Louis A. Tartaglia

Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance

Insulin resistance arises from the inability of insulin to act normally in regulating nutrient metabolism in peripheral tissues. Increasing evidence from human population studies and animal research has established correlative as well as causative links between chronic inflammation and insulin resistance. However, the underlying molecular pathways are largely unknown. In this report, we show that many inflammation and macrophage-specific genes are dramatically upregulated in white adipose tissue (WAT) in mouse models of genetic and high-fat diet-induced obesity (DIO). The upregulation is progressively increased in WAT of mice with DIO and precedes a dramatic increase in circulating-insulin level. Upon treatment with rosiglitazone, an insulin-sensitizing drug, these macrophage-originated genes are downregulated. Histologically, there is evidence of significant infiltration of macrophages, but not neutrophils and lymphocytes, into WAT of obese mice, with signs of adipocyte lipolysis and formation of multinucleate giant cells. These data suggest that macrophages in WAT play an active role in morbid obesity and that macrophage-related inflammatory activities may contribute to the pathogenesis of obesity-induced insulin resistance. We propose that obesity-related insulin resistance is, at least in part, a chronic inflammatory disease initiated in adipose tissue.

Identification and Expression Cloning of a Leptin Receptor, OB-R

The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.

Evidence That the Diabetes Gene Encodes the Leptin Receptor: Identification of a Mutation in the Leptin Receptor Gene in db/db Mice

OB-R is a high affinity receptor for leptin, an important circulating signal for the regulation of body weight. We identified an alternatively spliced transcript that encodes a form of mouse OB-R with a long intracellular domain. db/db mice also produce this alternatively spliced transcript, but with a 106 nt insertion that prematurely terminates the intracellular domain. We further identified G --> T point mutation in the genomic OB-R sequence in db/db mice. This mutation generates a donor splice site that converts the 106 nt region to a novel exon retained in the OB-R transcript. We predict that the long intracellular domain form of OB-R is crucial for initiating intracellular signal transduction, and as a corollary, the inability to produce this form of OB-R leads to the severe obese phenotype found in db/db mice.

The leptin receptor.

Of the many genetic obesity syndromes, none have been as intensively studied as ob/ob and db/db mice. These two mutant mice were originally identified over 30 years ago (1, 2) and shown to be a result of two distinct single gene mutations residing on mouse chromosomes 6 (ob) and 4 (db). The phenotypes and pathophysiologies of these two mice have been studied for decades and described in well over 1000 publications. However, the nature of the lesions or primary defects was not revealed until very recently. Perhaps the most informative early studies on the nature of the ob and db primary defects were the parabiosis experiments (partial connection of the circulatory systems of animals through grafting) performed throughout the 1970s (3) (reviewed in Ref. 4). Parabiosis of an ob/obmouse and a lean control resulted in partial normalization of body weight in the ob/obmutant mouse. This led to the proposal that ob/obmice were deficient in a circulating factor that could be restored through the blood of the lean animal. However, db/db mice that underwent parabiosis with lean controls did not exhibit body weight normalization. This suggested that db/db mice may be defective in their ability to respond to the putative satiety factor, perhaps because they were defective in the receptor for this molecule. The Obese (ob) Gene and Its Product, Leptin Despite intensive interest in the nature of the putative satiety factor missing in ob/ob mice, biochemical strategies failed to identify it. It was not until a genetic/positional cloning strategy was employed that the gene corresponding to the ob locus and its gene product were ultimately identified (5). The wild type ob gene encodes a protein of about 16 kDa that is preceded by a secretory hydrophobic signal peptide. It is expressed in adipose tissue in multiple mammalian species including mice and humans. The development of antibody reagents confirmed that this factor (leptin) is found at high levels in blood, consistent with the previous parabiosis studies (6). Since the cloning of the ob gene numerous studies have described the regulation of the leptin mRNA and protein. Although the purpose of this review is not to comprehensively examine the growing literature on the regulation of the leptin ligand, it is important to briefly summarize a few aspects of leptin expression and regulation that are key in interpreting the biology of the leptin receptor. The leptin transcript appears to be expressed fairly specifically in adipose tissue (5), although it is also detectable in human placenta on poly(A) Northern blots. Steady state levels of the leptin mRNA and protein are elevated in a variety of rodent obesity models (6–9). These observations have led to the proposal that leptin serves as an “adipostat,” informing the body of the status of energy storage in the adipose tissue so that appropriate changes in appetite, metabolism, and nutrient partitioning can be signaled via the leptin receptor. Dramatic regulation of the leptin transcript and protein has also been observed in response to short term alterations in food intake (7, 9–11); fasting results in dramatic down-regulation, and excessive caloric intake results in up-regulation. It is therefore plausible that an important role for leptin is mediating the response to starvation (12). Acute effects on leptin mRNA and protein have also been observed in response to a variety of stimuli including glucocorticoids, cytokines, and insulin (10, 13, 14). There has now also been considerable analysis of leptin regulation in humans. The leptin mRNA is regulated in humans by both changes in the percentage of body fat as well as changes in food intake (6, 15–18). However, the degree of mRNA regulation in humans is less impressive than that seen in rodents. Importantly, at the protein level human leptin is dramatically regulated, with changes approaching those seen in the rodent obesities and fasting (6, 15). The protein is much higher in individuals with an increased percentage of body fat and is down-regulated during body weight loss. This parallel regulation in mice and humans may imply that leptin is functioning in humans as it is in rodents, although further studies are required to directly address this. An obvious and important question has been whether a significant portion of human obesity can be due to mutations in the ob gene. The ob coding region has been sequenced from hundreds of human individuals, but mutations have not been found (16, 19, 20). Although mutations affecting mRNA levels can reside outside of the coding region, individuals with severely reduced leptin mRNA levels have also not yet been described (15–18), suggesting that the number of such individuals will not be high. On the other hand, genotyping of microsatellite markers that span the ob gene region has suggested linkage of this region with extreme human obesity (21, 22). Considerable excitement has been generated by the observations that administration of recombinant leptin to rodents results in food intake reduction and weight loss (23–26). Although the potency of leptin is highest in mice that are completely deficient in this protein (ob/ob), significant effects can be seen at higher doses in normal mice and mice with diet-induced obesity. Such studies have brought hope that leptin may be an effective treatment even in some obesities that are not due to leptin deficiencies. Of particular interest are studies that have investigated the effects of centrally administered leptin. These studies showed that leptin injection into the lateral or third brain ventricle produced reduction in food intake and weight loss, strongly implying that leptin could act directly on receptors within the central nervous system (23, 26). Weight loss in rodents following leptin administration appears to be due to not only decreases in food intake but also increases in energy expenditure (24, 25). Although the mechanisms of increased energy utilization are likely to be complex, one important component may involve the activation of brown adipose tissue (27). The observation that leptin deficiencies are not common in human obesity and, in fact, that leptin levels appear to be up-regulated as the percentage of body fat increases has suggested that resistance to normal or elevated levels of leptin may be more important than inadequate leptin production in human obesity (15). This line of thought has been further strengthened by the parallel situation of type II diabetics, many of whom exhibit severe insulin resistance while producing elevated levels of insulin. These observations further stimulated interest in the identification of the receptor for leptin and the analysis of leptin signal reception. They also heightened interest in the db/dbmouse, a model of total leptin resistance (23–26) and elevated leptin levels (8). Cloning of the Leptin Receptor (OB-R) The identification of the receptor for the leptin protein was realized through an expression cloning strategy (28). Tagged versions of leptin were generated either through a traditional iodination strategy or by generating recombinant fusion proteins between leptin and secreted placental alkaline phosphatase. These tagged reagents were then used to identify a tissue source expressing a cell surface leptin binding activity (28, 29). Significant and specific leptin binding was detected in the mouse choroid plexus. To clone this leptin binding activity a murine choroid plexus cDNA library was constructed, and cells transfected with this library were screened with a leptin-alkaline phosphatase fusion protein. From this screen, cDNAs were identified that encoded a cell surface * This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. ‡ To whom correspondence should be addressed: Millennium Pharmaceuticals, 640 Memorial Dr., Cambridge, MA 02139. Tel.: 617-679-7110; Fax: 617-679-7370; E-mail: tartaglia@mpi.com. 1 X. Weng and L. A. Tartaglia, unpublished observations. Minireview THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 10, Issue of March 7, pp. 6093–6096, 1997

Phenotypes of Mouse diabetes and Rat fatty Due to Mutations in the OB (Leptin) Receptor

Mice harboring mutations in the obese (ob) and diabetes (db) genes display similar phenotypes, and it has been proposed that these genes encode the ligand and receptor, respectively, for a physiologic pathway that regulates body weight. The cloning of ob, and the demonstration that it encodes a secreted protein (leptin) that binds specifically to a receptor (OB-R) in the brain, have validated critical aspects of this hypothesis. Here it is shown by genetic mapping and genomic analysis that mouse db, rat fatty (a homolog of db), and the gene encoding the OB-R are the same gene.

PTP1B Regulates Leptin Signal Transduction In Vivo

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.

Cloning and Characterization of an Uncoupling Protein Homolog: A Potential Molecular Mediator of Human Thermogenesis

We have identified a novel cDNA encoding a protein highly homologous to the mammalian brown fat uncoupling protein (UCP). Unlike the known UCP, which is expressed specifically in brown adipose tissue, the UCP homolog (UCPH) mRNA is expressed in a variety of tissues, with predominant expression in human white adipose tissue and skeletal muscle. In the white adipose tissue of ob/ob and db/db mice, the UCPH transcript is induced approximately fivefold relative to lean littermate controls. Expression of murine UCPH in yeast results in growth inhibition under conditions that require aerobic respiration, but does not affect growth under anaerobic conditions. Furthermore, UCPH expression in yeast causes a decrease in the mitochondrial membrane potential, as judged by staining with the potential-sensitive dye DiOC6. These observations suggest that UCPH, like UCP, uncouples oxidative phosphorylation. The possibility that the UCPH protein is an important mediator of human thermogenesis is discussed.

Medicinal strategies in the treatment of obesity.

When prevention fails, medicinal treatment of obesity may become a necessity. Any strategic medicinal development must recognize that obesity is a chronic, stigmatized and costly disease that is increasing in prevalence. Because obesity can rarely be cured, treatment strategies are effective only as long as they are used, and combined therapy may be more effective than monotherapy. For a drug to have significant impact on body weight it must ultimately reduce energy intake, increase energy expenditure, or both. Currently approved drugs for long-term treatment of obesity include sibutramine, which inhibits food intake, and orlistat, which blocks fat digestion.

Identification of the Major Intestinal Fatty Acid Transport Protein

While intestinal transport systems for metabolites such as carbohydrates have been well characterized, the molecular mechanisms of fatty acid (FA) transport across the apical plasmalemma of enterocytes have remained largely unclear. Here, we show that FATP4, a member of a large family of FA transport proteins (FATPs), is expressed at high levels on the apical side of mature enterocytes in the small intestine. Further, overexpression of FATP4 in 293 cells facilitates uptake of long chain FAs with the same specificity as enterocytes, while reduction of FATP4 expression in primary enterocytes by antisense oligonucleotides inhibits FA uptake by 50%. This suggests that FATP4 is the principal fatty acid transporter in enterocytes and may constitute a novel target for antiobesity therapy.

Transport Function and Regulation of Mitochondrial Uncoupling Proteins 2 and 3

Uncoupling protein 1 (UCP1) dissipates energy and generates heat by catalyzing back-flux of protons into the mitochondrial matrix, probably by a fatty acid cycling mechanism. If the newly discovered UCP2 and UCP3 function similarly, they will enhance peripheral energy expenditure and are potential molecular targets for the treatment of obesity. We expressed UCP2 and UCP3 inEscherichia coli and reconstituted the detergent-extracted proteins into liposomes. Ion flux studies show that purified UCP2 and UCP3 behave identically to UCP1. They catalyze electrophoretic flux of protons and alkylsulfonates, and proton flux exhibits an obligatory requirement for fatty acids. Proton flux is inhibited by purine nucleotides but with much lower affinity than observed with UCP1. These findings are consistent with the hypothesis that UCP2 and UCP3 behave as uncoupling proteins in the cell.