Adaptive thermogenesis is an important component of energy homeostasis and a metabolic defense against obesity. We have cloned a novel transcriptional coactivator of nuclear receptors, termed PGC-1, from a brown fat cDNA library. PGC-1 mRNA expression is dramatically elevated upon cold exposure of mice in both brown fat and skeletal muscle, key thermogenic tissues. PGC-1 greatly increases the transcriptional activity of PPARgamma and the thyroid hormone receptor on the uncoupling protein (UCP-1) promoter. Ectopic expression of PGC-1 in white adipose cells activates expression of UCP-1 and key mitochondrial enzymes of the respiratory chain, and increases the cellular content of mitochondrial DNA. These results indicate that PGC-1 plays a key role in linking nuclear receptors to the transcriptional program of adaptive thermogenesis.

Previously, we have isolated and characterized an enhancer from the 5'-flanking region of the adipocyte P2 (aP2) gene that directs high-level adipocyte-specific gene expression in both cultured cells and transgenic mice. The key regulator of this enhancer is a cell type-restricted nuclear factor termed ARF6. Target sequences for ARF6 in the aP2 enhancer exhibit homology to a direct repeat of hormone response elements (HREs) spaced by one nucleotide; this motif (DR-l) has been demonstrated previously to be the preferred binding site for heterodimers of the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). We have cloned a novel member of the peroxisome proliferator-activated receptor family designated mPPART2, and we demonstrate that a heterodimeric complex of mPPAR~/2 and RXR~ constitute a functional ARF6 complex. Expression of mPPAR,/2 is induced very early during the differentiation of several cultured adipocyte cell lines and is strikingly adipose-specific in vivo. mPPAR-/2 and RXRc~ form heterodimers on ARF6-binding sites in vitro, and antiserum to RXRc~ specifically inhibits ARF6 activity in adipocyte nuclear extracts. Moreover, forced expression of mPPAR~/2 and RXR~ activates the adipocyte-specific aP2 enhancer in cultured fibroblasts, and this activation is potentiated by peroxisome proliferators, fatty acids, and 9-cis retinoic acid. These results identify mPPAR~/2 as the first adipocyte-specific transcription factor and suggest mechanisms whereby fatty acids, peroxisome proliferators, 9-cis retinoic acid, and other lipids may regulate adipocyte gene expression and differentiation.

The levels of histone mRNA are rapidly reduced after treatment of cultured cells with hydroxyurea or cytosine arabinonucleoside. The histone mRNA for the replicative histone variants is destroyed rapidly, with a half-life of [10][11][12][13][14][15] min. The levels of mRNA coding for the replacement histone variant H3.3 were unchanged after treatment with DNA synthesis inhibitors. In addition to the rapid destruction of histone mRNA, there was a reduction to 1/5th in the rate of transcription of the histone genes. Lymphoma cells (S49) arrested in GI by cyclic AMP produce and contain significant levels of histone mRNA. Hydroxyurea reduces the rate of transcription and the levels of histone mRNA in the GI-arrested cells.Histones are the basic proteins that are complexed with DNA to form the eukaryotic chromosome. It is generally thought that histones are synthesized when DNA is synthesized (1, 2). However, in one mammalian cell line, S49 mouse lymphoma cells, histones are also synthesized in G1 and incorporated into chromosomes during the subsequent S phase (3).The levels of histone mRNA are also regulated, both during the cell cycle and in response to DNA The isolation of mouse histone genes (14) allows us to directly measure both synthesis and degradation of histone mRNA. We report here that treatment of mouse cells with inhibitors of deoxynucleotide synthesis reduces the level of replication variant histone mRNAs, but not the level of a replacement variant mRNA, H3.3. This reduction is due to both a decreased rate of histone mRNA synthesis and an increased rate of degradation. In G1 lymphoma cells, which are not synthesizing DNA but are synthesizing histone mRNA, the inhibitors also reduce the rate of histone mRNA synthesis. METHODSCell Growth. Mouse myeloma 66-2 cells were grown, synchronized by isoleucine starvation (15), and treated with DNA synthesis inhibitors as described in the figure legends. When double drug treatments with cycloheximide and a DNA synthesis inhibitor were performed the cycloheximide was added 5 min prior to the addition of the DNA synthesis inhibitor. The final concentration of cycloheximide was 0.1 mM. Cells washed with fresh medium after drug treatment retained their viability. "Deathless" S49 mouse lymphoma cells were grown and synchronized as described by Lemaire and Coffino (16).Nuclear Transcription. Nuclei were isolated and incubated for RNA synthesis essentially as described by Marzluff (17). The final salt concentrations in the transcription were 70 mM KCI and 6 mM MgCl2. We used 100 uCi (1 Ci = 3. with an equal volume of DNase I (Sigma) at 25 /g/ml, 0.6 M NaCl, 50 mM Tris-HCl at pH 7.5, and 20 mM MgCl2. NaDodSO4 and EDTA were then added to final concentrations of 1% and 10 mM, respectively, followed by the addition of ammonium acetate to 0.3 M. RNA was extracted with an equal volume of water-saturated phenol at 55°C for 5 min, precipitated with ethanol, and washed three times with 75% (vol/vol) ethanol. The RNA was separated from unincorporated nucleoside triphosph...

No abstract

Previously, we identified a novel transcription factor, ARF6, as a key regulator of the tissue-specific adipocyte P2 (aP2) enhancer. In order to identify the proteins which comprise the adipocyte ARF6 complex, we have purified this DNA binding activity from a cultured adipocyte cell line. We have developed a system for growth and differentiation of HIB-1B brown adipocytes in suspension culture that facilitates the production of large quantities of adipocyte nuclear extract. ARF6 was purified from HIB-1B nuclear extract by a combination of conventional and sequence-specific DNA affinity chromotography. Chemical sequencing and mass spectral analysis of tryptic peptides derived from the purified polypeptides identifies the ARF6 complex as a heterodimer of the retinoid X receptor alpha (RXR alpha) and the murine peroxisome proliferator activated receptor gamma (PPAR gamma). Of the known PPAR gamma isoforms, PPAR gamma is the predominant form expressed in adipose tissue. These results suggest that PPAR gamma 2 serves a unique function among PPAR family members as an important regulator of adipocyte-specific gene expression.

We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL-and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor ␥ mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue. ( J. Clin. Invest. 1997. 100:2900-2908.)

Uncoupling protein (UCP) is expressed only in brown adipocytes and is responsible for the unique thermogenic properties of this cell type. The novel brown preadipocyte cell line, HIB-1B, expresses UCP in a strictly differentiation-dependent manner. Transgenic mice studies have shown that a region from kb ؊2.8 to ؊1.0 of the murine UCP gene is required for brown adipocyte-specific expression. Subsequent analysis identified a potent 220-bp enhancer from kb ؊2.5 to ؊2.3. We show that this enhancer is active only in differentiated HIB-1B adipocytes, and we identify a peroxisome proliferator-activated receptor ␥ (PPAR␥) response element, referred to as UCP regulatory element 1 (URE1), within the enhancer. URE1 has differentiation-dependent enhancing activity in HIB-1B cells and is required for enhancer action, since mutations of URE1 that block protein binding abolish enhancer activity. We also show that PPAR␥ antibodies block binding to URE1 of nuclear extracts from cultured brown adipocytes and from the brown adipose tissue of cold-exposed mice. Protein binding to URE1 increases substantially during differentiation of HIB-1B preadipocytes, and PPAR␥ mRNA levels increase correspondingly. Although forced expression of PPAR␥ and retinoid X receptor ␣ activates the enhancer in HIB-1B preadipocytes, these receptors are not capable of activating the enhancer in NIH 3T3 fibroblasts. Our results show that PPAR␥ is a regulator of the differentiation-dependent expression of UCP and suggest that there are additional factors in HIB-1B cells required for brown adipocyte-specific UCP expression.

The murine gene for adipocyte P2 encodes an adipocyte-specific member of the family of intracellular lipid binding proteins. The region upstream from the start of transcription of this gene has been found to contain binding sites for the transcription factors c-jun/c-fos and C/EBP (CCAAT/enhancer binding protein) and several short sequence elements found in other adipocyte gene promoters, termed fat-specific elements. To identify DNA sequences that were responsible for the high level of transcription of the gene for adipocyte P2 in vivo, we made a series of transgenic mice containing 168 base pairs (bp), 247 bp, 1.7 kilobases (kb), and 5.4 kb of 5' flanking sequence linked to the bacterial gene chloramphenicol acetyltransferase. Although plasmids containing only 168 bp of 5' sequence including the C/EBP and AP-1 (activation protein 1) binding sites were expressed well in cultured adipocytes, high levels of chloramphenicol acetyltransferase activity in the adipose tissue oftransgenic mice were not observed until the 5' flanking region was extended to kb -5.4. An enhancer mapping between kb -4.9 and kb -5.4 upstream from the start of transcription was identified by transfection of further deletions into cultured adipocytes. This enhancer, when linked to a bp -63 promoter fragment from the gene for adipocyte P2, directed very high level chloramphenicol acetyltransferase expression specifically to adipose tissue in transgenic mice. These results identify a functional adipose-specific enhancer and indicate that it is the major determinant of tissue specificity of the gene for adipocyte P2. These results also demonstrate that the proximal-promoter binding sites for AP-1 and C/EBP are not sufficient or necessary to give adipose-tissue-specific expression in vivo, though they may play an important role in the response of this promoter to glucocorticoids.The major role of the adipocyte in higher eukaryotes is the storage of nutritional energy in the form of triglycerides. Disordered gene expression in the adipocyte may result in pathological conditions such as lipodystrophy and obesity, the latter of which contributes heavily to morbidity and mortality through associated cardiovascular disorders and diabetes. In addition, the ability to genetically alter the expression of adipocyte genes and thus control the fatness of feed animals remains an important goal of agricultural research. Thus, the regulation of adipocyte gene expression is of interest for biological, medical, and agricultural reasons.