Search PubMed⌕ Search

Biomedical subjects

M D Lane

Publications and source records attributed to M D Lane.

At least 55 records · Page 3Linked to original sources
PubMed1995

Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes.

A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expressed concomitant with mRNAs encoding adipocyte marker proteins. A factor(s) present in calf serum markedly activated expression of leptin by fully differentiated 3T3-L1 adipocytes. A 16-hr fast decreased (by approximately 85%) the leptin mRNA level of adipose tissue of lean (ob/+ or +/+) mice but had no effect on the approximately 4-fold higher level in obese (ob/ob) littermates. Since the mutation at the ob locus fails to produce the functional protein, yet its cognate mRNA is overproduced, it appears that leptin is necessary for its own downregulation. Leptin mRNA was also suppressed in adipose tissue of rats during a 16-hr fast and was rapidly induced during a 4-hr refeeding period. Insulin deficiency provoked by streptozotocin also markedly down-regulated leptin mRNA and this suppression was rapidly reversed by insulin. These results suggest that insulin may regulate the expression of leptin.

3T3 Cells↗
PubMed1995

The blockade of preadipocyte differentiation by protein-tyrosine phosphatase HA2 is reversed by vanadate.

A tyrosine phosphatase, i.e. PTPase HA2, was previously isolated from 3T3-L1 cells and characterized using O-phospho Tyrosine19-422/aP2 protein (a target of the insulin receptor tyrosine kinase) as substrate. The nucleotide sequence of a PTPase HA2 cDNA showed it to be a homologue of PTPase 1B. When induced to differentiate into adipocytes, confluent 3T3-L1 preadipocytes undergo mitotic clonal expansion followed by growth arrest and then coordinate expression of adipocyte genes. During clonal expansion, expression of PTPase HA2 increases abruptly and then decreases concomitant with the transcriptional activation of adipocyte genes. Constitutive expression of the PTPase by 3T3-L1 preadipocytes using a PTPase HA2 expression vector prevents adipocyte gene expression and differentiation into adipocytes. Appropriately timed exposure of transfected preadipocytes to vanadate (a PTPase inhibitor), just as clonal expansion ceases restores their capacity to differentiate. Treatment of transfected preadipocytes with vanadate prior to or during clonal expansion fails to reverse PTPase HA2-blocked differentiation, whereas treatment of untransfected preadipocytes during mitotic clonal expansion blocks differentiation. Vanadate added following clonal expansion has no effect on differentiation. Thus, a critical tyrosine phosphorylation event(s) occurs between termination of clonal expansion and initiation of adipocyte gene expression while a critical tyrosine dephosphorylation event(s) occurs during clonal expansion.

3T3 Cells↗
PubMed1995

High fat diet-induced hyperglycemia: prevention by low level expression of a glucose transporter (GLUT4) minigene in transgenic mice.

High-fat intake leading to obesity contributes to the development of non-insulin-dependent diabetes mellitus (NIDDM, type 2). Similarly, mice fed a high-fat (safflower oil) diet develop defective glycemic control, hyperglycemia, and obesity. To assess the effect of a modest increase in the expression of GLUT4 (the insulin-responsive glucose transporter) on impaired glycemic control caused by fat feeding, transgenic mice harboring a GLUT4 minigene were fed a high-fat diet. Low-level tissue-specific (heart, skeletal muscle, and adipose tissue) expression of the GLUT4 minigene in transgenic mice prevented the impairment of glycemic control and accompanying hyperglycemia, but not obesity, caused by fat feeding. Thus, a small increase (< or = 2-fold) in the tissue level of GLUT4 prevents a primary symptom of the diabetic state in a mouse model, suggesting a possible target for intervention in the treatment of NIDDM.

Animals↗
PubMed1995

Expression of a novel insulin-activated amino acid transporter gene during differentiation of 3T3-L1 preadipocytes into adipocytes.

A cDNA encoding a novel insulin-activated adipocyte amino acid transporter (designated AAAT) was cloned from a mouse 3T3-L1 adipocyte library. The deduced amino acid sequence of the cDNA corresponds to a protein of 553 amino acids that possesses 56% amino acid sequence identity to the human neutral amino acid transporter and 42% identity to the rat brain glutamate transporter. Transient transfection of 3T3-L1 preadipocytes with an AAAT expression vector led to insulin-dependent uptake of L-serine and to a lesser extent, uptake of L-alanine and L-glutamate. Expression of the AAAT message is tissue-specific, with the highest level occurring in mouse adipose tissue and a lower level in lung. Unlike other sodium-dependent amino acid transporter mRNAs, the AAAT message is not expressed in brain, kidney, liver or heart and only traces are detected in spleen, thymus and skeletal muscle. Consistent with its high level in adipose tissue, expression of the AAAT message is markedly increased when 3T3-L1 preadipocytes are induced to differentiate into adipocytes.

3T3 Cells↗
PubMed1995

Expression of an insulin-responsive glucose transporter (GLUT4) minigene in transgenic mice: effect of exercise and role in glucose homeostasis.

The effects of a GLUT4 mini-transgene (containing 7 kb of 5' flanking and 1 kb of 3' flanking sequence and all exons and introns of the GLUT4 gene as well as a small foreign DNA tag) and of exercise training on expression of GLUT4 and glycemic control in mice were investigated. Transgenic mice harboring the minigene expressed < or = 2-fold the normal level of GLUT4 mRNA and protein in skeletal (gastrocnemius) muscle and adipose tissue. This modest tissue-specific increase in GLUT4 expression led to an unexpectedly rapid blood glucose clearance rate following oral glucose administration. In nontransgenic animals exercise caused a 1.5-fold increase in expression of GLUT4 mRNA and protein as well as a significant improvement of glycemic control. In transgenic animals harboring the minigene exercise increased expression of GLUT4 mRNA and protein derived from the minigene and endogenous gene and led to a further improvement of glycemic control. These findings indicate that the cis-regulatory element(s) controlling exercise-induced expression of the GLUT4 gene is located within the nucleotide sequence encompassed by the GLUT4 minigene. The fact that glycemic control is markedly improved by a relatively low level of expression of GLUT4 caused by the transfected minigene and is further enhanced by exercise in transgenic animals demonstrates that GLUT4 plays a pivotal role in glucose homeostasis in vivo. Of the effectors--i.e., cAMP, insulin, and arachidonic acid--known to down-regulate expression of GLUT4 by 3T3-L1 adipocytes in culture, only the decline in circulating arachidonate level in vivo correlated with up-regulation of GLUT4 caused by exercise.

Animals↗
PubMed1995

Insulin regulates transcription of the CCAAT/enhancer binding protein (C/EBP) alpha, beta, and delta genes in fully-differentiated 3T3-L1 adipocytes.

The effect of insulin on expression of CCAAT/enhancer binding protein (C/EBP) alpha, beta, and delta was investigated in fully-differentiated 3T3-L1 adipocytes. Treatment of adipocytes with insulin stimulated rapid dephosphorylation of C/EBP alpha, and repressed the expression of C/EBP alpha within 2-4 h, with > 90% suppression occurring at 24 h. While insulin induced expression of C/EBP beta and C/EBP delta within 1 h and caused a > 20-fold increase by 4 h, expression returned to nearly pretreatment levels by 24 h. The insulin concentration dependence of these effects was consistent with involvement of the insulin receptor. Gel shift analysis revealed that 6 h of insulin treatment decreased the binding of nuclear C/EBP alpha while increasing binding of nuclear C/EBP beta and C/EBP delta. The reciprocal effects of insulin on the steady-state levels of C/EBP transcription factors can be accounted for kinetically and quantitatively by changes in their mRNA levels, which can be accounted for by effects on gene transcription. The effects of insulin on adipocyte gene transcription (e.g. GLUT4) may be mediated, at least in part, by down-regulation of C/EBP alpha and/or its dephosphorylation.

3T3 Cells↗
PubMed1995

Transcriptional regulation of gene expression during adipocyte differentiation.

Cell culture models (e.g. 3T3-L1 cells) have been developed for studying the process of adipocyte differentiation. Differentiation can be induced by adding insulin-like growth factor I, glucocorticoid, fatty acids, and an agent that increases intracellular cAMP level. The adipocyte differentiation program is regulated by transcriptional activators such as CCAAT/enhancer binding protein alpha (C/EBP alpha), peroxisomal proliferator activated receptor gamma 2 (PPAR gamma 2), fatty acid activated receptor (FAAR), and transcriptional repressors such as preadipocyte repressor element binding protein (PRE) and C/EBP undifferentiated protein (CUP). These transcription factors coordinate the expression of genes involved in creating and maintaining the adipocyte phenotype including the insulin-responsive glucose transporter (GLUT4), stearoyl CoA desaturase 1 (SCD1), and the fatty acid binding protein (422/aP2).

Adipose Tissue↗
PubMed1994

CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program.

Previous studies showed that CCAAT/enhancer binding protein alpha (C/EBP alpha) is required for differentiation of 3T3-L1 preadipocytes induced by exogenous hormonal agents. It was not possible to ascertain, however, whether C/EBP alpha alone is sufficient to induce differentiation because its antimitogenic activity precluded propagating 3T3-L1 cell lines that constitutively express C/EBP alpha at high levels. This problem was circumvented by using 3T3-L1 preadipocytes stably transfected with an isopropyl beta-D-thiogalactoside (IPTG)-inducible p42 C/EBP alpha expression vector system. IPTG-induced expression of the 42-kDa isoform of C/EBP alpha in preadipocytes caused expression of several endogenous adipocyte-specific genes (genes encoding the 422 adipose P2 protein, glucose transporter 4, and C/EBP alpha) and the accumulation of cytoplasmic triglyceride. Thus, C/EBP alpha is not only necessary but also is sufficient to trigger differentiation of growth-arrested 3T3-L1 preadipocytes without use of exogenous hormonal agents.

3T3 Cells↗
PubMed1994

Glucocorticoids reciprocally regulate expression of the CCAAT/enhancer-binding protein alpha and delta genes in 3T3-L1 adipocytes and white adipose tissue.

Glucocorticoid agonists, i.e. dexamethasone or triamcinolone acetonide, rapidly induce expression of CCAAT/enhancer-binding protein (C/EBP) delta and repress expression of C/EBP alpha in fully differentiated 3T3-L1 adipocytes. Within 30 min of glucocorticoid treatment, the cellular level of C/EBP delta rises dramatically, increasing > 100-fold within 6 h. Concurrently, the level of C/EBP alpha decreases, reaching a minimum within 4 h. The dexamethasone concentration dependence and steroid specificity of these responses suggest that both processes are mediated by the glucocorticoid receptor. The reciprocal effects of dexamethasone on the steady-state levels of C/EBP alpha and C/EBP delta can be accounted for kinetically and quantitatively by changes in their mRNA levels and by the transcription rates of their respective genes. The glucocorticoid-induced changes in expression of the C/EBP isoforms are correlated with the transcriptional activation of the SCD1 gene, an adipocyte gene known to be transactivated by C/EBP isoforms. Glucocorticoids also regulate expression of the C/EBP isoforms in vivo. Within 4 h of administration of dexamethasone or triamcinolone acetonide to adult rats, expression of C/EBP delta is induced in white adipose tissue while expression of C/EBP alpha is repressed. Like the response in 3T3-L1 adipocytes, the effects of dexamethasone on C/EBP alpha in white adipose tissue are rapid and transient.

3T3 Cells↗
PubMed1994

Transcriptional control of the stearoyl-CoA desaturase-1 gene by polyunsaturated fatty acids.

The effect of exogenous fatty acids on expression of the stearoyl-CoA desaturase-1 (SCD1) gene was assessed both in vivo and ex vivo. Mice fed a fat-free diet or a diet containing a largely monounsaturated (18:1) fat, i.e., olive oil, expressed high levels of hepatic SCD1 mRNA. In contrast, in mice fed diets containing primarily polyunsaturated (18:2 and 18:3) fats, expression of the hepatic SCD1 message was markedly suppressed. Similar experiments with pure fatty acid esters showed that arachidonate (20:4) and linoleate (18:2) were far more potent in down-regulating expression of the hepatic SCD1 message than oleate (18:1), an end-product (as its CoA thioester) of the SCD1-catalyzed reaction. The reduction of hepatic SCD1 mRNA appears to be primarily due to inhibition of SCD1 gene transcription since polyunsaturated fatty acids caused a decrease in run-on transcription of the gene comparable to the decrease in message level. Consistent with the effects observed in vivo, unsaturated fatty acids suppressed the expression of SCD1 mRNA by rat hepatocytes cultured in serum-free medium. Suppression increased with degree of unsaturation with arachidonic (20:4) and eicosapentaenoic (20:5) acids, causing a > or = 90% reduction in the level of SCD1 message. Thus, the SCD1 gene, like the fatty acid synthase and S14 genes, undergoes coordinate transcriptional down-regulation in response to unsaturated fatty acids.

Animals↗
PubMed1993

A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity.

Full-length (42 kDa) CCAAT/enhancer binding protein alpha (C/EBP alpha) (p42) has been implicated in the transcriptional activation of adipocyte genes including the 422(aP2) and C/EBP alpha genes during differentiation of 3T3-L1 preadipocytes. We have identified a 30-kDa isoform (p30) of C/EBP alpha that is expressed by 3T3-L1 adipocytes, mouse adipose tissue, and rat liver. In vitro translation of wild-type C/EBP alpha mRNA or transient transfection with a wild-type C/EBP alpha vector gave rise to similar levels of p42 and p30. Mutational analysis revealed that p30 is an alternative translation product initiated at the third in-frame methionine codon of the C/EBP alpha message. p30C/EBP alpha binds to the C/EBP sites within and activates reporter gene expression driven by the 422(aP2) and C/EBP alpha gene promoters. Although transfection of 3T3-L1 preadipocytes with a strong p30C/EBP alpha expression vector is insufficient to induce differentiation, this vector advances the differentiation program. Unlike p42C/EBP alpha, which inhibits cell proliferation, p30C/EBP alpha is not antimitotic. Thus, the N-terminal 12-kDa segment of full-length C/EBP alpha contains an amino acid sequence necessary for antimitotic activity. During differentiation of 3T3-L1 preadipocytes and during hepatocyte development, the cellular p42C/EBP alpha/p30C/EBP alpha ratio changes, raising the possibility of a regulatory role.

3T3 Cells↗
PubMed1993

Cyclic AMP-induced transcriptional repression of the insulin-responsive glucose transporter (GLUT4) gene: identification of a promoter region required for down-regulation of transcription.

The mechanism(s) by which cyclic AMP represses transcription of the GLUT4 gene was investigated. 3T3-L1 preadipocytes were stably transfected with a series of 5' deletion mutants of the mouse GLUT4 gene promoter fused to the bacterial CAT gene and then were induced to differentiate into adipocytes. A method based on reverse transcription/polymerase chain reaction (PCR) amplification was developed and optimized to quantitate expression of CAT mRNA transcripts. Treatment with 8-bromo-cAMP down-regulated the level of CAT mRNA in adipocytes transfected with the -7000/CAT, -785/CAT and -469/CAT constructs, but not the -78/CAT construct. Thus, the regulatory element(s) which mediates transcriptional repression by cAMP resides in the proximal promoter of the GLUT4 gene between positions -469 and -78. Since down-regulation of GLUT4 mRNA is unaffected by inhibitors of protein synthesis, cAMP (and insulin) may activate phosphorylation or dephosphorylation of an existing transcription factor that interacts with the GLUT4 proximal promoter.

3T3 Cells↗
PubMed1993

CCAAT/enhancer binding protein alpha (C/EBP alpha) undifferentiated protein: a developmentally regulated nuclear protein that binds to the C/EBP alpha gene promoter.

During differentiation of 3T3-L1 preadipocytes into adipocytes, transcription of the C/EBP alpha (CCA-AT/enhancer binding protein alpha) gene is activated. The promoter of the C/EBP alpha gene contains a bipartite cis element with binding sites for C/EBP alpha undifferentiated protein (CUP) and an Sp1-like GT box binding protein. Binding of CUP to this element is markedly enhanced by its interaction with the Sp1-like protein. CUP, purified approximately 100,000-fold from HeLa cell nuclear extracts, appears to be composed of at least two types of subunit. Evidence is presented that a CUP-containing protein complex bridges between the CUP/Sp1-like GT box element and a downstream cis element, which contains a C/EBP binding site. During differentiation of 3T3-L1 preadipocytes into adipocytes, CUP activity or expression decreases as expression of C/EBP alpha increases. It is suggested that bridging by the CUP-containing protein complex may play a role in transcriptional regulation of the C/EBP alpha gene.

3T3 Cells↗
PubMed1993

Regulated expression of an insulin-responsive glucose transporter (GLUT4) minigene in 3T3-L1 adipocytes and transgenic mice.

Preliminary studies showed that up to 7 kb of 5' flanking sequence of the insulin-responsive glucose transporter (GLUT4) gene are insufficient to mediate differentiation-induced reporter gene expression in mouse 3T3-L1 preadipocytes. To locate the regulatory element(s) responsible for this function, a minigene containing the entire GLUT4 gene with substantial 5' and 3' flanking sequence and a short segment of foreign DNA (for transcript identification) was constructed and transfected into mice and 3T3-L1 preadipocytes at relatively low copy number. In transgenic mice the GLUT4 minigene exhibited a pattern of tissue-specific expression similar, but not identical, to that of the endogenous gene. In 3T3-L1 cells expression of minigene mRNA occurred upon differentiation into adipocytes, with kinetics virtually identical to that of endogenous GLUT4 mRNA. In both cultured adipocytes and transgenic mice, the level of expression of the minigene was low relative to that of the endogenous gene. Treatment of minigene-transfected 3T3-L1 adipocytes with 8-bromo-cAMP, which represses transcription of the endogenous GLUT4 gene, also repressed expression of the GLUT4 minigene. However, insulin, which down-regulates transcription of the endogenous GLUT4 gene, failed to normally down-regulate expression of the GLUT4 minigene. These findings indicate that the cis-acting elements required for directing tissue-specific expression (in heart, skeletal muscle, and brown adipose tissue), differentiation-induced activation of transcription, and cAMP-induced repression of transcription are located within the 14-kb GLUT4 minigene. However, the cis elements necessary for maximal tissue-specific expression and for insulin-induced down-regulation of expression are not located in the minigene.

3T3 Cells↗
PubMed1993

Insulin down-regulates expression of the insulin-responsive glucose transporter (GLUT4) gene: effects on transcription and mRNA turnover.

Insulin rapidly represses expression of the gene encoding the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 mouse adipocytes. Upon exposure to the hormone the cellular level of GLUT4 mRNA falls (t1/2 approximately 2.5 hr) to 20-30% of its initial level within 10 hr. This is followed by a similar decrease in the level of GLUT4 protein. Down-regulation of GLUT4 mRNA is a result of both rapid repression of transcription of the GLUT4 gene and an increased rate of turnover of the GLUT4 message. As a consequence of prolonged exposure to insulin, 3T3-L1 adipocytes lose their capacity for acute stimulation of hexose uptake by insulin. These findings provide an explanation for the resistance of glucose uptake to insulin in adipose tissue observed in non-insulin-dependent (type 2) diabetes mellitus, particularly that associated with hyperinsulinemia and obesity.

3T3 Cells↗
PubMed1993

Trans-acting factors involved in adipogenic differentiation.

The differentiation of preadipocytes into adipocytes in culture is accompanied by the coordinate transcriptional activation of adipose-specific genes. Recent studies have identified cis-acting elements that are involved in activating (or derepressing) the transcription of many of these genes during differentiation. The identification of key trans-acting nuclear factors that interact with certain of these elements makes it possible to formulate models for the regulatory network governing the adipogenic differentiation program.

3T3 Cells↗