Biomedical subjects

M D Lane

Publications and source records attributed to M D Lane.

At least 19 recordsLinked to original sources

PubMed2000

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 alpha and peroxisome proliferator-activated receptor gamma. 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 beta 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.

3T3 Cells↗

PubMed2000

Role of C/EBP homologous protein (CHOP-10) in the programmed activation of CCAAT/enhancer-binding protein-beta during adipogenesis.

Hormone induction of growth-arrested preadipocytes triggers mitotic clonal expansion followed by expression of CCAAT/enhancer-binding protein (C/EBP)alpha and differentiation into adipocytes. The order of these events is critical because C/EBPalpha is antimitotic and its expression prematurely would block the mitotic clonal expansion required for differentiation. C/EBPbeta, a transcriptional activator of the C/EBPalpha gene, is expressed early in the differentiation program, but lacks DNA-binding activity and fails to localize to centromeres until preadipocytes traverse the G(1)-S checkpoint of mitotic clonal expansion. Evidence is presented that dominant-negative CHOP-10 expressed by growth-arrested preadipocytes transiently sequesters C/EBPbeta by heterodimerization. As preadipocytes reach S phase, CHOP-10 is down-regulated, apparently releasing C/EBPbeta from inhibitory constraint and allowing transactivation of the C/EBPalpha gene. In support of these findings, up-regulation of CHOP-10 with the protease inhibitor N-acetyl-Leu-Leu-norleucinal prevents activation of C/EBPbeta, expression of C/EBPalpha, and adipogenesis.

3T3 Cells↗

PubMed2000

Sequential repression and activation of the CCAAT enhancer-binding protein-alpha (C/EBPalpha ) gene during adipogenesis.

CCAAT enhancer-binding protein-alpha (C/EBPalpha) functions as a pleiotropic transcriptional activator of adipocyte genes during adipogenesis. Nuclear factor C/EBP undifferentiated protein (CUP), an isoform of activator protein-2alpha (AP-2alpha), binds to repressive elements in the C/EBPalpha gene promoter, silencing the gene until late in the differentiation program. The CUP regulatory element overlaps a Sp (GT-box) element in the promoter to which Sp3 (or Sp1) can bind. Binding by Sp3 or Sp1 and CUP/AP2-alpha is mutually exclusive. Sp3 is a strong transcriptional activator of the C/EBPalpha gene promoter in 3T3-L1 preadipocytes and Schneider cells, this activation being repressed by CUP/AP-2alpha. Sp3 is expressed throughout differentiation, whereas CUP/AP-2alpha, which is expressed only by preadipocytes, is down-regulated during differentiation coincident with transcription of the C/EBPalpha gene. Thus, CUP/AP-2alpha delays access of Sp3 to the Sp regulatory element, preventing premature expression of C/EBPalpha and thereby interference by C/EBPalpha (which is antimitotic) with mitotic clonal expansion, an essential early event in the differentiation program.

3T3 Cells↗

PubMed2000

c-Crk, a substrate of the insulin-like growth factor-1 receptor tyrosine kinase, functions as an early signal mediator in the adipocyte differentiation process.

Differentiation of 3T3-L1 preadipocytes into adipocytes is induced by a combination of inducers, including a glucocorticoid, an agent that elevates cellular cAMP, and a ligand of the insulin-like growth factor-1 receptor. Previous studies have implicated protein-tyrosine phosphatase (PTPase) HA2, a homologue of PTPase 1B, in the signaling cascade initiated by the differentiation inducers. Vanadate, a potent PTPase inhibitor, blocks adipocyte differentiation at an early stage in the program, but has no effect on the mitotic clonal expansion required for differentiation. Exposure of preadipocytes to vanadate along with the inducing agents led to the accumulation of pp35, a phosphotyrosyl protein that is a substrate for PTPase HA2. pp35 was purified to homogeneity and shown by amino acid sequence and mass analyses of tryptic peptides to be c-Crk, a known cytoplasmic target of the insulin-like growth factor-1 receptor tyrosine kinase. Transfection of 3T3-L1 preadipocytes with a c-Crk antisense RNA expression vector markedly reduced c-Crk levels and prevented differentiation into adipocytes. Studies with C3G, a protein that binds to the SH3 domain in c-Crk, showed that phosphorylation of c-Crk rendered the SH3 domain inaccessible to C3G. Taken together, these findings indicate that locking c-Crk in the phosphorylated state with vanadate prevents its participation in the signaling system that initiates adipocyte differentiation.

3T3 Cells↗

PubMed2000

Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors.

With the escalation of obesity-related disease, there is great interest in defining the mechanisms that control appetite and body weight. We have identified a link between anabolic energy metabolism and appetite control. Both systemic and intracerebroventricular treatment of mice with fatty acid synthase (FAS) inhibitors (cerulenin and a synthetic compound C75) led to inhibition of feeding and dramatic weight loss. C75 inhibited expression of the prophagic signal neuropeptide Y in the hypothalamus and acted in a leptin-independent manner that appears to be mediated by malonyl-coenzyme A. Thus, FAS may represent an important link in feeding regulation and may be a potential therapeutic target.

Acetyl-CoA Carboxylase↗

PubMed2000

Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27.

Evidence is presented that calpain, a calcium-activated protease, degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation. Calpain activity is required during an early stage of the adipocyte differentiation program. Thus, inhibition of calpain with N-acetyl-Leu-Leu-norleucinal (ALLN) blocks clonal expansion and acquisition of the adipocyte phenotype only when added between 12 and 24 h after the induction of differentiation. Likewise, inhibition of calpain by overexpression of calpastatin, the specific endogenous inhibitor of calpain, prevents 2-day post-confluent preadipocytes from reentering the cell cycle triggered by the differentiation inducers. Inhibition of calpain with ALLN causes preadipocytes to arrest just prior to S phase and prevents phosphorylation of the retinoblastoma gene product, DNA replication, clonal expansion, and subsequent adipocyte differentiation but does not affect the expression of immediate early genes (i.e. fos, jun, C/EBPbeta, and C/EBPdelta). Inhibition of calpain by either ALLN or by overexpression of calpastatin blocks the degradation of p27. p27 is degraded in vitro by cell-free extracts from clonally expanding preadipocytes that contain "active" calpain but not by extracts from pre-mitotic preadipocytes that do not. This action is inhibited by calpastatin or ALLN. Likewise, p27 in preadipocyte extracts is a substrate for purified calpain; this proteolytic action was inhibited by heat inactivation, EGTA, or ALLN. Thus, extracellular signals from the differentiation inducers appear to activate calpain, which degrades p27 allowing density-dependent inhibited preadipocytes to reenter the cell cycle and undergo mitotic clonal expansion.

3T3 Cells↗

PubMed1999

Role of the CCAAT enhancer binding proteins (C/EBPs) in adipocyte differentiation.

Members of the C/EBP family of transcription factors play essential roles in the adipocyte differentiation program. Treatment of growth-arrested 3T3-L1 preadipocytes with appropriate hormonal agents causes the cells to synchronously reenter the cell cycle and to undergo mitotic clonal expansion. Expression of C/EBPbeta and delta occur early in clonal expansion, later followed by C/EBPalpha (which is anti-mitotic) as the cells exit the cell cycle begin to express adipocyte genes. C/EBPalpha serves as transcriptional activator of many adipocyte genes whose expression produce the adipocyte phenotype. Recent work in this laboratory has focussed on the roles of C/EBPbeta and delta in the differentiation program, in particular the mechanisms by which they activate transcription of the C/EBPalpha gene. Several regulatory elements, both repressive and activating, in proximal promoter of the gene have been identified. The cognate transacting factors that interact with these elements have been characterized and their functions elucidated. These factors have been incorporated into a model for a cascade that leads to transcriptional activation of the C/EBPalpha gene and the terminal steps in the differentiation program.

Adipocytes↗

PubMed1999

Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation.

Hormonal induction of 3T3-L1 preadipocytes triggers a cascade of events that initiate differentiation into adipocytes. CCAAT/enhancer-binding proteins beta and delta (C/EBPbeta/delta) are expressed early in the differentiation program, but are not immediately active. After a long lag, C/EBPbeta/delta become competent to bind to the C/EBP regulatory element in the C/EBPalpha gene promoter, C/EBPalpha being a transcriptional activator of numerous adipocyte genes. As C/EBPbeta/delta acquire binding activity, they become localized to centromeres as preadipocytes synchronously enter S phase at the onset of mitotic clonal expansion. Localization to centromeres occurs through C/EBP consensus-binding sites in centromeric satellite DNA. C/EBPalpha, which is antimitotic, becomes centromere-associated much later in the differentiation program as mitotic clonal expansion ceases and the cells become terminally differentiated.

3T3 Cells↗

PubMed1999

Transcription factor NF1 mediates repression of the GLUT4 promoter by cyclic-AMP.

Prolonged treatment of 3T3-L1 adipocytes with 8-Br-cAMP decreases expression of GLUT4, the insulin-responsive glucose transporter. Expression of a promoter-reporter gene construct that contained 785 base pairs of 5'-flanking region of the murine GLUT4 gene was down regulated by 8-Br-cAMP (p < 0.001), whereas expression of constructs that contained 641 or 469 base pairs of 5'-flanking region was not. A reporter gene construct in which bases -706 to -676 were deleted was not repressed by 8-Br-cAMP, thereby identifying a 30 bp region as necessary for repression of the GLUT4 promoter by 8-Br-cAMP. Mutations in this regulatory element that disrupt binding of the transcription factor NF1 abolish the 8-Br-cAMP-induced repression of the gene. Although insulin and cAMP both repress the GLUT4 promoter through this cis-element, they appear to do this through different mechanisms, as treatment with 8-Br-cAMP does not induce the phosphorylation of NF1 that is induced by insulin treatment.

3T3 Cells↗

PubMed1999

Up-regulation of uncoupling protein-3 by fatty acid in C2C12 myotubes.

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple oxidative phosphorylation by dissipating the proton gradient across the membrane. We have investigated regulation of the UCP3 gene in skeletal muscle and C2C12 muscle cells. UCP3 mRNA in mouse skeletal muscle is markedly increased by fasting and rapidly (within 4 h) decreased by re-feeding. Methyl palmoxirate, which inhibits fatty acid uptake by mitochondria and increases blood free fatty acids, prevents the fall in UCP3 message level induced by re-feeding. These findings suggest that fatty acid or a metabolite thereof, activates the UCP3 gene. Proof that fatty acid per se up-regulates UCP3 mRNA was obtained with C2C12 muscle cells in culture. Thus, oleic acid activated expression of UCP3 mRNA in differentiated C2C12 myotubes in a time and concentration-dependent manner. Moreover, BRL49653, a ligand for the nuclear hormone receptor PPARgamma induces expression of UCP3 mRNA suggesting that PPARgamma may regulate transcription of the UCP3 gene.

Animals↗

PubMed1999

The transcription factor nuclear factor I mediates repression of the GLUT4 promoter by insulin.

Insulin represses GLUT4 expression in 3T3-L1 adipocytes through an insulin response element located at bases -706 to -676 in the 5'-flanking sequence. Nuclear proteins related to the nuclear factor I (NF1) family of transcription factors bind to this insulin response element. Mutations that disrupt binding of NF1 proteins to the insulin response element impair the insulin response in reporter gene assays. Insulin treatment of 3T3-L1 adipocytes induces a rapid change in the level of phosphorylation of NF1 proteins, providing a potential mechanism for insulin's ability to regulate gene expression through NF1. Another as yet unidentified protein, not related to NF1, also binds to the GLUT4 insulin response element and is able to mediate partial repression of the GLUT4 promoter in reporter gene assays.

3T3 Cells↗

PubMed1999

Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle.

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.

Adipose Tissue, Brown↗

PubMed1999

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.

Adenosine Triphosphate↗

PubMed1999

Role of calpain in adipocyte differentiation.

Evidence is presented that the calcium-activated protease, calpain, is required for differentiation of 3T3-L1 preadipocytes into adipocytes induced by methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin. Calpain is expressed by preadipocytes and its level falls during differentiation. Exposure of preadipocytes to the calpain inhibitor N-acetyl-Leu-Leu-norleucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, blocks expression of adipocyte-specific genes, notably the CCAAT/enhancer-binding protein (C/EBP)alpha gene, and acquisition of the adipocyte phenotype. The inhibitor disrupts the differentiation-inducing effect of methylisobutylxanthine (by means of the cAMP-signaling pathway), but is without effect on differentiation induced by dexamethasone or insulin. N-acetyl-Leu-Leu-norleucinal, or overexpression of calpastatin, inhibits reporter gene expression mediated by the C/EBPalpha gene promoter by preventing C/EBPbeta, a transcriptional activator of the C/EBPalpha gene, from binding to the promoter. These findings implicate calpain in the transcriptional activation of the C/EBPalpha gene, a process required for terminal adipocyte differentiation.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Repressive effect of Sp1 on the C/EBPalpha gene promoter: role in adipocyte differentiation.

Expression of C/EBPalpha is required for differentiation of 3T3-L1 preadipocytes into adipocytes. Previous investigations indicated that transcription of the C/EBPalpha gene is sequentially activated during differentiation, initially by C/EBPbeta and C/EBPdelta and later by C/EBPalpha (autoactivation). These events are mediated by a C/EBP regulatory element in the promoter of the C/EBPalpha gene. This article presents evidence that members of the Sp family, notably Sp1, act repressively on the C/EBPalpha promoter prior to the induction of differentiation. Sp1 was shown to bind to a GC box at the 5' end of the C/EBP regulatory element in the C/EBPalpha promoter and, in so doing, to competitively prevent binding to and transactivation of the promoter by the C/EBPs. One of the differentiation inducers methylisobutylxanthine (a cAMP phosphodiesterase inhibitor) or Forskolin, both of which increase the cellular cAMP level, causes down-regulation of Sp1. This decrease in Sp1 level early in the differentiation program appears to facilitate access of C/EBPbeta and/or C/EBPdelta to the C/EBP regulatory element and, thereby, derepression of the C/EBPalpha gene.

3T3 Cells↗

PubMed1998

Insulin depletion leads to adipose-specific cell death in obese but not lean mice.

Mutation of the obese gene produces obesity, hyperinsulinemia, and compensatory "overexpression" of the defective gene. As insulin activates obese gene expression, it seemed possible that hyperinsulinemia might be responsible for overexpression of the gene. To address this question we rapidly neutralized circulating insulin by injection of an insulin antibody. Unexpectedly, insulin depletion in obese (ob/ob or db/db) mice caused massive adipose RNA degradation confirmed by histological analysis to result from adipocyte cell death by a largely necrotic mechanism. This effect was not observed in lean littermates and was completely corrected by coadministration of insulin. Comparison of multiple tissues demonstrated that the effect was restricted to adipose tissue. Insulin depletion in obese mice by administration of streptozotocin also led to cell death, but this death was less extensive and appeared to be apoptotic in mechanism. Thus insulin may promote the survival side of the physiological balance between adipocyte survival and death.

3T3 Cells↗

PubMed1998

Bacterial lipopolysaccharide induces uncoupling protein-2 expression in hepatocytes by a tumor necrosis factor-alpha-dependent mechanism.

The liver is a target for bacterial lipopolysaccharide (LPS) and participates in the metabolic response to endotoxemia. Recently published evidence indicates that LPS increases the expression of mitochondrial uncoupling protein-2 (UCP-2) mRNAs in several tissues, including the liver. Because hepatocytes in the healthy liver do not express UCP-2, LPS was thought to induce UCP-2 in liver macrophages, which express UCP-2 constitutively. However, the present studies of cultured peritoneal macrophages indicate that LPS reduces steady state levels of UCP-2 mRNAs in these cells. In contrast, UCP-2 mRNAs are induced in hepatocytes isolated from LPS treated rats and transfection of these hepatocytes with UCP-2 promoter-reporter constructs demonstrates substantial increases in UCP-2 promoter activity. LPS induction of hepatocyte UCP-2 expression is virtually abolished by prior treatment of rats with neutralizing antibodies to tumor necrosis factor alpha (TNF). Futhermore, TNFalpha treatment induces UCP-2 mRNA accumulation in primary cultures of hepatocytes from healthy rats. Thus, hepatocytes are likely to be important contributors to endotoxin-related increases in liver UCP-2 via a mechanism that involves the LPS-inducible cytokine, TNFalpha.

Animals↗

PubMed1998

Expression and function of insulin/insulin-like growth factor I hybrid receptors during differentiation of 3T3-L1 preadipocytes.

During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50-70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (alpha2,beta2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor beta subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.

3T3 Cells↗