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Biomedical subjects

M D Lane

Publications and source records attributed to M D Lane.

At least 19 recordsLinked 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

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↗
PubMed1992

Identification of a transcriptional repressor down-regulated during preadipocyte differentiation.

During differentiation of 3T3-L1 preadipocytes into adipocytes, the transcription of adipocyte genes, including the stearoyl-CoA desaturase 2 (SCD2) gene, is activated. Transfection experiments with chimeric SCD2 promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs revealed a preadipocyte repressor element (PRE) capable of repressing transcription of the reporter gene in preadipocytes but not in adipocytes. DNase I protection and gel retardation analyses were used to localize the PRE site between nucleotides -435 and -410 of the SCD2 promoter and to identify a nuclear PRE binding protein present at high levels in preadipocytes and HeLa cells but lacking or inactive in adipocytes. Southwestern blot analysis indicated that the PRE binding protein has an apparent molecular mass of approximately 58 kDa. A single copy of the PRE site, inserted upstream of the simian virus 40 enhancer/promoter of pSV2CAT, was capable of strongly repressing transcription of the reporter gene in preadipocytes and HeLa cells but not in adipocytes. Taken together these results suggest that the PRE site and binding protein may regulate transcription of SCD2 and possibly other adipocyte genes by inhibiting their transcription in preadipocytes.

3T3 Cells↗
PubMed1992

Iodophenylarsine oxide and arsenical affinity chromatography: new probes for dithiol proteins. Application to tubulins and to components of the insulin receptor-glucose transporter signal transduction pathway.

In our studies of the effects of the trivalent arsenical phenylarsine oxide on insulin-dependent hexose uptake in 3T3-L1 adipocytes, we needed direct methods to study arsenical-protein interactions. In this report, we describe two such new tools. The first is the radiolabeled covalent affinity reagent 4-[125I]iodophenylarsine oxide. This compound has effects on 3T3-L1 adipocytes similar to those of phenylarsine oxide both with respect to effects of hexose uptake and the accumulation of pp15, a phosphotyrosine-containing putative mediator of insulin action. Iodophenylarsine oxide labels numerous proteins in intact cells in a concentration-dependent, but apparently insulin-independent fashion. The second tool is trivalent arsenical affinity chromatography, which we use to show novel direct interactions between trivalent arsenicals and several proteins from 3T3-L1 adipocytes including the insulin-responsive glucose transporter GLUT4, the insulin proreceptor, and both the alpha and beta subunits of tubulin. The non-insulin-dependent glucose transporter GLUT1, the mature insulin receptor, and the fatty acid-binding protein 422(aP2) do not show strong interactions with arsenical resin. These results provide a new chemical approach to the study of both insulin-dependent hexose transport and tubulin function.

3T3 Cells↗
PubMed1992

Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.

Heterologous proteins can be expressed in Xenopus laevis oocytes by cytoplasmic microinjection of mRNA. To circumvent limitations inherent in this approach we investigate direct nuclear injection of strong viral expression vectors to drive transcription and subsequent translation of cDNAs encoding cytoplasmic, secreted, and plasma membrane proteins. After several viral promoters had been tested, the pMT2 vector was found to be a superior expression vector for X. laevis oocytes capable of directing expression of high levels of functional heterologous proteins. Typically the amount of protein derived from transcription-translation of the microinjected cDNA accounts for approximately 1% of total non-yolk protein. Moreover, the inefficiency usually associated with nuclear injections was overcome by coinjection of pMT2 driving expression of a secreted alkaline phosphatase as an internal control to select positive-expressing oocytes. Using this method, we have successfully expressed high levels of chloramphenicol acetyltransferase, the adipocyte-specific cytosolic 422(aP2) protein, and the membrane-associated glucose transporter GLUT1. The system described should be applicable to a wide variety of proteins for which cDNAs are available. Hence, the cumbersome and often inefficient in vitro synthesis of mRNA for studying ion channels, receptors, and transporters as well as for expression cloning in Xenopus oocytes should no longer be necessary.

Alkaline Phosphatase↗
PubMed1992

Antisense CCAAT/enhancer-binding protein RNA suppresses coordinate gene expression and triglyceride accumulation during differentiation of 3T3-L1 preadipocytes.

Previous studies suggest that the CCAAT/enhancer-binding protein (C/EBP) functions in the coordinate expression of adipocyte genes during differentiation of 3T3-L1 preadipocytes. We sought to block expression of C/EBP selectively using a bovine papilloma virus (BPV) vector to direct transcription of a approximately 0.4-kb segment of C/EBP cDNA (in antisense orientation) containing translated sequence 5' to that encoding the basic and leucine zipper regions of the protein. Vector-directed expression of antisense C/EBP RNA in 3T3-L1 preadipocytes inhibited expression of C/EBP mRNA and protein, as well as several adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the "adipocyte phenotype" was accomplished by transfection of cells expressing antisense RNA with a modified BPV vector that directs transcription of the complementary sense C/EBP RNA.

3T3 Cells↗
PubMed1991

Cell-free transcription directed by the 422 adipose P2 gene promoter: activation by the CCAAT/enhancer binding protein.

Previous investigations have shown that CCAAT/enhancer binding protein (C/EBP) can function as a trans-activator of the promoters of several adipocyte-specific genes--i.e., the 422 adipose P2 (422/aP2), stearoyl-CoA desaturase 1 (SCD1), and glucose transporter 4 (GLUT4) genes, in 3T3-L1 mouse preadipocytes. We now describe a cell-free system prepared from nuclei of 3T3-L1 cells that carries out transcription directed by these promoters. To measure transcript formation, we employed a polymerase chain reaction-assisted analysis. Nuclear extract from 3T3-L1 adipocytes that express C/EBP supports a higher rate of transcription of chimeric 422(aP2) promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs than nuclear extract from preadipocytes that lack C/EBP. A competitor oligonucleotide containing the C/EBP binding site sequence and antibodies raised against C/EBP inhibit transcription directed by the 422(aP2) promoter. The factor limiting transcription by nuclear extract from preadipocytes appears to be C/EBP, since recombinant C/EBP (rC/EBP) markedly activates transcription of the 422(aP2) promoter-CAT gene with preadipocyte extract but not with adipocyte extract. rC/EBP also activates cell-free transcription of SCD1 promoter-CAT and GLUT4 promoter-CAT chimeric genes. Point mutations within the C/EBP binding site in the 422(aP2) promoter markedly decrease transcription activated by rC/EBP. Consistent with activation by cAMP of the 422(aP2) promoter in intact preadipocytes, cAMP-dependent protein kinase activates transcription through this promoter with the cell-free system, this effect being independent of C/EBP. Thus, regulation of transcription directed by the 422(aP2) promoter in the cell-free system resembles that which occurs in intact 3T3-L1 cells.

Adipose Tissue↗
PubMed1991

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes.

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.

Animals↗
PubMed1991

Phosphotyrosyl turnover in insulin signaling. Characterization of two membrane-bound pp15 protein tyrosine phosphatases from 3T3-L1 adipocytes.

It was shown previously that 422 (aP2) protein, a 15-kDa fatty acid binding protein, is phosphorylated on Tyr19 both in vitro by the insulin receptor tyrosine kinase and in intact 3T3-L1 adipocytes treated with insulin and phenylarsine oxide (PAO). Phospho-422(aP2) protein (pp15) accumulates in cells treated with insulin and PAO because the arsenical blocks turnover of the phosphoryl group of pp15. These findings suggest that a PAO-sensitive enzyme mediates turnover of the pp15 tyrosine phosphoryl group. We have purified and characterized two membrane protein tyrosine phosphatases (PTPases) from 3T3-L1 adipocytes that catalyze hydrolysis of phospho-Tyr19 of authentic pp15. These enzymes, designated PTPases HA1 and HA2, were purified approximately 20,000-fold and approximately 15,000-fold, respectively, and shown to differ markedly in their sensitivity to both vanadate and phosphotyrosine. Both enzymes are inhibited by PAO and accordingly can be labeled with 4-[125I]iodo-PAO. By this method, it was demonstrated that PTPases HA1 and HA2 have molecular masses of approximately 60 kDa and approximately 38 kDa, respectively. Both enzymes exhibit substrate preference for pp15 when compared with other phosphotyrosine-containing protein substrates. Proteins containing phosphoserine and phosphothreonine do not serve as substrates for the enzymes. The pp15 PTPase HA2 is expressed both in 3T3-L1 preadipocytes and adipocytes, whereas pp15 PTPase HA1 is expressed only in 3T3-L1 adipocytes.

Adipose Tissue↗