown to be associated with gestational FPG levels (p = 0.04). Our data also suggested that, the LD pattern of these variants in the studied women conformed to that in the general populations: rs1387153 and rs2166706 were in high LD, they linked moderately with rs10830963, but might not linked with rs1447352;rs10830963 might not link with rs1447352, either. In addition, the MTNR1B variants were not found to be associated with any other traits tested.

Conclusions

The MTNR1B is likely to be involved in the regulation of glucose homeostasis during pregnancy.

Background

HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity.

Methodology/Principal Findings

We demonstrate in primary cortical cell cultures that 17β-estradiol or isoflavones (genistein or daidzein) attenuate Tat1–86-induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERα or ERβ specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERβ selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling.

Conclusions/Significance

Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons.

Backgrounds

We compared the usefulness of fasting plasma glucose (FPG), or hemoglobin A1c (HbA1c), or both in predicting type 2 diabetes.

Methods

This retrospective cohort study investigated 9,322 Japanese adults (4,786 men and 4,536 women), aged 19–69 yrs, free of diabetes at baseline. Usefulness was assessed by predictive values (PV), sensitivity, specificity, and the area under the receiver operating characteristic curve (AUROC) maximised under the best cut-off point.

Results

During the average 6 years of follow-up, 221 men (4.6%) and 92 women (2%) developed diabetes. The best cut-off points for FPG (i.e., 5.67 mmol/l for men and 5.5 mmol/l for women) gave excellent AUROC, and the highest positive PV (13% for men and 9% for women) in predicting diabetes. In high risk subjects with FPG 6.1–6.9 mmol/l, 119 men (26.8%) and 39 women (28.3%) developed diabetes. Under the best cut-off points of FPG 6.39 mmol/l and A1c 5.8, AUROC and positive PV for FPG slightly decreased indicating FPG became less useful and were statistically indistinguishable from those for HbA1c in men. In fact, HbA1c was the most useful in women: HbA1c of 6.0% gave the highest positive likelihood ratio of 2.74 and larger AUROC than did FPG. Although AUROC for HbA1c was acceptable and indistinguishable from that for the combined use, HbA1c had higher specificity and positive LR than did the combined use.

Conclusions

This study demonstrated that FPG was the most useful to predict diabetes in the general population. However, in subjects with FPG 6.1–6.9 mmol/l, FPG became less useful and diagnostic performance of FPG was indistinguishable from that of HbA1c in men whereas HbA1c was the most useful in women. Thus, a two-step screening, measurement of HbA1c in association with FPG, may be useful in predicting diabetes.

Pulmonary fibrosis is characterized by lung fibroblast proliferation and collagen secretion. In lipopolysaccharide (LPS)-induced acute lung injury (ALI), aberrant proliferation of lung fibroblasts is initiated in early disease stages, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) expression in cultured mouse lung fibroblasts using TLR4-siRNA-lentivirus in order to investigate the effects of LPS challenge on lung fibroblast proliferation, phosphoinositide3-kinase (PI3K)-Akt pathway activation, and phosphatase and tensin homolog (PTEN) expression. Lung fibroblast proliferation, detected by BrdU assay, was unaffected by 1 mug/mL LPS challenge up to 24 hours, but at 72 hours, cell proliferation increased significantly. This proliferation was inhibited by siRNA-mediated TLR4 knockdown or treatment with the PI3K inhibitor, Ly294002. In addition, siRNA-mediated knockdown of TLR4 inhibited the LPS-induced up-regulation of TLR4, down-regulation of PTEN, and activation of the PI3K-Akt pathway (overexpression of phospho-Akt) at 72 hours, as detected by real-time PCR and Western blot analysis. Treatment with the PTEN inhibitor, bpV(phen), led to activation of the PI3K-Akt pathway. Neither the baseline expression nor LPS-induced down-regulation of PTEN in lung fibroblasts was influenced by PI3K activation state. PTEN inhibition was sufficient to exert the LPS effect on lung fibroblast proliferation, and PI3K-Akt pathway inhibition could reverse this process. Collectively, these results indicate that LPS can promote lung fibroblast proliferation via a TLR4 signaling mechanism that involves PTEN expression down-regulation and PI3K-Akt pathway activation. Moreover, PI3K-Akt pathway activation is a downstream effect of PTEN inhibition and plays a critical role in lung fibroblast proliferation. This mechanism could contribute to, and possibly accelerate, pulmonary fibrosis in the early stages of ALI/ARDS.

Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of μ-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and μ-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform (tau-441; HEK293/tau441 cells). We examined whether DEX, a synthetic GCs, induces tau phosphorylation and μ-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated μ-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and μ-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation.

IL-7 signalling is important in regulating both survival and cellular size (growth) of T cells. While glucose metabolism has previously been implicated in the mechanism of IL-7 induced survival and growth, the role of amino acids has not before been reported. Here, we show IL-7 dependent T cell survival does not require either exogenous glucose or amino acids. In contrast, maintenance of cell size and IL-7 induced growth were specifically dependent on amino acids. Furthermore, cellular amino acid uptake was implicated in the mechanism of IL-7 induced growth. Analysis of IL-7 regulated gene expression revealed that neutral and cationic amino acid transporters were specific transcriptional targets of IL-7 signalling. In contrast, none of the four glucose transporters expressed in T cells were modulated. Taken together, these data reveal for the first time the central importance of amino acid homeostasis for IL-7 regulated T cell growth.

Background and Aims

Determinants of fatty liver (FL) might be predictive for further deterioration in insulin resistance (IR) in women with previous gestational diabetes (pGDM). The aim was to evaluate the association between pGDM, FL and future manifestation of type 2 diabetes (T2DM) by a detailed pathophysiological characterization early after pregnancy.

Methods

68 pGDM and 29 healthy controls were included 3–6 months after delivery and underwent specific metabolic assessments: status of IR was determined via oral- and intravenous-glucose-tolerance-tests with analysis of proinflammatory factors and kinetics of free-fatty-acids (FFA). According to the fatty-liver-index (FLI), pGDMs were categorized into three groups with low (FLI≤20), intermediate (20

Results

FL was strongly associated with IR in pGDM. pGDM with FLI≥60 showed significantly increased interleukin-6, plasminogen-activator-inhibitor-1, tissue-plasminogen-activator, fibrinogen and increased ultrasensitive-C-reactive-protein compared to the low risk group (FLI≤20). Analysis of FFA indicated a less pronounced decrease of plasma FFA levels during the oral-glucose-tolerance-test in subjects with FLI≥60. History of GDM plus FLI≥60 conferred a high risk for the manifestation of diabetes over 10 years of observation as compared to pGDMs with FLI≤20 (HR:7.85, Cl:2.02–30.5, p = 0.003).

Conclusion

FL is closely linked to GDM, especially to IR and inflammation. Most interestingly, subjects with the highest FLI values showed significant alterations in FFA kinetics and a higher risk to develop T2DM in future.