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Biomedical subjects

M D Lane

Publications and source records attributed to M D Lane.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

Derepression of the C/EBPalpha gene during adipogenesis: identification of AP-2alpha as a repressor.

During adipogenesis, CCAAT/enhancer binding protein alpha (C/EBPalpha) serves as a pleiotropic transcriptional activator of adipocyte genes. Previously, we identified dual repressive elements in the C/EBPalpha gene and a putative transacting factor (C/EBPalpha undifferentiated protein, or CUP) expressed by preadipocytes, but not adipocytes, that bind to these elements. In the present investigation, CUP was purified 17,000-fold from nuclear extracts of 3T3-L1 preadipocytes. Amino acid sequence and mass spectral analysis of tryptic peptides derived from purifed CUP (molecular mass approximately 50 kDa) revealed that the repressor is (or contains) an isoform of the transcription factor, AP-2alpha. Electrophoretic mobility shift and Western blot analysis on purified CUP and preadipocyte nuclear extracts confirmed the identity of CUP as AP-2alpha. Both AP-2alpha protein and CUP binding activity are expressed by preadipocytes and then decrease concomitantly during differentiation of 3T3-L1 preadipocytes into adipocytes. Consistent with a repressive role of AP-2alpha/CUP, an AP-2alpha1 expression vector, cotransfected with a C/EBPalpha promoter-reporter construct into 3T3-L1 adipocytes, inhibited reporter gene transcription. Taken together with previous results, these findings suggest that in preadipocytes the C/EBPalpha gene is repressed by AP-2alpha/CUP, which, upon induction of differentiation, is down-regulated, allowing expression of the gene.

Adipocytes↗
PubMed1998

A sequence element in the GLUT4 gene that mediates repression by insulin.

Prolonged treatment of 3T3-L1 adipocytes decreases expression of GLUT4, the insulin-responsive glucose transporter. Expression of promoter-reporter gene constructs that contained 2900 or 785 base pairs of 5'-flanking region of the murine GLUT4 gene was down-regulated by insulin (p < 0.0005), whereas expression of constructs that contained 641, 469, or 78 base pairs of 5'-flanking region was not. Nuclear extract from 3T3-L1 adipocytes protected the region from -707 to -681 in the GLUT4 5'-flanking region from DNase I digestion. Using an oligonucleotide probe that corresponded to this footprinted region, two major protein-DNA complexes were identified by a gel mobility shift assay. Southwestern analysis identified four protein bands with molecular masses from 38 to 46 kDa that bound to the insulin-responsive region probe. A reporter gene construct in which bases -706 to -676 were deleted was not repressed by insulin treatment, confirming that this sequence is necessary for the repression of the GLUT4 promoter by insulin in 3T3-L1 adipocytes. This sequence does not show homology to previously described insulin response elements and thus represents a distinct mechanism of gene regulation by insulin.

Adipocytes↗
PubMed1998

Leptin regulates proinflammatory immune responses.

Obesity is associated with an increased incidence of infection, diabetes, and cardiovascular disease, which together account for most obesity-related morbidity and mortality. Decreased expression of leptin or of functional leptin receptors results in hyperphagia, decreased energy expenditure, and obesity. It is unclear, however, whether defective leptin-dependent signal transduction directly promotes any of the conditions that frequently complicate obesity. Abnormalities in tumor necrosis factor alpha expression have been noted in each of the above comorbid conditions, so leptin deficiency could promote these complications if leptin had immunoregulatory activity. Studies of rodents with genetic abnormalities in leptin or leptin receptors revealed obesity-related deficits in macrophage phagocytosis and the expression of proinflammatory cytokines both in vivo and in vitro. Exogenous leptin up-regulated both phagocytosis and the production of proinflammatory cytokines. These results identify an important and novel function for leptin: up-regulation of inflammatory immune responses, which may provide a common pathogenetic mechanism that contributes to several of the major complications of obesity.

Animals↗
PubMed1997

Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein alpha gene.

During adipocyte differentiation, the expression of C/EBPalpha is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPalpha gene led to the identification of a potential repressive element within the proximal 5' flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPalpha undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPalpha promoter-luciferase constructs containing both the proximal 5' flanking and the entire 5' untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPalpha gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5' untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5' untranslated region. Although mutation of either CUP element in constructs containing both the 5' flanking and 5' untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPalpha gene prior to induction of the adipocyte differentiation program.

3T3 Cells↗
PubMed1997

Regulated expression of 5'-deleted mouse GLUT4 minigenes in transgenic mice: effects of exercise training and high-fat diet.

Fourteen kb murine GLUT4 minigene (= -7395 GLUT4) contains DNA sequence that confers tissue specific, exercise-induced up-regulation of the GLUT4 gene in skeletal muscle and high-fat diet induced-down-regulation in white adipose tissue. To identify the DNA sequences required for regulated expression, we generated GLUT4 minigene transgenic mice harboring 3237, 2000, 1000, and 442 bp of 5'-flanking region, all exons and introns, and 1 kb of 3'-flanking sequence of the mouse GLUT4 gene. The -3237-, -2000-, and -1000-GLUT4 constructs were expressed in a tissue-specific manner identical to the endogenous GLUT4. Exercise-induced up-regulation and high-fat diet-induced down-regulation of these constructs also paralleled those of the endogenous GLUT4 gene. In contrast, the -442 GLUT4 construct was expressed substantially in skeletal muscle (gastrocnemius and quadriceps) and heart, but was only expressed very weakly in white adipose tissue and was not expressed in brown adipose tissue. Furthermore, this -442 GLUT4 construct failed to respond to exercise or a high-fat diet in either muscle or adipose tissue. These results indicate that brown and white adipocyte-specific enhancer(s) and exercise- and high-fat diet-responsive elements are located between bases -1000 and -442 of the murine GLUT4 5'-flanking region.

Animals↗
PubMed1997

Obese gene expression at in vivo levels by fat pads derived from s.c. implanted 3T3-F442A preadipocytes.

3T3-F442A preadipocytes implanted s.c. into athymic mice develop into fat pads that are indistinguishable from normal adipose tissue. Implanted preadipocytes harboring a beta-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed beta-galactosidase. This finding proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed "adipose tissue." 3T3-F442A preadipocytes, when differentiated into adipocytes in cell culture, express the obese gene at an unexpectedly low level, i.e., </=1% the level in adipose tissue. However, adipose tissue derived from s.c. implanted 3T3-F442A preadipocytes expressed leptin mRNA at a level comparable to that in epididymal adipose tissue. These findings indicate that a factor(s) or condition, present in the tissue context and necessary for maximal obese gene expression, is lacking in cell culture. Furthermore, adipocytes derived from the implanted cells were hormonally responsive in that leptin mRNA levels were up-regulated 3- to 8-fold by glucocorticoid injection into the host animal. Thus, these findings indicate that adipose-specific promoter-reporter constructs, transfected into 3T3-F442A preadipocytes, can be tested in an in vivo context during and after development of these cells into adipose tissue. Furthermore, the effect of transgenes on the adipogenic development of the implanted preadipocytes can be assessed. Thus, this approach offers a faster and less costly alternative to the transgenic mouse method for assessing adipose gene function.

Adipocytes↗
PubMed1997

A concentrated and stable aerosol formulation of cationic lipid:DNA complexes giving high-level gene expression in mouse lung.

Advances in gene therapy vectors and techniques hold promise for treatment of many inherited and acquired diseases. For lung indications, especially those involving the epithelium, delivery of the gene therapy vehicle ideally will involve the use of an aerosol. Aerosol delivery of transgenes using cationic lipids is currently limited by the ability to generate highly concentrated formulations of lipid:DNA complexes that are stable and retain their activity following aerosolization. We have examined many of the variables inherent in aerosolizing cationic lipid gene delivery vehicles and have devised a new formulation that incorporates small amounts of a polyethylene glycol-containing lipid. This formulation has allowed the preparation of concentrated dispersions of cationic lipid:plasmid DNA (pDNA) complexes (> 20 mM pDNA) at approximately 10-fold higher concentrations than previously reported. Most of the pDNA in these formulations was bound to the lipid component and thereby protected from nebulizer-induced shearing; the pDNA also maintained full biological activity both in vitro and in vivo. This new formulation thus represents a significant improvement over current methods to prepare concentrated, active cationic lipid gene delivery vectors, and provides a new tool with which to test gene transfer to the lung.

Administration, Inhalation↗
PubMed1997

Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis.

Genetically obese fatty/fatty rats and obese/obese mice exhibit increased sensitivity to endotoxin hepatotoxicity, quickly developing steatohepatitis after exposure to low doses of lipopolysaccharide (LPS). Among obese animals, females are more sensitive to endotoxin liver injury than males. LPS induction of tumor necrosis factor alpha (TNF alpha), the proven affecter of endotoxin liver injury, is no greater in the livers, white adipose tissues, or sera of obese animals than in those of lean controls. Indeed, the lowest serum concentrations of TNF occur in female obese rodents, which exhibit the most endotoxin-induced liver injury. Several cytokines that modulate the biological activity of TNF are regulated abnormally in the livers of obese animals. After exposure to LPS, mRNA of interferon gamma, which sensitizes hepatocytes to TNF toxicity, is overexpressed, and mRNA levels of interleukin 10, a TNF inhibitor, are decreased. The phagocytic activity of liver macrophages and the hepatic expression of a gene encoding a macrophage-specific receptor are also decreased in obesity. This new animal model of obesity-associated liver disease demonstrates that hepatic macrophage dysfunction occurs in obesity and suggests that this might promote steatohepatitis by sensitizing hepatocytes to endotoxin.

Animals↗
PubMed1997

Modulating the transcriptional control of adipogenesis.

Current evidence indicates that much of the regulation of adipocyte differentiation serves to modulate a common adipogenic transcriptional control pathway, comprising members of the C/EBP and PPAR families. Hormonal regulators have been found to control expression of these factors and to alter their activity through ligand binding, post-transcriptional modification, and protein-protein interactions.

Adipocytes↗
PubMed1997

Adipocyte differentiation and leptin expression.

Adipose tissue has long been known to house the largest energy reserves in the animal body. Recent research indicates that in addition to this role, the adipocyte functions as a global regulator of energy metabolism. Adipose tissue is exquisitely sensitive to a variety of endocrine and paracrine signals, e.g. insulin, glucagon, glucocorticoids, and tumor necrosis factor (TNF), that combine to control both the secretion of other regulatory factors and the recruitment and differentiation of new adipocytes. The process of adipocyte differentiation is controlled by a cascade of transcription factors, most notably those of the C/EBP and PPAR families, which combine to regulate each other and to control the expression of adipocyte-specific genes. One such gene, i.e. the obese gene, was recently identified and found to encode a hormone, referred to as leptin, that plays a major role in the regulation of energy intake and expenditure. The hormonal and transcriptional control of adipocyte differentiation is discussed, as is the role of leptin and other factors secreted by the adipocyte that participate in the regulation of adipose homeostasis.

Adipocytes↗
PubMed1996

Tumor necrosis factor alpha promotes nuclear localization of cytokine-inducible CCAAT/enhancer binding protein isoforms in hepatocytes.

Hepatocytes were cultured in the presence of recombinant tumor necrosis factor (TNF) alpha or mutated TNF alpha peptides that specifically activate either p55 or p75 TNF receptors to determine if TNF alpha can activate cytokine-inducible CCAAT/enhancer binding protein (C/EBP) isoforms by post-transcriptional mechanisms that are initiated by TNF receptors. Within 5-10 min after treatment with any of these agents, nuclear concentrations of C/EBP beta and C/EBP delta double and remain 2-4-fold greater than control cultures for 30 min (p < 0.01). Consistent with these results, gel mobility shift assays demonstrate 3-fold increased nuclear C/EBP beta- and C/EBP delta-DNA binding activity in TNF alpha-treated cells, and immunocytochemistry confirms rapid redistribution of these C/EBP isoforms into the nucleus. In contrast, mRNA and whole cell protein concentrations of C/EBP beta and delta are not altered by TNF alpha exposure, and nuclear concentrations of another C/EBP isoform, C/EBP alpha, are decreased by 80%. This novel evidence that TNF alpha initiates post-transcriptional activation of cytokine-inducible C/EBP isoforms identifies a mechanism that enables hepatocytes to respond immediately to inflammatory stress.

Amino Acid Sequence↗
PubMed1996

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.

Animals↗
PubMed1996

Control of adipocyte differentiation by CCAAT/enhancer binding protein alpha (C/EBP alpha).

Upon differentiation of 3T3-L1 preadipocytes into adipocytes transcription of many adipose-specific genes is coordinately activated. A differentiation-induced factor, later identified as C/EBP alpha, binds to and transactivates the promoters of these genes. Vector-directed expression of antisense C/EBP alpha RNA in preadipocytes blocked expression of C/EBP alpha, as well as adipose-specific mRNAs, and also prevented cytoplasmic triglyceride accumulation. Rescue of the 'adipocyte phenotype' was accomplished by transfection of the antisense cells with a complementary sense C/EBP alpha RNA expression vector. Using an IPTG-inducible double-vector LacSwitch C/EBP alpha expression system, it was found that differentiation can be induced without exogenous hormone inducers. These findings indicate that C/EBP alpha is not only required, but is sufficient, to trigger differentiation of 3T3-L1 preadipocytes. The C/EBP alpha gene promoter possesses a C/EBP binding site through which C/EBP alpha autoactivates its own expression. A nuclear protein referred to as CUP (C/EBP undifferentiated protein) that binds to a bipartite element in the C/EBP alpha promoter just 5' to the C/EBP binding site has been purified and characterized. During differentiation of preadipocytes, expression of CUP activity decreases as expression of C/EBP alpha increases. Evidence suggests that a CUP-containing protein complex bridges between the CUP (repression) and C/EBP (autoactivation) elements in the promoter and may maintains the C/EBP alpha gene in the repressed state prior to differentiation.

3T3 Cells↗