The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, we have established viable and fertile knock-in mice, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6). Here we show that contrary to the widely accepted model, this mutation does not affect the translational control of TOP mRNAs. rpS6 P−/− mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6 P+/+ MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6 P−/− MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, as it also selectively characterizes pancreatic ␤-cells in adult rpS6 P−/− mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance.

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 ␥ (PPAR␥) 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.

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A sequential pattern of interactions of trans-acting factors in rat liver with the phosphoenolpyruvate carboxykinase promoter during late development was observed. A liver-enriched factor, possibly AF1, interacted with the promoter in fetal liver, whereas a factor with the characteristics of C/EBP bound the promoter after birth with the onset of the gene expression.

The hepatic transcriptional regulation by glucocorticoids of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) gene is coordinated by interactions of specific transcription factors at the glucocorticoid regulatory unit (GRU). We propose an extended GRU that consists of four accessory sites, two proximal AF1 and AF2 sites and their distal counterpart dAF1 (؊993) and a new site, dAF2 (؊1365); together, these four sites form a palindrome. Sequencing and gel shift binding assays of hepatic nuclear proteins interacting with these sites indicated similarity of dAF1 and dAF2 sites to the GRU proximal AF1 and AF2 sites. Chromatin immunoprecipitation assays demonstrated that glucocorticoids enhanced the binding of FOXO1 and peroxisome proliferator-activated receptor-␣ to AF2 and dAF2 sites and not to dAF1 site but enhanced the binding of hepatic nuclear transcription factor-4␣ only to the dAF1 site. Insulin inhibited the binding of these factors to their respective sites but intensified the binding of phosphorylated FOXO1. Transient transfections in HepG2 human hepatoma cells showed that glucocorticoid receptor interacts with several non-steroid nuclear receptors, yielding a synergistic response of the PEPCK-C gene promoter to glucocorticoids. The synergistic stimulation by glucocorticoid receptor together with peroxisome proliferator-activated receptor-␣ or hepatic nuclear transcription factor-4␣ requires all four accessory sites, i.e. a mutation of each of these markedly affects the synergistic response. Mice with a targeted mutation of the dAF1 site confirmed this requirement. This mutation inhibited the full response of hepatic PEPCK-C gene to diabetes by reducing PEPCK-C mRNA level by 3.5-fold and the level of circulating glucose by 25%.

To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-lclc7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the a-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor la (HNF-lae) and HNF-4 and the late-expressed factor C/EBPa, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene.Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-la but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1a resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.The liver is specialized to perform a large variety of metabolic functions in accordance with its central role in mammalian metabolism. This is due to the selective expression in the liver of a subset of genes which code for key proteins that participate in these functions. In recent years, it became apparent that the genes in this subset share some specific, nonubiquitous, cis-regulatory elements which, by binding cognate liver-enriched transcription factors (for reviews, see references 18, 37, and 75), direct their liver-specific expression. To this date, four families of liver-enriched transcription factors have been characterized (for reviews, see references 18, 37, and 75). Analyses of a number of genes have shown that each contains a combination of some or all of the liver-specific cis-regulatory elements. It appears that the combination of cis-regulatory elements, rather than a single cis-regulatory element, is required for liver-specific gene expression (18,37,75). It follows that the corresponding cognate transcription activators of the combinatory sites cooperate to regulate the liver-specific transcription of a given gene and that this process requires that the factors in this subset coexist in the cells that express the gene. Direct evidence for cooperation between factors has been shown in a few instances (43,44,72).The onsets of expression of various liver-specific genes do not occur simultaneo...

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.

The gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) [PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32], a key enzyme in gluconeogenesis and glyceroneogenesis, is expressed in tissues that arise from different embryonal origins: the gluconeogenic liver arises from endoderm, whereas the gluconeogenic kidney cortex and glyceroneogenic adipose tissue arise from the mesoderm. To identify the cis-regulatory elements conferring the differential gene expression, PEPCK chimeric genes were transfected into two rat hepatoma cell lines (H4IIEC3 and HTC-M1.1) and mouse adipocytes (3T3F442A), which express the endogenous gene, and into myoblasts and preadipocytes, which do not express it. The results demonstrate that 597 base pairs of the 5' flanking region of the PEPCK gene are sufficient to confer cell-speciflic gene expression in the PEPCK-expressing hepatoma cells and adipocytes. However, different elements within this 597-base-pair region enhance the gene expression in the hepatoma cells (endoderm) and adipocytes (mesoderm). In the hepatocytes, expression is conferred by two elements-one 5' of position -362 and the other 3' of position -98 with respect to the transcription start site. The region in between these two elements (from -362 to -98), which seems to inhibit the gene expression in the hepatocytes, confers enhanced expression in the adipocytes. Moreover, the distal positive regulatory element ofthe hepatocytes seems to be orientation and PEPCK promoter dependent. In contrast, the positive regulatory element of the adipocytes seems to act as a more typical enhancer. These results suggest that separate cis-regulatory elements confer cell-speciflic expression of the PEPCK gene.