Reduced islet function contributes to impaired glucose homeostasis in fructose-fed mice.

Increased sugar consumption, particularly fructose, in the form of sweetened beverages and sweeteners in our diet adversely affects metabolic health. Because these effects are associated with features of the metabolic syndrome in humans, the direct effect of fructose on pancreatic islet function is unknown. Therefore, we examined the islet phenotype of mice fed excess fructose. Fructose-fed mice exhibited fasting hyperglycemia and glucose intolerance but not hyperinsulinemia, dyslipidemia, or hyperuricemia. Islet function was impaired, with decreased glucose-stimulated insulin secretion and increased glucagon secretion and high fructose consumption leading to α-cell proliferation and upregulation of the fructose transporter GLUT5, which was localized only in α-cells. Our studies demonstrate that excess fructose consumption contributes to hyperglycemia by affecting both ß- and α-cells of islets in mice.

Diet-induced obesity impairs endometrial stromal cell decidualization: a potential role for impaired autophagy.

STUDY QUESTION: What effect does diet-induced obesity have on endometrial stromal cell (ESC) decidualization? SUMMARY ANSWER: Diet-induced obesity impairs ESC decidualization. WHAT IS KNOWN ALREADY: Decidualization is important for successful implantation and subsequent health of the pregnancy. Compared with normal-weight women, obese women have lower pregnancy rates (both spontaneous and by assisted reproductive technology), higher rates of early pregnancy loss and poorer oocyte quality. STUDY DESIGN, SIZE, DURATION: Beginning at 6 weeks of age, female C57Bl/6J mice were fed either a high-fat/high-sugar diet (HF/HS; 58% Fat Energy/Sucrose) or a diet of standard mouse chow (CON; 13% Fat) for 12 weeks. At this point, metabolic parameters were measured. Some of the mice (n = 9 HF/HS and 9 CON) were mated with reproductively competent males, and implantation sites were assessed. Other mice (n = 11 HF/HS and 10 CON) were mated with vasectomized males, and artificial decidualization was induced. For in vitro human studies of primary ESCs, endometrial tissue was obtained via biopsy from normo-ovulatory patients without history of infertility (obese = BMI > 30 kg/m(2), n = 11 and lean = BMI < 25 kg/m(2), n = 7) and from patients consented for hysterectomies for a benign indication (n = 4). In vitro studies were also performed with immortalized human ESCs. ESCs were decidualized in culture for nine 9 days in the presence or absence of palmitic acid (PA), and the degree of decidualization was assessed by measuring expression of decidualization markers. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sizes of implantation sites and fetuses were analyzed in mice mated with reproductively competent males. In mice mated with vasectomized males, decidualization was induced, and uterine tissues were analyzed via hematoxylin and eosin staining, quantitative RT-PCR (RT-qPCR), and western blots. Human ESCs were cultured in vitro and induced to decidualize by treatment with cAMP and medroxyprogesterone. The level of expression of decidualization markers was assessed by RT-qPCR (mRNA) and western blotting (protein). ATP content of ESCs was measured, and levels of autophagy were assessed by western blotting of the autophagy regulators acetyl coa carboxylase (ACC) and ULK1 (Ser 317). Autophagic flux was measured by western blot of the marker LC3b-II. MAIN RESULTS AND THE ROLE OF CHANCE: Mice exposed to an HF/HS diet became obese and metabolically impaired. HF/HS-exposed mice mated to reproductively competent males had smaller implantation sites in early pregnancy (P <0.001) and larger fetuses at term (P <0.05) than CON-exposed mice. In the artificial decidualization experiments, mice exposed to the HF/HS diet developed 50% smaller deciduomas than mice exposed to CON diet (P< 0.001). Human ESCs cultured in the presence of PA had markedly decreased mRNA expression of the decidualization markers, decidual prolactin (PRL) (P< 0.0001) and insulin-like growth factor binding protein 1 (IGFBP1) (P< 0.0001). Expression of PRL and IGFBP1 by mRNA were also significantly lower in early follicular phase ESCs of obese women than in those of normal-weight women (P< 0.05). Protein expression of phosphorylated ACC and phosphorylated ULK1, both activated forms, were lower in deciduomas of HF/HS mice than in those of control mice (P < 0.01). In immortalized human ESCs, LC3b-II levels were higher in decidualized cells than in controls, indicating increased autophagy. PA treatment abrogated this increase. LIMITATIONS, REASONS FOR CAUTION: Many aspects of obesity and metabolic impairment could contribute to the decidualization defects observed in the HF/HS-exposed mice. Although our findings suggest that both autophagy and decidualization are impaired by exposure to PA, the underlying mechanisms should be elucidated. Finally, our human patient sample size was small. WIDER IMPLICATIONS OF THE FINDINGS: Although many factors contribute to poor reproductive outcome and early pregnancy loss in obese women, our study suggests the importance of decidualization defects. Such defects may contribute to compromised endometrial receptivity and poor implantation. If defects in autophagy contribute to impaired decidualization, therapeutics could be developed to improve this process and thus improve implantation and pregnancy outcomes in obese women. STUDY FUNDING/COMPETING INTERESTS: Grants include NIH 5T32HD040135-12 (J.S.R.), R01 HD065435 (K.H.M.), NIH T32 HD049305 (J.L.S.) and ACOG Research Grant (M.B.S.). The authors report no conflicts of interest.

Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.

The clonal isolation of putative adult pancreatic precursors has been an elusive goal of researchers seeking to develop cell replacement strategies for diabetes. We report the clonal identification of multipotent precursor cells from the adult mouse pancreas. The application of a serum-free, colony-forming assay to pancreatic cells enabled the identification of precursors from pancreatic islet and ductal populations. These cells proliferate in vitro to form clonal colonies that coexpress neural and pancreatic precursor markers. Upon differentiation, individual clonal colonies produce distinct populations of neurons and glial cells, pancreatic endocrine beta-, alpha- and delta-cells, and pancreatic exocrine and stellate cells. Moreover, the newly generated beta-like cells demonstrate glucose-dependent Ca(2+) responsiveness and insulin release. Pancreas colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. These cells represent a previously unidentified adult intrinsic pancreatic precursor population and are a promising candidate for cell-based therapeutic strategies.

Excess Maternal Fructose Consumption Increases Fetal Loss and Impairs Endometrial Decidualization in Mice.

The most significant increase in metabolic syndrome over the previous decade occurred in women of reproductive age, which is alarming given that metabolic syndrome is associated with reproductive problems including subfertility and early pregnancy loss. Individuals with metabolic syndrome often consume excess fructose, and several studies have concluded that excess fructose intake contributes to metabolic syndrome development. Here, we examined the effects of increased fructose consumption on pregnancy outcomes in mice. Female mice fed a high-fructose diet (HFrD) for 6 weeks developed glucose intolerance and mild fatty liver but did not develop other prominent features of metabolic syndrome such as weight gain, hyperglycemia, and hyperinsulinemia. Upon mating, HFrD-exposed mice had lower pregnancy rates and smaller litters at midgestation than chow-fed controls. To explain this phenomenon, we performed artificial decidualization experiments and found that HFrD consumption impaired decidualization. This appeared to be due to decreased circulating progesterone as exogenous progesterone administration rescued decidualization. Furthermore, HFrD intake was associated with decreased bone morphogenetic protein 2 expression and signaling, both of which were restored by exogenous progesterone. Finally, expression of forkhead box O1 and superoxide dismutase 2 [Mn] proteins were decreased in the uteri of HFrD-fed mice, suggesting that HFrD consumption promotes a prooxidative environment in the endometrium. In summary, these data suggest that excess fructose consumption impairs murine fertility by decreasing steroid hormone synthesis and promoting an adverse uterine environment.

Maternal Metabolic Syndrome Programs Mitochondrial Dysfunction via Germline Changes across Three Generations.

Maternal obesity impairs offspring health, but the responsible mechanisms are not fully established. To address this question, we fed female mice a high-fat/high-sugar diet from before conception until weaning and then followed the outcomes in the next three generations of offspring, all fed a control diet. We observed that female offspring born to obese mothers had impaired peripheral insulin signaling that was associated with mitochondrial dysfunction and altered mitochondrial dynamic and complex proteins in skeletal muscle. This mitochondrial phenotype persisted through the female germline and was passed down to the second and third generations. Our results indicate that maternal programming of metabolic disease can be passed through the female germline and that the transfer of aberrant oocyte mitochondria to subsequent generations may contribute to the increased risk for developing insulin resistance.

Maternal fructose drives placental uric acid production leading to adverse fetal outcomes.

Maternal metabolic diseases increase offspring risk for low birth weight and cardiometabolic diseases in adulthood. Excess fructose consumption may confer metabolic risks for both women and their offspring. However, the direct consequences of fructose intake per se are unknown. We assessed the impact of a maternal high-fructose diet on the fetal-placental unit in mice in the absence of metabolic syndrome and determined the association between maternal serum fructose and placental uric acid levels in humans. In mice, maternal fructose consumption led to placental inefficiency, fetal growth restriction, elevated fetal serum glucose and triglyceride levels. In the placenta, fructose induced de novo uric acid synthesis by activating the activities of the enzymes AMP deaminase and xanthine oxidase. Moreover, the placentas had increased lipids and altered expression of genes that control oxidative stress. Treatment of mothers with the xanthine oxidase inhibitor allopurinol reduced placental uric acid levels, prevented placental inefficiency, and improved fetal weights and serum triglycerides. Finally, in 18 women delivering at term, maternal serum fructose levels significantly correlated with placental uric acid levels. These findings suggest that in mice, excess maternal fructose consumption impairs placental function via a xanthine oxidase/uric acid-dependent mechanism, and similar effects may occur in humans.

Leptin improves insulin resistance and hyperglycemia in a mouse model of type 2 diabetes.

Leptin has metabolic effects on peripheral tissues including muscle, liver, and pancreas, and it has been successfully used to treat lipodystrophic diabetes, a leptin-deficient state. To study whether leptin therapy can be used for treatment of more common cases of type 2 diabetes, we used a mouse model of type 2 diabetes (MKR mice) that show normal leptin levels and are diabetic due to a primary defect in both IGF-I and insulin receptors signaling in skeletal muscle. Here we show that leptin administration to the MKR mice resulted in improvement of diabetes, an effect that was independent of the reduced food intake. The main effect of leptin therapy was enhanced hepatic insulin responsiveness possibly through decreasing gluconeogenesis. In addition, the reduction of lipid stores in liver and muscle induced by enhancing fatty acid oxidation and inhibiting lipogenesis led to an improvement of the lipotoxic condition. Our data suggest that leptin could be a potent antidiabetic drug in cases of type 2 diabetes that are not leptin resistant.

Peroxisome proliferator-activated receptor-alpha agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis.

Abnormalities in insulin action are the characteristics of type 2 diabetes. Dominant-negative muscle-specific IGF-I receptor (MKR) mice exhibit elevated lipid levels at an early age and eventually develop type 2 diabetes. To evaluate the role of elevated lipids in the progression of the diabetic state, MKR mice were treated with WY14,643, a peroxisome proliferator-activated receptor (PPAR)-alpha agonist. WY14,643 treatment markedly reduced serum fatty acid and triglyceride levels within a few days, as well as muscle triglyceride levels, and subsequently normalized glucose and insulin levels in MKR mice. Hyperinsulinemic-euglycemic clamp analysis showed that WY14,643 treatment enhanced muscle and adipose tissue glucose uptake by improving whole-body insulin sensitivity. Insulin suppression of endogenous glucose production by the liver of MKR mice was also improved. The expression of genes involved in fatty acid oxidation was increased in liver and skeletal muscle, whereas gene expression levels of hepatic gluconeogenic enzymes were decreased in WY14,643-treated MKR mice. WY14,643 treatment also improved the pattern of glucose-stimulated insulin secretion from the perfused pancreata of MKR mice and reduced the beta-cell mass. Taken together, these findings suggest that the reduction in circulating or intracellular lipids by activation of PPAR-alpha improved insulin sensitivity and the diabetic condition of MKR mice.

Muscle-specific overexpression of CD36 reverses the insulin resistance and diabetes of MKR mice.

Insulin resistance is one of the primary characteristics of type 2 diabetes. Mice overexpressing a dominant-negative IGF-I receptor specifically in muscle (MKR mice) demonstrate severe insulin resistance with high levels of serum and tissue lipids and eventually develop type 2 diabetes at 5-6 wk of age. To determine whether lipotoxicity plays a role in the progression of the disease, we crossed MKR mice with mice overexpressing a fatty acid translocase, CD36, in skeletal muscle. The double-transgenic MKR/CD36 mice showed normalization of the hyperglycemia and the hyperinsulinemia as well as a marked improvement in liver insulin sensitivity. The MKR/CD36 mice also exhibited normal rates of fatty acid oxidation in skeletal muscle when compared with the decreased rate of fatty acid oxidation in MKR. With the reduction in insulin resistance, beta-cell function returned to normal. These and other results suggest that the insulin resistance in the MKR mice is associated with increased muscle triglycerides levels and that whole-body insulin resistance can be, at least partially, reversed in association with a reduction in muscle triglycerides levels, although the mechanisms are yet to be determined.

Glucose-regulated glucagon secretion requires insulin receptor expression in pancreatic alpha-cells.

The insulin receptor (IR) and its signaling appear to be essential for insulin secretion from pancreatic beta-cells. However, much less is known about the role of the IR in alpha-cells. To assess the role of the IR in glucagon and insulin secretion, we engineered adeno-viruses for high efficiency small interference RNA (siRNA)-IR expression in isolated mouse pancreatic islets and lentiviruses for siRNA-IR expression in pancreatic alpha- and beta-cell lines (alpha-TC6 and MIN6) with specific, long term stable IR knockdown. Western blot analysis showed that these strategies resulted in 60-80% reduction of IR protein in islets and alpha- and beta-cell lines. Cell growth was reduced by 35-50% in alpha-TC and MIN6 cells stably expressing siRNA-IR, respectively. Importantly, glucagon secretion, in response to glucose (25 to 2.8 mm), was completely abolished in islets expressing siRNA-IR, whereas secretion increased 1.7-fold in islets expressing control siRNA. In contrast, there was no difference in glucose-stimulated insulin secretion when comparing siRNA-IR and siRNA control, with both groups showing a 1.7-fold increase. Islet glucagon and insulin content were also unaffected by IR knockdown. To further explore the role of the IR, siRNA-IR was stably expressed in pancreatic cell lines, which dramatically suppressed glucose-regulated glucagon secretion in alpha-TC6 cells (3.4-fold) but did not affect GSIS in MIN6 cells. Defects in siRNA-IR-expressing alpha-cells were associated with an alteration in the activity of Akt and p70S6K where insulin-induced phosphorylation of protein kinase B/AKt was greatly reduced while p70S6K activation was enhanced, suggesting that the related pathways play important roles in alpha cell function. This study provides direct evidence that appropriate expression of the IR in alpha-cells is required for glucose-dependent glucagon secretion.