Michael A. Lomax

C/EBPβ Reprograms White 3T3-L1 Preadipocytes to a Brown Adipocyte Pattern of Gene Expression

cAMP-dependent protein kinase induction of PPARγ coactivator-1α (PGC-1α) and uncoupling protein 1 (UCP1) expression is an essential step in the commitment of preadipocytes to the brown adipose tissue (BAT) lineage. We studied the molecular mechanisms responsible for differential expression of PGC-1α in HIB1B (BAT) and 3T3-L1 white adipose tissue (WAT) precursor cell lines. In HIB1B cells PGC-1α and UCP1 expression is cAMP-inducible, but in 3T3-L1 cells, expression is reduced and is cAMP-insensitive. A proximal 264-bp PGC-1α reporter construct was cAMP-inducible only in HIB1B cells and was suppressed by site-directed mutagenesis of the proximal cAMP response element (CRE). In electrophoretic mobility shift assays, the transcription factors CREB and C/EBPβ, but not C/EBPα and C/EBPδ, bound to the CRE on the PGC-1α promoter region in HIB1B and 3T3-L1 cells. Chromatin immunoprecipitation studies demonstrated that C/EBPβ and CREB bound to the CRE region in HIB1B and 3T3-L1 cell lysates. C/EBPβ expression was induced by cAMP only in HIB1B cells, and overexpression of C/EBPβ rescued cAMP-inducible PGC-1α and UCP1 expression in 3T3-L1 cells. These data demonstrate that differentiation of preadipocytes toward the BAT rather than the WAT phenotype is controlled in part by the action of C/EBPβ on the CRE in PGC-1α proximal promoter.

ADIPOSE TISSUE DEVELOPMENT DURING EARLY POSTNATAL LIFE IN EWE-REARED LAMBS

This study examines the precise time course that brown adipose tissue (BAT) takes to adopt the characteristics of white adipose tissue in postnatal lambs. Perirenal adipose tissue was sampled from ewe‐reared lambs within 1 h of birth and at 1, 2, 4, 7, 14, 21 and 30 days of age and analysed for the amount of mRNA for uncoupling protein (UCP), the amount and activity of UCP, and protein, mitochondrial protein and lipid content. This was combined with measurements of colonic temperature and jugular venous plasma concentrations of thyroid hormones and insulin‐like growth factor‐1 (IGF‐1). Over the first 4‐7 days of age, large quantities of UCP mRNA were associated with a peak in plasma triiodothyronine concentration at 2 days of age followed by a maximal amount and activity of UCP at 4 days and a basal colonic temperature of 39.3 degrees C. Between 7 and 30 days there was a large increase in lipid deposition as the amount and activity of UCP and the amount of UCP mRNA declined to basal values and colonic temperature was maintained at 40 degrees C. A significant positive relationship between perirenal adipose tissue lipid content and plasma IGF‐1 concentration was observed throughout the study period. It is concluded that ovine adipose tissue maturation occurs in two distinct phases over the first month of life. The precise time scale of this process could be regulated in part by the lambs body temperature which determines whether adipose tissue is required for heat production (i.e. BAT) or as an endogenous energy source (i.e. white adipose tissue).

Nutrition, temperature and homeostasis during perinatal development

Introduction 907 The maternal environment and fetal development 908 Maternal nutrition and fetal lung growth 911 Fetal metabolism 913 Placetal inhibitory factors 915 Adenosine 915 Prostaglandin E2 916 Fetal stimulatory factors 917 Thyroid hormones 917 Catecholamines 917 Ontogeny of fetal brown adipose tissue 918 Birth 920 Caesarean section delivery 922 Environmental temperature and metabolic adaptation after birth 923 Postnatal development 925 Thyroid hormones and the control of metabolism during postnatal life 926 Nutritional and environmental influences on postnatal metabolism and growth 929 Postnatal metabolism and breathing control 931 Conclusion 932 References 933

Combinatorial Transcription Factor Regulation of the Cyclic AMP-response Element on the Pgc-1α Promoter in White 3T3-L1 and Brown HIB-1B Preadipocytes

Cold stress in rodents increases the expression of UCP1 and PGC-1α in brown and white adipose tissue. We have previously reported that C/EBPβ specifically binds to the CRE on the proximal Pgc-1α promoter and increases forskolin-sensitive Pgc-1α and Ucp1 expression in white 3T3-L1 preadipocytes. Here we show that in mice exposed to a cold environment for 24 h, Pgc-1α, Ucp1, and C/ebpβ but not C/ebpα or C/ebpδ expression were increased in BAT. Conversely, expression of the C/EBP dominant negative Chop10 was increased in WAT but not BAT during cold exposure. Reacclimatization of cold-exposed mice to a warm environment for 24 h completely reversed these changes in gene expression. In HIB-1B, brown preadipocytes, forskolin increased expression of Pgc-1α, Ucp1, and C/ebpβ early in differentiation and inhibited Chop10 expression. Employing chromatin immunoprecipitation, we demonstrate that C/EBPβ, CREB, ATF-2, and CHOP10 are bound to the Pgc-1α proximal CRE, but CHOP10 does not bind in HIB-1B cell lysates. Forskolin stimulation and C/EBPβ overexpression in 3T3-L1 cells increased C/EBPβ and CREB but displaced ATF-2 and CHOP10 binding to the Pgc-1α proximal CRE. Overexpression of ATF-2 and CHOP10 in 3T3-L1 cells decreased Pgc-1α transcription. Knockdown of Chop10 in 3T3-L1 cells using siRNA increased Pgc-1α transcription, whereas siRNA against C/ebpβ in HIB-1B cells decreased Pgc-1α and Ucp1 expression. We conclude that the increased cAMP stimulation of Pgc-1α expression is regulated by the combinatorial effect of transcription factors acting at the CRE on the proximal Pgc-1α promoter.

Consequences of exposure to serum, with or without vitamin E supplementation, in terms of the fatty acid content and viability of bovine blastocysts produced in vitro

To determine whether serum supplementation influenced fatty acid content of bovine blastocysts and whether vitamin E addition to culture medium containing serum could improve development in vitro, cleaved eggs were cultured in synthetic oviduct fluid supplemented with bovine serum albumin (BSA, 0.4% w/v, fraction V) (SVBSA), fetal calf serum (FCS, 10% v/v) (SFCS) or FCS (10% v/v) plus 100 micro M vitamin E (SFCS + E). Blastocyst yields were recorded and fatty acid composition was determined by gas chromatography. Day 7 blastocysts were incubated with [2-(14)C] pyruvate for 3 h and then fixed for cell counts. Yields of good quality blastocysts were greatest from cleaved eggs cultured in serum-free conditions (P < 0.01). In the presence of serum, supplementation with vitamin E increased both total and good quality blastocyst yields (P < 0.01). Presence of serum increased fatty acid content (mean +/- SEM) of blastocysts (SVBSA v. SFCS = 57 +/- 2 v. 74 +/- 2 ng embryo(-1); P < 0.001). In contrast, pyruvate metabolism was greater in blastocysts produced without serum (27 +/- 3 v. 21 +/- 3 picomoles embryo(-1) 3h(-1); P < 0.01) but, on a per cell basis, no differences were detected. Addition of vitamin E to the serum-supplemented formulation did not alter either the fatty acid content (73 +/- 2 ng embryo(-1)) or pyruvate metabolism index (19 +/- 1 pmol embryo(-1) 3h(-1)) of SFCS + E blastocysts. Thus, despite lipid accumulation, supplementary vitamin E improved blastocyst yields in embryos exposed to serum.

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue

Aims/hypothesisIncreasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues.MethodsMethylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding.ResultsWe show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter–reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment.Conclusions/interpretationThe results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails.

Adipose tissue development--impact of the early life environment.

Increasing experimental and observational evidence in both animals and humans suggests that early life events are important in setting later fat mass. This includes both the number of adipocytes and the relative distribution of both brown and white adipose tissue. Brown adipose tissue is characterised as possessing a unique uncoupling protein (UCP)1 which enables the rapid generation of large amounts of heat and is most abundant in the newborn. In large mammals such as sheep and humans, brown fat that is located around the major internal organs, is largely lost during the postnatal period. However, it is retained in small and discrete areas into adulthood when it is sensitive to environmental cues such as changes in ambient temperature or day length. The extent to which brown adipose tissue is lost or replaced by white adipose tissue and/or undergoes a process of transdifferentiation remains controversial. Small amounts of UCP1 can also be present in skeletal muscle which now appears to share the same common precursor cell as brown adipose tissue. The functional consequences of UCP1 in muscle remain to be confirmed but it could contribute to dietary induced thermogenesis. Challenges in elucidating the primary mechanisms regulating adipose tissue development include changes in methylation status of key genes during development in different species, strains and adipose depots. A greater understanding of the mechanisms by which early life events regulate adipose tissue distribution in young offspring are likely to provide important insights for novel interventions that may prevent excess adiposity in later life.

Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

BackgroundThe ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysisResultsIncubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA), relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p < 0.05). The proteasome inhibitor MG132 blocked the rise in proteolysis observed in the 0.2 × PC AA media. Addition of insulin to the medium inhibited proteolysis at both 0.2 and 1.0× PC AA and the expression of 14-kDa E2 proteins and C2 sub unit of 20 S proteasome (p < 0.05). Incubation of myotubes with increasing concentrations of leucine in the 0.2 × PC AA media inhibited proteolysis but only in the presence of insulin. Incubation of rapamycin (inhibitor of mTOR) inhibited amino acid or insulin-dependent p70 S6 kinase phosphorylation, blocked (P < 0.05) the inhibitory effects of 1.0 × PC AA on protein degradation, but did not alter the inhibitory effects of insulin or leucineConclusionIn a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.

Cold-induced changes in gene expression in brown adipose tissue, white adipose tissue and liver

Cold exposure imposes a metabolic challenge to mammals that is met by a coordinated response in different tissues to prevent hypothermia. This study reports a transcriptomic analysis in brown adipose tissue (BAT), white adipose (WAT) and liver of mice in response to 24 h cold exposure at 8°C. Expression of 1895 genes were significantly (P<0.05) up- or down-regulated more than two fold by cold exposure in all tissues but only 5 of these genes were shared by all three tissues, and only 19, 14 and 134 genes were common between WAT and BAT, WAT and liver, and BAT and liver, respectively. We confirmed using qRT-PCR, the increased expression of a number of characteristic BAT genes during cold exposure. In both BAT and the liver, the most common direction of change in gene expression was suppression (496 genes in BAT and 590 genes in liver). Gene ontology analysis revealed for the first time significant (P<0.05) down regulation in response to cold, of genes involved in oxidoreductase activity, lipid metabolic processes and protease inhibitor activity, in both BAT and liver, but not WAT. The results reveal an unexpected importance of down regulation of cytochrome P450 gene expression and apolipoprotein, in both BAT and liver, but not WAT, in response to cold exposure. Pathway analysis suggests a model in which down regulation of the nuclear transcription factors HNF4α and PPARα in both BAT and liver may orchestrate the down regulation of genes involved in lipoprotein and steroid metabolism as well as Phase I enzymes belonging to the cytochrome P450 group in response to cold stress in mice. We propose that the response to cold stress involves decreased gene expression in a range of cellular processes in order to maximise pathways involved in heat production.

Pigs fed saturated fat/cholesterol have a blunted hypothalamic-pituitary-adrenal function, are insulin resistant and have decreased expression of IRS-1, PGC1α and PPARα.

The increasing incidence of insulin resistance has been linked to both increased intake of saturated fatty acids and disruption of the hypothalamic-pituitary-adrenal (HPA) axis. We tested the hypothesis that adding saturated fat/cholesterol to the diet of growing pigs would both disrupt HPA function and cause insulin resistance. Three-month-old pigs were fed either a control (13% energy from fat) or a high saturated fatty acid cholesterol (HSFC) diet (44% energy from fat; 2% cholesterol). After 10 weeks on the diets, intravenous ACTH, insulin and glucose challenges were performed, and after 12 weeks, tissue samples were taken for measurement of mRNA and for lipid-rich aortic lesions. Plasma total, HDL- and LDL-cholesterol were significantly increased in pigs fed the HSFC diet. Cortisol release during the ACTH challenge was suppressed in HSFC-fed pigs which were also more insulin resistant and glucose intolerant than controls. The HSFC diet decreased the expression of insulin receptor (IR) and insulin receptor substrate-1 in muscle and adipose tissue as well as adiponectin and adiponectin receptor 2 expression in fat. The HSFC diet decreased PGC-1α and PPARα expression in muscle but increased PPARα expression in liver. There was a trend for an increase in lipid-stained lesion frequency around the abdominal branches of the aorta in HSFC-fed pigs. We conclude that feeding increased saturated fat to pigs causes disruption in the HPA axis, insulin resistance and decreased muscle and adipose expression of genes controlling insulin signalling and mitochondrial oxidative capacity.