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.     

PCK1 and PCK2 as candidate diabetes and obesity genes

The PCK1 gene (Pck1 in rodents) encodes the cytosolic isozyme of phosphoenolpyruvate carboxykinase (PEPCK-C), which is well-known for its function as a gluconeogenic enzyme in the liver and kidney. Mouse studies involving whole body and tissue-specific Pck1 knockouts as well as tissue-specific over-expression of PEPCK-C have resulted in type 2 diabetes as well as several surprising phenotypes including obesity, lipodystrophy, fatty liver, and death. These phenotypes arise from perturbations not only in gluconeogenesis but in two additional metabolic functions of PEPCK-C: (1) cataplerosis which maintains metabolic flux through the Krebs cycle by removing excess oxaloacetate, and (2) glyceroneogenesis which produces glycerol-3-phosphate as a precursor for fatty acid esterification into triglycerides. PEPCK-C catalyzes the conversion of oxaloacetate + GTP to phosphoenolpyruvate + GDP + CO₂. It is in part the tissue-specificity of this simple reaction that results in the variety of phenotypes listed above. Briefly: (1) A 7-fold over-expression of PEPCK-C in the livers of mice causes excessive glucose production. (2) Mice with a whole-body knockout of Pck1 die within 2–3 days of birth, not from hypoglycemia, but probably because the Krebs cycle slows to approximately 10% of normal in the absence of cataplerosis. (3) Mice with a liver-specific knockout have an inability to remove oxaloacetate from the Krebs cycle, which leads to a fatty liver following a fast. (4) An adipose-specific knockout of Pck1 results in a fraction of the mice developing lipodystrophy due to lost glyceroneogenesis and a consequent decrease in fatty acid re-esterification. (5) Finally, disregulated over-expression of PEPCK-C in adipose tissue increases fatty acid re-esterification leading to obesity. These varied experimental phenotypes in mice have led us to postulate that abnormal production of PEPCK isozymes encoded by two PEPCK genes, PCK1 and PCK2, in humans could have similar consequences (Beale, E. G. et al. (2004). Trends in Endocrinology and Metabolism, 15, 129–135). The purpose of this review is to further explore these possibilities.     

Lens Fiber Cell Differentiation and Denucleation Are Disrupted through Expression of the N-Terminal Nuclear Receptor Box of Ncoa6 and Result in p53-dependent and p53-independent Apoptosis

Nuclear receptor coactivator 6 (NCOA6) is a multifunctional protein implicated in embryonic development, cell survival, and homeostasis. An 81-amino acid fragment, dnNCOA6, containing the N-terminal nuclear receptor box (LXXLL motif) of NCOA6, acts as a dominant-negative (dn) inhibitor of NCOA6. Here, we expressed dnNCOA6 in postmitotic transgenic mouse lens fiber cells. The transgenic lenses showed reduced growth; a wide spectrum of lens fiber cell differentiation defects, including reduced expression of γ-crystallins; and cataract formation. Those lens fiber cells entered an alternate proapoptotic pathway, and the denucleation (karyolysis) process was stalled. Activation of caspase-3 at embryonic day (E)13.5 was followed by double-strand breaks (DSBs) formation monitored via a biomarker, γ-H2AX. Intense terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) signals were found at E16.5. Thus, a window of ∼72 h between these events suggested prolonged though incomplete apoptosis in the lens fiber cell compartment that preserved nuclei in its cells. Genetic experiments showed that the apoptotic-like processes in the transgenic lens were both p53-dependent and p53-independent. Lens-specific deletion of Ncoa6 also resulted in disrupted lens fiber cell differentiation. Our data demonstrate a cell-autonomous role of Ncoa6 in lens fiber cell differentiation and suggest novel insights into the process of lens fiber cell denucleation and apoptosis.     

Development of hepatocellular adenomas and carcinomas in mice with liver-specific G6Pase-α deficiency

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α), and is characterized by impaired glucose homeostasis and a high risk of developing hepatocellular adenomas (HCAs). A globally G6Pase-α-deficient (G6pc^−/−) mouse model that shows pathological features similar to those of humans with GSD-1a has been developed. These mice show a very severe phenotype of disturbed glucose homeostasis and rarely live beyond weaning. We generated liver-specific G6Pase-α-deficient (LS‑G6pc^−/−) mice as an alternative animal model for studying the long-term pathophysiology of the liver and the potential treatment strategies, such as cell therapy. LS‑G6pc^−/− mice were viable and exhibited normal glucose profiles in the fed state, but showed significantly lower blood glucose levels than their control littermates after 6 hours of fasting. LS‑G6pc^−/− mice developed hepatomegaly with glycogen accumulation and hepatic steatosis, and progressive hepatic degeneration. Ninety percent of the mice analyzed developed amyloidosis by 12 months of age. Finally, 25% of the mice sacrificed at age 10–20 months showed the presence of multiple HCAs and in one case late development of hepatocellular carcinoma (HCC). In conclusion, LS‑G6pc^−/− mice manifest hepatic symptoms similar to those of human GSD-1a and, therefore, represent a valid model to evaluate long-term liver pathogenesis of GSD-1a.KEY WORDS: Glycogen storage disease type 1a, Glucose-6-phosphatase-α, Animal model, Hepatomegaly, Hepatic steatosis, Hepatocellular adenoma, Hepatocellular carcinoma     

Generation of mice with a conditional allele for G6pc

Glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC) catalyzes the hydrolysis of glucose‐6‐phosphate to glucose and is a key enzyme in interprandial glucose homeostasis. Mutations in the human G6PC gene, expressed primarily in the liver, kidney, and intestine, cause glycogen storage disease Type Ia (GSD‐Ia), an autosomal recessive disorder characterized by a disturbed glucose homeostasis. For better understanding of the roles of G6Pase‐α in different tissues and in pathological conditions, we have generated mice harboring a conditional null allele for G6pc by flanking Exon 3 of the G6pc gene with loxP sites. We confirmed the null phenotype by using the EIIa‐Cre transgenic approach to generate mice lacking Exon 3 of the G6pc gene. The resulting homozygous Cre‐recombined null mice manifest a phenotype mimicking G6Pase‐α‐deficient mice and human GSD‐Ia patients. This G6pc conditional null allele will be valuable to examine the consequence of tissue‐specific G6Pase‐α deficiency and the mechanisms of long‐term complications in GSD‐Ia. genesis 47:590–594, 2009. Published 2009 Wiley‐Liss, Inc.     

Alteration of De Novo Glucose Production Contributes to Fasting Hypoglycaemia in Fyn Deficient Mice

Previous studies have demonstrated that glucose disposal is increased in the Fyn knockout (FynKO) mice due to increased insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable to compensate for the increased basal glucose utilization. The present study investigates the basis for the reduction in plasma glucose levels and the reduced ability for the liver to produce glucose in response to gluconeogenic substrates. FynKO mice had a 5-fold reduction in phosphoenolpyruvate carboxykinase (PEPCK) gene and protein expression and a marked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, de novo glucose production was also blunted using gluconeogenic substrates that bypass the PEPCK step. Impaired conversion of glycerol to glucose was observed in both glycerol tolerance test and determination of the conversion of ¹³C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were not changed. Fructose-driven glucose production was also diminished without alteration of fructokinase expression levels. The normal levels of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triose kinase function. Fructose-bisphosphate aldolase (aldolase) mRNA or protein levels were normal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold) and fructose-1,6-bisphosphate (7-fold) levels as well as a reduction in glucose-6-phosphate (2-fold) levels. These data suggest a mechanistic defect in the allosteric regulation of aldolase activity.     

Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism

Metformin is widely used as a hypoglycemic agent for the treatment of type 2 diabetes. Both metformin and rotenone, an inhibitor of respiratory chain complex I, suppressed glucose-6-phosphatase (G6pc), a rate limiting enzyme of liver glucose production, mRNA expression in a rat hepatoma cell line accompanied by a reduction of intracellular ATP concentration and an activation of AMP-activated protein kinase (AMPK). When yeast NADH-quinone oxidoreductase 1 (NDI1) gene was introduced into the cells, neither inhibition of ATP synthesis nor activation of AMPK was induced by these agents. Interestingly, in contrast to rotenone treatment, G6pc mRNA down-regulation was observed in the NDI1 expressing cells after metformin treatment. Since NDI1 can functionally complement the complex I under the presence of metformin or rotenone, our results indicate that metformin induces down-regulation of G6pc expression through an inhibition of complex I and an activation of AMPK-independent mechanism.     

Plasmodium berghei: gluconeogenesis in the infected mouse liver studied by 13C nuclear magnetic resonance

Using 13C nuclear magnetic resonance, we have compared the gluconeogenic activity of perfused livers isolated from normal starved mice and mice highly parasitized with Plasmodium berghei, using [2-13C]pyruvate as substrate. In both types of livers, 13C labeling of glucose carbons occurred in positions 1, 2, 5, and 6. The equal proportions of [1,6-13C]- and [2,5-13C]glucose in livers from malarial and normal mice suggests that pyruvate enters the gluconeogenic pathway directly and, to an equal extent, via the tricarboxylic acid cycle. The normalized signal heights indicated that at a given time after the addition of [2-13C]pyruvate the degree of 13C labeling in glucose carbons was reduced in livers from malarial animals, when compared to livers from normal animals. During the course of the perfusion experiment, the [2-13C]lactate resonance signal was always more intense from livers of malarial animals than from normal animals. A reduced activity of hepatic gluconeogenesis in malarial animals was further confirmed by a separate set of perfusion experiments which showed a 56% reduction of the measured rate of glucose production in livers from malarial animals, with respect to that of normal animals. A lowered NAD/NADH ratio in livers from malarial animals would explain the increased proportion of lactate observed in the spectra and be related to a decreased gluconeogenic rate. A more reduced oxidoreduction level in the hepatocytes of a malarial animal would result from a defect in the oxidative phosphorylation activity of mitochondria.     

Systemic oscillator-driven and nutrient-responsive hormonal regulation of daily expression rhythms for gluconeogenic enzyme genes in the mouse liver

Gluconeogenesis is de novo glucose synthesis from substrates such as amino acids and is vital when glucose is lacking in the diurnal nutritional fluctuation. Accordingly, genes for hepatic gluconeogenic enzymes exhibit daily expression rhythms, whose detailed regulations under nutritional variations remain elusive. As a first step, we performed general systematic characterization of daily expression profiles of gluconeogenic enzyme genes for phosphoenolpyruvate carboxykinase (PEPCK), cytosolic form (Pck1), glucose-6-phosphatase (G6Pase), catalytic subunit (G6pc), and tyrosine aminotransferase (TAT) (Tat) in the mouse liver. On a standard diet fed ad libitum, mRNA levels of these genes showed robust daily rhythms with a peak or an elevation phase during the late sleep-fasting period in the diurnal feeding/fasting (wake/sleep) cycle. The rhythmicity was preserved in constant darkness, modulated with prolonged fasting, attenuated by Clock mutation, and entrained to varied photoperiods and time-restricted feedings. These results are concordant with the notion that gluconeogenic enzyme genes are under the control of the intrinsic circadian oscillator, which is entrained by the light/dark cycle, and which in turn entrains the feeding/fasting cycle and also drives systemic signaling pathways such as the hypothalamic-pituitary-adrenal axis. On the other hand, time-restricted feedings also showed that the ingestion schedule, when separated from the light/dark cycle, can serve as an independent entrainer to daily expression rhythms of gluconeogenic enzyme genes. Moreover, nutritional changes dramatically modified expression profiles of the genes. In addition to prolonged fasting, a high-fat diet and a high-carbohydrate (no-protein) diet caused modification of daily expression rhythms of the genes, with characteristic changes in profiles of glucoregulatory hormones such as corticosterone, glucagon, and insulin, as well as their modulators including ghrelin, leptin, resistin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1). Remarkably, high-protein (60% casein or soy-protein) diets activated the gluconeogenic enzyme genes atypically during the wake-feeding period, with paradoxical up-regulation of glucagon, which frequently formed correlation networks with other humoral factors. Based on these results, we propose that daily expression rhythms of gluconeogenic enzyme genes are under the control of systemic oscillator-driven and nutrient-responsive hormones.     

COH-SR4 Reduces Body Weight,Improves Glycemic Control and Prevents Hepatic Steatosis in High Fat Diet-Induced Obese Mice

Obesity is a chronic metabolic disorder caused by imbalance between energy intake and expenditure, and is one of the principal causative factors in the development of metabolic syndrome, diabetes and cancer. COH-SR4 (“SR4”) is a novel investigational compound that has anti-cancer and anti-adipogenic properties. In this study, the effects of SR4 on metabolic alterations in high fat diet (HFD)-induced obese C57BL/J6 mice were investigated. Oral feeding of SR4 (5 mg/kg body weight.) in HFD mice for 6 weeks significantly reduced body weight, prevented hyperlipidemia and improved glycemic control without affecting food intake. These changes were associated with marked decreases in epididymal fat mass, adipocyte hypertrophy, increased plasma adiponectin and reduced leptin levels. SR4 treatment also decreased liver triglycerides, prevented hepatic steatosis, and normalized liver enzymes. Western blots demonstrated increased AMPK activation in liver and adipose tissues of SR4-treated HFD obese mice, while gene analyses by real time PCR showed COH-SR4 significantly suppressed the mRNA expression of lipogenic genes such as sterol regulatory element binding protein-1c (Srebf1), acetyl-Coenzyme A carboxylase (Acaca), peroxisome proliferator-activated receptor gamma (Pparg), fatty acid synthase (Fasn), stearoyl-Coenzyme A desaturase 1 (Scd1), carnitine palmitoyltransferase 1a (Cpt1a) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (Hmgcr), as well as gluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) in the liver of obese mice. In vitro, SR4 activates AMPK independent of upstream kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Together, these data suggest that SR4, a novel AMPK activator, may be a promising therapeutic compound for treatment of obesity, fatty liver disease, and related metabolic disorders.     

In vivo hepatic lipid quantification using MRS at 7 Tesla in a mouse model of glycogen storage disease type 1a

The assessment of liver lipid content and composition is needed in preclinical research to investigate steatosis and steatosis-related disorders. The purpose of this study was to quantify in vivo hepatic fatty acid content and composition using a method based on short echo time proton magnetic resonance spectroscopy (MRS) at 7 Tesla. A mouse model of glycogen storage disease type 1a with inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc^−/−) mice and control mice were fed a standard diet or a high-fat/high-sucrose (HF/HS) diet for 9 months. In control mice, hepatic lipid content was found significantly higher with the HF/HS diet than with the standard diet. As expected, hepatic lipid content was already elevated in L-G6pc^−/− mice fed a standard diet compared with control mice. L-G6pc^−/− mice rapidly developed steatosis which was not modified by the HF/HS diet. On the standard diet, estimated amplitudes from olefinic protons were found significantly higher in L-G6pc^−/− mice compared with that in control mice. L-G6pc^−/− mice showed no noticeable polyunsaturation from diallylic protons. Total unsaturated fatty acid indexes measured by gas chromatography were in agreement with MRS measurements. These results showed the great potential of high magnetic field MRS to follow the diet impact and lipid alterations in mouse liver.     

Impact of Peripheral Ketolytic Deficiency on Hepatic Ketogenesis and Gluconeogenesis during the Transition to Birth

Preservation of bioenergetic homeostasis during the transition from the carbohydrate-laden fetal diet to the high fat, low carbohydrate neonatal diet requires inductions of hepatic fatty acid oxidation, gluconeogenesis, and ketogenesis. Mice with loss-of-function mutation in the extrahepatic mitochondrial enzyme CoA transferase (succinyl-CoA:3-oxoacid CoA transferase, SCOT, encoded by nuclear Oxct1) cannot terminally oxidize ketone bodies and develop lethal hyperketonemic hypoglycemia within 48 h of birth. Here we use this model to demonstrate that loss of ketone body oxidation, an exclusively extrahepatic process, disrupts hepatic intermediary metabolic homeostasis after high fat mother''s milk is ingested. Livers of SCOT-knock-out (SCOT-KO) neonates induce the expression of the genes encoding peroxisome proliferator-activated receptor γ co-activator-1a (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase, and glucose-6-phosphatase, and the neonate''s pools of gluconeogenic alanine and lactate are each diminished by 50%. NMR-based quantitative fate mapping of ¹³C-labeled substrates revealed that livers of SCOT-KO newborn mice synthesize glucose from exogenously administered pyruvate. However, the contribution of exogenous pyruvate to the tricarboxylic acid cycle as acetyl-CoA is increased in SCOT-KO livers and is associated with diminished terminal oxidation of fatty acids. After mother''s milk provokes hyperketonemia, livers of SCOT-KO mice diminish de novo hepatic β-hydroxybutyrate synthesis by 90%. Disruption of β-hydroxybutyrate production increases hepatic NAD⁺/NADH ratios 3-fold, oxidizing redox potential in liver but not skeletal muscle. Together, these results indicate that peripheral ketone body oxidation prevents hypoglycemia and supports hepatic metabolic homeostasis, which is critical for the maintenance of glycemia during the adaptation to birth.