Plasma obestatin is lower at fasting and not suppressed by insulin in insulin-resistant humans

Obestatin, a recently discovered 23-amino acid peptide, is involved in the regulation of appetite and body weight in antagonistic fashion to ghrelin, both deriving from a common precursor peptide. Ghrelin was shown to be associated with insulin resistance, which may also affect obestatin. We investigated the association between insulin resistance and plasma concentrations of obestatin and ghrelin in nondiabetic individuals with high (IS; n = 18, 13 females and 5 males, age 47 +/- 2 yr, BMI = 25.5 +/- 0.9 kg/m(2)) and low (IR; n = 18, 12 females and 6 males, age 45 +/- 2 yr, P = 0.49, BMI = 27.5 +/- 1.1 kg/m(2), P = 0.17) insulin-stimulated glucose disposal (M), measured by 2-h hyperinsulinemic (40 mU.min(-1).m(-2)) isoglycemic clamp tests. M(100-120 min) was higher in IS (10.7 +/- 0.7) than in IR (4.4 +/- 0.2 mg.min(-1).kg(-1), P < 10(-9)), whereas insulin-dependent suppression of free fatty acids (FFA) in plasma was reduced in IR (71 +/- 6% vs. IS: 82 +/- 5%, P < 0.02). In both groups, plasma ghrelin concentrations were comparable at fasting and similarly reduced by 24-28% during insulin infusion. IR had lower fasting plasma obestatin levels (383 +/- 26 pg/ml vs. IS: 469 +/- 23 pg/ml, P < 0.02). Clamp insulin infusion reduced plasma obestatin to approximately 81% of basal values in IS (P < 0.00002), but not in IR. Fasting plasma obestatin was correlated positively with M (r = 0.34, P = 0.04), HDL cholesterol (r = 0.45, P = 0.01), and plasma ghrelin concentrations (r = 0.80, P < 0.000001) and negatively with measures of adiposity, plasma FFA during clamp (r = -0.42, P < 0.01), and systolic blood pressure (r = -0.33, P < 0.05). In conclusion, fasting plasma concentrations of obestatin, but not of ghrelin, are reduced in insulin resistance and are positively associated with whole body insulin sensitivity in nondiabetic humans. Furthermore, plasma obestatin is reduced by insulin in insulin-sensitive but not in insulin-resistant persons.     

Polymorphisms in the ABCG5 and ABCG8 genes associate with cholesterol absorption and insulin sensitivity

The roles of polymorphisms of the sitosterolemia genes ABCG5 and ABCG8 in the regulation of cholesterol metabolism and insulin sensitivity were studied in mildly hypercholesterolemic noncoronary subjects (n = 263, 144 men and 119 women) divided into tertiles by baseline serum cholestanol-to-cholesterol ratio (< or = 118.3 and > or = 147.7 10(2) x mmol/mol cholesterol), a surrogate marker of cholesterol absorption efficiency. The lowest cholestanol tertile was associated with high body mass index (BMI), plasma glucose, serum insulin and triglycerides, and cholesterol synthesis markers (cholestenol, desmosterol, lathosterol) and low HDL cholesterol and cholesterol absorption markers (campesterol, sitosterol) (P < 0.01 for all). The 19H allele of the ABCG8 gene accumulated in the lowest cholestanol tertile (P < 0.001) and was associated with low total and LDL cholesterol and absorption markers and with high synthesis markers (P < 0.05 for all). The 604E allele of the ABCG5 gene in men was associated with high BMI, plasma insulin, low serum sitosterol, and high serum cholestenol levels (P < 0.05 for all). In a subgroup of 71 men, the 604E allele was associated with insulin resistance measured with the hyperinsulinemic euglycemic clamp. In conclusion, low cholesterol absorption efficiency was associated with characteristics of the metabolic syndrome. Low serum cholesterol and cholesterol absorption were linked to the D19H polymorphism of the ABCG8 gene, and characteristics of the insulin resistance syndrome in men were linked with the Q604E polymorphism of the ABCG5 gene.     

In vivo 2H2O administration reveals impaired triglyceride storage in adipose tissue of insulin-resistant humans

Indirect evidence suggests that impaired triglyceride storage in the subcutaneous fat depot contributes to the development of insulin resistance via lipotoxicity. We directly tested this hypothesis by measuring, in vivo, TG synthesis, de novo lipogenesis (DNL), adipocyte proliferation, and insulin suppression of lipolysis in subcutaneous adipose tissue of BMI-matched individuals classified as insulin resistant (IR) or insulin sensitive (IS). Nondiabetic, moderately obese subjects with BMI 25–35 kg/m², classified as IR or IS by the modified insulin suppression test, consumed deuterated water (²H₂O) for 4 weeks. Deuterium incorporation into glycerol, palmitate, and DNA indicated TG synthesis, DNL, and adipocyte proliferation, respectively. Net TG synthesis and DNL in adipose cells were significantly lower in IR as compared with IS subjects, whereas adipocyte proliferation did not differ significantly. Plasma FFAs measured during an insulin suppression test were 2.5-fold higher in IR subjects, indicating resistance to insulin suppression of lipolysis. Adipose TG synthesis correlated directly with DNL but not with proliferation. These results provide direct in vivo evidence for impaired TG storage in subcutaneous adipose tissue of IR as compared with IS. Relative inability to store TG in the subcutaneous depot may represent a mechanism contributing to the development of insulin resistance in the setting of obesity.     

Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.     

Does magnesium dysbalance participate in the development of insulin resistance in early stages of renal disease?

We investigated the potential role of magnesium (Mg) dysbalance in the pathogenesis of insulin resistance (IR) in patients with mildly-to-moderately decreased renal function (creatinine: 142.8+/-11.0 mmol/l). The data were compared to those of 8 age- and sex-matched healthy controls (CTRL). The standard oral glucose tolerance test (oGTT) was performed in 61 patients. Twenty-two patients were classified as IR according to their values on fasting and after-load immunoreactive insulin concentrations. Serum and total erythrocyte Mg (tErMg) (atomic absorption spectro-photometry) and free erythrocyte Mg (fErMg) concentrations ((31) P NMR spectroscopy) were determined prior to and two hours after the glucose load. Ten out of 39 insulin-sensitive (IS) patients, but only one out of 22 insulin-resistant (IR) patients, had a low basal fErMg concentration (<162.2 micromol/l, chi2, p<0.01). IR patients had higher serum Mg, total erythrocyte Mg and bound erythrocyte Mg (bErMg) concentrations (both before and after glucose load) when compared with the IS group. Both groups responded to the glucose load with a significant decrease in serum Mg concentration (within the normal range), while the IR group also exhibited a decline in tErMg and bErMg. The mean sum of insulin needed to metabolize the same glucose load correlated positively with tErMg (r=0.545, p<0.01) and bErMg (r=0.560, p<0.01) in the IR patients. It is concluded that, at an early stage of renal dysfunction, IR is not associated with the decline in free erythrocyte Mg concentration, but the magnesium handling in red blood cells is altered.     

S-adenosyl-L-methionine decreases the elevated hepatotoxicity induced by Fas agonistic antibody plus acute ethanol pretreatment in mice

The current study was designed to investigate the effect and potential mechanism of exogenous administration of S-adenosyl-l-methionine (SAM) on the enhanced hepatotoxicity induced by the Fas agonistic Jo2 antibody plus acute ethanol pretreatment in C57BL/6 mice. Acute ethanol plus Jo2 treatment produces liver toxicity under conditions in which ethanol alone or Jo2 alone do not. SAM significantly attenuated this elevated hepatotoxicity in mice as manifested by a decrease of serum aminotransferases and morphological amelioration. Levels of SAM and activity of methionine adenosyltransferase were lowered by the ethanol plus Jo2 treatment but restored by administration of SAM. The ethanol plus Jo2 treatment increased activity and content of CYP2E1, iNOS content and TNF-α levels; these increases were blunted by SAM. SAM also protected against the elevated oxidative and nitrosative stress found after ethanol plus Jo2, likely due to the decreases in CYP2E1, iNOS and TNF-α. Calcium-induced swelling of mitochondria was enhanced by the ethanol plus Jo2 treatment and this was prevented by SAM. JNK and P38 MAPK were activated by the ethanol plus Jo2 treatment; JNK activation was partially prevented by SAM. It is suggested that SAM protects against the ethanol plus Jo2 toxicity by restoring hepatic SAM levels, preventing the increase in iNOS, CYP2E1 and TNF-α and there by lowering the elevated oxidative/nitrosative stress and activation of the JNK signal pathway, ultimately preventing mitochondrial damage.     

Dietary monounsaturated fat in early life regulates IGFBP2: implications for fat mass accretion and insulin sensitivity

The aim of this study was to investigate effects of dietary supplementation with fat or sugar on body composition (BC) and insulin sensitivity (IS) in maturing pigs. Fifty newborn pigs randomized to a control diet or 18% saturated fat (SF), 18% monounsaturated fat (MUF), 18% mixed fat (MF), or 50% sucrose (SUC), from 1 to 16 weeks of age. Outcomes included weight gain, BC (dual energy X-ray absorptiometry, DXA), IS (fasting insulin and hyperinsulinaemic-euglycaemic clamps), fasting Non-Esterified Fatty Acid (NEFA) concentrations, and mRNA expression of genes involved in lipogenesis and IS in skeletal muscle (SM), subcutaneous (SAT), and visceral adipose tissue (VAT). In vitro studies examined direct effects of fatty acids on insulin-like growth factor-binding protein 2 (IGFBP2) mRNA in C2C12 myotubes. While SUC-fed pigs gained most weight (due to larger quantities consumed; P < 0.01), those fed fat-enriched diets exhibited more weight gain per unit energy intake (P < 0.001). Total (P = 0.03) and visceral (P = 0.04) adiposity were greatest in MUF-fed pigs. Whole-body IS was decreased in those fed fat (P = 0.04), with fasting insulin increased in MUF-fed pigs (P = 0.03). SM IGFBP2 mRNA was increased in MUF-fed pigs (P = 0.009) and, in all animals, SM IGFBP2 mRNA correlated with total (P = 0.007) and visceral (P = 0.001) fat, fasting insulin (r = 0.321; P = 0.03) and change in NEFA concentrations (r = 0.285; P = 0.047). Furthermore, exposure of in vitro cultured myotubes to MUF, but not SF, reduced IGFBP2 mRNA suggesting a converse direct effect. In conclusion, diets high in fat, but not sugar, promote visceral adiposity and insulin resistance in maturing pigs, with evidence that fatty acids have direct and indirect effects on IGFBP2 mRNA expression in muscle.     

The relationship between fasting hyperglycemia and insulin secretion in subjects with normal or impaired glucose tolerance

To assess the relationship between the fasting plasma glucose (FPG) concentration and insulin secretion in normal glucose tolerance (NGT) and impaired glucose tolerance (IGT) subjects, 531 nondiabetic subjects with NGT (n = 293) and IGT (n = 238; 310 Japanese and 232 Mexican Americans) received an oral glucose tolerance test (OGTT) with measurement of plasma glucose, insulin, and C-peptide every 30 min. The insulin secretion rate was determined by plasma C-peptide deconvolution. Insulin sensitivity (Matsuda index) was measured from plasma insulin and glucose concentrations. The insulin secretion/insulin resistance (IS/IR) or disposition index was calculated as DeltaISR/DeltaG / IR. As FPG increased in NGT subjects, the IS/IR index declined exponentially over the range of FPG from 70 to 125 mg/dl. The relationship between the IS/IR index and FPG was best fit with the equation: 28.8 exp(-0.036 FPG). For every 28 mg/dl increase in FPG, the IS/IR index declined by 63%. A similar relationship between IS/IR index and FPG was observed in IGT. However, the decay constant was lower than in NGT. The IS/IR index for early-phase insulin secretion (0-30 min) was correlated with the increase in FPG in both NGT and IGT (r = -0.43, P < 0.0001 and r = -0.20, P = 0.001, respectively). However, the correlation between late-phase insulin secretion (60-120 min) and FPG was not significant. In conclusion, small increments in FPG, within the "normal" range, are associated with a marked decline in glucose-stimulated insulin secretion and the decrease in insulin secretion with increasing FPG is greater in subjects with NGT than IGT and primarily is due to a decline in early-phase insulin secretion.     

The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation

Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.     

Insulin signaling is inhibited by micromolar concentrations of H(2)O(2). Evidence for a role of H(2)O(2) in tumor necrosis factor alpha-mediated insulin resistance.

Both hyperglycemia and tumor necrosis factor alpha (TNFalpha) were found to induce insulin resistance at the level of the insulin receptor (IR). How this effect is mediated is, however, not understood. We investigated whether oxidative stress and production of hydrogen peroxide could be a common mediator of the inhibitory effect. We report here that micromolar concentrations of H(2)O(2) dramatically inhibit insulin-induced IR tyrosine phosphorylation (pretreatment with 500 microM H(2)O(2) for 5 min inhibits insulin-induced IR tyrosine phosphorylation to 8%), insulin receptor substrate 1 phosphorylation, as well as insulin downstream signaling such as activation of phosphatidylinositol 3-kinase (inhibited to 57%), glucose transport (inhibited to 36%), and mitogen-activated protein kinase activation (inhibited to 7.2%). Both sodium orthovanadate, a selective inhibitor of tyrosine-specific phosphatases, as well as the protein kinase C inhibitor G?6976 reduced the inhibitory effect of hydrogen peroxide on IR tyrosine phosphorylation. To investigate whether H(2)O(2) is involved in hyperglycemia- and/or TNFalpha-induced insulin resistance, we preincubated the cells with the H(2)O(2) scavenger catalase prior to incubation with 25 mM glucose, 25 mM 2-deoxyglucose, 5.7 nM TNFalpha, or 500 microM H(2)O(2), respectively, and subsequent insulin stimulation. Whereas catalase treatment completely abolished the inhibitory effect of H(2)O(2) and TNFalpha on insulin receptor autophosphorylation, it did not reverse the inhibitory effect of hyperglycemia. In conclusion, these results demonstrate that hydrogen peroxide at low concentrations is a potent inhibitor of insulin signaling and may be involved in the development of insulin resistance in response to TNFalpha.     

Effect of progesterone and/or estradiol-17beta on sperm penetration in vitro of bovine oocytes

The effect of progesterone (P4) and estradiol-17beta (E2) on sperm penetration was evaluated by in vitro fertilization technique. When spermatozoa were treated with modified Tyrode's medium (mTALP) alone, mTALP + 1.0 microg/ml heparin (H), mTALP + 0.1 microg/ml P4, mTALP + 0.1 microg/ml E2, or mTALP + 0.1 microg/ml P4 + 0. 1 microg/ml E2, the percentages of penetrated oocytes were 11% (8/74), 94% (54/60), 19% (12/64), 10% (6/63) and 13% (8/62), respectively. The penetration rates by spermatozoa treated with H, H + P4, H + E2 and H + P4 + E2 were 94% (118/125), 100% (138/138), 95% (129/136), and 94% (100/106), respectively. However, the oocyte penetration rates significantly increased (P < 0.01) 4, 5 and 6 h after insemination, respectively, when the spermatozoa were treated with H + P4, H + E2 and H + P4 + E2 compared with that of the control (H). Cleavage rates also increased significantly (P < 0.01) 24 and 30 h following insemination, respectively, when spermatozoa were treated with H + P4, H + E2 and H + P4 + E2 compared with that of H. Nevertheless, There was no difference in the production of > or = 32 cell stage embryos among the 4 treatments (19% = 28/149, 18% = 32/176, 18% = 23/128 and 22% = 26/120, respectively). These results indicate that the time course of capacitated sperm penetration was accelerated by progesterone and estradiol-17beta but it did not affect subsequent early embryonic development.     

High doses of vitamin E improve mitochondrial dysfunction in rat hippocampus and frontal cortex upon aging

Rat aging from 4 to 12 mo was accompanied by hippocampus and frontal cortex mitochondrial dysfunction, with decreases of 23 to 53% in tissue and mitochondrial respiration and in the activities of complexes I and IV and of mitochondrial nitric oxide synthase (mtNOS) (P < 0.02). In aged rats, the two brain areas showed mitochondria with higher content (35-78%) of oxidation products of phospholipids and proteins and with higher (59-95%) rates of O(2)(-) and H(2)O(2) production (P < 0.02). Dietary supplementation with vitamin E (2.0 or 5.0 g/kg of food) from 9 to 12 mo of rat age, restored in a dose-dependent manner, the decreases in tissue and mitochondrial respiration (to 90-96%) and complexes I and IV and mtNOS activities (to 86-88%) of the values of 4-mo-old rats (P < 0.02). Vitamin E prevented, by 73-80%, the increases in oxidation products, and by 62-68%, the increases in O(2)(-) and H(2)O(2) production (P < 0.05). High resolution histochemistry of cytochrome oxidase in the hippocampal CA1 region showed higher staining in vitamin E-treated rats than in control animals. Aging decreased (19%) hippocampus mitochondrial mass, an effect that was restored by vitamin E. High doses of vitamin E seem to sustain mitochondrial biogenesis in synaptic areas.     

Relative hypoadiponectinemia, insulin resistance, and increased visceral fat in euthyroid prepubertal girls with low-normal serum free thyroxine

A lower activity of the thyroid axis within the clinical reference range is related to a dysmetabolic phenotype in adult populations. We posited that such an association is already present as early as in prepubertal childhood. Serum thyroid stimulating hormone (TSH) and free T4, body fat (bioelectric impedance), insulin resistance (homeostasis model assessment of insulin resistance (HOMA(IR))), total and high molecular weight (HMW)-adiponectin and serum lipids were assessed in 234 euthyroid prepubertal children (113 boys and 121 girls) attending primary care clinics. Visceral fat (abdominal ultrasound) was measured in a subset of these subjects (n = 147; 74 boys and 73 girls). Explants of visceral adipose tissue from an additional six prepubertal children (three boys and three girls) were used to study the regulation of total and HMW-adiponectin by thyroid hormone. Serum free T4 was in girls independently associated with HMW-adiponectin, HOMA(IR) and visceral fat, so that circulating HMW-adiponectin decreased by 30% (β = 0.305 P < 0.005, R(2) = 0.13) and HOMA(IR) and visceral fat increased, respectively, by 90% (β = -0.255 P < 0.01, R(2) = 0.05) and 30% (β = -0.369, P < 0.005, R(2) = 0.12) from the highest to the lowest tertile of serum free T4. Nonsignificant differences in these parameters were found in boys. Treatment of visceral fat explants with thyroid hormone increased total and HMW-adiponectin by 70% and 53%, respectively, above control values (P < 0.01). In conclusion, a dysmetabolic phenotype, consisting of relative hypoadiponectinemia, insulin resistance and increased visceral fat, is associated with low-normal serum free thyroxine in euthyroid prepubertal girls. These associations may be partly explained by a positive regulation of HMW-adiponectin secretion by thyroid hormone.     

Variants at the endocannabinoid receptor CB1 gene (CNR1) and insulin sensitivity, type 2 diabetes, and coronary heart disease

Inhibition of the endocannabinoid receptor CB1 improves insulin sensitivity, lowers glycemia, and slows atherosclerosis. We analyzed whether common variants in the gene encoding CB1, CNR1, are associated with insulin resistance, risk of type 2 diabetes (T2D) or coronary heart disease (CHD). We studied 2,411 participants of the Framingham Offspring Study (mean age 60 years, 52% women) for quantitative traits and CHD, and the Framingham SHARe database for T2D risk. We genotyped 19 single-nucleotide polymorphisms (SNPs) that tagged 85% (at r(2) = 0.8) of common (>5%) CNR1 SNPs. Fasting blood glucose and insulin at the 7th (1999-2001) exam were collected. We used age-, sex-, BMI-adjusted models to test additive associations of genotype with homeostasis model assessment of insulin resistance (HOMA(IR)) (linear mixed-effect models), T2D, or CHD. To account for multiple tests of SNPs, we generated empirical P values. The C allele at SNP rs806365 (frequency, 57.4%), ~4.1 kb 3' from CNR1, was associated with increased HOMA(IR) (n = 2,261, β = 0.05 per C, empirical P = 0.01), risk of T2D (674 cases, odds ratio = 1.19 per C, nominal P = 0.01) and CHD (237 cases, hazard ratio = 1.23 per C, nominal P = 0.04). The association of rs806365 with HOMA(IR) was replicated in a meta-analysis of two independent cohorts (National Health and Nutrition Examination Survey III genetic cohort (NHANES-III) plus Partners Case-Control Diabetes Study; 2,540 white individuals, β = 0.037, nominal P = 0.007), but not in the large Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) Consortium (n = 29,248, nominal P = 0.74). The association of rs806365 was not replicated either with T2D in Diabetes Genetics Replication and Meta-analysis (DIAGRAM) (n = 10,128, nominal P = 0.31), or with CHD in PROCARDIS (n = 13,614, nominal P = 0.37). Although supported by initial results, we found no reproducible statistical association of common variation at CNR1 with insulin resistance, T2D, or CHD.     

Effects of follicle-stimulating hormone with and without luteinizing hormone on serum hormone concentrations, follicle growth, and intrafollicular estradiol and aromatase activity in gonadotropin-releasing hormone-immunized heifers

To evaluate the roles of FSH and LH in follicular growth, GnRH-immunized anestrous heifers (n = 17) were randomly assigned (Day 0) to one of three groups (n = 5 or 6). Group 1 received i.m. injections of 1.5 mg porcine FSH (pFSH) 4 times/day for 2 days; group 2 received i.v. injections of 150 microg pLH 6 times/day for 6 days; group 3 received both pFSH and pLH as described for groups 1 and 2. After slaughter on Day 6, measurements were made of follicle number and size, and follicular fluid concentrations of progesterone (P(4)), estradiol (E(2)), and aromatase activity. Injection of pFSH increased (P: < 0.01) the serum concentrations of FSH between 12 and 54 h. Infusion of pLH increased (P: < 0.05) mean and basal concentrations of LH and LH pulse frequency. Serum E(2) concentrations were higher (P: < 0.05) for heifers given pFSH + pLH than those given either pFSH or pLH alone. There was no difference (P: > or = 0.24) between treatments in the number of small follicles (<5 mm). Heifers given pFSH or pFSH + pLH had more (P: < or = 0.02) medium follicles (5.0-9.5 mm) than those that were given pLH alone (none present). Heifers given pFSH + pLH had more (P: = 0.04) large follicles (> or =10 mm) than those given either pLH or pFSH alone (none present). Overall, only 1 of 35 small follicles and 2 of 96 medium follicles were E(2)-active (i.e., E(2):P(4) >1.0), whereas 18 of 21 large follicles (all in the pFSH + pLH treatment) were E(2)-active; of these, 8 of 18 had aromatase activity. Concentrations of E(2) and E(2) activity in follicular fluid were correlated (r > or = 0.57; P: < 0.0001) with aromatase activity in heifers given pLH + pFSH. In conclusion, pLH failed to stimulate follicle growth greater than 5 mm; pFSH stimulated growth of medium follicles that were E(2)-inactive at slaughter and failed to increase serum E(2) concentrations; whereas pFSH + pLH stimulated growth of medium follicles and E(2)-active large follicles, and a 10- to 14-fold increase in serum E(2) concentrations.     

Longitudinal changes in energy expenditure and body composition in obese women with normal and impaired glucose tolerance

Our primary objective was to evaluate changes in energy expenditure and body composition in women with normal glucose tolerance (NGT) and gestational diabetes mellitus (GDM). A secondary objective was to examine the relationship between maternal leptin and nutrient metabolism. Fifteen obese women, eight with NGT and seven with GDM, were evaluated before conception (P), at 12-14 wk (E), and at 34-36 wk (L). Energy expenditure and glucose and fat metabolism were measured using indirect calorimetry. Basal hepatic glucose production was measured using [6,6-2H2]glucose and insulin sensitivity by euglycemic clamp. There was a significant increase (6.6 kg, P = 0.0001) in fat mass from P to L. There was a 30% (P = 0.0001) increase in basal O2 consumption (VO2, ml/min). There were no significant changes in carbohydrate oxidation during fasting or storage from P to L. There was, however, a significant (P = 0.0001) 150% increase in basal fat oxidation (mg/min) from P to L. Under hyperinsulinemic conditions, there were similar 25% increases in VO2 (P = 0.0001) from P to L in both groups. Because of the significant increases in insulin resistance from P to L, there was a significant (P = 0.0001) decrease in carbohydrate oxidation and storage. There was a net change from lipogenesis to lipolysis, i.e., fat oxidation (30-40 mg/min, P = 0.0001) from P to L. Serum leptin concentrations had a significant positive correlation with fat oxidation at E (r = 0.76, P = 0.005) and L (r = 0.72, P = 0.009). Pregnancy in obese women is associated with significant increases in fat mass and basal metabolic rate and an increased reliance on lipids both in the basal state and during the clamp. These modifications are similar in women with NGT and GDM. The increased reliance on fat metabolism is accompanied by a concomitant decrease in carbohydrate metabolism during hyperinsulinemia. The increase in fat oxidation may be related to increased maternal serum leptin.     

A study of the metabolism of apolipoprotein B100 in relation to insulin resistance in African American males.

The purpose of this study was to determine the relationship between insulin resistance and apoB100 metabolism in African American males. Fifteen subjects, 33 +/- 7.6 years old, were divided into two groups, insulin-resistant (IR) or insulin-sensitive (IS), based on the sum of the plasma insulin concentrations during an oral glucose tolerance test. The IR group (n = 8) differed significantly from the IS group (n = 7) with respect to body mass index (BMI) (30.1 vs 23.1 kg/m2; P = 0.0003), fasting triglycerides, (118 vs 54 mg/dl, P = 0. 013), and total plasma apolipoprotein B100 (80 vs 59 mg/dl, P = 0.014). Significantly elevated apoB100 levels in the IR group were seen in very low density lipoprotein (VLDL) (5.1 vs 3.4 mg/dl, P = 0.045) and intermediate density lipoprotein (IDL) (18 vs 12 mg/dl, P = 0.017) but not in low density lipoprotein (LDL) (57 vs 46 mg/dl, P = 0.19). Total cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apolipoprotein A-I, and blood pressure were not significantly different between the two groups. There was a high correlation between the sum of insulins during the oral glucose tolerance test and the BMI (rho = 0.88, P = 0.0001). In five IR and five IS subjects, apoB100 kinetics were determined in the fasting state using a bolus dose of deuteroleucine and multicompartmental modeling. IR subjects had significantly lower fractional catabolic rates (FCR) in the larger VLDL1 (-70%), the smaller VLDL2 (-71%), and the IDL (-53%) fractions. No significant differences in production rates were observed for any lipoprotein class. There was a significant correlation between the sum of insulins and the FCR of the apoB100 of VLDL1 (rho = -0.65, P = 0.05) and of IDL (rho = -0.85, P = 0.004). The correlation coefficient of the sum of insulins and the FCR of VLDL2 was -0.61 with P = 0.067. We conclude that in this population of African American males, IR is correlated with a decreased FCR of apoB100 in VLDL and IDL and elevated plasma levels of apoB and triglycerides (TG). These changes might be explained by decreased clearance of the TG-rich lipoproteins. We postulate that this may reflect decreased lipoprotein and/or hepatic lipase activity related to insulin resistance and its association with obesity.     

Decreased islet sensitivity to insulin in hamsters with dietary-induced insulin resistance

In the present study, we evaluated the autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from hamsters with insulin resistance (IR) induced by administration of a sucrose-rich diet (SRD) during 5 weeks. We used an approach of two metabolic pathways (glucose oxidation and utilization) based on the measurement of 14CO2 and 3H2O production from D-[U-14C]-glucose and D-[5-(3)H]-glucose, respectively, in isolated islets incubated with 3.3 and 16.7 mM glucose alone, or with 5 or 15 mU/ml insulin, anti-insulin guinea-pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured by radioimmunoassay in islets incubated with 3.3 or 16.7 mM glucose, with or without 75, 150, and 300 nM wortmannin. Results showed that the stimulatory effect of insulin upon 14CO2 and 3H2O production in control islets was not observed in SRD islets. Addition of anti-insulin serum, nifedipine or wortmannin to the medium with 16.7 mM glucose decreased 14CO2 and 3H2O production in control but not in SRD islets. Whereas wortmannin did not decrease insulin release induced by 16.7 mM glucose in SRD hamsters, it did in controls. We can conclude that the autocrine stimulatory effect of insulin upon glucose metabolism observed in normal islets is attenuated or even absent in islets from IR animals. Such decreased islet sensitivity to insulin did not prevent the compensatory secretion of insulin from maintaining glucose homeostasis, suggesting that, at least in this model, the islets can put forward alternative mechanisms to overcome such defect.     

Effects of metformin on the expression of GLUT4 in endometrium of obese women with polycystic ovary syndrome

The objective was to explore the effects of metformin on the expression of endometrial glucose transporter 4 (GLUT4) and analyze the related factors of GLUT4 in patients with polycystic ovary syndrome (PCOS). This study included 20 obese patients with PCOS (PCOS group) and 20 obese patients who had infertility caused by oviducal or pelvic factors but had no PCOS (control group). Follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol-2 (E(2)), testosterone (T), fasting serum glucose (FSG), fasting insulin serum (FINS), homeostasis model assessment-insulin resistance (HOMA-IR), and endometrial GLUT4 expression were determined in the two groups. In PCOS group, patients were given 500 mg of metformin three times per day for 3 mo, and then the parameters above were determined again and compared with that before metformin treatment. The parameters above also were compared between PCOS and control groups. The correlation of GLUT4 with its related factors was analyzed. The levels of T, FINS, and HOMA-IR were higher in PCOS group than in the control group (P < 0.01). The levels of protein and mRNA of endometrial GLUT4 were lower in the PCOS group than in the control group (P < 0.001). The expression of protein and mRNA of endometrial GLUT4 increased after metformin treatment (P < 0.001). HOMA-IR was negatively correlated with GLUT4 expression (P = 0.027). In patients with PCOS, the levels of protein and mRNA of endometrial GLUT4 were lower compared with that in non-PCOS women, and HOMA-IR was strongly associated with endometrial GLUT4 expression. Metformin may up-regulate endometrial GLUT4 expression to improve endometrial IR.