Colleen M. Croniger

A mutation in the peroxisome proliferator-activated receptor γ-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice

Regulation of the turnover of triglycerides in adipose tissue requires the continuous provision of 3-glycerophosphate, which may be supplied by the metabolism of glucose or by glyceroneogenesis, the de novo synthesis of 3-glycerophosphate from sources other than hexoses or glycerol. The importance of glyceroneogenesis in adipose tissue was assessed in mice by specifically eliminating the expression of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C), an enzyme that plays a pivotal role in the pathway. To accomplish this, we mutated the binding site for the peroxisome proliferator-activated receptor γ (PPARγ) called the peroxisome proliferator-activated receptor element (PPARE), in the 5′ flanking region of the PEPCK-C gene in the mouse by homologous recombination. The mutation abolished expression of the gene in white adipose tissue and considerably reduced its expression in brown adipose tissue, whereas the level of PEPCK-C mRNA in liver and kidney remained normal. Epididymal white adipose tissue from these mice had a reduced triglyceride deposition, with 25% of the animals displaying lipodystrophy. There was also a greatly reduced level of lipid accumulation in brown adipose tissue. A strong correlation between the hepatic content of triglycerides and the size of the epididymal fat pad in PPARE−/− mice suggests that hepatic triglyceride synthesis predominantly utilizes free fatty acids derived from the adipose tissue. Unlike other models, PPARE−/− mice with lipodystrophy did not exhibit the lipodystrophy-associated features of diabetes and displayed only moderate hyperglycemia. These studies establish the importance of the PPARE site for PEPCK-C gene expression in adipose tissue and the role of PEPCK-C in the regulation of glyceroneogenesis, a pathway critical for maintaining the deposition of triglycerides in adipose tissue.

Role of the Isoforms of CCAAT/Enhancer-binding Protein in the Initiation of Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription at Birth

The gene for phosphoenolpyruvate carboxykinase (PEPCK), a target of CCAAT/enhancer-binding protein-α (C/EBPα) and -β (C/EBPβ), begins to be expressed in the liver at birth. Mice homozygous for a deletion in the gene for CEBPα (C/EBPα−/− mice) die shortly after birth of hypoglycemia, with no detectable hepatic PEPCK mRNA and negligible hepatic glycogen stores. Half of the mice homozygous for a deletion in the gene for CEBPβ (C/EBPβ−/−mice) have normal glucose homeostasis (phenotype A), and the other half die at birth of hypoglycemia due to a failure to express the gene for PEPCK and to mobilize hepatic glycogen (phenotype B). Insulin deficiency induces C/EBPα and PEPCK gene transcription in the livers of 19-day fetal rats, whereas dibutyryl cyclic AMP (Bt2cAMP) increases the expression of the gene for C/EBPβ and causes a transient burst of PEPCK mRNA. Bt2cAMP induces PEPCK mRNA in the livers of fetal C/EBPα−/− mice, but at only 20% of the level of control animals; however, there is no induction of PEPCK mRNA if the cyclic nucleotide is injected into C/EBPα−/− mice immediately after delivery. The expression of the gene for C/EBPβ is markedly induced in the livers of C/EBPα−/− mice within 2 h after the administration of Bt2cAMP. C/EBPβ−/− mice injected at 20 days of fetal life with Bt2cAMP have a normal pattern of induction of hepatic PEPCK mRNA. In C/EBPβ−/− mice with phenotype B, the administration of Bt2cAMP immediately after delivery induces PEPCK mRNA, causes the mobilization of hepatic glycogen, and maintains normal glucose homeostasis for up to 4 h (duration of the experiment). We conclude that C/EBPα is required for the cAMP induction of PEPCK gene expression in the liver and that C/EBPβ can compensate for the loss of C/EBPα if its concentration is induced to appropriate levels.

Functional cooperation between CCAAT/enhancer-binding proteins and the vitamin D receptor in regulation of 25-hydroxyvitamin D3 24-hydroxylase

ABSTRACT 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces the synthesis of 25-hydroxyvitamin D3 24-hydroxylase [24(OH)ase], an enzyme involved in its catabolism, thereby regulating its own metabolism. Here we demonstrate that CCAAT enhancer binding protein β (C/EBPβ) is induced by 1,25(OH)2D3 in kidney and in osteoblastic cells and is a potent enhancer of vitamin D receptor (VDR)-mediated 24(OH)ase transcription. Transfection studies indicate that 1,25(OH)2D3 induction of 24(OH)ase transcription is enhanced a maximum of 10-fold by C/EBPβ. Suppression of 1,25(OH)2D3-induced 24(OH)ase transcription was observed with dominant negative C/EBP or osteoblastic cells from C/EBPβ−/− mice. A C/EBP site was identified at positions −395 to −388 (−395/−388) in the rat 24(OH)ase promoter. Mutation of this site inhibited C/EBPβ binding and markedly attenuated the transcriptional response to C/EBPβ. We also report the cooperation of CBP/p300 with C/EBPβ in regulating VDR-mediated 24(OH)ase transcription. We found that not only 1,25(OH)2D3 but also parathyroid hormone (PTH) can induce C/EBPβ expression in osteoblastic cells. PTH potentiated the induction of C/EBPβ and 24(OH)ase expression in response to 1,25(OH)2D3 in osteoblastic cells. Data with the human VDR promoter (which contains two putative C/EBP sites) indicate a role for C/EBPβ in the protein kinase A-mediated induction of VDR transcription. From this study a fundamental role has been established for the first time for cooperative effects and cross talk between the C/EBP family of transcription factors and VDR in 1,25(OH)2D3-induced transcription. These findings also indicate a novel role for C/EBPβ in the cross talk between PTH and 1,25(OH)2D3 that involves the regulation of VDR transcription.

Retinoic acid upregulates preadipocyte genes to block adipogenesis and suppress diet-induced obesity.

Retinoic acid (RA) protects mice from diet-induced obesity. The activity is mediated in part through activation of the nuclear receptors RA receptors (RARs) and peroxisome proliferator–activated receptor β/δ and their associated binding proteins cellular RA binding protein type II (CRABP-II) and fatty acid binding protein type 5 in adipocytes and skeletal muscle, leading to enhanced lipid oxidation and energy dissipation. It was also reported that RA inhibits differentiation of cultured preadipocytes. However, whether the hormone suppresses adipogenesis in vivo and how the activity is propagated remained unknown. In this study, we show that RA inhibits adipocyte differentiation by activating the CRABP-II/RARγ path in preadipose cells, thereby upregulating the expression of the adipogenesis inhibitors Pref-1, Sox9, and Kruppel-like factor 2 (KLF2). In turn, KLF2 induces the expression of CRABP-II and RARγ, further potentiating inhibition of adipocyte differentiation by RA. The data also indicate that RA suppresses adipogenesis in vivo and that the activity significantly contributes to the ability of the hormone to counteract diet-induced obesity.

C/EBP and the control of phosphoenolpyruvate carboxykinase gene transcription in the liver.

In 1989, shortly after the discovery of CAAT/enhancer-binding protein (C/EBP) and in a period before it was clear that there was more than one form of C/EBP, McKnight et al. (1) published a review entitled: “Is C/EBP a Central Regulator of Energy Metabolism?” This prediction of a critical metabolic role for this transcription factor was based on the very slim evidence that C/EBP was involved in the transcription of a number of metabolically important genes such as 422/aP2, phosphoenolpyruvate carboxykinase (PEPCK), and fatty acid synthase, in addition to its role in the differentiation of adipocytes (2, 3). Over the decade since this article was published, the prediction has proven to be remarkably accurate. C/EBP is now known to comprise a gene family with a number of closely related members, the biology of which has been detailed in the first minireview in this series by Lekstrom-Himes and Xanthopoulos (4). These C/EBP isoforms can stimulate or inhibit transcription from a growing list of genes in a variety of tissues in animals as diverse as chickens and rats. One of the critical aspects of the biology of C/EBP that has emerged over the past 10 years is the key role that members of the family of transcription factors play in both the development and maintenance of metabolically important processes (1, 5, 6). This review will focus on the effects of C/EBP isoforms on the control of transcription of the gene for the key gluconeogenic enzyme PEPCK (GTP) (EC 4.1.1.32) as a model for its regulation of other genes that code for enzymes of metabolic importance.

The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin A homeostasis in tissues other than the eye.

Background: STRA6 transports retinol into cells and activates cell signaling. Results: Ablation of Stra6 does not impair vitamin A homeostasis in tissues other than the eye but protects mice against RBP-induced insulin resistance. Conclusion: One major function of STRA6 is to control cell signaling. Significance: The data point at a new function for vitamin A and its blood carrier RBP. The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.

THE TRANSCRIPTION FACTOR CCAAT/ENHANCER-BINDING PROTEIN BETA REGULATES GLUCONEOGENESIS AND PHOSPHOENOLPYRUVATE CARBOXYKINASE (GTP) GENE TRANSCRIPTION DURING DIABETES

CCAAT/enhancer-binding protein (C/EBP) β and C/EBPα are members of the c/ebp gene family and are highly expressed in mammalian liver and adipose tissue. C/EBPα is essential for adipogenesis and neonatal gluconeogenesis, as shown by the C/EBPα knockout mouse. C/EBPβ binds to several sequences of the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter with high affinity, and C/EBPβ protein is increased 200% in the livers of streptozotocin-diabetic mice, concurrent with increased PEPCK mRNA. To elucidate the role of C/EBPβ in the control of gluconeogenesis during diabetes, we studied the levels of plasma metabolites and hormones related to energy metabolism during diabetes in adult mice heterozygous and homozygous for a null mutation of the gene for C/EBPβ. We also examined the expression of PEPCK and glucose 6-phosphatase mRNAs and regulation of blood glucose, including the contribution of gluconeogenesis to blood glucose inc/ebpβ−/− mice. C/EBPβ was not essential to basal PEPCK mRNA levels. However, C/EBPβ deletion affected streptozotocin-diabetic response by: (a) delaying hyperglycemia, (b) preventing the increase of plasma free fatty acids, (c) limiting the full induction of PEPCK and glucose 6-phosphatase genes, and (d) preventing the increase in gluconeogenesis rate. Gel supershifts of transcription factor C/EBPα, bound to CRE, P3I, and AF-2 sites of the PEPCK promoter, was not increased in diabeticc/ebpβ−/− mouse liver nuclei, suggesting that C/EBPα does not substitute for C/EBPβ in the diabetic response of liver gene transcription. These results link C/EBPβ to the metabolic and gene regulatory responses to diabetes and implicate C/EBPβ as an essential factor underlying glucocorticoid-dependent activation of PEPCK gene transcription in the intact animal.

Mice with a Deletion in the Gene for CCAAT/Enhancer-binding Protein β 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 β (C/EBPβ−/− 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/EBPβ−/− 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, β-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/EBPβ−/− 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 RIα and 27% increase in RIIα in the particulate fraction of the livers of C/EBPβ−/− mice relative to wild type mice, with no change in the catalytic subunit (Cα). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBPβ−/− mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBPβ−/− 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/EBPβ influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.

Differential Control of the CCAAT/Enhancer-binding Protein β (C/EBPβ) Products Liver-enriched Transcriptional Activating Protein (LAP) and Liver-enriched Transcriptional Inhibitory Protein (LIP) and the Regulation of Gene Expression during the Response to Endoplasmic Reticulum Stress

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response program that protects cells early in the response and can lead to apoptosis during prolonged stress. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein β (C/EBPβ), is one of the genes with increased expression during ER stress. Translation of the C/EBPβ mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP-1 and -2) and a transcriptional repressor (LIP). The LIP/LAP ratio is a critical factor in C/EBPβ-mediated gene transcription. It is shown here that the LIP/LAP ratio decreased by 5-fold during the early phase of ER stress and increased by 20-fold during the late phase, mostly because of changes in LIP levels. The early decrease in LIP required degradation via the proteasome pathway and phosphorylation of the translation initiation factor, eIF2α. The increased LIP levels during the late phase were due to increased synthesis and increased stability of the protein. It is proposed that regulation of synthesis and degradation rates during ER stress controls the LIP/LAP ratio. The importance of C/EBPβ in the ER-stress response program was demonstrated using C/EBPβ-deficient mouse embryonic fibroblasts. It is shown that C/EBPβ attenuates expression of pro-survival ATF4 target genes in late ER stress and enhances expression of cell death-associated genes downstream of CHOP. The inhibitory effect of LIP on ATF4-induced transcription was demonstrated for the cat-1 amino acid transporter gene. We conclude that regulation of LIP/LAP ratios during ER stress is a novel mechanism for modulating the cellular stress response.

Mice With a Deletion in the Gene for CCAAT/Enhancer-Binding Protein β Are Protected Against Diet-Induced Obesity

The CCAAT/enhancer-binding protein β (C/EBPβ) is required for adipocyte differentiation and maturation. We have studied the role of the transcription factor, C/EBPβ, in the development of diet-induced obesity. Mice with a deletion in the gene for C/EBPβ (C/EBPβ−/−) and wild-type mice were fed a high-fat diet (60% fat) for 12 weeks. The C/EBPβ−/− mice lost body fat, whereas the wild-type mice increased their total body fat on a high-fat diet. The C/EBPβ−/− mice had lower levels of blood triglycerides, free fatty acids, cholesterol, and hepatic triglyceride accumulation compared with the wild-type mice, thus protecting them from diet-induced obesity and fatty liver on a high-fat diet. Deletion of C/EBPβ gene resulted in greatly reducing hepatic lipogenic genes, acetyl CoA carboxylase, and fatty acid synthase and increasing the expression of β-oxidation genes in the brown adipose tissue. CO2 production was significantly higher in the C/EBPβ−/− mice as was the level of uncoupling protein (UCP)-1 and UCP-3 in the muscle. In conclusion, the transcription factor C/EBPβ is an important regulator in controlling lipid metabolism and in the development of diet-induced obesity.