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The discovery of genetic mechanisms for resistance to obesity and diabetes may illuminate new therapeutic strategies for the treatment of this global health challenge. We used the polygenic 'lean' mouse model, which has been selected for low adiposity over 60 generations, to identify mitochondrial thiosulfate sulfurtransferase (Tst; also known as rhodanese) as a candidate obesity-resistance gene with selectively increased expression in adipocytes. Elevated adipose Tst expression correlated with indices of metabolic health across diverse mouse strains. Transgenic overexpression of Tst in adipocytes protected mice from diet-induced obesity and insulin-resistant diabetes. Tst-deficient mice showed markedly exacerbated diabetes, whereas pharmacological activation of TST ameliorated diabetes in mice. Mechanistically, TST selectively augmented mitochondrial function combined with degradation of reactive oxygen species and sulfide. In humans, TST mRNA expression in adipose tissue correlated positively with insulin sensitivity in adipose tissue and negatively with fat mass. Thus, the genetic identification of Tst as a beneficial regulator of adipocyte mitochondrial function may have therapeutic significance for individuals with type 2 diabetes N. Morton was supported by a Career Development Fellowship, Institutional Strategic Support Fund award and a New Investigator Award from the Wellcome Trust (100981/Z/13/Z), a Research Councils UK Fellowship and a British Heart Foundation Centre of Research Excellence exchange award. We thank the Slovenian Research Agency (core funding P4-0220, project N5-0003 Syntol and J4-6804 to S.H.); and a Young Scientist Fellowship to J. Beltram. We acknowledge support of the British Heart Foundation Research Excellence Award in support of the Bioinformatics Core contribution. T. Stulnig received funding from the Federal Ministry of Economy, Family and Youth and the Austrian National Foundation for Research, Technology and Development. G. Churchill was supported by the US National Institutes of Health grant R01GM 070683. J.M. Fernandez-Real acknowledges funding from FIS PI11/00214. A. Vidal-Puig was funded by the UK Medical Research Council (MRC) MDU, an MRC Programme grant, MRC DMC Core and MITIN (HEALTH-F4-2008-223450)

Background The maternal hypothalamic-pituitary-adrenal-axis (HPAA) undergoes dramatic activation during pregnancy. Increased cortisol and corticotrophin-releasing-hormone (CRH) associate with low birthweight and preterm labor. In non-pregnant obesity, the HPAA is activated but circulating cortisol levels are normal or lower than in lean women. We hypothesized that maternal cortisol levels would be lower in obese pregnancy, and would associate with increased fetal size and length of gestation. Method Fasting serum cortisol was measured at 16, 28 and 36 weeks gestation and at 3–6 months postpartum in 276 severely obese and 135 lean women. In a subset of obese ( n = 20) and lean ( n = 20) we measured CRH, hormones that regulate bioavailable cortisol (corticosteroid-binding-globulin, estradiol, estriol, and progesterone). Urinary glucocorticoid metabolites were measured in pregnant (obese n = 6, lean n = 5) and non-pregnant (obese n = 7, lean n = 7) subjects. Results Maternal cortisol and HPAA hormones were lower in obese pregnancy. Total urinary glucocorticoid metabolites increased significantly in lean pregnancy, but not in obese. Lower maternal cortisol in obese tended to be associated with increased birthweight ( r = −0.13, p = 0.066). In obese, CRH at 28 weeks correlated inversely with gestational length ( r = −0.49, p = 0.04), and independently predicted gestational length after adjustment for confounding factors (mean decrease in CRH of −0.25 pmol/L (95% CI −0.45 to −0.043 pmol/L) per/day increase in gestation). Conclusion In obese pregnancy, lower maternal cortisol without an increase in urinary glucocorticoid clearance may indicate a lesser activation of the HPAA than in lean pregnancy. This may offer a novel mechanism underlying increased birthweight and longer gestation in obese pregnancy.

Nat. Med. 22, 771–779 (2016); published online 6 June 2016; corrected after print 7 March 2018 In the version of this article initially published, the colors of the lines were switched in the graph that shows the glucose infusion rates for wild-type mice and Adipoq-Tst transgenic mice in Figure 3b. The top line should be purple, and the bottom line should be black.

Selenium (Se), a micronutrient essential for human health, is incorporated into at least 25 selenoproteins including selenoprotein P (SePP), which transports Se within the body. This research identified two single nucleotide polymorphisms (SNPs) in the SePP gene, one in the coding region (position 24731, causing an Ala to Thr change) and one in the 3'untranslated region (position 25191). Their frequency was similar in Caucasian, Chinese, and South Asian populations. Prospectively genotyped volunteers were supplemented for 6 wk with 100 microg sodium selenite/day. Blood samples were analyzed for plasma Se and selenoprotein biomarkers at baseline, after supplementation, and during a washout period. Plasma Se, SePP, and glutathione peroxidase 3 (GPx3) levels increased with supplementation. Baseline plasma Se content depended on both SePP genotypes and body mass index. Presupplementation SePP concentration was associated with gender and genotype at SNP 24731 and postsupplementation concentration with SNP 25191. Both SNPs and gender were associated with differences in GPx3 activity, plasma, and erythrocyte thioredoxin reductase 1 concentrations and lymphocyte glutathione peroxidase 1 and 4 activities and concentrations. In conclusion, the data reveal two common functional SNPs within the human SePP gene that may predict behavior of biomarkers of Se status and response to supplementation and thus susceptibility to disease.

Damage to the endothelium by reactive oxygen species favours atherogenesis. Such damage can be prevented by selenium, which is thought to exert its actions through the expression of selenoproteins. The family of glutathione peroxidases (GPXs) may have antioxidant roles in the endothelium but other intracellular and extracellular selenoproteins with antioxidant actions may also be important. The selenoproteins expressed by cultured human umbilical-vein endothelial cells (HUVECs) were labelled with [75Se]selenite and separated using SDS/PAGE. HUVECs secreted no extracellular selenoproteins. There were distinct differences between the intracellular selenoprotein profile of 75Se-labelled HUVECs and those of other tissues. A single selenoprotein with a molecular mass of 58 kDa accounted for approx. 43% of the intracellular 75Se-labelled proteins in HUVECs. This protein was identified by Western blotting as the redox-active lipid-hydroperoxide-detoxifying selenoprotein, thioredoxin reductase (TR). TR expression in HUVECs was down-regulated by transiently exposing cells to the phorbol ester PMA for periods as short as 1 min. However, there was a delay of 48 h after PMA exposure before maximal down-regulation of TR was observed. The protein kinase C (PKC) inhibitor bisindolylmaleimide I hydrochloride had no effect on TR expression when added alone, but the agent prevented the down-regulation of TR expression seen with PMA. The calcium ionophore A23187 increased TR expression in HUVECs after a 12-h exposure, but the maximal effect was only observed after a 35-h exposure. These findings suggest that TR may be an important factor in the known ability of Se to protect HUVECs from peroxidative damage. Furthermore, the results also suggest that TR expression can be negatively regulated through PKC. It is possible that TR expression may be positively regulated by the calcium-signalling cascade, although TR induction by A23187 may be due to toxicity.

Human thyrocytes incubated with the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10−5–10−8 mol/L) and the calcium ionophore A23187 (10−5–10−8 mol/L) showed a marked increase in the expression of a 57-kDa selenoprotein identified as thioredoxin reductase (TR). After the addition of A23187 with PMA, a significant induction in TR expression was observed after 6 h, with maximal induction occurring by 24 h. The addition of 8-bromo-cAMP (10−4 mol/L) or TSH (10 U/L) alone had no effect on TR expression, nor did these agents influence the induction of TR brought about by the addition of A23187 and PMA. These data show that the calcium-phosphoinositol second messenger cascade that controls hydrogen peroxide generation in the human thyrocyte is also an important stimulator of TR expression. The role of TR in the thyrocyte is unclear, but the selenoenzyme has a high capacity to detoxify compounds, such as hydrogen peroxide and lipid hydroperoxides, that are produced in high concentration during thyroid hormo...

The generation of reactive oxygen species has been implicated as part of the mechanism responsible for UVB-radiation-induced skin damage. In mice, evidence suggests that increased dietary selenium intake may protect skin from many of the harmful effects of UVB radiation. We sought to determine the selenoprotein profile of cultured human skin cells and whether selenium supplementation could protect keratinocytes and melanocytes from the lethal effects of UVB radiation. Labelling experiments using [75Se]selenite showed qualitative and quantitative differences in selenoprotein expression by human fibroblasts, keratinocytes and melanocytes. This was most noticeable for thioredoxin reductase (60 kDa) and phospholipid glutathione peroxidase (21 kDa); these proteins were identified by Western blotting. Despite these differences, we found that a 24 h preincubation with sodium selenite or selenomethionine protected both cultured human keratinocytes and melanocytes from UVB-induced cell death. With primary keratinocytes, the greatest reduction in cell death was found with 10 nM sodium selenite (79% cell death reduced to 21.7%; P< 0.01) and with 50 nM selenomethionine (79% cell death reduced to 13.2%; P< 0.01). Protection could be obtained with concentrations as low as 1 nM with sodium selenite and 10 nM with selenomethionine. When selenium was added after UVB radiation, little protection could be achieved, with cell death only being reduced from 88.5% to about 50% with both compounds. In all of the experiments sodium selenite was more potent than selenomethionine at providing protection from UVB radiation.

Dietary isothiocyanates and selenium are chemopreventive agents and potent inducers of antioxidant enzymes. It has been previously shown that sulforaphane and selenium have a synergistic effect on the upregulation of thioredoxin reductase-1 (TrxR-1) in human hepatoma HepG2 cells. In this paper, further evidence is presented to show that sulforaphane and selenium synergistically induce TrxR-1 expression in immortalised human hepatocytes. Sulforaphane was found to be more toxic toward hepatocytes than HepG2 cells with IC50=25.1 and 56.4 μM, respectively. Sulforaphane can protect against hydrogen peroxide-induced cell death and this protection was enhanced by co-treatment with selenium. Using siRNA to knock down TrxR-1 or Nrf2, sulforaphane (5 μM)-protected cell viability was reduced from 73% to 46% and 34%, respectively, suggesting that TrxR-1 is an important enzyme in protection against hydrogen peroxide-induced cell death. Sulforaphane-induced TrxR-1 expression was positively associated with significant levels of Nrf2 translocation into the nucleus, but co-treatment with selenium showed no significant increase in Nrf2 translocation. Moreover, MAPK (ERK, JNK and p38) and PI3K/Akt signalling pathways were found to play no significant role in sulforaphane-induced Nrf2 translocation into the nucleus. However, blocking ERK and JNK signalling pathways decreased sulforaphane-induced TrxR-1 mRNA by about 20%; whereas blocking p38 and PI3K/AKT increased TrxR-1 transcription. In summary, a combination of sulforaphane and selenium resulted in a synergistic upregulation of TrxR-1 that contributed to the enhanced protection against free radical-mediated oxidative damage in human hepatocytes.

Background Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and hepatic steatosis. Non-alcoholic steatohepatitis (NASH) is a serious consequence of NAFLD where chronic tissue damage and inflammation result in fibrosis which may progress to cirrhosis. Transforming growth factor β1 (TGFβ1), proinflammatory cytokines and oxidative stress are thought to play crucial roles in the pathogenesis of these conditions. The contributions of individual liver cell types to fibrogenesis remain controversial and the influence of selenium status has not been investigated. Methods In this study we have used a cell culture model of fat-loading using oleate-treated human hepatoblastoma (C3A) cells to investigate how fat-loading and selenium status might influence the production of collagen in response to TGFβ1. The secretion of inflammatory cytokines was also investigated, together with the epithelial character of the treated cells. Results We found that in response to treatment with TGFβ1, C3A cells produced mRNA encoding the pro-αI chain of procollagen I, secreted procollagen I peptide, up-regulated production of the proinflammatory cytokine interleukin-8 (IL-8) and the mesenchymal marker vimentin, and down-regulated albumin production. Most of these responses were considerably enhanced when cells were fat-loaded with oleate and were attenuated by selenium addition at a dose that optimised the expression of thioredoxin reductase and glutathione peroxidase. Conclusions Our data establish that both fat-loading and suboptimal selenium status enhance collagen and IL-8 production by C3A hepatocytes in response to TGFβ1, possibly as part of an epithelial to mesenchymal transition. General significance These findings suggest that the hepatocyte may be an important contributor to the pathogenesis of fibrosis associated with NAFLD.

3beta-Hydroxysteroid dehydrogenase (3betaHSD) is a key enzyme in the synthesis of bioactive steroid hormones. Objectives of the present study were to clone canine 3betaHSD and to investigate its expression in dog corpora lutea (CL) covering the periods of their formation, early and late regression (days 5, 15, 25, 35, 45, 65 after ovulation). Complete complementary DNA sequence was amplified by RACE PCR. Subsequent cloning revealed that the canine ovarian 3betaHSD transcript was composed of a 5'-untranslated region (5'-UTR) of 126 nucleotides, an open reading frame (ORF) of 1122 nucleotides and a 3'-UTR of 441 nucleotides. The putative ORF encoded a 374 amino acid protein which remains highly conserved (79-85% identity) between species. The transient expression of the cloned canine 3betaHSD in a mammalian heterologous cell expression system (HEK293T cells) identified the 3betaHSD activity as the only activity of this canine enzyme (absence of any detectable 17-hydroxysteroid dehydrogenase activity). Qualitative RT-PCR revealed expression of 3betaHSD on all days investigated and the signals were strongest on days 5 and 15, with day 25 intensity tending to decrease. However, variability between individual animals was high. The significant decrease in the expression of 3betaHSD towards the end of diestrus as indicated by Real Time PCR (p

The human endothelial cell line EAhy926 was used to determine the importance of selenium in preventing oxidative damage induced by tert-butyl hydroperoxide (tert-BuOOH) or oxidised low density lipoprotein (LDLox). In cells grown in a low selenium medium, tert-BuOOH and LDLox killed cells in a dose-dependent manner. At 555 mg/l LDLox or 300 microM tert-BuOOH,80% of cells were killed after 20 h. No significant cell kill was achieved by these agents if cells were pre-incubated for 48 h with 40 nM sodium selenite, a concentration that maximally induced the activities of cytoplasmic glutathione peroxidase (cyGPX; 5.1-fold), phospholipid hydroperoxide glutathione peroxidase (PHGPX;1.9-fold) and thioredoxin reductase (TR; 3.1-fold). Selenium-deficient cells pre-treated with 1 microM gold thioglucose (GTG) (a concentration that inhibited 25% of TR activity but had no inhibitory effect on cyGPX or PHGPX activity) were significantly (P0.05) more susceptible to tert-BuOOH toxicity (LC(50) 110 microM) than selenium-deficient cells (LC(50) 175 microM). This was also the case for LDLox. In contrast, cells pre-treated with 40 nM selenite prior to exposure to GTG were significantly more resistant to damage from tert-BuOOH and LDLox than Se-deficient cells. Treatment with GTG or selenite had no significant effect on intracellular total glutathione concentrations. These results suggest that selenium supplementation, acting through induction of TR and GPX, has the potential to protect the human endothelium from oxidative damage.

Background Increased hypothalamic-pituitary-adrenal (HPA) axis activity has been associated with low birthweight and preterm labour. Maternal obesity is associated with macrosomia (birthweight ≥4000 g) and prolonged pregnancy (≥41 weeks). We hypothesised altered HPA axis activity may underlie these pregnancy outcomes and aimed to characterise HPA axis hormones in obese pregnancy. Method Fasting serum cortisol was measured by radioimmunoassay at 16, 28 and 36 weeks in 286 obese (BMI 44.05 ± 3.98 kg/m 2 ) and 137 lean (BMI 22.71 ± 1.66 kg/m 2 ) pregnant women. In subsets (n = 20 obese, 20 lean) we measured corticosteroid-binding-globulin (CBG) and corticotrophin-releasing-hormone (CRH) by radioimmunoassay. Free cortisol was calculated using Coolen’s equation. Results Cortisol was significantly lower throughout pregnancy in obese (Table 1), and lower free cortisol at 16 weeks was associated with higher birthweight (r = -0.46, p Conclusion Decreased HPA axis activity in obese pregnancy may underlie macrosomia and prolonged pregnancy.

The developmental expression of the alpha, mu and pi class glutathione S-transferases has been defined in human liver using radioimmunoassay and immunohistochemistry. Expression of alpha and mu class isoenzymes increased significantly at birth, while that of the pi isoenzyme declined during the first trimester. Mu-class isoenzymes (GST1 1, GST1 2, GST1 2-1) were expressed in hepatocytes but not in other liver cell types.

The relative value of fructosamine as an alternative to glycosylated hemoglobin (HbA1) and other measures of glycemic control was assessed in 100 insulin-dependent (IDDM) and 104 non-insulin-dependent (NIDDM) diabetic patients. We measured HbA1 (by electrophoretic and affinity methods), plasma glucose, glycosylated plasma proteins, and fructosamine in blood taken at a single clinic visit. The values were compared both by correlation analysis and by considering whether the various indices of glycemic control placed the patients in the same clinical decision categories as they were in by the HbA1 (affinity) result. Fructosamine correlated moderately well with HbA1 (affinity; r = .8) and placed 71% of IDDM and 72% of NIDDM patients in the same clinical category of good, moderate, or poor control. Differences can probably be partly attributed to the different periods over which HbA1 and fructosamine reflect average glycemia and partly to imprecision.

A method is described for the measurement of glycosylated albumin in human plasma. Purification of albumin is achieved by a simple one-step chromatographic method, adapted for use as a batch analysis technique. The glycosylated albumin fraction is subsequently measured by a modification of the fructosamine assay (alkaline reduction of nitroblue tetrazolium). The method correlates with the plasma fructosamine assay (r = 0.85; p less than 0.001) and with an electrophoretic method for the measurement of HbA1 (r = 0.78; p less than 0.001).