Human cord plasma proteomic analysis reveals sexually dimorphic proteins associated with intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is associated with increased risk of cardiometabolic disease later in life and has been shown to affect female and male offspring differently, but the mechanisms remain unclear. The purpose of this study was to identify proteomic differences and metabolic risk markers in IUGR male and female neonates when compared to appropriate for gestational age (AGA) babies that will provide a better understanding of IUGR pathogenesis and its associated risks. Our results revealed alterations in IUGR cord plasma proteomes with most of the differentially abundant proteins implicated in peroxisome pathways. This effect was evident in females but not in males. Furthermore, we observed that catalase activity, a peroxisomal enzyme, was significantly increased in females (p < 0.05) but unchanged in males. Finally, we identified risk proteins associated with obesity, type-2 diabetes, and glucose intolerance such as EGF containing fibulin extracellular matrix protein 1 (EFEMP1), proprotein convertase subtilisin/kexin type 9 (PCSK9) and transforming growth factor beta receptor 3 (TGFBR3) proteins unique to females while coagulation factor IX (C9) and retinol binding protein 4 (RBP4) are unique in males. In conclusion, IUGR may display sexual dimorphism which may be associated with differences in lifelong risk for cardiometabolic disease between males and females.

Postnatal weaning to different diets leads to different reproductive phenotypes in female offspring following perinatal exposure to high levels of dietary advanced glycation end products.

OBJECTIVE: To examine, following perinatal exposure to a diet high in advanced glycation end products (AGEs), whether the use of standard AGE-free mouse chow during the postweaning period alters metabolism and reproduction differently than exposure to a diet low in AGEs. DESIGN: Experimental animal study. SETTING: University-based research laboratory. ANIMAL(S): Female CD1 mice. INTERVENTION(S): Seven-week-old mice were placed on a diet either low or high in AGEs perinatally, before mating and then during pregnancy and lactation. All offspring were weaned onto an AGE-free normal chow. MAIN OUTCOME MEASURE(S): Growth curve, liver and abdominal fat weight, insulin and glucose tolerance tests, vaginal opening, estrous cyclicity, and serum levels of antimüllerian hormone, leptin, and adiponectin were assessed. Ovarian histologic examination for follicular count and gene expression was also performed. RESULT(S): Compared with the mice exposed to a diet low in AGEs, the mice exposed to a diet high in AGEs showed lower body weight in pups, lower liver weight, delayed vaginal opening, higher serum antimüllerian hormone levels, lower primordial and secondary follicle pools, and higher ovarian Fshr messenger RNA levels. CONCLUSION(S): Following weaning, perinatal AGEs can target puberty onset and folliculogenesis differently to standard mouse chow.

Perinatal exposure to high dietary advanced glycation end products affects the reproductive system in female offspring in mice.

Maternal nutrition and the intrauterine environment are important in determining susceptibility to reproductive and metabolic disturbances. Advanced glycation end products (AGEs) are widely consumed in Western diet. The purpose of this study was to determine whether perinatal exposure to a high levels of dietary AGEs affect metabolic and reproductive parameters in female mice offspring. Female CD1 mice, 7 weeks old, were placed on either a diet low (L-AGE) or high (H-AGE) in AGEs before mating and then during pregnancy and lactation. All offspring were weaned onto the L-AGE diet and studied through to 16 weeks of age; they were counted and weighed at birth and then every week for a total of 11 weeks. Vaginal opening, litter size, growth curve, liver and abdominal fat weights, serum levels of anti-Mullerian hormone, leptin and adiponectin, as well as insulin and glucose tolerance tests were compared. Ovaries were harvested for follicular count and gene expression by real-time polymerase chain reaction. Compared to perinatal exposure to the L-AGE diet, perinatal exposure to the H-AGE diet caused lower body weight at birth, and adult offspring exhibited delayed growth, lower serum leptin and adiponectin levels, delayed vaginal opening, irregular oestrous cyclicity, arrested follicular development and significant alterations in the expression of genes involved in folliculogenesis (Amh and Amhr2) and steroidogenesis (Cyp19a1). These results indicate that perinatal exposure to a diet elevated in AGEs causes deficits in perinatal growth, pubertal onset, and reproductive organ development in female mice. Whether these findings translate to humans remains to be determined in future studies.

Monocyte chemotactic protein-1 plays a role in ovarian dysfunction related to high-fat diet-induced obesity.

Obesity, known to cause a systemic elevation in monocyte chemotactic protein-1 (MCP-1), adversely affects normal ovarian function. The aim of this study was to determine whether MCP-1 plays a role in ovarian dysfunction that is related to obesity induced by high-fat (HF) diet intake. Wild type (WT) C57BL/6J mice were fed either normal chow (NC) (Group 1, control group) or HF diet (Group 2). To assess whether MCP-1 is involved in HF-diet-induced ovarian dysfunction, MCP-1 knock-out mice were fed HF diet (Group 3). Body weight, body fat composition, number of oocytes collected following ovarian superovulation with gonadotropins, ovarian macrophage markers and expression of genes important in folliculogenesis and steroidogenesis were quantified in the 3 groups of animals. Animals in Group 2 gained significant body weight and body mass, produced the fewest number of oocytes following superovulation, and had significant alterations in ovarian genes involved in folliculogenesis and steroidogenesis as well as genes involved in inflammation. Although animals in Group 3 had the highest body weight and body fat composition, they produced similar number of oocytes compared to animals in Group 1 but had different ovarian gene expression compared to Group 2. These findings suggest that MCP-1 gene knockout could reverse some of the adverse effects of obesity induced by HF diet intake. Future studies assessing ovarian histology in MCP-1 knock out mouse model will confirm our findings. MCP-1 inhibition could represent a future therapeutic target to protect ovarian health from the adverse effects of HF diet ingestion.

Antioxidant Effects of N-Acetylcysteine Prevent Programmed Metabolic Disease in Mice.

An adverse maternal in utero and lactation environment can program offspring for increased risk for metabolic disease. The aim of this study was to determine whether N-acetylcysteine (NAC), an anti-inflammatory antioxidant, attenuates programmed susceptibility to obesity and insulin resistance in offspring of mothers on a high-fat diet (HFD) during pregnancy. CD1 female mice were acutely fed a standard breeding chow or HFD. NAC was added to the drinking water (1 g/kg) of the treatment cohorts from embryonic day 0.5 until the end of lactation. NAC treatment normalized HFD-induced maternal weight gain and oxidative stress, improved the maternal lipidome, and prevented maternal leptin resistance. These favorable changes in the in utero environment normalized postnatal growth, decreased white adipose tissue (WAT) and hepatic fat, improved glucose and insulin tolerance and antioxidant capacity, reduced leptin and insulin, and increased adiponectin in HFD offspring. The lifelong metabolic improvements in the offspring were accompanied by reductions in proinflammatory gene expression in liver and WAT and increased thermogenic gene expression in brown adipose tissue. These results, for the first time, provide a mechanistic rationale for how NAC can prevent the onset of metabolic disease in the offspring of mothers who consume a typical Western HFD.

Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones.

Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide(-/-) mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE's physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation.

High-fat intake during pregnancy and lactation exacerbates high-fat diet-induced complications in male offspring in mice.

Altered fetal environments, such as a high-fat milieu, induce metabolic abnormalities in offspring. Different postnatal environments reveal the predisposition for adult diseases that occur during the fetal period. This study investigates the ability of a maternal high-fat diet (HFD) to program metabolic responses to HFD reexposure in offspring after consuming normal chow for 23 weeks after weaning. Wild-type CD1 females were fed a HFD (H) or control (C) chow during pregnancy and lactation. At 26 weeks of age, offspring were either reexposed (H-C-H) or newly exposed (C-C-H) to the HFD for 19 weeks. Body weight was measured weekly, and glucose and insulin tolerance were measured after 10 and 18 weeks on the HFD. The metabolic profile of offspring on a HFD or C diet during pregnancy and lactation and weaned onto a low-fat diet was similar at 26 weeks. H-C-H offspring gained more weight and developed larger adipocytes after being reintroduced to the HFD later in life than C-C-H. H-C-H mice were glucose and insulin intolerant and showed reduced gene expression of cox6a2 and atp5i in muscle, indicating mitochondrial dysfunction. In adipocytes, the expression of slc2a4, srebf1, and adipoq genes was decreased in H-C-H mice compared with C-C-C, indicating insulin resistance. H-C-H showed extensive hepatosteatosis, accompanied by increased gene expression for cd36 and serpin1, compared with C-C-H. Perinatal exposure to a HFD programs a more deleterious response to a HFD challenge later in life even after an interval of normal diet in mice.

Hypoglycemia, hyperglucagonemia, and fetoplacental defects in glucagon receptor knockout mice: a role for glucagon action in pregnancy maintenance.

Alterations in insulin signaling as well as insulin action predispose to infertility as well as adverse pregnancy outcomes; however, little is known about the role of glucagon signaling in reproduction. The glucagon receptor knockout (Gcgr(-/-)) mouse created by our laboratory was used to define the role of glucagon signaling in maintaining normal reproduction. In this mouse model, lack of glucagon signaling did not alter the hypothalamic-pituitary-ovarian axis. Pregnant Gcgr(-/-) female mice displayed persistent hypoglycemia and hyperglucagonemia. Gcgr(-/-) pregnancies were associated with decreased fetal weight, increased late-gestation fetal demise, and significant abnormalities of placentation. Gcgr(-/-) placentas contained areas of extensive mineralization, fibrinoid necrosis, narrowing of the vascular channels, and a thickened interstitium associated with trophoblast hyperplasia. Absent glucagon signaling did not alter glycogen content in Gcgr(-/-) placentas but significantly downregulated genes that control growth, adrenergic signaling, vascularization, oxidative stress, and G protein-coupled receptors. Our data suggest that, similarly to insulin, glucagon action contributes to normal female reproductive function.

Glucagon receptor knockout prevents insulin-deficient type 1 diabetes in mice.

OBJECTIVE: To determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency. RESEARCH DESIGN AND METHODS: We compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr(-/-)) mice and wild-type (Gcgr(+/+)) controls after equivalent destruction of ß-cells. We used a double dose of streptozotocin to maximize ß-cell destruction. RESULTS: Gcgr(+/+) mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable ß-cell destruction in Gcgr(-/-) mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (~1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr(+/+) mice with diabetes--evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting ß-hydroxy butyrate levels were lower. CONCLUSIONS: We conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice.

Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass.

In addition to its primary role in regulating glucose production from the liver, glucagon has many other actions, reflected by the wide tissue distribution of the glucagon receptor (Gcgr). To investigate the role of glucagon in the regulation of insulin secretion and whole body glucose homeostasis in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release in response to glucagon and glucose, the glucose excursion resulting from both a glucagon challenge and intraperitoneal glucose tolerance test (IPGTT) was significantly reduced in RIP-Gcgr mice compared with controls. However, RIP-Gcgr mice display similar glucose responses to an insulin challenge. beta-Cell mass and pancreatic insulin content were also increased (20 and 50%, respectively) in RIP-Gcgr mice compared with controls. When fed a high-fat diet (HFD), both control and RIP-Gcgr mice developed similar degrees of obesity and insulin resistance. However, the severity of both fasting hyperglycemia and impaired glucose tolerance (IGT) were reduced in RIP-Gcgr mice compared with controls. Furthermore, the insulin response of RIP-Gcgr mice to an IPGTT was twice that of controls when fed the HFD. These data indicate that increased pancreatic beta-cell expression of the Gcgr increased insulin secretion, pancreatic insulin content, beta-cell mass, and, when mice were fed a HFD, partially protected against hyperglycemia and IGT.

Hepatic response to restoration of GLUT4 in skeletal muscle of GLUT4 null mice.

Expression of GLUT4 in fast-twitch skeletal muscle fibers of GLUT4 null mice (G4-MO) normalized glucose uptake in muscle and restored peripheral insulin sensitivity. GLUT4 null mice exhibit altered carbohydrate and lipid metabolism in liver and skeletal muscle. To test the hypothesis that increased glucose utilization by G4-MO muscle would normalize the changes seen in the GLUT4 null liver, serum metabolites and hepatic metabolism were compared in control, GLUT4 null, and G4-MO mice. The fed serum glucose and triglyceride levels of G4-MO mice were similar to those of control mice. In addition, the alternations in liver metabolism seen in GLUT4 nulls including increased GLUT2 expression and fatty acid synthesis accompanied by an increase in the oxidative arm of the pentose phosphate pathway were absent in G4-MO mice. The transgene used for GLUT4 restoration in muscle was specific for fast-twitch muscle fibers. The mitochondria hypertrophy/hyperplasia in all GLUT4 null skeletal muscles was absent in transgene-positive extensor digitorum longus muscle but present in transgene-negative soleus muscle of G4-MO mice. Results of this study suggest that the level of muscle GLUT4 expression influences mitochondrial biogenesis. These studies also demonstrate that the type and amount of substrate that muscle takes up and metabolizes, determined in part by GLUT4 expression levels, play a major role in directing hepatic carbohydrate and lipid metabolism.