Two recently identified glucose transporters, GLUT8 and GLUT12, are expressed in human skeletal muscle and may be involved in insulin-mediated sugar transport. Purpose: The purpose of this study was to measure GLUT8 and GLUT12 mRNA levels in endurance-trained versus sedentary individuals in an effort to determine the effect of repeated days of contractile activity on gene expression. Methods: GLUT 4, 8, and 12 mRNA were measured in biopsies from the vastus lateralis using quantitative real-time PCR in endurance-trained (N = 16, age = 22.0 ± 0.9 yr, V̇O2max (L·min−1) = 4.13 ± 0.25) and sedentary (N = 15, age = 21.3 ± 0.8 yr, V̇O2max (L·min−1) = 3.21 ± 0.24) subjects. Results: GLUT12 mRNA was lower (40 ± 14%, P < 0.05) in the exercise-trained compared with the sedentary subjects. There was no difference between groups in GLUT8 mRNA content. mRNA of the insulin-sensitive glucose transporter (GLUT4) was 78 ± 27% (P < 0.05) higher in skeletal muscle from endurance-trained compared with sedentary individuals. Conclusion: These findings suggest an isoform-specific effect on the mRNA of the glucose transporters in human skeletal muscle with repeated days of contractile activity.
Fetal adaptations to high fat (HF) diet in utero (IU) that may predispose to Metabolic Syndrome (MetS) in adulthood include changes in fetal hepatic gene expression. Studies were performed to determine whether maternal exposure to HF diet at different stages during pregnancy had different effects on the fetus, including hepatic gene expression. Female wild type mice were fed either a HF or breeding chow (C) for 2 wks prior to mating. The experimental groups were composed of embryonic day (e) 18.5 fetuses obtained from WT female mice that were fed HF (HF, 35.5% fat) or breeding chow (C, 9.5% fat) for 2 wk before mating until e9.5 of pregnancy (periconception-midpregnancy). At e9.5 dams were switched to the opposite diet (C-HF or HF-C). Exposure to HF diet throughout pregnancy reduced maternal weight gain compared to C diet (p < 0.02 HF vs. C). HF-C dams had significantly decreased adiponectin levels and litter size when compared to C-HF (p < 0.02 HF-C vs C-HF). Independent of the timing of exposure to HF, fetal weight and length were significantly decreased when compared to C diet (HF, C-HF and HF-C vs. C p < 0.02). HF diet during the second half of pregnancy increased expression of genes in the fetal liver associated with fetal growth (C-HF vs C p < 0.001), glucose production (C-HF vs C p < 0.04), oxidative stress and inflammation (C-HF vs C p < 0.01) compared to C diet. This model defines that there are critical periods during gestation in which the fetus is actively shaped by the environment. Early exposure to a HF diet determines litter size while exposure to HF during the second half of pregnancy leads to dysregulation of expression of key genes responsible for fetal growth, hepatic glucose production and oxidative stress. These findings underscore the importance of future studies designed to clarify how these critical periods may influence future risk of developing MetS later in life.
Inhibiting the synthesis of endogenous prostaglandins with nonsteroidal anti-inflammatory drugs exacerbates arterial hypertension. We hypothesized that the converse, i.e., raising the level of endogenous prostaglandins, might have anti-hypertensive effects. To accomplish this, we focused on inhibiting the prostaglandin transporter PGT (SLCO2A1), which is the obligatory first step in the inactivation of several common PGs. We first examined the role of PGT in controlling arterial blood pressure blood pressure using anesthetized rats. The high-affinity PGT inhibitor T26A sensitized the ability of exogenous PGE2 to lower blood pressure, confirming both inhibition of PGT by T26A and the vasodepressor action of PGE2 T26A administered alone to anesthetized rats dose-dependently lowered blood pressure, and did so to a greater degree in spontaneously hypertensive rats than in Wistar-Kyoto control rats. In mice, T26A added chronically to the drinking water increased the urinary excretion and plasma concentration of PGE2 over several days, confirming that T26A is orally active in antagonizing PGT. T26A given orally to hypertensive mice normalized blood pressure. T26A increased urinary sodium excretion in mice and, when added to the medium bathing isolated mouse aortas, T26A increased the net release of PGE2 induced by arachidonic acid, inhibited serotonin-induced vasoconstriction, and potentiated vasodilation induced by exogenous PGE2. We conclude that pharmacologically inhibiting PGT-mediated prostaglandin metabolism lowers blood pressure, probably by prostaglandin-induced natriuresis and vasodilation. PGT is a novel therapeutic target for treating hypertension.
To determine the phase of NUDT15 sequence variants for more comprehensive star (*) allele diplotyping, we developed a novel long-read single-molecule real-time HiFi amplicon sequencing method. A 10.5 kb NUDT15 amplicon assay was validated using reference material positive controls and additional samples for specimen type and blinded accuracy assessment. Triplicate NUDT15 HiFi sequencing of two reference material samples had nonreference genotype concordances of >99.9%, indicating that the assay is robust. Notably, short-read genome sequencing of a subset of samples was unable to determine the phase of star (*) allele-defining NUDT15 variants, resulting in ambiguous diplotype results. In contrast, long-read HiFi sequencing phased all variants across the NUDT15 amplicons, including a *2/*9 diplotype that previously was characterized as *1/*2 in the 1000 Genomes Project v3 data set. Assay throughput was also tested using 8.5 kb amplicons from 100 Ashkenazi Jewish individuals, which identified a novel NUDT15 *1 suballele (c.-121G>A) and a rare likely deleterious coding variant (p.Pro129Arg). Both novel alleles were Sanger confirmed and assigned as *1.007 and *20, respectively, by the PharmVar Consortium. Taken together, NUDT15 HiFi amplicon sequencing is an innovative method for phased full-gene characterization and novel allele discovery, which could improve NUDT15 pharmacogenomic testing and subsequent phenotype prediction.
Exposure to a high-fat (HF) diet in utero is associated with increased incidence of cardiovascular disease, diabetes, and metabolic syndrome later in life. However, the molecular basis of this enhanced susceptibility for metabolic disease is poorly understood. Gene expression microarray and genome-wide DNA methylation analyses of mouse liver revealed that exposure to a maternal HF milieu activated genes of immune response, inflammation, and hepatic dysfunction. DNA methylation analysis revealed 3360 differentially methylated loci, most of which (76%) were hypermethylated and distributed preferentially to hotspots on chromosomes 4 [atherosclerosis susceptibility quantitative trait loci (QTLs) 1] and 18 (insulin-dependent susceptibility QTLs 21). Interestingly, we found six differentially methylated genes within these hotspot QTLs associated with metabolic disease that maintain altered gene expression into adulthood (Arhgef19, Epha2, Zbtb17/Miz-1, Camta1 downregulated; and Ccdc11 and Txnl4a upregulated). Most of the hypermethylated genes in these hotspots are associated with cardiovascular system development and function. There were 140 differentially methylated genes that showed a 1.5-fold increase or decrease in messenger RNA levels. Many of these genes play a role in cell signaling pathways associated with metabolic disease. Of these, metalloproteinase 9, whose dysregulation plays a key role in diabetes, obesity, and cardiovascular disease, was upregulated 1.75-fold and hypermethylated in the gene body. In summary, exposure to a maternal HF diet causes DNA hypermethylation, which is associated with long-term gene expression changes in the liver of exposed offspring, potentially contributing to programmed development of metabolic disease later in life.
Peroxisome proliferatior-activated receptor-γ (PPARγ) is a transcription factor that modulates lipid and glucose metabolism in mammals. The aim of the present study was to investigate whether chicken PPARγ is expressed in tissues in a similar manner to mammalian PPAR and whether it is involved in the regulation of lipid metabolism, particularly in the regulation of fat accumulation in adipose tissue and ovaries. In 30-wk-old chickens, PPARγ mRNA was detected in most tissues that were examined. Of those tissues expressing chicken PPARγ mRNA, the lowest expression levels were found in adipose tissue, the tissue that in mammals was shown to express the highest levels of PPARγ mRNA. Chicken PPARγ mRNA expression in abdominal adipose tissue tended to increase with age, as shown by higher expression levels at 6 wk than at 1 and 2 wk of age. With regard to nutritional modulation, PPARγ mRNA levels in abdominal adipose tissue were significantly higher in broiler chickens fed for 7 d a diet containing 8% safflower oil (18:2-rich) or linseed oil (18:3-rich) compared with chickens fed a diet containing olive oil (18:1-rich). In contrast, feeding a 3% cholesterol-supplemented diet for 7 d resulted in no changes to adipose PPARγ mRNA expression. In broiler chickens orally administered troglitazone, a PPARγ ligand, abdominal fat pad weight and PPARγ and lipoprotein lipase (LPL) mRNA levels were significantly increased relative to those of control chickens. Levels of PPARγ mRNA in liver, skeletal muscle, and ovaries were increased with the onset of egg laying, whereas in adipose tissue the level of PPARγ mRNA was decreased. These findings suggest that PPARγ plays an important role in the regulation of fat deposition and egg production and the characteristic pattern of PPARγ mRNA expression may be indicative of specific differences in the lipid and glucose metabolism of chickens compared with mammals.
Genetic and environmental factors, including the in utero environment, contribute to Metabolic Syndrome. Exposure to high fat diet exposure in utero and lactation increases incidence of Metabolic Syndrome in offspring. Using GLUT4 heterozygous (G4+/−) mice, genetically predisposed to Type 2 Diabetes Mellitus, and wild-type littermates we demonstrate genotype specific differences to high fat in utero and lactation. High fat in utero and lactation increased adiposity and impaired insulin and glucose tolerance in both genotypes. High fat wild type offspring had increased serum glucose and PAI-1 levels and decreased adiponectin at 6 wks of age compared to control wild type. High fat G4+/− offspring had increased systolic blood pressure at 13 wks of age compared to all other groups. Potential fetal origins of adult Metabolic Syndrome were investigated. Regardless of genotype, high fat in utero decreased fetal weight and crown rump length at embryonic day 18.5 compared to control. Hepatic expression of genes involved in glycolysis, gluconeogenesis, oxidative stress and inflammation were increased with high fat in utero. Fetal serum glucose levels were decreased in high fat G4+/− compared to high fat wild type fetuses. High fat G4+/−, but not high fat wild type fetuses, had increased levels of serum cytokines (IFN-γ, MCP-1, RANTES and M-CSF) compared to control. This data demonstrates that high fat during pregnancy and lactation increases Metabolic Syndrome male offspring and that heterozygous deletion of GLUT4 augments susceptibility to increased systolic blood pressure. Fetal adaptations to high fat in utero that may predispose to Metabolic Syndrome in adulthood include changes in fetal hepatic gene expression and alterations in circulating cytokines. These results suggest that the interaction between in utero-perinatal environment and genotype plays a critical role in the developmental origin of health and disease.
Chickens have a blood glucose level that is twice as high as that in most mammals and are regarded as an insulin resistant animal. We previously reported that the major insulin responsive glucose transporter gene, GLUT4, is deficient in broiler chickens, therefore insulin regulation of blood glucose level in chickens is not well understood. In the present study, we characterized gene expressions of GLUT, hexokinase (HK) and glycogen synthase (GS) in insulin-stimulated state in chicken skeletal muscles and cultured chicken myotubes. In a study in vivo, 3-week-old male chickens were injected with insulin (400μg/kg body weight) or 0.9% of NaCl. Skeletal muscles were collected at 1 and 3 h after insulin injection. Expressions of GLUT1, GLUT3, GLUT8, HK I, HK II, GS mRNA and 18S rRNA were determined by Real-Time PCR. GLUT1, GLUT3 and HK II mRNA expression were significantly increased at 3 h after insulin injection in skeletal muscle but no significant increase in GLUT8 and HKI mRNA expression was observed. In a study in vitro, myotubes derived from muscle satellite cells were incubated with serum-free medium for 1 or 3 h in presence or absence of insulin (0, 0.2, 1, 5 μg/ml). GLUT1 and HK II mRNA expression of cultured chicken myotubes at both 1 and 3 h after the incubation were lineally increased with an increase of insulin added to cultures. GLUT3 mRNA expression showed significantly increase at 3 h after incubation with insulin. No significant increase by insulin was observed in expression of HK1 mRNA. Although GLUT1 is not regarded as an insulin-responsive GLUT in mammals, our in vivo and in vitro studies show that insulin stimulates GLUT1 and HK II mRNA expressions in chicken muscles, suggesting that glucose uptake and glucose phosphorylation is regulated in species-specific manner in chicken skeletal muscles.
Lipoperoxide concentration in erythrocytes from workers occupationally exposed to lead (mean blood lead concentration 57.1 (SD 17.6) micrograms/dl) was significantly higher than that in controls. It was not different in plasma from the two groups. The activity of superoxide dismutase (SOD) and catalase in erythrocytes from workers exposed to lead was significantly lower than that of control subjects. The effect of lead was also seen in the glutathione concentration of erythrocytes from lead exposed workers, which was reduced to 69% of that found in erythrocytes from control workers. The increase in methaemoglobin content of erythrocytes from workers exposed to lead was less than expected and not significantly different from that of controls. A positive correlation between lipoperoxide concentration in erythrocytes and lead concentration in blood and a negative correlation between glutathione concentration in erythrocytes and blood lead concentration were found. Incubation of erythrocytes for 24 hours at 37 degrees C in the presence of lead (100 micrograms/dl) produced no changes in glutathione and lipoperoxide concentrations, although there was inhibition of activity of SOD (14.3%), catalase (10.1%), and glutathione peroxidase (35.1%). A similar experiment with heparinised whole blood showed increased haemolysis with no changes in membrane lipid peroxidation of erythrocytes. It is postulated that the lowered concentration of glutathione and decreased activity of SOD, catalase, and glutathione peroxidase in erythrocytes from workers exposed to lead may play a part in the increased membrane lipid peroxidation. Furthermore, the results suggest the possibility that leucocytes, or platelets, or both, may induce haemolysis in the presence of lead.
