Obesity alters the uterine environment before pregnancy

Obesity is a metabolic disorder that predisposes to numerous diseases and has become a major global public health concern. Cafeteria diet (CAF) is the animal model used for the study of obesity that more closely reflects Western diet habits. Previously, we described that CAF administration for 60 days induces obesity in female rats and their fetuses develop macrosomia. Given that, in our model, rats are not genetically modified and that obese mothers were fed standard chow during pregnancy, the aim of the current study was to test the hypothesis that obesity alters the intrauterine environment prior to pregnancy, and this may explain the exacerbated fetal weight gain. We found that uteri from obese rats during the estrous phase developed insulin resistance through mechanisms that involve the induction of uterine hypoxia and the down-regulation of the insulin receptor gene. Moreover, uterine cell proliferation was induced by obesity concomitantly with the reduction in the uterine contractile response to a β2 AR agonist, salbutamol, and this may be consequence of the down-regulation in the uterine β2 AR expression. We conclude that CAF-induced obesity alters the uterine environment in rats during the estrous phase and may cause the fetal macrosomia previously described by us in obese animals. The lower sensitivity of the uterus to a relaxation stimulus (salbutamol) is not a minor fact given that for implantation to occur the uterus must be relaxed for embryo nidation. Thus, the alteration in the uterine quiescence may impair implantation and, consequently, the foregoing pregnancy.     

Effects Of A Post-Weaning Cafeteria Diet In Young Rats: Metabolic Syndrome,Reduced Activity And Low Anxiety-Like Behaviour

Among adolescents, overweight, obesity and metabolic syndrome are rapidly increasing in recent years as a consequence of unhealthy palatable diets. Animal models of diet-induced obesity have been developed, but little is known about the behavioural patterns produced by the consumption of such diets. The aim of the present study was to determine the behavioural and biochemical effects of a cafeteria diet fed to juvenile male and female rats, as well as to evaluate the possible recovery from these effects by administering standard feeding during the last week of the study. Two groups of male and female rats were fed with either a standard chow diet (ST) or a cafeteria (CAF) diet from weaning and for 8 weeks. A third group of males (CAF withdrawal) was fed with the CAF diet for 7 weeks and the ST in the 8th week. Both males and females developed metabolic syndrome as a consequence of the CAF feeding, showing overweight, higher adiposity and liver weight, increased plasma levels of glucose, insulin and triglycerides, as well as insulin resistance, in comparison with their respective controls. The CAF diet reduced motor activity in all behavioural tests, enhanced exploration, reduced anxiety-like behaviour and increased social interaction; this last effect was more pronounced in females than in males. When compared to animals only fed with a CAF diet, CAF withdrawal increased anxiety in the open field, slightly decreased body weight, and completely recovered the liver weight, insulin sensitivity and the standard levels of glucose, insulin and triglycerides in plasma. In conclusion, a CAF diet fed to young animals for 8 weeks induced obesity and metabolic syndrome, and produced robust behavioural changes in young adult rats, whereas CAF withdrawal in the last week modestly increased anxiety, reversed the metabolic alterations and partially reduced overweight.     

Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice

Background  

Women with polycystic ovary syndrome (PCOS) are often treated with insulin-sensitizing agents, e.g. thiazolidinediones (TZD), which have been shown to reduce androgen levels and improved ovulatory function. Acting via peroxisome proliferator-activated receptor (PPAR) gamma, TZD alter the expression of a large variety of genes. Lethal yellow (LY; C57BL/6J Ay/a) mice, possessing a mutation (Ay) in the agouti gene locus, exhibit progressive obesity, reproductive dysfunction, and altered metabolic regulation similar to women with PCOS. The current study was designed to test the hypothesis that prolonged treatment of aging LY mice with the TZD, pioglitazone, alters the ovarian expression of genes that may impact reproduction.     

Ovarian development and insulin-signaling pathways during reproductive differentiation in the queenless ponerine ant Diacamma sp.

In many social hymenopteran species, workers possess functional ovaries that are physiologically inactive in the presence of queens. We investigated the ovarian regulatory mechanism of workers and reproductives in a queenless ponerine ant, Diacamma sp., using histological and molecular techniques. In this ant, clear reproductive differentiation occurs via a highly sophisticated dominance behavioral interaction called “gemmae mutilation”. This clear and rapid bifurcation of reproductive physiology allows us to elucidate the detailed ovarian differentiation process. Histological characteristics of functional ovaries (fusomes and ring canals) were found in both workers and reproductives, suggesting that early oogenesis is not blocked in workers. Since insulin/insulin-like growth factor signaling (IIS) is known to control insect reproduction, orthologs of 2 positive IIS regulators, insulin receptor and serine-threonine kinase Akt (protein kinase B), were cloned in Diacamma (DiaInR, DiaAkt); their expression patterns during reproductive differentiation were examined by real-time quantitative polymerase chain reaction; DiaInR and DiaAkt were strongly expressed in the gasters of reproductives. Whole-mount in situ hybridization of ovaries indicated that DiaInR and DiaAkt were expressed in nurse cells, oocytes, and upper germarial regions of reproductives but not of workers. Our data suggest that the IIS pathway accounts for reproductive differentiation in late oogenesis.     

Remarkable features of ovarian morphology and reproductive hormones in insulin-resistant Zucker fatty (fa/fa) rats

Background

Zucker fatty (fa/fa) rats are a well-understood model of obesity and hyperinsulinemia. It is now thought that obesity/hyperinsulinemia is an important cause of endocrinological abnormality, but to date there have been no reports on the changes in ovarian morphology or the ovarian androgen profile in rat models of obesity and insulin resistance.

Methods

In this study we investigated the effects of obesity and hyperinsulinemia on ovarian morphology and the hormone profile in insulin-resistant Zucker fatty rats (5, 8, 12 and 16 weeks of age, n = 6-7).

Results

Ovaries from 5-week-old fatty rats had significantly greater total and atretic follicle numbers, and higher atretic-to-total follicle ratios than those from lean rats. Ovaries from 12- and 16-week-old fatty rats showed interstitial cell hyperplasia and numerous cysts with features of advanced follicular atresia. In addition, serum testosterone and androstenedione levels significantly declined in fatty rats from age 8 to 16 weeks, so that fatty rats showed significantly lower levels of serum testosterone (12 and 16 weeks) and androstenedione (all weeks) than lean rats. This may reflect a reduction of androgen synthesis during follicular atresia. Serum adiponectin levels were high in immature fatty rats, and although the levels declined significantly as they matured, it remained significantly higher in fatty rats than in lean rats. On the other hand, levels of ovarian adiponectin and its receptors were significantly lower in mature fatty rats than in lean mature rats or immature fatty rats.

Conclusions

Our findings indicate that ovarian morphology and hormone profiles are significantly altered by the continuous insulin resistance in Zucker fatty rats. Simultaneously, abrupt reductions in serum and ovarian adiponectin also likely contribute to the infertility seen in fatty rats.     

Impaired Glucose Metabolism in Response to High Fat Diet in Female Mice Conceived by In Vitro Fertilization (IVF) or Ovarian Stimulation Alone

Individuals conceived by in vitro fertilization (IVF) may be at increased risk of cardio-metabolic disorders. We recently reported that IVF conceived male mice displayed impaired glucose metabolism at normal and high body weights. In this study, we examined glucose metabolism in mature female C57BL/6J mice that were conceived by natural conception (NC), by ovarian stimulation (OS) or by IVF following chow or high-fat diet (HFD) for 8 weeks. By design, litter size was comparable between groups, but interestingly the birth weight of IVF and OS females was lower than NC females (p≤0.001). Mature IVF female mice displayed increased fasting glucose as compared to NC and OS mice, irrespective of diet. Mature IVF and OS mice were also more susceptible to the metabolic consequences of high fat diet as compared with NC females, with impaired glucose tolerance (p≤0.01), whereas peripheral insulin resistance and increased hepatic expression of gluconeogenic genes Ppargc1α, Pck1 and G6pc was observed in IVF mice only (p<0.05). This study suggests that ovarian stimulation alone and IVF program distinct metabolic effects in females, but that high fat diet may be required to unmask these effects. This study adds to the growing body of literature that assisted reproduction procedures may increase the risk of developing type 2 diabetes in an obesity prone environment.     

Effects of excess metabolizable protein on ovarian function and circulating amino acids of beef cows: 1. Excessive supply from corn gluten meal or soybean meal

In the dairy industry, excess dietary CP is consistently correlated with decreased conception rates. However, the source from which excess CP is derived and how it affects reproductive function in beef cattle is largely undefined. The objective of this experiment was to determine the effects of feeding excess metabolizable protein (MP) from feedstuffs differing in rumen degradability on ovulatory follicular dynamics, subsequent corpus luteum (CL) development, steroid hormone production and circulating amino acids (AA) in beef cows. Non-pregnant, non-lactating mature beef cows (n=18) were assigned to 1 of 2 isonitrogenous diets (150% of MP requirements) designed to maintain similar BW and body condition score (BCS) between treatments. Diets consisted of ad libitum corn stalks supplemented with corn gluten meal (moderate rumen undegradable protein (RUP); CGM) or soybean meal (low RUP; SBM). After a 20-day supplement adaptation period, cows were synchronized for ovulation. After 10 days of synchronization, gonadotropin releasing hormone (GnRH) was administered to reset ovarian follicular growth. Starting at GnRH administration and daily thereafter until spontaneous ovulation, transrectal ultrasonography was used to diagram ovarian follicular growth, and blood samples were collected for hormone, metabolite and AA analyses. After 7 days of visual detection of estrus, CL size was determined via ultrasound. Data were analyzed using the MIXED procedures of SAS. As designed, cow BW and BCS were not different (P⩾0.33). Ovulatory follicular wavelength, antral follicle count, ovulatory follicle size at dominance and duration of dominance were not different (P>0.13) between treatments. Cows supplemented with CGM had greater post-dominance ovulatory follicle growth, larger dominant follicles at spontaneous luteolysis, shorter proestrus, and larger ovulatory follicles (P⩽0.03) than SBM cows. No differences (P⩾0.44) in peak estradiol, ratio of estradiol to ovulatory follicle volume, or plasma urea nitrogen were observed. While CL volume and the ratio of progesterone to CL volume were not affected by treatment (P⩾0.24), CGM treated cows tended to have decreased (P=0.07) circulating progesterone 7 days post-estrus compared with SBM cows. Although total circulating plasma AA concentration did not differ (P=0.70) between treatments, CGM cows had greater phenylalanine (P=0.03) and tended to have greater leucine concentrations (P=0.07) than SBM cows. In summary, these data illustrate that excess MP when supplemented to cows consuming a low quality forage may differentially impact ovarian function depending on ruminal degradability of the protein source.     

Antioxidant enzymes gene expression and antihypertensive effects of seaweeds Ulva linza and Lessonia trabeculata in rats fed a high-fat and high-sucrose diet

The objective of this work was to evaluate the antihypertensive and antioxidant effect of seaweeds (Ulva linza and Lessonia trabeculata) in rats which were fed a hypercaloric diet. Seaweed at 400 mg kg^?1 of body weight was administered for 8 weeks to Wistar rats that were fed with a standard diet or a hypercaloric diet. Intra-abdominal fat, insulin resistance, and lipid profile of the rats were determined. Liver was isolated to determine antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)] activity and gene expression. The administration of seaweed to the rats reduced the levels of intra-abdominal fat, arterial blood pressure, insulin resistance, and cholesterol and triglyceride serum levels. U. linza reduced the GPx activity in control animals but increased it in animals with MS, which could be reduced by using L. trabeculata. Both seaweeds diminished the SOD and GPx expression and increased CAT in control group. Both seaweeds reduced the CAT expression in animals with metabolic syndrome. Combined effects of the different compounds found in the seaweeds explain the regulating effect over different antioxidant enzymes and metabolic pathways that protect the animals fed a hypercaloric diet against the development of hypertension, hyperglycemia, and obesity.     

Improved n-3 fatty acid status does not modulate insulin resistance in fa/fa Zucker rats

The objective was to examine the effect of polyunsaturated fatty acid type (plant vs fish oil-derived n-3, compared to n-6 fatty acids in the presence of constant proportions of saturated, monounsaturated and polyunsaturated fatty acids) on obesity, insulin resistance and tissue fatty acid composition in genetically obese rats. Six-week-old fa/fa and lean Zucker rats were fed with a 10% (w/w) mixed fat diet containing predominantly flax-seed, menhaden or safflower oils for 9 weeks. There was no effect of dietary lipid on obesity, oral glucose tolerance (except t=60 min insulin), pancreatic function or molecular markers related to insulin, glucose and lipid metabolism, despite increased n-3 fatty acids in muscle and adipose tissue. The menhaden oil diet reduced fasting serum free fatty acids in both fa/fa and lean rats. These data suggest that n-3 composition does not alter obesity and insulin resistance in the fa/fa Zucker rat model when dietary lipid classes are balanced.     

Dietary soy protein induces hepatic lipogenic enzyme gene expression while suppressing hepatosteatosis in obese female Zucker rats bearing DMBA-initiated mammary tumors

Fatty liver is associated with obesity and breast cancer. We used an obese rat model of mammary cancer to examine whether hepatosteatosis is modifiable by diet and associated with altered expression of hepatic lipogenic enzyme genes, thyroid hormone system genes and cholesterol metabolism-related genes. Beginning at the age of 5 weeks, lean and obese female Zucker rats were fed high-isoflavone soy protein- or casein (control protein)-containing diets. Rats were euthanized at 200 days of age [corresponding to 147 days after administration of carcinogen to induce mammary tumors; (Hakkak et al. in, Oncol Lett 2:29–36, 2011)]. Obese rats had a greater degree of liver steatosis than lean rats. Obese casein-fed rats had marked steatosis with small foci of mononuclear infiltration, whereas obese soy protein-fed rats had a significantly lower steatosis index. Comparisons between lean and obese casein-fed rats showed that obesity was associated with significant reductions in hepatic mRNA abundance for Glucose 6-Phosphate Dehydrogenase (G6PD), 6-Phosphogluconate Dehydrogenase (6PGD), Thyroid Receptor Alpha 1 (TRα1), Thyroid Receptor Beta 1 (TRβ1) and Iodothyronine Deiodinase 1 (DIO1). The soy protein diet was associated with increased expression of Fatty Acid Synthase (FASN), Malic Enzyme 1 (ME1), 6PGD, Sterol Regulatory Element Binding Protein-1c (SREBP-1c) and SREBP-2 genes in the livers of obese but not lean rats. Western blot analysis showed a significant induction of ME1 protein expression in the livers of obese, soy protein-fed rats, which paralleled the increased serum insulin level in this group. Long-term soy protein consumption can counter hepatic steatosis while coincidently promoting hepatic lipogenic gene expression, the latter likely a consequence of elevated serum insulin. We suggest that elevations in serum insulin, hepatic lipogenesis and cholesterol synthesis all contributed to the increased tumorigenesis previously observed for the obese, soy protein-fed rats.     

Development of an in vitro system for functional studies of ovarian follicular cells in European sea bass (Dicentrarchus labrax)

The layers of follicular cells surrounding the oocyte and the interactions among them and the germ cells are critical for the successful maintenance of the ovarian functions. We have set up the isolation procedure and culture conditions of sea bass ovarian follicular cells. Their behaviour at three different physiological temperatures (25, 18 and 15 °C) was evaluated by verifying their steroidogenic capacity along time together with the expression of follicular specific genes (cyp19a1, fshr, lhr and star). These characteristics revealed this culture as a good in vitro alternative to short term in vivo studies at the level of the ovarian follicle. Moreover, to evaluate the suitability of this system for gene function studies conditions for transient transfection of plasmid DNA were optimized. Finally, the characteristics of the follicular culture were not affected by freezing and thawing cycles what facilitates the performance of experiments independently of the reproductive season. In conclusion, we have developed an in vitro homologous system that enables functional and gene expression studies and resembles the in vivo situation in the ovarian follicle.     

The insulin-like receptor gene expression in the tissues synthesizing gonadotropic hormones at sexual maturation of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> females

The insulin/insulin-like growth factor signaling pathway is involved in the regulation of the synthesis of insect gonadotropic hormones, juvenile (JH) and 20-hydroxyecdysone (20E). We carried out the immunohistochemical analysis of the insulin receptor (InR) expression in the corpus allatum (the JH-producing gland) and in the ovarian follicular cells (a site for the synthesis of 20E precursor, ecdysone) in the process of sexual maturation of D. melanogaster females and examined the influence of exogenous JH on the InR expression in these tissues. For the first time, it was demonstrated that InR was expressed in follicular cells and that its expression in corpus allatum and follicular cells of Drosophila females was stage-specific, i.e., the expression intensity in young females greatly exceeded that in mature individuals. We also found a negative feedback loop in the regulation of JH levels by the insulin signaling pathway in Drosophila adults: the experimental increase in the JH titers in young females dramatically reduced the InR expression intensity in corpus allatum and follicular cells.     

Conserved insulin signaling in the regulation of oocyte growth,development, and maturation

Insulin signaling regulates various aspects of physiology, such as glucose homeostasis and aging, and is a key determinant of female reproduction in metazoans. That insulin signaling is crucial for female reproductive health is clear from clinical data linking hyperinsulinemic and hypoinsulinemic condition with certain types of ovarian dysfunction, such as altered steroidogenesis, polycystic ovary syndrome, and infertility. Thus, understanding the signaling mechanisms that underlie the control of insulin‐mediated ovarian development is important for the accurate diagnosis of and intervention for female infertility. Studies of invertebrate and vertebrate model systems have revealed the molecular determinants that transduce insulin signaling as well as which biological processes are regulated by the insulin‐signaling pathway. The molecular determinants of the insulin‐signaling pathway, from the insulin receptor to its downstream signaling components, are structurally and functionally conserved across evolution, from worms to mammals—yet, physiological differences in signaling still exist. Insulin signaling acts cooperatively with gonadotropins in mammals and lower vertebrates to mediate various aspects of ovarian development, mainly owing to evolution of the endocrine system in vertebrates. In contrast, insulin signaling in Drosophila and Caenorhabditis elegans directly regulates oocyte growth and maturation. In this review, we compare and contrast insulin‐mediated regulation of ovarian functions in mammals, lower vertebrates, C. elegans, and Drosophila, and highlight conserved signaling pathways and regulatory mechanisms in general while illustrating insulin's unique role in specific reproductive processes.     

Sex hormone-binding globulin overexpression protects against high-fat diet-induced obesity in transgenic male mice

Obese subjects of all ages and sex have reduced plasma SHBG levels. Whether these low plasma SHBG levels play a role in obesity development is unknown. In the present work we wanted to explore if SHBG overexpression could prevent obesity development induced by high fat diet (HFD). To do so, we fed humanized SHBG transgenic male mice and their wild-type littermates with control diet (CD) or HFD over the course of 8 weeks. The results showed that SHBG overexpression protected against body weight gain and fat accumulation induced by HFD. In addition, SHBG overexpression also abrogated the increase in insulin, leptin and resistin levels, as well as the reduction in adiponectin, induced by HFD. Mechanistically, the SHBG protection against HFD-induced obesity was achieved by stimulating lipolysis in white adipose tissue. Furthermore, we have demonstrated the SHBG cell-autonomous effect using human primary visceral adipocytes. Taking together, our results demonstrate that SHBG overexpression protects against diet-induced obesity and improves the metabolic profile of male mice fed a HFD diet.     

Insulin and Leptin Signaling Interact in the Mouse Kiss1 Neuron during the Peripubertal Period

Reproduction requires adequate energy stores for parents and offspring to survive. Kiss1 neurons, which are essential for fertility, have the potential to serve as the central sensors of metabolic factors that signal to the reproductive axis the presence of stored calories. Paradoxically, obesity is often accompanied by infertility. Despite excess circulating levels of insulin and leptin, obese individuals exhibit resistance to both metabolic factors in many neuron types. Thus, resistance to insulin or leptin in Kiss1 neurons could lead to infertility. Single deletion of the receptors for either insulin or the adipokine leptin from Kiss1 neurons does not impair adult reproductive dysfunction. However, insulin and leptin signaling pathways may interact in such a way as to obscure their individual functions. We hypothesized that in the presence of genetic or obesity-induced concurrent insulin and leptin resistance, Kiss1 neurons would be unable to maintain reproductive function. We therefore induced a chronic hyperinsulinemic and hyperleptinemic state in mice lacking insulin receptors in Kiss1 neurons through high fat feeding and examined the impact on fertility. In an additional, genetic model, we ablated both leptin and insulin signaling in Kiss1 neurons (IR/LepR^Kiss mice). Counter to our hypothesis, we found that the addition of leptin insensitivity did not alter the reproductive phenotype of IR^Kiss mice. We also found that weight gain, body composition, glucose and insulin tolerance were normal in mice of both genders. Nonetheless, leptin and insulin receptor deletion altered pubertal timing as well as LH and FSH levels in mid-puberty in a reciprocal manner. Our results confirm that Kiss1 neurons do not directly mediate the critical role that insulin and leptin play in reproduction. However, during puberty kisspeptin neurons may experience a critical window of susceptibility to the influence of metabolic factors that can modify the onset of fertility.     

Lycopus lucidus Turcz Water Extract Ameliorates the Metabolic Disorder by Up-Regulated Major Urinary Protein Expression in High-Fat Diet-Induced Obesity

Despite a century of research on obesity, metabolic disorders and their complications, including dyslipidemia, insulin resistance, and fatty liver disease remain a serious global health problem. Lycopus lucidus Turcz (LT) is a traditional medicine used for its anti-inflammatory properties that has not been evaluated for its efficacy in improving obesity. In this study, mice were fed a normal diet (n = 10) or obesity was induced with a high-fat diet (HFD, n = 20, 60% kcal from fat) for 4 weeks. The HFD mice were then divided into two groups, one of which received LT supplementation with water extract for 13 weeks [HFD (n = 10) or HFD with LT water extract (n = 10, 1.5%)]. LT reduced body and adipose tissue weight by elevating energy expenditure by increasing fatty oxidation in epididymal white adipose tissue (eWAT) and muscle. LT ameliorated dyslipidemia and hepatic steatosis by restricting lipogenesis. Additionally, LT normalized the impaired glucose homeostasis by diet-induced obesity to improve pancreatic islet dysfunction with increasing hepatic major urinary protein expression. Moreover, LT attenuated the inflammation and collagen accumulation in the liver and eWAT. In conclusion, these results suggest that LT can treat obesity-related metabolic disorders such as adiposity, dyslipidemia, hepatic steatosis, insulin resistance, and inflammation.