Nicole K. MacLennan

Increased Hepatic Peroxisome Proliferator-Activated Receptor-γ Coactivator-1 Gene Expression in a Rat Model of Intrauterine Growth Retardation and Subsequent Insulin Resistance

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) increase the risk of type 2 diabetes in humans and rats. Unsuppressed endogenous hepatic glucose production is a common component of the insulin resistance associated with type 2 diabetes. Peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1) mediates hepatic glucose production by controlling mRNA levels of glucose-6-phosphatase (G-6-Pase), phosphoenolpyruvate carboxykinase (PEPCK), and fructose-1,6-bisphosphatase (FBPase). We therefore hypothesized that gene expression of PGC-1 would be increased in juvenile IUGR rat livers, and this increase would directly correlate with hepatic mRNA levels of PEPCK, G-6-Pase, and FBPase, but not glucokinase. We found that IUGR hepatic PGC-1 protein levels were increased to 230 ± 32% and 310 ± 47% of control values at d 0 and d 21 of life, respectively. Similarly, IUGR hepatic PGC-1 mRNA levels were significantly elevated at both ages. Concurrent with the increased PGC-1 gene...

IUGR Alters Postnatal Rat Skeletal Muscle Peroxisome Proliferator-Activated Receptor-γ Coactivator-1 Gene Expression in a Fiber Specific Manner

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) increase the risk of insulin resistance in humans and rats. Aberrant skeletal muscle lipid metabolism contributes to the pathogenesis of insulin resistance. Peroxisome proliferator-activated receptor-γ co-activator-1 (PGC-1) is a transcriptional co-activator that affects gene expression of key lipid metabolizing enzymes such as carnitine palmitoyl-transferase I (mCPTI). Because gene expression of lipid metabolizing enzymes is altered in IUGR postnatal skeletal muscle, and we hypothesized that PGC-1 expression would be similarly affected. To prove this hypothesis, bilateral uterine artery ligation and sham surgery were used to produce IUGR and control rats respectively. Western Blotting demonstrated that PGC-1 hind limb skeletal muscle protein levels were increased in perinatal and postnatal IUGR rats. Conventional RT-PCR demonstrated that PGC-1 mRNA levels were similarly increased in perinatal hind limb skeletal muscle and juvenile extensor digitorum longus (EDL), but were decreased in juvenile soleus. Because a gender specific trend was noted in PGC-1 mRNA levels, real time RT-PCR was used for further differentiation. Real time RT-PCR revealed that changes in postnatal skeletal muscle PGC-1 expression were more marked in male IUGR rats versus female IUGR rats. Down stream targets of PGC-1 followed a similar pattern of expression. We conclude that PGC-1 expression is altered in rat IUGR skeletal muscle and speculate that it contributes to the pathogenesis of insulin resistance in the IUGR rat.

Glycerol kinase deficiency alters expression of genes involved in lipid metabolism, carbohydrate metabolism, and insulin signaling.

Glycerol kinase (GK) is at the interface of fat and carbohydrate metabolism and has been implicated in insulin resistance and type 2 diabetes mellitus. To define GKs role in insulin resistance, we examined gene expression in brown adipose tissue in a glycerol kinase knockout (KO) mouse model using microarray analysis. Global gene expression profiles of KO mice were distinct from wild type with 668 differentially expressed genes. These include genes involved in lipid metabolism, carbohydrate metabolism, insulin signaling, and insulin resistance. Real-time polymerase chain reaction analysis confirmed the differential expression of selected genes involved in lipid and carbohydrate metabolism. PathwayAssist analysis confirmed direct and indirect connections between glycerol kinase and genes in lipid metabolism, carbohydrate metabolism, insulin signaling, and insulin resistance. Network component analysis (NCA) showed that the transcription factors (TFs) PPAR-γ, SREBP-1, SREBP-2, STAT3, STAT5, SP1, CEBPα, CREB, GR and PPAR-α have altered activity in the KO mice. NCA also revealed the individual contribution of these TFs on the expression of genes altered in the microarray data. This study elucidates the complex network of glycerol kinase and further confirms a possible role for glycerol kinase deficiency, a simple Mendelian disorder, in insulin resistance, and type 2 diabetes mellitus, a common complex genetic disorder.

Weighted gene co-expression network analysis identifies biomarkers in glycerol kinase deficient mice

Symptomatic glycerol kinase deficiency (GKD) is associated with episodic metabolic and central nervous system deterioration. We report here the first application of weighted gene co-expression network analysis (WGCNA) to investigate a knockout (KO) murine model of a human genetic disease. WGCNA identified networks and key hub transcripts from liver mRNA of glycerol kinase (Gyk) KO and wild-type (WT) mice. Day of life 1 (dol1) samples from KO mice contained a network module enriched for organic acid metabolism before Gyk KO mice develop organic acidemia and die on dol3-4. Furthermore, the module containing Gyk was enriched with apoptotic genes. We used causal testing to elucidate the causal relationships between intramodular hub genes Acot, Psat and Plk3. Important causal relationships are confirmed in cell cultures. We provide evidence that GK may have an apoptotic moonlighting role that is lost in GKD. This first application of WGCNA to mouse knockout data provides insights into the molecular mechanisms of GKD pathogenesis. The resulting systems-genetic gene screening method identifies candidate biomarkers for GKD.

Uteroplacental insufficiency alters liver and skeletal muscle branched-chain amino acid metabolism in intrauterine growth-restricted fetal rats

Uteroplacental insufficiency causes intrauterine growth restriction (IUGR) and decreases plasma levels of the branched-chain amino acids in both humans and rats. Increased fetal oxidation of these amino acids may contribute to their decline in the IUGR fetus. The rate-limiting step of branched-chain amino acid oxidation is performed by the mitochondrial enzyme branched-chain α-keto acid dehydrogenase (BCKAD), which is regulated by a deactivating kinase. We therefore hypothesized that uteroplacental insufficiency increases BCKAD activity through altered mRNA and protein levels of BCKAD and/or the BCKAD kinase. In IUGR fetal liver, BCKAD activity was increased 3-fold, though no difference in hepatic BCKAD protein or mRNA levels were noted. Hepatic BCKAD kinase mRNA and protein levels were significantly decreased in association with the increase in BCKAD activity. In IUGR fetal skeletal muscle, BCKAD mRNA levels were significantly increased. IUGR skeletal muscle BCKAD protein levels as well as BCKAD kinase mRNA and protein levels were unchanged. We also quantified mRNA levels of two amino acid transporters: LAT1 (system L) and rBAT (cysteine and dibasic amino acids). Both hepatic and muscle LAT1 mRNA levels were significantly increased in the IUGR fetus. We conclude that uteroplacental insufficiency significantly increases hepatic BCKAD activity in association with significantly decreased mRNA and protein levels of the deactivating kinase. We speculate that these changes contribute to the decreased serum levels of branched-chain amino acids seen in the IUGR fetus and may be an adaptation to the deprived milieu associated with uteroplacental insufficiency.

Uncoupling Protein 3 (UCP3) but Not UCP2 Gene Expression Is Increased in the Human Glucose Transporter - 4 Overexpressing Suckling Mice

Uncoupling Protein 3 (UCP3) but Not UCP2 Gene Expression Is Increased in the Human Glucose Transporter - 4 Overexpressing Suckling Mice

Prenatal Glucocorticoid Induced Fetal and Postnatal Growth Restriction is Mediated by Obese Gene (OB) or Leptin Receptors † 1559

Prenatal glucocorticoid (GC) therapy is an accepted practice in the prevention of respiratory distress syndrome in the neonate. Concerns regarding its use in early gestation and its side effects upon fetal and postnatal growth and metabolism persist. The recent cloning of the obese gene and obese gene receptors (OB-R) has shed some light upon mechanisms by which altered metabolism (anorexic/catabolic) and diminution in growth occurs. To determine if the ob gene (leptin) receptors mediate the growth reducing effects of GC, we intraperitoneally administered 2 doses of 0.3 mg/kg/day dexamethasone (GC) or vehicle (V) beginning d7, d9, d11, d13, d15, d17, and d19 (term ≈21d; n=7/age/group), and examined the binding of125 I-leptin (10-9M), in the presence and absence of 0.6 X 10-6M unlabeled leptin, to the d9, d11, d13, d15, d17, d19, and d21 frozen saggital embryonic liver sections. In addition, postnatal livers were studied on d15 and d30 (n=7/age/group) subsequent to GC or V administration on d19 gestation. Specific 125I-leptin binding was assessed by quantitation of the autoradiographs and phoshoimages by microdensitometry(Molecular Analyst Microdensitometry Program-1.4.1, NIH). GC reduced body weight at all ages (p < 0.001), and increased hepatic 125I-leptin binding at all ages with a maximal 3-fold increase noted at postnatal d15 (p< 0.005). This is in contrast to a 2-fold decline in hepatic125 I-insulin binding (p < 0.01) at postnatal d15, which served as a known control. We conclude that prenatal GC 1] increases hepatic leptin receptors from mid- to late gestation, 2] has a persistent enhancing effect upon postnatal hepatic leptin receptors. We speculate that prenatal GC increases the biological action of leptin peripherally, thereby affecting the metabolic mileu and the growth potential of the fetus and the newborn.

Obese Gene (Leptin) Receptors are Widely Distributed in Embryonic Tissues • 293

Leptin receptors (LR) mediate central (satiety) and peripheral metabolic actions of the translated and secreted product of the obese (ob) gene. While two isoforms as products of RNA splicing have been characterized, both of them bind leptin on the same epitope. Even though LR has been reported in adult tissues, limited information exists with respect to its presence and tissue-distribution in the embryo. We examined the spatial and temporal distribution of LR in the rat conceptus and placenta from d9 to d21 at 2 day intervals (term≈d21), and postnatal d15, and d30 (n=7/age/group). Employing frozen saggital embryonic and postnatal rat sections, 125I-leptin binding in the presence and absence of 0.6 × 10-6M unlabeled leptin was assessed by autoradiography and microdensitometry (Molecular Analyst Microdensitometry Program - 1.4.1, NIH). On d9, the placenta revealed LR while the known control (125I-insulin binding [IR]) was noted in amniotic membranes. At d11, LR was noted in the liver, while liver IR was present at d13. LR was present in the brain [d13], spine and long bones [d13], heart [d15], and kidney [d17]. The LR increased 2.5 fold in density from d9 to d21 (p < 0.01), with a subsequent 3-fold rise postnatally reaching a peak at d15 (p < 0.05). In contrast, IR demonstrated a 1.5-fold increase prenatally followed by a peak at postnatal d15. We conclude that 1] LR are present in embryonic tissues although the ontogenic peak is observed postnatally at d15, 2] LR are distributed in various tissues, with the earliest appearance being limited to the placenta and liver even before its appearance in the brain. We speculate that LR mediate the biological effects of leptin both centrally and peripherally in the embryo thereby regulating metabolism and growth.