Improved menstrual function in obese women with polycystic ovary syndrome after behavioural modification intervention-A randomized controlled trial.

OBJECTIVE: Lifestyle intervention is the recommended first-line treatment for overweight women with polycystic ovary syndrome (PCOS). However, the efficacy of lifestyle change in improving reproductive function is still unclear. DESIGN: A randomized controlled trial (RCT) with allocation to a behavioural modification programme (intervention) or minimal intervention (control) for 4 months with a follow-up at 12 months. PATIENTS: Sixty-eight women, aged 18-40 years, body mass index (BMI) ≥ 27 kg/m2 , fulfilling all Rotterdam PCOS criteria were randomized to treatment. MEASUREMENTS: The primary outcome was improved menstrual regularity. Secondary outcomes were ovulation and pregnancy rates. RESULTS: At 4 months, the weight loss was significant in the intervention group (-2.1%, P = 0.002) and nonsignificant in the control group (-1.0%). A higher proportion of patients in the intervention group improved menstrual regularity compared to the control group, mean difference 35% (95% CI: 16-60), P = 0.003. There was no difference in ovulation rate between groups. Logistic regression analysis showed that intervention was the only predictor of improved menstrual function, OR 3.9 (95% CI: 1.3-11.9). At 12 months, a total of 54% of the women improved menstrual regularity compared to baseline (P = 0.000) and 43% (P = 0.000) had confirmed ovulation. 38% of the women wishing to become pregnant succeeded within 1 year of study completion. CONCLUSIONS: This is the first RCT in overweight women with PCOS showing efficacy in improving reproductive function following behavioural modification intervention in comparison with minimal intervention. Although extensive weight loss is difficult to achieve in these women, behavioural modification intervention can help improve reproductive function.

Effects of oestrogen and testosterone therapy on serum metabolites in postmenopausal women.

OBJECTIVES: Metabolite profiles of body fluids or tissue extracts can be regarded as important indicators of physiological or pathological states. Whether hormone-specific alterations of the serum metabolome can be identified using this technique has not been tested yet. The aim of this study was to investigate metabolic responses during hormone therapy in postmenopausal women by a nontargeted metabolomics approach. METHODS: Sixty naturally postmenopausal women were randomly assigned to treatment with testosterone undecanoate 40 mg every second day; estradiol valerate 2 mg daily; or the combination of both. Serum metabolites were determined by gas chromatography-mass spectrometry (GC-MS) before and after 3 months of treatment. Metabolites affected by the treatment were identified and correlated with changes in insulin sensitivity and lipid profiles. RESULTS: Treatment-dependent and hormone-specific effects on serum metabolites were observed, ranging between 69% reduction and 184% increase, but the metabolites that best explained the differences could not be structurally identified. Effects on annotated metabolites were less associated with clinical parameters as compared to established serum markers for adverse lipid and carbohydrate metabolism, such as cholesterol and triglycerides. However, cystine, lysine and tyrosine were shown to change in correlation with insulin sensitivity and high-density lipoprotein cholesterol levels in response to testosterone, indicating that those responses were somehow related to each other. CONCLUSIONS: Oestrogen- and androgen-specific alterations in the serum metabolome could be identified using GC-MS, reflecting hormone-specific effects on whole body metabolism. New knowledge regarding steroid-mediated metabolic responses within different tissues might be obtained using a similar approach on tissue extracts.

Sex-different hepaticglycogen content and glucose output in rats.

BACKGROUND: Genes involved in hepatic metabolism have a sex-different expression in rodents. To test whether male and female rat livers differ regarding lipid and carbohydrate metabolism, whole-genome transcript profiles were generated and these were complemented by measurements of hepatic lipid and glycogen content, fatty acid (FA) oxidation rates and hepatic glucose output (HGO). The latter was determined in perfusates from in situ perfusion of male and female rat livers. These perfusates were also analysed using nuclear magnetic resonance (NMR) spectroscopy to identify putative sex-differences in other liver-derived metabolites. Effects of insulin were monitored by analysis of Akt-phosphorylation, gene expression and HGO after s.c. insulin injections. RESULTS: Out of approximately 3 500 gene products being detected in liver, 11% were significantly higher in females, and 11% were higher in males. Many transcripts for the production of triglycerides (TG), cholesterol and VLDL particles were female-predominant, whereas genes for FA oxidation, gluconeogenesis and glycogen synthesis were male-predominant. Sex-differences in mRNA levels related to metabolism were more pronounced during mild starvation (12 h fasting), as compared to the postabsorptive state (4 h fasting). No sex-differences were observed regarding hepatic TG content, FA oxidation rates or blood levels of ketone bodies or glucose. However, males had higher hepatic glycogen content and higher HGO, as well as higher ratios of insulin to glucagon levels. Based on NMR spectroscopy, liver-derived lactate was also higher in males. HGO was inhibited by insulin in parallel with increased phosphorylation of Akt, without any sex-differences in insulin sensitivity. However, the degree of Thr172-phosphorylated AMP kinase (AMPK) was higher in females, indicating a higher degree of AMPK-dependent actions. CONCLUSIONS: Taken together, males had higher ratios of insulin to glucagon levels, higher levels of glycogen, lower degree of AMPK phosphorylation, higher expression of gluconeogenic genes and higher hepatic glucose output. Possibly these sex-differences reflect a higher ability for the healthy male rat liver to respond to increased energy demands.

Sex-different and growth hormone-regulated expression of microRNA in rat liver.

BACKGROUND: MicroRNAs (miRNAs) are short non-coding RNAs playing an important role in post-transcriptional regulation of gene expression. We have previously shown that hepatic transcript profiles are different between males and females; that some of these differences are under the regulation of growth hormone (GH); and that mild starvation diminishes some of the differences. In this study, we tested if hepatic miRNAs are regulated in a similar manner. RESULTS: Using microarrays, miRNA screening was performed to identify sex-dependent miRNAs in rat liver. Out of 324 unique probes on the array, 254 were expressed in the liver and eight (3% of 254) of those were found to be different between the sexes. Among the eight putative sex-different miRNAs, only one female-predominant miRNA (miR-29b) was confirmed using quantitative real-time PCR. Furthermore, 1 week of continuous GH-treatment in male rats reduced the levels of miR-451 and miR-29b, whereas mild starvation (12 hours) raised the levels of miR-451, miR-122a and miR-29b in both sexes. The biggest effects were obtained on miR-29b with GH-treatment. CONCLUSION: We conclude that hepatic miRNA levels depend on the hormonal and nutritional status of the animal and show that miR-29b is a female-predominant and GH-regulated miRNA in rat liver.

Hormonal and nutritional regulation of alternative CD36 transcripts in rat liver--a role for growth hormone in alternative exon usage.

BACKGROUND: CD36 is a multiligand receptor involved in various metabolic pathways, including cellular uptake of long-chain fatty acids. Defect function or expression of CD36 can result in dyslipidemia or insulin resistance. We have previously shown that CD36 expression is female-predominant in rat liver. In the present study, hormonal and nutritional regulation of hepatic CD36 expression was examined in male and female rats. Since alternative transcription start sites have been described in murine and human Cd36, we investigated whether alternative CD36 transcripts are differentially regulated in rat liver during these conditions. RESULTS: Sequence information of the rat Cd36 5'-UTR was extended, showing that the gene structure of Cd36 in rat is similar to that previously described in mouse with at least two alternative first exons. The rat Cd36 exon 1a promoter was sequenced and found to be highly similar to murine and human Cd36. We show that alternative first exon usage is involved in the female-predominant expression of CD36 in rat liver and during certain hormonal states that induce CD36 mRNA abundance. Estrogen treatment or continuous infusion of growth hormone (GH) in male rats induced CD36 expression preferentially through the exon 1a promoter. Old age was associated with increased CD36 expression in male rats, albeit without any preferential first exon usage. Intermittent GH treatment in old male rats reversed this effect. Mild starvation (12 hours without food) reduced CD36 expression in female liver, whereas its expression was increased in skeletal muscle. CONCLUSION: The results obtained in this study confirm and extend our previous observation that GH is an important regulator of hepatic CD36, and depending on the mode of treatment (continuous or intermittent) the gene might be either induced or repressed. We suggest that the effects of continuous GH secretion in females (which is stimulatory) and intermittent GH secretion in males (which is inhibitory) explains the sex-different expression of this gene. Furthermore, a female-specific repression of hepatic CD36 in response to food deprivation was found, which was in contrast to a stimulatory effect in skeletal muscle. This demonstrates a tissue-specific regulation of Cd36.

Suppressor of cytokine signaling-2 deficiency induces molecular and metabolic changes that partially overlap with growth hormone-dependent effects.

Suppressor of cytokine signaling-2 (SOCS2)-deficient (SOCS2-/-) mice grow significantly larger than their littermates, suggesting that SOCS2 is important in the negative regulation of the actions of GH and/or IGF-I. The aim of this study was to identify genes and metabolic parameters that might contribute to the SOCS2-/- phenotype. We demonstrate that although SOCS2 deficiency induces significant changes in hepatic gene expression, only a fraction of these overlap with known GH-induced effects in the liver, suggesting that SOCS2 might be an important regulator of other growth factors and cytokines acting on the liver. However, an important role of GH and IGF-I in the phenotype of these animals was demonstrated by an overexpression of IGF-binding protein-3 mRNA in the liver and increased levels of circulating IGF-binding protein-3. Other GH-like effects included diminished serum triglycerides and down-regulation of lipoprotein lipase in adipose tissue. Interestingly, SOCS2-/- mice did not differ from their wild-type littermates in glucose or insulin tolerance tests, which is in contrast with the known diabetogenic effects of GH. Furthermore, there was no evidence of impaired insulin signaling in primary hepatocytes isolated from SOCS2-/- mice. Moreover, increased expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA was detected in skeletal muscle, which might contribute to normal glycemic control despite the apparent overactivity of the GH/IGF-I axis. Our data indicate that SOCS2 deficiency partially mimics a state of increased GH activity, but also results in changes that cannot be related to known GH effects.

Immunohistochemical analysis of cannabinoid receptor 1 expression in steatotic rat livers.

The primary aim of the present study was to determine the expression levels of cannabinoid receptor type 1 (CB1) in steatotic rat livers. The secondary aim was to clarify whether steatosis and inflammation are more marked in areas with increased CB1 overexpression. For ethical and economic reasons, the present study investigated tissue from archived liver blocks, which were obtained from 38 rats that had been euthanized during the course of previous research at the Karolinska Institute of the Karolinska University Hospital (Stockholm, Sweden) and Lund University (Malmö, Sweden). Liver tissue fixed in formalin and embedded in paraffin was used that had been sourced from 36 male Sprague Dawley rats (age, 7 weeks) and 2 rats (age, 180 days) lacking normal leptin receptors. The rat liver tissue was stained with antibodies against CB1 and counterstained with hematoxylin. The expression of CB1 and the number of cells overexpressing CB1 were determined. Steatosis was scored according to the Dixon scoring system. CB1 overexpression and steatosis were detected in hepatocytes from all 38 livers sampled. The expression of CB1 was more marked in hepatocytes localized next to portal triads. Near the central veins, the expression was significantly weaker. Steatosis was more marked in areas of increased CB1 overexpression. Lymphocyte infiltration was more commonly observed in areas of increased CB1 overexpression. Therefore, the present results indicate that CB1 receptors are overexpressed in areas with steatosis, and indicate that CB1 in hepatocytes contributes to the formation of steatosis in rats, even prior to its progression to steatohepatitis. These results are consistent with publications reporting that CB1 in hepatocytes increases lipogenesis and contributes to inflammation.

Exploring hepatic hormone actions using a compilation of gene expression profiles.

BACKGROUND: Microarray analysis is attractive within the field of endocrine research because regulation of gene expression is a key mechanism whereby hormones exert their actions. Knowledge discovery and testing of hypothesis based on information-rich expression profiles promise to accelerate discovery of physiologically relevant hormonal mechanisms of action. However, most studies so-far concentrate on the analysis of actions of single hormones and few examples exist that attempt to use compilation of different hormone-regulated expression profiles to gain insight into how hormone act to regulate tissue physiology. This report illustrates how a meta-analysis of multiple transcript profiles obtained from a single tissue, the liver, can be used to evaluate relevant hypothesis and discover novel mechanisms of hormonal action. We have evaluated the differential effects of Growth Hormone (GH) and estrogen in the regulation of hepatic gender differentiated gene expression as well as the involvement of sterol regulatory element-binding proteins (SREBPs) in the hepatic actions of GH and thyroid hormone. RESULTS: Little similarity exists between liver transcript profiles regulated by 17-alpha-ethinylestradiol and those induced by the continuos infusion of bGH. On the other hand, strong correlations were found between both profiles and the female enriched transcript profile. Therefore, estrogens have feminizing effects in male rat liver which are different from those induced by GH. The similarity between bGH and T3 were limited to a small group of genes, most of which are involved in lipogenesis. An in silico promoter analysis of genes rapidly regulated by thyroid hormone predicted the activation of SREBPs by short-term treatment in vivo. It was further demonstrated that proteolytic processing of SREBP1 in the endoplasmic reticulum might contribute to the rapid actions of T3 on these genes. CONCLUSION: This report illustrates how a meta-analysis of multiple transcript profiles can be used to link knowledge concerning endocrine physiology to hormonally induced changes in gene expression. We conclude that both GH and estrogen are important determinants of gender-related differences in hepatic gene expression. Rapid hepatic thyroid hormone effects affect genes involved in lipogenesis possibly through the induction of SREBP1 proteolytic processing.

Growth hormone-mediated alteration of fuel metabolism in the aged rat as determined from transcript profiles.

Age-related changes in body composition and serum lipids resemble symptoms of adult-onset growth hormone (GH) deficiency. GH treatment has been shown to normalize these changes in both GH-deficient adult patients and elderly subjects. The aim of this study was to identify GH-responsive genes that might mediate positive effects of GH treatment on fuel metabolism and body composition. cDNA microarrays were used to analyze age- and GH-induced changes in gene expression patterns in male rats. Tissues analyzed were liver, adipose tissue, and skeletal muscle from animals on or off GH treatment. A value of 1.5 was chosen to denote differences (increased or decreased expression) in the level of mRNA expression. In the liver, 7.3% of the expressed genes were affected by age and 6.5% by GH. Similar values for the other tissues were 8.3% and 5.3% (fat), and 7.9% and 9.6% (muscle), respectively. Among the differentially expressed genes, we identified several that encode proteins involved in fuel metabolism. Old rats were shown to have induced expression of genes involved in hepatic glucose oxidation and lipid synthesis, whereas these pathways were reduced in adipose tissue. GH treatment induced the expression of genes for lipid oxidation in liver and for glucose oxidation in skeletal muscle. In adipose tissue, GH reduced the expression of genes involved in lipogenesis even further. Changes in transcript levels were reflected in serum in terms of altered lipid profiles. Serum levels of triglycerides, high-density lipoprotein (HDL) cholesterol, and total cholesterol were higher in the old animals than in the young and normalized by GH treatment.

Female-predominant expression of fatty acid translocase/CD36 in rat and human liver.

The aim of this study was to identify genes for hepatic fuel metabolism with a gender-differentiated expression and to determine which of these that might be regulated by the female-specific secretion of GH. Effects of gender and continuous infusion of GH to male rats were studied in the liver using cDNA microarrays representing 3200 genes. Sixty-nine transcripts displayed higher expression levels in females, and 177 displayed higher expression in males. The portion of GH-regulated genes was the same (30%) within the two groups of gender-specific genes. The male liver had a higher expression of genes involved in fuel metabolism, indicating that male rats might have a greater capacity for high metabolic turnover, compared with females. Most notable among the female-predominant transcripts was fatty acid translocase/CD36, with 18-fold higher mRNA levels in the female liver and 4-fold higher mRNA levels in males treated with GH, compared with untreated males. This gender-differentiated expression was confirmed at mRNA and protein levels in the rat and at the mRNA level in human livers. Although purely speculative, it is possible that higher levels of fatty acid translocase/CD36 in human female liver might contribute to the sexually dimorphic development of diseases resulting from or characterized by disturbances in lipid metabolism, such as arteriosclerosis, hyperlipidemia, and insulin resistance.

Metabolomics: a tool for the diagnosis of GH deficiency and for monitoring GH replacement?

The diagnostic value of insulin-like growth factor 1 (IGF1) for GH deficiency (GHD) in adults is not optimal. Molecular profiling could be used for biomarker discovery. The aim of this pilot study was to compare the serum metabolome between GHD patients and healthy controls, and identification of potential markers for diagnosis and/or for individual GH dosing. A total of ten patients with GHD, median age of 55 years and BMI of 27 kg/m(2), were compared with ten healthy age- and gender-matched controls. The serum metabolic profiles were generated using gas chromatography-coupled mass spectroscopy on fasting samples taken in the morning from the controls and at baseline and during 6 months of GH replacement in the patients with GHD. The difference in low-molecular weight compounds (LMC) distinguished the healthy controls from GHD patients. Among 285 measured metabolites, 13 were identified as being most important in differentiating GHD patients from controls. Of these, 11 could not be structurally annotated but many were classified as lipids. The difference in the LMC pattern persisted despite normalisation of IGF1 following GH replacement. GH replacement increased the levels of specific fatty acid compounds and decreased the levels of certain amino acids. No metabolite changed in response to GH treatment, to the same extent as IGF1. The measurement of 285 metabolites resulted in a unique pattern in GHD, but changes in the metabolite patterns during GH treatment were limited. The utility of metabolomics to find new markers in GHD and GH replacement remains to be further elucidated.

Sex-dependent hepatic transcripts and metabolites in the development of glucose intolerance and insulin resistance in Zucker diabetic fatty rats.

Male Zucker diabetic fatty (mZDF) rats spontaneously develop type 2 diabetes, whereas females only become diabetic when fed a diabetogenic high-fat diet (high-fat-fed female ZDF rat, HF-fZDF). The aim of this study was to investigate if differences in liver functions could provide clues to this sex difference. Non-diabetic obese fZDF rats were compared with either mZDF or HF-fZDF regarding hepatic molecular profiles, to single out those components that might be protective in the females. High-fat feeding in fZDF led to enhanced weight gain, increased blood glucose and insulin levels, reduced insulin sensitivity and a trend towards reduced glucose tolerance, indicative of a prediabetic state. mZDF rats were diabetic, with low levels of insulin, high levels of glucose, reduced insulin sensitivity and impaired glucose tolerance. Transcript profiling and capillary electrophoresis time-of-flight mass spectrometry were used to indentify hepatic transcripts and metabolites that might be related to this. Many diet-induced alterations in transcript and metabolite levels in female rats were towards a 'male-like' phenotype, including reduced lipogenesis, increased fatty acid (FA) oxidation and increased oxidative stress responses. Alterations detected at the level of hepatic metabolites, indicated lower capacity for glutathione (GSH) production in male rats, and higher GSH turnover in females. Taken together, this could be interpreted as if anabolic pathways involving lipogenesis and lipid output might limit the degree of FA oxidation and oxidative stress in female rats. Together with a greater capacity to produce GSH, these hepatic sex differences might contribute to the sex-different development of diabetes in ZDF rats.

Cocoa butter and safflower oil elicit different effects on hepatic gene expression and lipid metabolism in rats.

The aim of this study was to compare the effects of cocoa butter and safflower oil on hepatic transcript profiles, lipid metabolism and insulin sensitivity in healthy rats. Cocoa butter-based high-fat feeding for 3 days did not affect plasma total triglyceride (TG) levels or TG-rich VLDL particles or hepatic insulin sensitivity, but changes in hepatic gene expression were induced that might lead to increased lipid synthesis, lipotoxicity, inflammation and insulin resistance if maintained. Safflower oil increased hepatic beta-oxidation, was beneficial in terms of circulating TG-rich VLDL particles, but led to reduced hepatic insulin sensitivity. The effects of safflower oil on hepatic gene expression were partly overlapping with those exerted by cocoa butter, but fewer transcripts from anabolic pathways were altered. Increased hepatic cholesterol levels and increased expression of hepatic CYP7A1 and ABCG5 mRNA, important gene products in bile acid production and cholesterol excretion, were specific effects elicited by safflower oil only. Common effects on gene expression included increased levels of p8, DIG-1 IGFBP-1 and FGF21, and reduced levels of SCD-1 and SCD-2. This indicates that a lipid-induced program for hepatic lipid disposal and cell survival was induced by 3 days of high-fat feeding, independent on the lipid source. Based on the results, we speculate that hepatic TG infiltration leads to reduced expression of SCD-1, which might mediate either neutral, beneficial or unfavorable effects on hepatic metabolism upon high-fat feeding, depending on which fatty acids were provided by the diet.

Iron load and redox stress in skeletal muscle of aged rats.

Loss of skeletal muscle mass (sarcopenia) is a major contributor to disability in old age. We used two-dimensional gel electrophoresis and mass spectrometry to screen for changes in proteins, and cDNA profiling to assess transcriptional regulations in the gastrocnemius muscle of adult (4 months) and aged (30 months) male Sprague-Dawley rats. Thirty-five proteins were differentially expressed in aged muscle. Proteins and mRNA transcripts involved in redox homeostasis and iron load were increased, representing novel components that were previously not associated with sarcopenia. Tissue iron levels were elevated in senescence, paralleling an increase in transferrin. Proteins involved in redox homeostasis showed a complex pattern of changes with increased SOD1 and decreased SOD2. These results suggest that an elevated iron load is a significant component of sarcopenia with the potential to be exploited clinically, and that mitochondria of aged striated muscle may be more vulnerable to radicals produced in cell respiration.

Differentially expressed genes in visceral or subcutaneous adipose tissue of obese men and women.

There is growing evidence that the distribution of adipose tissue in the body is of importance in the development of metabolic complications of obesity, such as diabetes, hypertension, and hyperlipidemia. The aim of this study was to identify differentially expressed genes in subcutaneous and omental human adipose tissue in obese men, using a subtractive hybridization strategy. From the obtained set of differentially expressed transcripts, we also aimed to identify genes that have a sex-specific pattern of expression in omental or subcutaneous adipose tissue. Representational difference analysis (RDA) was performed on cDNA from subcutaneous and omental fat tissue from a man with extreme abdominal obesity. Forty-four putatively differentially expressed genes were identified. The obtained RDA products were spotted onto glass slides to screen for differential expression in other obese patients by using a microarray hybridization procedure. Five genes were confirmed to be differentially expressed in subcutaneous or omental adipose tissue from male or female obese patients. One gene was detected only in males and was found to be upregulated in subcutaneous tissue. The findings extend previous knowledge that different fat depots have differential gene expression and indicate that sex differences exist in adipose gene expression patterns.