Genetic control of obesity, glucose homeostasis, dyslipidemia and fatty liver in a mouse model of diet-induced metabolic syndrome.

BACKGROUND/OBJECTIVES: Both genetic and dietary factors contribute to the metabolic syndrome (MetS) in humans and animal models. Characterizing their individual roles as well as relationships among these factors is critical for understanding MetS pathogenesis and developing effective therapies. By studying phenotypic responsiveness to high-risk versus control diet in two inbred mouse strains and their derivatives, we estimated the relative contributions of diet and genetic background to MetS, characterized strain-specific combinations of MetS conditions, and tested genetic and phenotypic complexity on a single substituted chromosome. METHODS: Ten measures of metabolic health were assessed in susceptible C57BL/6 J and resistant A/J male mice fed either a control or a high-fat, high-sucrose (HFHS) diet, permitting estimates of the relative influences of strain, diet and strain-diet interactions for each trait. The same traits were measured in a panel of C57BL/6 J (B6)-Chr(A/J) chromosome substitution strains (CSSs) fed the HFHS diet, followed by characterization of interstrain relationships, covariation among metabolic traits and quantitative trait loci (QTLs) on Chromosome 10. RESULTS: We identified significant genetic contributions to nine of ten metabolic traits and significant dietary influence on eight. Significant strain-diet interaction effects were detected for four traits. Although a range of HFHS-induced phenotypes were observed among the CSSs, significant associations were detected among all traits but one. Strains were grouped into three clusters based on overall phenotype and specific CSSs were identified with distinct and reproducible trait combinations. Finally, several Chr10 regions were shown to control the severity of MetS conditions. CONCLUSIONS: Generally strong genetic and dietary effects validate these CSSs as a multifactorial model of MetS. Although traits tended to segregate together, considerable phenotypic heterogeneity suggests that underlying genetic factors influence their co-occurrence and severity. Identification of multiple QTLs within and among strains highlights both the complexity of genetically regulated, diet-induced MetS and the ability of CSSs to prioritize candidate loci for mechanistic studies.

Sexual dimorphism in developmental and diet-dependent circulating retinol binding protein 4.

OBJECTIVE: Retinol binding protein 4 (RBP4) transports vitamin A (Retinol) in the blood and contributes mechanistically to the linkage between obesity, insulin resistance and associated comorbidities including type 2 diabetes mellitus, coronary artery and neoplastic diseases. Circulating RBP4 levels have variably been associated with body mass and gender differences. Many of these differences have been demonstrated after limited dietary interventions, and/or at single unique time points. This study investigated the impact of sex and age as biologic variables as well as high versus low fat diets on development of obesity, RBP4 levels and insulin resistance in C57BL/6J mice. METHODS: Male and female C57BL/6J mice were fed for 400 days with either low or high fat diets. Female mice were also evaluated on same diets after ovariectomy or sham ovariectomy. Mice were monitored for changes in weight, circulating levels RBP4, glucose and insulin at 100-day intervals and also by 2-hour glucose tolerance tests. RESULTS: All mice on low or high fat diets gained weight. Mice on high fat diets showed significantly greater weight gain than those on low fat. Male mice showed significantly greater weight gain compared with females on corresponding diet. Male mice compared with females already showed significantly higher RBP4 levels even before starting diets. Sex differences were maintained for more than 1 year. Gender differences in RBP4 were associated with significant differences in development of glucose intolerance and insulin resistance. CONCLUSIONS: Male compared with female C57BL/6J mice show significant gender differences in circulating RBP4 levels from 6 weeks of age, extending more than 1 year. Gender differences in RBP4 may be mechanistically associated with protection against glucose intolerance and insulin resistance. Targeting RBP4 pathways could be useful to disrupt gender differences in insulin resistance and disparities in comorbidities.

Glucocorticoid receptor, C/EBP, HNF3, and protein kinase A coordinately activate the glucocorticoid response unit of the carbamoylphosphate synthetase I gene.

A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPalpha-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPbeta knockout mice and in HNF3alpha and -gamma double-knockout mice. (The role of HNFbeta could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway.

Structure and expression of a muscle specific gene which is adjacent to the Drosophila myosin heavy-chain gene and can encode a cytochrome b related protein.

We report the characterization of a transcription unit at the second chromosome locus 36B, designated TU-36B which is adjacent to the 3' end of the Drosophila myosin heavy-chain (Mhc) gene. We have isolated and sequenced a complementary DNA clone and the region of genomic DNA which represents this gene. The sequencing studies reveal that this gene contains one intron, the mRNA is 1480 nucleotides in length, the TU-36B mRNA is transcribed in an orientation opposite to the Mhc mRNAs, and the poly(A) addition site of this gene is 99 nucleotides downstream of poly(A) addition site A-2 of the Drosophila Mhc gene. The mRNA contains a continuous open reading frame which can encode a protein product of 47,000 daltons in molecular weight. The proposed protein shares homology with cytochrome b proteins. Comparison of in situ hybridization of Mhc specific and TU-36B specific probes to tissue sections demonstrates that both mRNAs are predominantly transcribed in the same muscle tissues of the developing fly.

Animal models for studying the genetic basis of metabolic regulation.

Modern genetics has developed methods to modify the expression of genes in animals to study the factors responsible for the tissue-specific expression and hormonal and dietary regulation of metabolic processes. As these methods are applied to genes that code for critical proteins in metabolic pathways, a new insight into the control of metabolism is emerging. There are three general approaches currently in use. First, is the introduction of genes into the germ line to create transgenic animal models in which the gene of interest is over-expressed. This model is of particular value for promoter analysis because it is possible to introduce specific mutations into a putative regulatory region of a transgene and study its transcriptional control in the intact animal. Second, the developmental function of a gene product and its effect on various metabolic processes in a mouse can be directly determined by deleting a gene of interest by homologous recombination. Gene "knockout" mice are currently available with deletions in the genes for a variety of transcription factors and other biologically active proteins, permitting a critical analysis of the proteins responsible for the metabolic patterning of the animal. Third, the metabolic role of a gene of interest in a specific tissue can be studied by ablating its mRNA by the introduction of a transgene that codes for antisense mRNA targeted against the gene transcript. Because it is possible to use a transgene with a tissue-specific promoter, this procedure allows the isolation of the metabolic effect to a selected tissue in the transgenic animal. Taken together, these procedures provide a unique set of metabolic models for an in-depth study of metabolic regulation. This review will present examples of selected animal models currently available and will outline the challenge these animals present for investigators in the nutritional sciences.

The use of transgenic mice to analyze the role of accessory factor two in the regulation of phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes.

The regulation of transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (4.1.1.32) during diabetes is a complex process that involves a number of regulatory elements in the PEPCK-C gene promoter. The accessory factor 2 (AF2)-binding region that is contained within the glucocorticoid regulatory unit of the PEPCK-C gene promoter (-451 to -353) has been implicated in the action of both insulin and glucocorticoids on PEPCK-C gene transcription. To determine the role of AF2 in these processes, we have generated a mouse model bearing a transgene that contains the PEPCK-C gene promoter with a mutation in the AF2-binding region. This promoter is linked to the structural gene for human growth hormone that is biologically inactive (AF2-2000/hGx). In the absence of the AF2 regulatory element, the transcription of the transgene in the liver is not induced by diabetes but is inhibited by the administration of insulin. There is also a marked reduction in the response of the AF2-2000/hGx gene in the kidney to the administration of glucocorticoids. The AF2-2000/hGx gene in the liver responds normally to a high carbohydrate diet with a marked decrease in gene transcription. This suggests that insulin is not exerting its usual negative effect on the PEPCK-C gene promoter through the AF2 site. In contrast, the response of this transgene to a high fat/carbohydrate-free diet is severely blunted. Our results support a role for the AF2 site in the PEPCK-C gene promoter in the effect of glucocorticoids, but not insulin, on PEPCK-C gene transcription in the liver.

Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism.

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.