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Biomedical subjects

M Daniel Lane

Publications and source records attributed to M Daniel Lane.

At least 19 recordsLinked to original sources
PubMed2006

BMP-4 treatment of C3H10T1/2 stem cells blocks expression of MMP-3 and MMP-13.

Microarray gene expression profiling was used to identify bone morphogenetic protein-4 (BMP-4) responsive factors involved in late stages of adipocyte commitment in C3H10T1/2 cells. The analysis revealed that the matrix metalloproteinase-3 (MMP-3) gene decreased 100-fold after BMP-4 treatment, and expression of MMP-13 decreased 19.5-fold. Uncommitted C3H10T1/2 cells exhibit dramatic up-regulation of MMP-3 and MMP-13 genes as cells become confluent. Real-time RT-PCR demonstrated that BMP-4 blocks expression of both transcripts. Likewise, a stable committed preadipocyte line derived from C3H10T1/2 cells did not express MMP-3 or MMP-13 at confluence, despite never receiving BMP-4. Active forms of both proteins were detected in media from confluent C3H10T1/2 cells but not in BMP-4 treated cells. Addition of BMP-4 to confluent C3H10T1/2 cells repressed the expression of both genes but did not induce adipocyte differentiation. The findings indicate that BMP-4-induced down-regulation of MMP-3 and MMP-13 is associated with commitment, but is insufficient to induce adipogenesis.

3T3-L1 Cells↗
PubMed2006

Hypothalamic malonyl-CoA triggers mitochondrial biogenesis and oxidative gene expression in skeletal muscle: Role of PGC-1alpha.

Previous investigations show that intracerebroventricular administration of a potent inhibitor of fatty acid synthase, C75, increases the level of its substrate, malonyl-CoA, in the hypothalamus. The "malonyl-CoA signal" is rapidly transmitted to skeletal muscle by the sympathetic nervous system, increasing fatty acid oxidation, uncoupling protein-3 (UCP3) expression, and thus, energy expenditure. Here, we show that intracerebroventricular or intraperitoneal administration of C75 increases the number of mitochondria in white and red (soleus) skeletal muscle. Consistent with signal transmission from the hypothalamus by the sympathetic nervous system, centrally administered C75 rapidly (< or =2 h) up-regulated the expression (in skeletal muscle) of the beta-adrenergic signaling molecules, i.e., norepinephrine, beta3-adrenergic receptor, and cAMP; the transcriptional regulators peroxisomal proliferator activator regulator gamma coactivator 1alpha (PGC-1alpha) and estrogen receptor-related receptor alpha (ERRalpha); and the expression of key oxidative mitochondrial enzymes, including pyruvate dehydrogenase kinase, medium-chain length fatty acyl-CoA dehydrogenase, ubiquinone-cytochrome c reductase, cytochrome oxidase, as well as ATP synthase and UCP3. The role of PGC-1alpha in mediating these responses in muscle was assessed with C2C12 myocytes in cell culture. Consistent with the in vivo response, adenovirus-directed expression of PGC-1alpha in C2C12 muscle cells provoked the phosphorylation/inactivation and reduced expression of acetyl-CoA carboxylase 2, causing a reduction of the malonyl-CoA concentration. These effects, coupled with an increased carnitine palmitoyltransferase 1b, led to increased fatty acid oxidation. PGC-1alpha also increased the expression of ERRalpha, PPARalpha, and enzymes that support mitochondrial fatty acid oxidation, ATP synthesis, and thermogenesis, apparently mediated by an increased expression of UCP3.

4-Butyrolactone↗
PubMed2006

The role of hypothalamic malonyl-CoA in energy homeostasis.

Energy balance is monitored by hypothalamic neurons that respond to peripheral hormonal and afferent neural signals that sense energy status. Recent physiologic, pharmacologic, and genetic evidence has implicated malonyl-CoA, an intermediate in fatty acid synthesis, as a regulatory component of this energy-sensing system. The level of malonyl-CoA in the hypothalamus is dynamically regulated by fasting and feeding, which alter subsequent feeding behavior. Fatty acid synthase (FAS) inhibitors, administered systemically or intracerebroventricularly to lean or obese mice, increase hypothalamic malonyl-CoA leading to the suppression of food intake. Conversely, lowering malonyl-CoA with an acetyl-CoA carboxylase (ACC) inhibitor or by the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus increases food intake and reverses inhibition by FAS inhibitors. Physiologically, the level of hypothalamic malonyl-CoA appears to be determined through phosphorylation/dephosphorylation of ACC by AMP kinase in response to changes in the AMP/ATP ratio, an indicator of energy status. Recent evidence suggests that the brain-specific carnitine:palmitoyl-CoA transferase-1 (CPT1c) may be a regulated target of malonyl-CoA that relays the "malonyl-CoA signal" in hypothalamic neurons that express the orexigenic and anorexigenic neuropeptides that regulate food intake and peripheral energy expenditure. Together these findings support a role for malonyl-CoA as an intermediary in the control of energy homeostasis.

Acetyl-CoA Carboxylase↗
PubMed2006

Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 gene.

Previous studies showed that exposure of C3H10T1/2 stem cells to bone morphogenetic protein-4 (BMP-4) produced cells that convert into adipocytes at high frequency when treated with differentiation inducers. In the present investigation, an independent approach shows that BMP-4 is required for stable commitment of pluripotent stem cells to the adipocyte lineage. Exposure of proliferating 10T1/2 stem cells to 5-azacytidine, a potent DNA methylation inhibitor, gave rise to a subpopulation of cells that can be cloned and that have the capacity to undergo conversion into adipocytes upon treatment with terminal differentiation inducers. Detailed studies performed with a cloned committed subline, the A33 line, verified stable adipocyte lineage determination in the absence of exogenous BMP-4. Remarkably, this cell line expresses and secretes BMP-4 during proliferation in the same time window that exogenous BMP-4 must be added to naïve 10T1/2 cells to induce maximal adipocyte commitment. Furthermore, exposure of A33 cells to noggin, a naturally occurring BMP-4-binding antagonist, during this critical time window blocks subsequent differentiation. The role of BMP-4 in adipocyte lineage commitment is further strengthened by gene expression profiling of proliferating 10T1/2 stem cells and A33 preadipocytes. These findings revealed changes in the molecular circuitry, specifically coordinated changes in the expression of members of the BMP-4 signaling pathway, that distinguish A33 preadipocytes from uncommitted parental 10T1/2 stem cells. Together, these studies provide compelling evidence for the participation of BMP-4 in adipocyte lineage determination.

Adipocytes↗
PubMed2006

The brain-specific carnitine palmitoyltransferase-1c regulates energy homeostasis.

Fatty acid synthesis in the central nervous system is implicated in the control of food intake and energy expenditure. An intermediate in this pathway, malonyl-CoA, mediates these effects. Malonyl-CoA is an established inhibitor of carnitine palmitoyltransferase-1 (CPT1), an outer mitochondrial membrane enzyme that controls entry of fatty acids into mitochondria and, thereby, fatty acid oxidation. CPT1c, a brain-specific enzyme with high sequence similarity to CPT1a (liver) and CPT1b (muscle) was recently discovered. All three CPTs bind malonyl-CoA, and CPT1a and CPT1b catalyze acyl transfer from various fatty acyl-CoAs to carnitine, whereas CPT1c does not. These findings suggest that CPT1c has a unique function or activation mechanism. We produced a targeted mouse knockout (KO) of CPT1c to investigate its role in energy homeostasis. CPT1c KO mice have lower body weight and food intake, which is consistent with a role as an energy-sensing malonyl-CoA target. Paradoxically, CPT1c KO mice fed a high-fat diet are more susceptible to obesity, suggesting that CPT1c is protective against the effects of fat feeding. CPT1c KO mice also exhibit decreased rates of fatty acid oxidation, which may contribute to their increased susceptibility to diet-induced obesity. These findings indicate that CPT1c is necessary for the regulation of energy homeostasis.

Animals↗
PubMed2006

Control of energy homeostasis: role of enzymes and intermediates of fatty acid metabolism in the central nervous system.

The regulation of energy homeostasis is critical for normal physiology and survival. Energy flux must be rigorously monitored and adjusted to ensure that fuel intake and expenditure remain within acceptable limits. The central nervous system (CNS) is, in large part, responsible for conducting this energy-monitoring function and for integrating the numerous inputs. It has become evident that neurons of the CNS monitor and respond to levels of metabolic intermediates that reflect peripheral energy status. Intermediates in the fatty acid biosynthetic pathway have been implicated as hypothalamic signaling mediators that sense and respond to changes in circulating fuels. Genetic and pharmacologic manipulation of the enzymes of fatty acid metabolism have led to the hypothesis that neuronal metabolic intermediates affect neural outputs that modify both feeding behavior and energy expenditure. This review focuses on the regulatory roles of these enzymes and intermediates in the regulation of food intake and energy balance.

Central Nervous System↗
PubMed2005

Effect of serum on the down-regulation of CHOP-10 during differentiation of 3T3-L1 preadipocytes.

Hormonal induction of growth-arrested 3T3-L1 preadipocytes in medium containing fetal bovine serum (FBS) triggers a signaling cascade that culminates in adipogenesis. The transcription factor, C/EBPbeta, is expressed early in this differentiation program, but lacks DNA-binding activity until much later as the preadipocytes traverse the G(1)-S checkpoint of mitotic clonal expansion. Dominant-negative CHOP-10 is initially expressed by growth-arrested preadipocytes and sequesters/inactivates C/EBPbeta by heterodimerization with its leucine zipper. After a lag period, CHOP-10 undergoes down-regulation releasing C/EBPbeta from inhibitory constraint allowing transactivation of the C/EBPalpha and PPARgamma genes, transcription factors required for terminal differentiation. We verify that following induction of differentiation in FBS-containing medium, CHOP-10 undergoes down-regulation and differentiation occurs normally. However, when differentiation is induced in calf serum-containing medium, CHOP-10 is not down-regulated resulting in delayed and incomplete differentiation. Under these conditions the expression of C/EBPalpha and PPARgamma and the accumulation of cytoplasmic triglyceride are attenuated. It appears that a factor(s) present in FBS is required to affect the down-regulation of CHOP-10 necessary for successful terminal differentiation.

3T3-L1 Cells↗
PubMed2005

A role for hypothalamic malonyl-CoA in the control of food intake.

The cellular level of malonyl-CoA, an intermediate in fatty acid biosynthesis, depends on its rate of synthesis catalyzed by acetyl-CoA carboxylase relative to its rate of utilization and degradation catalyzed by fatty acid synthase and malonyl-CoA decarboxylase, respectively. Recent evidence suggests that hypothalamic malonyl-CoA functions in the regulation of feeding behavior by altering the expression of key orexigenic and anorexigenic neuropeptides. Here we report that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a 5'-AMP kinase activator, rapidly lowers malonyl-CoA both in GT1-7 hypothalamic neurons and in the hypothalami of mice. These effects correlate closely with the phosphorylation of acetyl-CoA carboxylase, an established target of AMP kinase. Intracerebroventricular (i.c.v.) administration of AICAR rapidly lowers hypothalamic [malonyl-CoA] and increases food intake. Expression of an adenoviral cytosolic malonyl-CoA decarboxylase vector (Ad-cMCD) in hypothalamic GT1-7 cells decreases malonyl-CoA. When delivered by bilateral stereotaxic injection into the ventral hypothalamus (encompassing the arcuate nucleus) of mice, Ad-cMCD increases food intake and body weight. Ad-MCD delivered into the ventral hypothalamus also reverses the rapid suppression of food intake caused by i.c.v.-administered C75, a fatty acid synthase inhibitor that increases hypothalamic [malonyl-CoA]. Taken together these findings implicate malonyl-CoA in the hypothalamic regulation of feeding behavior.

Adenoviridae↗
PubMed2005

Inhibition of hypothalamic fatty acid synthase triggers rapid activation of fatty acid oxidation in skeletal muscle.

Malonyl-CoA functions as a mediator in the hypothalamic sensing of energy balance and regulates the neural physiology that governs feeding behavior and energy expenditure. The central administration of C75, a potent inhibitor of the fatty acid synthase (FAS), increases malonyl-CoA concentration in the hypothalamus and suppresses food intake while activating fatty acid oxidation in skeletal muscle. Closely correlated with the increase in muscle fatty acid oxidation is the phosphorylation/inactivation of acetyl-CoA carboxylase, which leads to reduced malonyl-CoA concentration. Lowering muscle malonyl-CoA, a potent inhibitor of carnitine/palmitoyl-CoA transferase 1 (CPT1), releases CPT1 from inhibitory constraint, facilitating the entry of fatty acids into mitochondria for beta oxidation. Also correlated with these events are C75-induced increases in the expression of skeletal muscle peroxisome proliferator-activated receptor alpha (PPARalpha), a transcriptional activator of fatty acid oxidizing enzymes, and uncoupling protein 3 (UCP3), a thermogenic mitochondrial uncoupling protein. Phentolamine, an alpha-adrenergic blocking agent, prevents the C75-induced increases of skeletal muscle UCP3 and whole body fatty acid oxidation and C75-induced decrease of skeletal muscle malonyl-CoA. Thus, the sympathetic nervous system is implicated in the transmission of the "malonyl-CoA signal" from brain to skeletal muscle. Consistent with the up-regulation of UCP3 and PPARalpha is the concomitant increase in the expression of PGC1alpha, transcriptional coactivator of the UCP3 and PPARalpha-activated genes. These findings clarify the mechanism by which the hypothalamic malonyl-CoA signal is communicated to metabolic systems in skeletal muscle that regulate fatty acid oxidation and energy expenditure.

4-Butyrolactone↗
PubMed2005

Perturbations in O-linked beta-N-acetylglucosamine protein modification cause severe defects in mitotic progression and cytokinesis.

The dynamic modification of nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification that is rapidly responsive to morphogens, hormones, nutrients, and cellular stress. Here we show that O-GlcNAc is an important regulator of the cell cycle. Increased O-GlcNAc (pharmacologically or genetically) results in growth defects linked to delays in G2/M progression, altered mitotic phosphorylation, and cyclin expression. Overexpression of O-GlcNAcase, the enzyme that removes O-GlcNAc, induces a mitotic exit phenotype accompanied by a delay in mitotic phosphorylation, altered cyclin expression, and pronounced disruption in nuclear organization. Overexpression of the O-GlcNAc transferase, the enzyme that adds O-GlcNAc, results in a polyploid phenotype with faulty cytokinesis. Notably, O-GlcNAc transferase is concentrated at the mitotic spindle and midbody at M phase. These data suggest that dynamic O-GlcNAc processing is a pivotal regulatory component of the cell cycle, controlling cell cycle progression by regulating mitotic phosphorylation, cyclin expression, and cell division.

3T3-L1 Cells↗
PubMed2005

Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK and glycogen synthase kinase 3beta is required for adipogenesis.

CCAAT enhancer-binding protein (C/EBP)beta, C/EBPalpha, and peroxisome proliferator activated receptor (PPAR)gamma act in a cascade where C/EBPbeta activates expression of C/EBPalpha and PPARgamma, which then function as pleiotropic activators of genes that produce the adipocyte phenotype. When growth-arrested 3T3-L1 preadipocytes are induced to differentiate, C/EBPbeta is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 3beta enters the nucleus, which correlates with hyperphosphorylation of C/EBPbeta and acquisition of DNA-binding activity. Concurrently, 3T3-L1 preadipocytes synchronously enter S phase and undergo mitotic clonal expansion, a prerequisite for terminal differentiation. Ex vivo and in vitro experiments with C/EBPbeta show that phosphorylation of Thr-188 by mitogen-activating protein kinase "primes" C/EBPbeta for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3beta, acquisition of DNA-binding function, and transactivation of the C/EBPalpha and PPARgamma genes. The delayed transactivation of the C/EBPalpha and PPARgamma genes by C/EBPbeta appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C/EBPalpha and PPARgamma are antimitotic.

3T3-L1 Cells↗
PubMed2005

Effect of centrally administered C75, a fatty acid synthase inhibitor, on ghrelin secretion and its downstream effects.

The central administration of the fatty acid synthase (FAS) inhibitor, C75, rapidly suppresses the expression of orexigenic neuropeptides [neuropeptide Y (NPY) and agouti-related protein (AgRP)] and activates expression of anorexigenic neuropeptides [proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART)] in the hypothalamus. The combined actions of these changes inhibit food intake and decrease body weight. Intracerebroventricular injection of C75 appears to rapidly inhibit the secretion of ghrelin by hypothalamic explants ex vivo and by the stomach in vivo. Ghrelin administered intracerebroventricularly reverses the anorexic effect of C75, suggesting that C75 acts upstream of ghrelin. Because ghrelin-producing neurons are known to form synapses onto NPY/AgRP neurons, we suggest that the reversal of C75-induced anorexia by ghrelin may be mediated by NPY/AgRP neurons. This hypothesis is supported by the finding that ghrelin reverses the C75-induced inactivation (assessed by c-Fos expression) of neurons in the arcuate nucleus that express NPY (assessed by immunohistochemical costaining). These effects closely correlate with appropriate changes downstream in the expression of the hypothalamic neuropeptides that regulate feeding behavior, i.e., down-regulation of the expression of NPY and AgRP and up-regulation of the expression of proopiomelanocortin/alpha-melanocyte-stimulating hormone, provoked by C75 and reversed by ghrelin. We propose a model in which ghrelin secretion plays an intermediary role between malonyl-CoA, the substrate of fatty acid synthase, and the neural circuitry regulating energy homeostasis.

4-Butyrolactone↗
PubMed2005

C/EBPalpha reverses the anti-adipogenic effects of the HIV protease inhibitor nelfinavir.

Nelfinavir and other HIV protease inhibitors effectively suppress morbidity and mortality in HIV-infected patients. However, therapeutic use of HIV protease inhibitors has been temporally associated with the development of a lipodystrophy/insulin resistance syndrome and some evidence suggests that HIV protease inhibitors may be the causative agent. We report here that nelfinavir promotes loss of cytoplasmic triglyceride from fully differentiated adipocytes and inhibits preadipocyte differentiation at biologically relevant concentrations. Forced expression of the adipogenic transcription factor C/EBPalpha rescues some of the anti-adipogenic effects of nelfinavir. These results suggest that nelfinavir may mediate an anti-adipogenic effect by antagonizing expression of C/EBPalpha.

3T3-L1 Cells↗
PubMed2005

Monitoring energy balance: metabolites of fatty acid synthesis as hypothalamic sensors.

Because energy balance is important for survival, a system is required to monitor energy status and to make appropriate adjustments in energy intake and energy expenditure. In higher animals, a centrally located system has evolved to accomplish this task. When caloric intake exceeds expenditure, the surplus is channeled into energy storage pathways, primarily the synthesis of fatty acids, which are converted into fat and stored in adipose tissue. Thus, metabolic flux through the pathway of fatty acid synthesis, located in the lipogenic tissues, reflects the "energy status" of the animal. The enzymatic machinery of this pathway is also expressed in the brain, notably the hypothalamus. In the hypothalamus, intermediates in this pathway appear to serve as energy sensors that signal higher brain centers to produce appropriate responses, e.g., altered food intake and energy expenditure.

Acyl Coenzyme A↗
PubMed2004

Nuclear factor 1 regulates adipose tissue-specific expression in the mouse GLUT4 gene.

Previous studies demonstrated that an adipose tissue-specific element(s) (ASE) of the murine GLUT4 gene is located between -551 and -506 in the 5'-flanking sequence and that a high-fat responsive element(s) for down-regulation of the GLUT4 gene is located between bases -701 and -552. A binding site for nuclear factor 1 (NF1), that mediates insulin and cAMP-induced repression of GLUT4 in 3T3-L1 adipocytes is located between bases -700 and -688. To examine the role of NF1 in the regulation of GLUT4 gene expression in white adipose tissues (WAT) in vivo, we created two types of transgenic mice harboring mutated either 5' or 3' half-site of NF1-binding sites in GLUT4 minigene constructs. In both cases, the GLUT4 minigene was not expressed in WAT, while expression was maintained in brown adipose tissue, skeletal muscle, and heart. This was an unexpected finding, since a -551 GLUT4 minigene that did not have the NF1-binding site was expressed in WAT. We propose a model that explains the requirement for both the ASE and the NF1-binding site for expression of GLUT4 in WAT.

Adipose Tissue↗
PubMed2004

Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage.

The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both ex vivo and in vivo. After treatment of pluripotent C3H10T1/2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1/2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1/2 stem cells to the adipocyte lineage.

3T3-L1 Cells↗