M. Daniel Lane

Mitotic clonal expansion: a synchronous process required for adipogenesis.

When induced to differentiate, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The preadipocytes traverse the G1/S checkpoint synchronously as evidenced by the expression/activation of cdk2-cyclin-E/A, turnover of p27/kip1, hyperphosphorylation of Rb, translocation of cyclin D1 from nuclei to cytoplasm and GSK-3β from cytoplasm to nuclei, and incorporation of [3H]thymidine into DNA. As the cells cross the G1/S checkpoint, C/EBPβ acquires DNA-binding activity, initiating a cascade of transcriptional activation that culminates in the expression of adipocyte proteins. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 delays, but does not block, MCE and differentiation, the extent of the delay causing a comparable delay in the expression of cell-cycle markers, MCE, and adipogenesis. The more potent and specific MEK inhibitor UO126 and the cyclin-dependent kinase inhibitor roscovitine, which inhibit the cell cycle at different points, block MCE, expression of cell cycle and adipocyte markers, as well as adipogenesis. These results show that MCE is a prerequisite for differentiation of 3T3-L1 preadipocytes into adipocytes.

Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes

Increased flux of glucose through the hexosamine biosynthetic pathway (HSP) is believed to mediate hyperglycemia-induced insulin resistance in diabetes. The end product of the HSP, UDPβ-N-acetylglucosamine (GlcNAc), is a donor sugar nucleotide for complex glycosylation in the secretory pathway and for O-linked GlcNAc (O-GlcNAc) addition to nucleocytoplasmic proteins. Cycling of the O-GlcNAc posttranslational modification was blocked by pharmacological inhibition of O-GlcNAcase, the enzyme that catalyzes O-GlcNAc removal from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc). PUGNAc treatment increased levels of O-GlcNAc and caused insulin resistance in 3T3-L1 adipocytes. Insulin resistance induced through the HSP by glucosamine and chronic insulin treatment correlated with increased O-GlcNAc levels on nucleocytoplasmic proteins. Whereas insulin receptor autophosphorylation and insulin receptor substrate 2 tyrosine phosphorylation were not affected by PUGNAc inhibition of O-GlcNAcase, downstream phosphorylation of Akt at Thr-308 and glycogen synthase kinase 3β at Ser-9 was inhibited. PUGNAc-induced insulin resistance was associated with increased O-GlcNAc modification of several proteins including insulin receptor substrate 1 and β-catenin, two important effectors of insulin signaling. These results suggest that elevation of O-GlcNAc levels attenuate insulin signaling and contribute to the mechanism by which increased flux through the HSP leads to insulin resistance in adipocytes.

OBESITY INDUCES EXPRESSION OF UNCOUPLING PROTEIN-2 IN HEPATOCYTES AND PROMOTES LIVER ATP DEPLETION

Uncoupling protein 2 (UCP2) uncouples respiration from oxidative phosphorylation and may contribute to obesity through effects on energy metabolism. Because basal metabolic rate is decreased in obesity, UCP2 expression is predicted to be reduced. Paradoxically, hepatic expression of UCP2 mRNA is increased in genetically obese (ob/ob) mice. In situ hybridization and immunohistochemical analysis of ob/ob livers demonstrate that UCP2 mRNA and protein expression are increased in hepatocytes, which do not express UCP2 in lean mice. Mitochondria isolated from ob/ob livers exhibit an increased rate of H+ leak which partially dissipates the mitochondrial membrane potential when the rate of electron transport is suppressed. In addition, hepatic ATP stores are reduced and these livers are more vulnerable to necrosis after transient hepatic ischemia. Hence, hepatocytes adapt to obesity by up-regulating UCP2. However, because this decreases the efficiency of energy trapping, the cells become vulnerable to ATP depletion when energy needs increase acutely.

CCAAT/enhancer-binding protein β is required for mitotic clonal expansion during adipogenesis

Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAAT/enhancer-binding protein (C/EBP)β is expressed immediately but gains DNA-binding activity only after a long lag as the cells synchronously begin mitotic clonal expansion (MCE). After MCE, a process required for adipogenesis, C/EBPβ activates expression of C/EBPα and peroxisome proliferator-activated receptor γ, which then transcriptionally activate genes that produce the adipocyte phenotype. When mouse embryo fibroblasts (MEFs) are subjected to the same differentiation protocol, a subset of the MEFs undergoes a similar program of events. Similar to 3T3-L1 preadipocytes, the MEFs reenter the cell cycle (as indicated by the synchronous expression of cyclin A) and undergo MCE as evidenced by the incorporation of BrdUrd into DNA and the formation of mitotic foci of cells that undergo adipogenesis. C/EBPβ is expressed immediately after induction but exhibits delayed acquisition of DNA-binding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from C/EBPβ(−/−) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of C/EBPβ (LAP) but not dominant-negative C/EBPβ (LIP) in C/EBPβ(−/−) MEFs restores MCE, expression of adipocyte markers, and the capacity to form mitotic foci of cells that undergo adipogenesis. These findings demonstrate that expression of C/EBPβ is a prerequisite for MCE in the adipocyte-differentiation program.

Hypothalamic malonyl-CoA as a mediator of feeding behavior

Previous studies showed that i.p. administration of C75, a potent inhibitor of fatty acid synthase (FAS), blocked fasting-induced up-regulation of orexigenic neuropeptides and down-regulation of anorexigenic neuropeptides in the hypothalami of mice. As a result, food intake and body weight were drastically reduced. Here we provide evidence supporting the hypothesis that hypothalamic malonyl-CoA, a substrate of FAS, is an indicator of global energy status and mediates the feeding behavior of mice. We use a sensitive recycling assay to quantify malonyl-CoA to show that the hypothalamic malonyl-CoA level is low in fasted mice and rapidly (≤2 h) increases (≈5-fold) on refeeding. Intracerebroventricular (i.c.v.) administration of C75 to fasted mice rapidly (≤2 h) increased (by 4-fold) hypothalamic malonyl-CoA and blocked feeding when the mice were presented with food. Moreover, prior i.c.v. administration of an acetyl-CoA carboxylase inhibitor, 5-(tetradecyloxy)-2-furoic acid, rapidly (although only partially) prevented the C75-induced rise of hypothalamic malonyl-CoA and prevented the C75-induced decrease of food intake. These effects correlated closely with the rapid (≤2 h) and reciprocal effects of i.c.v. C75 on the expression of hypothalamic orexigenic (NPY and AgRP) and anorexigenic (proopiomelanocortin) neuropeptide mRNAs. Previous results showing that C75 administered i.c.v. rapidly activates hypothalamic neurons of the arcuate and paraventricular nuclei are consistent with the results reported in this paper. Together these findings suggest that level of hypothalamic malonyl-CoA, which depends on the relative activities of acetyl-CoA carboxylase and FAS, is an indicator of energy status and mediates feeding behavior.

Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV protease inhibitors

Many human immunodeficiency virus (HIV)-infected patients taking combination antiretroviral therapy that includes HIV protease inhibitors experience atrophy of peripheral subcutaneous adipose tissue. We investigated the effects of HIV protease inhibitors on adipogenesis and adipocyte survival using the 3T3-L1 preadipocyte cell line. Several HIV protease inhibitors were found either to inhibit preadipocyte differentiation or to promote adipocyte cell death. One protease inhibitor, nelfinavir, elicited both of these effects strongly. When induced to differentiate in the presence of nelfinavir, 3T3-L1 preadipocytes failed to accumulate cytoplasmic triacylglycerol and failed to express normal levels of the adipogenic transcription factors CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ. The level of the proteolytically processed, active 68-kDa form of sterol regulatory element-binding protein-1, a transcription factor known to promote lipogenic gene expression, also was reduced markedly in nelfinavir-treated cells, whereas the level of the 125-kDa precursor form of this protein was unaffected. The inhibitory effect of nelfinavir occurred subsequent to critical early events in preadipocyte differentiation, expression of CCAAT/enhancer-binding protein β and completion of the mitotic clonal expansion phase, because these events were unaffected by nelfinavir treatment. In addition, nelfinavir treatment of fully differentiated 3T3-L1 adipocytes resulted in DNA strand cleavage and severe loss of cell viability. In contrast, cell proliferation and viability of preadipocytes were unaffected by nelfinavir treatment. Thus, molecular or cellular changes that occur during acquisition of the adipocyte phenotype promote susceptibility to nelfinavir-induced cell death. When considered together, these results suggest that nelfinavir may promote adipose tissue atrophy by compromising adipocyte viability and preventing replacement of lost adipocytes by inhibiting preadipocyte differentiation.

BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage

Obesity is accompanied by an increase in both adipocyte number and size. The increase in adipocyte number is the result of recruitment to the adipocyte lineage of pluripotent stem cells present in the vascular stroma of adipose tissue. These pluripotent cells have the potential to undergo commitment and then differentiate into adipocytes, as well as myocytes, osteocytes, and chondrocytes. In this article, we show that both bone morphogenetic protein (BMP)2 and BMP4 can induce commitment of C3H10T1/2 pluripotent stem cells into adipocytes. After treatment of C3H10T1/2 stem cells with these BMPs during proliferation followed by exposure to differentiation inducers at growth arrest, nearly all cells enter the adipose development pathway, express specific adipocyte markers, and acquire the adipocyte phenotype. Overexpression of constitutively active BMP receptor (CA)-BMPr1A or CA-BMPr1B induces commitment in the absence of BMP2/4, whereas overexpression of a dominant-negative receptor dominant-negative-BMPr1A suppresses commitment induced by BMP. Also, knockdown of the expression of Smad4 (coregulator in the BMP/Smad signaling pathway) with RNAi disrupts commitment by the BMPs. However, knockdown of expression of p38 MAPK (an intermediary in the BMP/MAPK signaling pathway) with RNAi had little effect on BMP-induced commitment. Together, these findings indicate that the BMP/Smad signaling pathway has a dominant role in adipocyte lineage determination. Proteomic analysis identified lysyl oxidase (LOX), a bona fide downstream target gene of the BMP signaling pathway. Expression of LOX is induced by BMP2/4 during adipocyte lineage commitment, and knockdown of its expression disrupts the commitment process.

Effect of a fatty acid synthase inhibitor on food intake and expression of hypothalamic neuropeptides

The fatty acid synthase inhibitor, C75, acts centrally to reduce food intake and body weight in mice. Here we report the effects of C75 on the expression of key orexigenic [neuropeptide Y (NPY), agouti-related protein (AgRP), and melanin-concentrating hormone] and anorexigenic [pro-opiomelanocortin (POMC) and cocaine-amphetamine-related transcript (CART)] neuropeptide messages in the hypothalami of lean and obese (ob/ob) mice. In lean mice, C75 rapidly and almost completely blocked food intake and prevented fasting-induced up-regulation of hypothalamic AgRP and NPY mRNAs, as well as down-regulation of CART and POMC mRNAs. Thus, in lean mice C75 seems to interrupt the fasting-induced signals that activate expression of NPY and AgRP and suppression of POMC and CART. In obese mice, C75 rapidly suppressed food intake, reduced body weight, and normalized obesity-associated hyperglycemia and hyperinsulinemia. Like its effect in lean mice, C75 prevented the fasting-induced increase of hypothalamic NPY and AgRP mRNAs in obese mice, but had no effect on the expression of POMC and CART mRNAs. The suppressive effect of C75 on food intake in lean mice seems to be mediated both by NPY/AgRP and POMC/CART neurons, whereas in obese mice the effect seems to be mediated primarily by NPY/AgRP neurons. In both lean and obese mice, C75 markedly increased expression of melaninconcentrating hormone and its receptor in the hypothalamus.

Brain fatty acid synthase activates PPARα to maintain energy homeostasis

Central nervous system control of energy balance affects susceptibility to obesity and diabetes, but how fatty acids, malonyl-CoA, and other metabolites act at this site to alter metabolism is poorly understood. Pharmacological inhibition of fatty acid synthase (FAS), rate limiting for de novo lipogenesis, decreases appetite independently of leptin but also promotes weight loss through activities unrelated to FAS inhibition. Here we report that the conditional genetic inactivation of FAS in pancreatic β cells and hypothalamus produced lean, hypophagic mice with increased physical activity and impaired hypothalamic PPARα signaling. Administration of a PPARα agonist into the hypothalamus increased PPARα target genes and normalized food intake. Inactivation of β cell FAS enzyme activity had no effect on islet function in culture or in vivo. These results suggest a critical role for brain FAS in the regulation of not only feeding, but also physical activity, effects that appear to be mediated through the provision of ligands generated by FAS to PPARα. Thus, 2 diametrically opposed proteins, FAS (induced by feeding) and PPARα (induced by starvation), unexpectedly form an integrative sensory module in the central nervous system to orchestrate energy balance.

Acetyl Coenzyme A Carboxylase

Publisher Summary This chapter discusses the molecular properties of acetyl-CoA carboxylases from animal, bacterial, and plant cells. It also focuses on the regulation of catalytic activity. The mode of regulation of acetyl-CoA carboxylase reflects its physiological role in animal and bacterial cells. The carboxylase reaction, being the first committed step of the major energy storage pathway in animals, is regulated through feed-forward activation, while the E. coli enzyme, being catalyst for the initial step of a biosynthetic pathway involved in membrane synthesis, is regulated by the inhibitory action of a growth modulator. The carboxylases from both animal and bacterial sources possess two catalytic sites: (1) a biotin carboxylase site at which the biotin prosthetic group is carboxylated, and (2) a carboxyl transferase site at which the carboxyl group is transferred to acetyl-CoA to form malonyl-CoA. Regulation of the animal acetyl-CoA carboxylases by the feed-forward allosteric activator, citrate, appears to be mediated through a conformational change that perfects the orientation of the biotin prosthetic group with respect to both catalytic sites. The regulation of the bacterial enzyme is exerted through the inhibitory action of ppGpp, a growth modulator, on the carboxyl transferase component of the acetyl-CoA carboxylase system.