Nutrient-dependent acetylation controls basic regulatory metabolic switches and cellular reprogramming.

Organisms must be able to selectively tailor their ability to use the macronutrients of carbohydrate, protein, and fat based on their availability. In different cell types, how the nutrient fluctuations are sensed and the mechanisms by which the pathways of central metabolism are switched to favor the use of one particular nutrient type over another are topics of intense interest. Protein acetylation is one major evolutionary conserved mechanism by which nutrient fluctuations are sensed within cells and subsequently coupled with metabolic switching. In this review, we present the case of PGC-1α acetylation and how the control of PGC-1α's activity by acetylation sets into motion a wide range of metabolic adaptations that makes this protein an exemplary model for acetylation-mediated mechanisms of nutrient sensing and communication.

Tissue-specific regulation of metabolic pathways through the transcriptional coactivator PGC1-alpha.

Metabolic pathways are controlled at different levels in response to environmental or hormonal stimuli. This control is achieved, at least in part, at the transcriptional level of gene expression. The regulation of gene expression is executed by specific transcription factors, but there is another level of regulation by a set of proteins that modulate these factors called transcriptional coactivators. In mammals, one of the most characterized examples of regulation of metabolic pathways by transcriptional coactivators is peroxisome proliferator-activated receptors gamma (PPARgamma) coactivator-1 alpha (PGC-1alpha). PGC-1alpha is activated by signals that control energy and nutrient homeostasis. Notably, PGC-1alpha induces and coordinates gene expression that stimulates mitochondrial biogenesis and a thermogenic program in brown fat, fiber-type switching in skeletal muscle, and metabolic pathways linked to the fasted response in the liver. PGC-1alpha activates gene expression through specific interaction with transcription factors that bind to the promoters of metabolic genes. These transcription factors can be ubiquitous such as the nuclear respiratory factors or tissue-enriched factors such as PPARgamma (brown fat), hepatocyte nuclear factor (HNF4alpha) (liver and pancreas) and muscle enhancer factor (MEF2s). The fact that PGC-1alpha controls important metabolic pathways in several tissues suggests that it can be a therapeutic target for antiobesity or diabetes drugs.

Cytokine stimulation of energy expenditure through p38 MAP kinase activation of PPARgamma coactivator-1.

Cachexia is a chronic state of negative energy balance and muscle wasting that is a severe complication of cancer and chronic infection. While cytokines such as IL-1alpha, IL-1beta, and TNFalpha can mediate cachectic states, how these molecules affect energy expenditure is unknown. We show here that many cytokines activate the transcriptional PPAR gamma coactivator-1 (PGC-1) through phosphorylation by p38 kinase, resulting in stabilization and activation of PGC-1 protein. Cytokine or lipopolysaccharide (LPS)-induced activation of PGC-1 in cultured muscle cells or muscle in vivo causes increased respiration and expression of genes linked to mitochondrial uncoupling and energy expenditure. These data illustrate a direct thermogenic action of cytokines and p38 MAP kinase through the transcriptional coactivator PGC-1.

Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1.

Blood glucose levels are maintained by the balance between glucose uptake by peripheral tissues and glucose secretion by the liver. Gluconeogenesis is strongly stimulated during fasting and is aberrantly activated in diabetes mellitus. Here we show that the transcriptional coactivator PGC-1 is strongly induced in liver in fasting mice and in three mouse models of insulin action deficiency: streptozotocin-induced diabetes, ob/ob genotype and liver insulin-receptor knockout. PGC-1 is induced synergistically in primary liver cultures by cyclic AMP and glucocorticoids. Adenoviral-mediated expression of PGC-1 in hepatocytes in culture or in vivo strongly activates an entire programme of key gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, leading to increased glucose output. Full transcriptional activation of the PEPCK promoter requires coactivation of the glucocorticoid receptor and the liver-enriched transcription factor HNF-4alpha (hepatic nuclear factor-4alpha) by PGC-1. These results implicate PGC-1 as a key modulator of hepatic gluconeogenesis and as a central target of the insulin-cAMP axis in liver.

CREB regulates hepatic gluconeogenesis through the coactivator PGC-1.

When mammals fast, glucose homeostasis is achieved by triggering expression of gluconeogenic genes in response to glucagon and glucocorticoids. The pathways act synergistically to induce gluconeogenesis (glucose synthesis), although the underlying mechanism has not been determined. Here we show that mice carrying a targeted disruption of the cyclic AMP (cAMP) response element binding (CREB) protein gene, or overexpressing a dominant-negative CREB inhibitor, exhibit fasting hypoglycaemia [corrected] and reduced expression of gluconeogenic enzymes. CREB was found to induce expression of the gluconeogenic programme through the nuclear receptor coactivator PGC-1, which is shown here to be a direct target for CREB regulation in vivo. Overexpression of PGC-1 in CREB-deficient mice restored glucose homeostasis and rescued expression of gluconeogenic genes. In transient assays, PGC-1 potentiated glucocorticoid induction of the gene for phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis. PGC-1 promotes cooperativity between cyclic AMP and glucocorticoid signalling pathways during hepatic gluconeogenesis. Fasting hyperglycaemia is strongly correlated with type II diabetes, so our results suggest that the activation of PGC-1 by CREB in liver contributes importantly to the pathogenesis of this disease.

Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1.

Muscle tissue is the major site for insulin-stimulated glucose uptake in vivo, due primarily to the recruitment of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane. Surprisingly, virtually all cultured muscle cells express little or no GLUT4. We show here that adenovirus-mediated expression of the transcriptional coactivator PGC-1, which is expressed in muscle in vivo but is also deficient in cultured muscle cells, causes the total restoration of GLUT4 mRNA levels to those observed in vivo. This increased GLUT4 expression correlates with a 3-fold increase in glucose transport, although much of this protein is transported to the plasma membrane even in the absence of insulin. PGC-1 mediates this increased GLUT4 expression, in large part, by binding to and coactivating the muscle-selective transcription factor MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can control the level of endogenous GLUT4 gene expression in muscle.

Regulation of adipogenesis and energy balance by PPARgamma and PGC-1.

There has been a great deal of recent progress in our understanding of the transcriptional control of adipogenesis. Current data suggest that fat cell differentiation involves an interplay between the C/EBP family of transcription factors and PPARgamma. The thermogenic program of brown fat cells may also include a contribution from a new coactivator, PGC-1. Recent data suggests that this coactivator is responsible for activation of thermogenesis and oxidative metabolism in both brown fat and muscle. The PGC-1 dependent program includes both mitochondrial biogenesis and tissue-specific expression of uncoupling proteins.

Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1.

Transcription and mRNA processing are coupled events in vivo, but the mechanisms that coordinate these processes are largely unknown. PGC-1 is a transcriptional coactivator that plays a major role in the regulation of adaptive thermogenesis. PGC-1 also has certain motifs characteristic of splicing factors. We demonstrate here that mutations in the serine- and arginine-rich domain and RNA recognition motif of PGC-1 interfere with the ability of PGC-1 to induce mRNAs of target genes. These mutations also disrupt the ability of PGC-1 to co-localize and associate with RNA processing factors. PGC-1 can alter the processing of an mRNA, but only when it is loaded onto the promoter of the gene. These data demonstrate the coordinated regulation of RNA transcription and processing through PGC-1.

Modulation of estrogen receptor-alpha transcriptional activity by the coactivator PGC-1.

A transcriptional coactivator of the peroxisome proliferator-activated receptor-gamma (PPARgamma), PPARgamma-coactivator-1(PGC-1) interacts in a constitutive manner with the hinge domain of PPARgamma and enhances its transcriptional activity. In this study we demonstrate that PGC-1 is a coactivator of estrogen receptor-alpha (ERalpha)-dependent transcriptional activity. However the mechanism by which PGC-1 interacts with ERalpha is different from that of PPARgamma. Specifically, it was determined that the carboxyl terminus of PGC-1 interacts in a ligand-independent manner with the ERalpha hinge domain. In addition, an LXXLL motif within the amino terminus of PGC-1 was shown to interact in an agonist-dependent manner with the AF2 domain within the carboxyl terminus of ERalpha. The ability of PGC-1 to associate with and potentiate the transcriptional activity of an ERalpha-AF2 mutant that is unable to interact with the p160 class of coactivators suggests that this coactivator may have a unique role in estrogen signaling. It is concluded from these studies that PGC-1 is a bona fide ERalpha coactivator, which may serve as a convergence point between PPARgamma and ERalpha signaling.

Transcriptional activation of adipogenesis.

Studies from the past several years have revealed that adipogenesis is controlled by an interplay of transcription factors, including members of the CCAAT/enhancer binding protein family and peroxisome proliferator activated receptor gamma. In addition to providing a new understanding of this aspect of the energy balance systems, these factors provide potential new targets for therapeutic intervention in metabolic diseases, such as obesity and type 2 diabetes mellitus.

Activation of PPARgamma coactivator-1 through transcription factor docking.

Transcriptional coactivators have been viewed as constitutively active components, using transcription factors mainly to localize their functions. Here, it is shown that PPARgamma coactivator-1 (PGC-1) promotes transcription through the assembly of a complex that includes the histone acetyltransferases steroid receptor coactivator-1 (SRC-1) and CREB binding protein (CBP)/p300. PGC-1 has a low inherent transcriptional activity when it is not bound to a transcription factor. The docking of PGC-1 to peroxisome proliferator-activated receptor gamma (PPARgamma) stimulates an apparent conformational change in PGC-1 that permits binding of SRC-1 and CBP/p300, resulting in a large increase in transcriptional activity. Thus, transcription factor docking switches on the activity of a coactivator protein.

Stimulation of uncoupling protein 1 expression in brown adipocytes by naturally occurring carotenoids.

OBJECTIVE: To assess the effect of naturally occurring carotenoids on brown adipocyte proliferation and differentiation. The rationale behind is that certain carotenoids have provitamin A activity in mammals, and that one of the active forms of vitamin A, (retinoic acid) is known to behave as a transcriptional activator of the key gene for brown fat thermogenesis, the one encoding the uncoupling protein thermogenin (UCP1). DESIGN: Confluent primary cultures of mice brown adipocytes were treated with various concentrations of carotenoids. Cell morphology, total culture protein content, the DNA synthesis rate, and the levels of UCP1, retinoic acid receptor alpha (RARalpha) and retinoid X receptor alpha (RXRalpha) were analysed. RESULTS: Treatment with beta-carotene, alpha-carotene and lutein promoted UCP1 expression in a dose-dependent manner, with an effectiveness that was related to their potency as vitamin A precursors. Cell morphology, total culture protein content at confluence and DNA synthesis rate were unaffected after carotenoid treatment up to 10 microM. CONCLUSION: The results indicate that carotenoids can positively affect the expression of UCP1 without altering brown adipocyte proliferation.

Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1.

Mitochondrial number and function are altered in response to external stimuli in eukaryotes. While several transcription/replication factors directly regulate mitochondrial genes, the coordination of these factors into a program responsive to the environment is not understood. We show here that PGC-1, a cold-inducible coactivator of nuclear receptors, stimulates mitochondrial biogenesis and respiration in muscle cells through an induction of uncoupling protein 2 (UCP-2) and through regulation of the nuclear respiratory factors (NRFs). PGC-1 stimulates a powerful induction of NRF-1 and NRF-2 gene expression; in addition, PGC-1 binds to and coactivates the transcriptional function of NRF-1 on the promoter for mitochondrial transcription factor A (mtTFA), a direct regulator of mitochondrial DNA replication/transcription. These data elucidate a pathway that directly links external physiological stimuli to the regulation of mitochondrial biogenesis and function.

Involvement of the retinoblastoma protein in brown and white adipocyte cell differentiation: functional and physical association with the adipogenic transcription factor C/EBPalpha.

We investigated the expression of the retinoblastoma protein (pRB) in adipocytes and its possible interaction with the adipogenic transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) in controlling the acquisition of the terminally differentiated adipocyte phenotype. The pRB was expressed (as measured by immunoblotting and/or immunofluorescence) in mice brown and white adipose tissue and in cultured adipocytes that showed lipid accumulation and expressed specific differentiation markers such as aP2 (measured using a specific cDNA probe) and in the case of brown adipocytes UCP-1 (measured using specific antibodies), but was undetectable in proliferative undifferentiated preadipocytes. Transient transfection experiments revealed a functional interaction between pRB and C/EBPalpha affecting transcription from the ucp-1 gene promoter. Thus, in immortalized brown adipocytes, co-transfection of both a C/EBPalpha and a pRB expression vectors maximally enhanced the expression of reporter chloramphenicol acetyltransferase driven by the ucp-1 promoter. Interestingly, C/EBPalpha inhibited reporter gene expression in CHO cells in an effect that was also potentiated in the presence of pRB. A positive effect of pRB on transcription from the ucp-1 promoter could be detected in C/EBPalpha-/-fibroblasts only after forced to overexpress C/EBPalpha, suggesting that the effect of pRB is dependent on its interaction with C/EBPalpha. We also found evidence that pRB and C/EBPalpha can directly bind to each other in vitro. Our results show that the expression of pRB is restricted to differentiated adipocytes, and provide evidence of a physical and functional interaction between pRB and C/EBPalpha that affects the transcriptional activity of the later on a brown adipocyte-specific gene.

A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.

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.

2-Methoxyestradiol, an endogenous metabolite of 17beta-estradiol, inhibits adipocyte proliferation.

The effects of 2-methoxyestradiol (2ME), a naturally occurring mammalian metabolite of 17beta-estradiol, on adipocyte growth has been investigated in mouse brown adipose tissue precursor cells developed in primary culture. 2ME inhibits brown adipocyte proliferation in a dose-response manner (IC50 = 1.7 x 10(-6) M for DNA synthesis), with much higher potency than its hormone precursor 17beta-estradiol, and cells acquire the typical differentiated morphology--more round with a higher content of triglycerides. 2ME causes similar effects in the immortal brown adipocyte tumor-derived hibernoma cell line HIB 1B and the immortal 3T3-F442A white adipocyte line. These findings suggest a possible role for 2ME in adipocyte proliferation, and probably in the differentiation process, entering the cells in the adipogenic program.

Diminished response to food deprivation of the rat brown adipose tissue mitochondrial uncoupling system with age.

The aim of the present work is to investigate the effect of starvation on brown adipose tissue thermogenic activity with aging. Interscapular brown adipose tissue from female Wistar rats of different ages was used; half of them were fed and the other half were starved for 24 hours. Mitochondria were isolated and mitochondrial protein content, GDP-binding, Cytochrome-c Oxidase activity and uncoupling protein levels were measured. Results show a decrease of all studied parameters, indicating a diminished thermogenic activity with age. The response to starvation is almost the same in all the parameters studied: a general reduction with starvation and a progressive disappearance of this response to starvation with aging. On the whole, these results would indicate a deficient regulation of brown adipose tissue thermogenic activity in old animals, as it happens in other animal models with an alterated thermogenesis.

Retinoic acid modulates retinoid X receptor alpha and retinoic acid receptor alpha levels of cultured brown adipocytes.

A novel potential regulatory pathway of brown adipose tissue (BAT) thermogenesis was recently recognized after identifying retinoic acid (RA) as a transcriptional activator of the uncoupling protein (UCP) gene. Here we provide evidence that the UCP responsiveness to RA in primary cultures of brown adipocytes involves RA receptor alpha (RAR alpha), and show, in the same system and also in CHO cells, that RA down-regulates the steady-state levels of RAR alpha and especially of retinoid X receptor alpha, suggesting autoregulation of the retinoid pathway and therefore supporting the idea of a physiological role for it in controlling the thermogenic capacity of BAT.

In vitro and in vivo induction of brown adipocyte uncoupling protein (thermogenin) by retinoic acid.

The effects of retinoic acid (RA) isomers (all-trans-RA and 9-cis-RA) on the appearance of uncoupling protein (UCP; thermogenin), the only unequivocal molecular marker of the brown adipocyte differentiated phenotype, have been investigated in primary cultures of brown adipocytes, in the brown adipocyte cell line HIB 1B and directly in intact mice. The results obtained with cultured cells indicate that retinoids function as inducers of the appearance of UCP and, at the same time, partially inhibit brown adipocyte cell proliferation. The two RA isomers displayed similar effectiveness as UCP inducers, their effect being comparable with that triggered by noradrenaline, so far considered to be the main modulator of UCP gene expression. The effectiveness of retinoids as UCP inducers was dependent on the stage of brown adipocyte differentiation, being maximal in confluent primary cells and in the medium-late differentiation stage of HIB 1B cells. Corroborating the results obtained in vitro, we show that administration of all-trans-RA or 9-cis-RA to mice leads to an increase in their brown adipose tissue specific UCP content. 9-cis-RA treatment also prevented the loss of UCP on cold deacclimation. To our knowledge, this is the first report of a stimulatory effect of retinoid compounds on UCP induction in vivo.