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.

An adipocyte-central nervous system regulatory loop in the control of adipose homeostasis.

Mounting evidence supports a 'lipostatic' model for the regulation of adipose mass. In such a model, signals are generated in the periphery in proportion to adipose mass that act on hypothalamic control centers in the brain to regulate food intake and energy expenditure. Two such signals, leptin and insulin, have been identified and found to dramatically lower food intake and body weight. Several signalling molecules in the effector pathways that mediate the response to these signals in the brain have also been identified. The regulation of these factors and the nature of the adipose-CNS regulatory loop will be discussed.

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.

Sources of NADPH and expression of mammalian NADP+-specific isocitrate dehydrogenases in Saccharomyces cerevisiae.

To compare roles of specific enzymes in supply of NADPH for cellular biosynthesis, collections of yeast mutants were constructed by gene disruptions and matings. These mutants include haploid strains containing all possible combinations of deletions in yeast genes encoding three differentially compartmentalized isozymes of NADP+-specific isocitrate dehydrogenase and in the gene encoding glucose-6-phosphate dehydrogenase (Zwf1p). Growth phenotype analyses of the mutants indicate that either cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p) or the hexose monophosphate shunt is essential for growth with fatty acids as carbon sources and for sporulation of diploid strains, a condition associated with high levels of fatty acid synthesis. No new biosynthetic roles were identified for mitochondrial (Idp1p) or peroxisomal (Idp3p) NADP+-specific isocitrate dehydrogenase isozymes. These and other results suggest that several major presumed sources of biosynthetic reducing equivalents are non-essential in yeast cells grown under many cultivation conditions. To develop an in vivo system for analysis of metabolic function, mammalian mitochondrial and cytosolic isozymes of NADP+-specific isocitrate dehydrogenase were expressed in yeast using promoters from the cognate yeast genes. The mammalian mitochondrial isozyme was found to be imported efficiently into yeast mitochondria when fused to the Idp1p targeting sequence and to substitute functionally for Idp1p for production of alpha-ketoglutarate. The mammalian cytosolic isozyme was found to partition between cytosolic and organellar compartments and to replace functionally Idp2p for production of alpha-ketoglutarate or for growth on fatty acids in a mutant lacking Zwf1p. The mammalian cytosolic isozyme also functionally substitutes for Idp3p allowing growth on petroselinic acid as a carbon source, suggesting partial localization to peroxisomes and provision of NADPH for beta-oxidation of that fatty acid.

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.

The QM protein associates with ribosomes in the rough endoplasmic reticulum.

QM is a human cDNA originally isolated as a transcript elevated in a nontumorigenic Wilms' tumor microcell hybrid, relative to the tumorigenic parental cell line. Homologs of this gene have been identified from a large number of diverse eukaryotic species which demonstrate a high degree of conservation. The functional importance implied by this strong conservation is supported by the observation that the disruption of the yeast homolog is lethal. In spite of its apparent importance, the function of the encoded protein remains elusive. Indirect immunofluorescent cell staining of cultured human, G401 cells with an antibody to the QM protein shows a punctate staining pattern in the cytoplasm with much of the signal in a perinuclear pattern. Subcellular fractionation demonstrated an association of QM protein with the rough endoplasmic reticulum. It was possible to disrupt this association by washing microsomal membranes with 1M NaCl, suggesting a peripheral association. Proteolytic latency studies showed the protein to be exposed on the cytoplasmic face of the membrane. In situ cross-linking followed by diagonal SDS gel analysis indicates that QM exists as a member of a large protein complex. In agreement with this, QM was found to copurify with the ribosome complex. Incubation with 1 M NaCl was found to disrupt this association while having no effect on the association of core ribosomal proteins.

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.,

The isolation, localization and characterization of the QM homolog in Drosophila melanogaster.

A fragment of 443 bp was amplified from a lambda ZAPII Drosophila central nervous system (CNS) cDNA library using minimally degenerate primers to very conserved regions of the QM gene. This fragment was used as a probe to screen the lambda ZAPII Drosophila CNS cDNA library. Two clones of the Drosophila QM homolog (pDQM-7A1 and pDQM-2B1), each containing the complete coding region, were isolated. The 5'-UTR of this gene was obtained by RACE PCR and ligated to the coding sequence to produce a the full-length copy of the Drosophila QM homolog (DQM) cDNA. The DQM cDNA measures 746 nucleotides in length and encodes a polypeptide of 218 residues. The amino acid sequence shows 76.1 percent identity with human QM and 69.1 percent identity with QSR1, the yeast homolog of QM. Unlike the human or mouse genome which contains multiple copies of the QM gene, the Drosophila genome has only a single copy as indicated by genomic Southern blot analysis. In situ hybridization confirms the presence of a single copy of DQM in the Drosophila genome and localizes it to the left arm of the third chromosome at the end of region 80A (80A-4).

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.

Isolation and characterization of the QM promoter.

This report describes the isolation, sequencing and preliminary characterization of the first 1 kb of the 5'-regulatory region of the human QM gene. This region and the 5' -half of the transcribed region of the QM gene are enriched for C and G nucleotides with no bias against CpG dinucleotides--indicative of a CpG island. Several consensus GC boxes are present within the sequence. Most are clustered at the distal end, with one site present in the proximal 200 bp of the promoter. Electrophoretic mobility shift experiments and luciferase assays done in insect cells transfected with an Sp1 expression construct suggest that most of these sites can bind Sp1 or a closely related factor. In addition, the promoter is shown to be responsive to cAMP via a response element (CRE) in the proximal promoter. Studies with 5'-end and internal deletion mutants suggest that elements in the distal promoter exert their positive effect through interactions with a proximal element(s). Candidate proximal elements include the proximal GC box and a 43 bp region between a KpnI site (at -182) and a Smal site (at -139).

Isolation, characterization, and disruption of the yeast gene encoding cytosolic NADP-specific isocitrate dehydrogenase.

The cytosolic isozyme of NADP-specific isocitrate dehydrogenase (IDP2) was purified from a Saccharomyces cerevisiae mutant containing a chromosomal disruption in the gene encoding the mitochondrial isozyme (IDP1). IDP2 was shown to be a homodimer with a subunit molecular weight of approximately 45,000 and an isoelectric point of 5.5. Amino acid sequences were obtained for tryptic peptides of IDP2 and used to plan polymerase chain reactions. A resulting 400 bp DNA fragment was used as a hybridization probe to isolate the IDP2 gene from a yeast genomic DNA library. The complete nucleotide sequence of the IDP2 coding region was determined and translated into a 412-residue amino acid sequence. IDP2 and IDP1 were found to be identical in 71% of the aligned residue positions. The identity of the IDP2 gene was confirmed by genomic replacement with a disrupted IDP2 coding region. Haploid yeast strains lacking either or both IDP2 and IDP1 were constructed by genetic crosses of mutant strains containing disruptions in chromosomal IDP2 and IDP1 loci. No dramatic differences in growth rates with common carbon sources could be attributed to these disruptions.

Extreme evolutionary conservation of QM, a novel c-Jun associated transcription factor.

QM is a 214 amino acid polypeptide, encoded by a gene (DXS648) in Xq28, that contains a high percentage of charged amino acids and has been found to bind c-Jun and DNA. Searches of the GenBank database revealed no matches between QM and any other known transcription factors. However, we and others have isolated QM homologs from a diverse array of eukaryotes. Alignment of these sequences indicated a high degree of conservation throughout the first 175 residues of the protein and revealed several interesting features. Most notable is the considerable conservation of charged amino acids within specific regions of the protein. Secondary structure analysis suggests that two of these regions form amphipathic alpha-helices, one basic and one acidic. A third conserved charged domain, comprising the N-terminal 30 amino acids, is both basic and proline rich. The rate of sequence divergence of the various homologs was found to be slow (of the order of 1% change every 22 million years), consistent with a critical role for QM in eukaryotic cells. A role for QM as a novel class of transcription regulatory protein is suggested.