ADIPOSE TISSUE HAS long been considered a simple

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1 /02/$15.00/0 Molecular Endocrinology 16(8): Printed in U.S.A. Copyright 2002 by The Endocrine Society doi: /me Cell Type-Specific Expression and Coregulation of Murine Resistin and Resistin-Like Molecule- in Adipose Tissue MICHAEL W. RAJALA, YING LIN, MOLLIE RANALLETTA, XIAO MAN YANG, HAO QIAN, RON GINGERICH, NIR BARZILAI, AND PHILIPP E. SCHERER Departments of Cell Biology (M.W.R., Y.L., P.E.S.), Biochemistry (M.R.), and Medicine (X.M.Y., N.B.), Albert Einstein College of Medicine, Bronx, New York 10461; Department of Research and Development (R.G., H.Q.), Linco Research, Inc., St. Charles, Missouri 63304; and Diabetes Research and Training Center (N.B., P.E.S.), Albert Einstein College of Medicine, Bronx, New York Adipocytes are the exclusive or predominant source of several secreted proteins that exert profound effects on systemic carbohydrate and lipid metabolism. Resistin, a 10-kDa adipose tissue specific secretory protein, has recently been implicated in exerting a negative effect on systemic insulin sensitivity. It is, however, not known how resistin mediates this insulin-desensitizing effect or what regulatory mechanisms control resistin expression. Resistinlike molecule- (RELM ), a homolog of resistin originally identified by its upregulation in asthmatic lung, is another secreted protein expressed in adipose tissue. The regulation of RELM in adipose tissue and its relationship to resistin expression has not been addressed so far. Here, we demonstrate that the expression of resistin and RELM are similarly regulated in adipose tissue despite the fact that RELM is exclusively expressed in the stromal vascular fraction of adipose tissue and not in adipocytes. Interestingly, this coregulation is limited to adipose tissue as the expression of RELM in lung is independent of metabolic regulation. Additionally, we show that resistin and RELM levels are not subject to regulation by proinflammatory stimuli. Finally, acute hyperglycemia leads to up-regulation of resistin and RELM transcription in various adipose depots. (Molecular Endocrinology 16: , 2002) ADIPOSE TISSUE HAS long been considered a simple energy storage depot of the body. Recently, the importance of adipose tissue as an endocrine organ has been demonstrated (1). We set out to identify novel adipose tissue-specific secretory proteins utilizing a subtractive hybridization approach. This search led to the discovery of a novel 10-kDa, cysteine-rich protein that is adipose specific, secreted constitutively and up-regulated during adipocyte differentiation. A search of the expressed sequence tag (EST) database identified other tissue-specific homologs with the same unique pattern of cysteines: CX 12 CX 8 CXCX 3 CX 10 CXCXCX 9 CC. Collectively, these small, related proteins comprise a new gene family that is approximately 70% identical and approximately 10% cysteine. Holcomb et al. (2) first described the gene family and its tissue specific distribution. By comparison of bronchiolar lavages from control mice with lavages from mice subjected to experimentally induced asthma, they identified a protein by microsequencing that was upregulated in the asthmatic lung. This novel protein, which they named FIZZ1 (found in inflammatory zone 1) (also known as RELM ), was used to identify two additional homologs found in the EST database. One of these, Abbreviations: EST, Expressed sequence tag; FIZZ, found in inflammatory zone; LPS, lipopolysaccharide; ORF, open reading frame; RELM, resistin-like molecule; SAA3, serum amyloid A3. FIZZ2 (also known as RELM ), was found to localize to proliferating epithelia at the base of the crypts in the intestinal tract. Steppan et al. (3) later provided supporting data that FIZZ2/RELM is also present in rapidly dividing epithelia by demonstrating a marked increase in intestinal tumors as compared with control epithelia. They also added that RELM, originally described as lung specific, is also expressed in adipose tissue. The third homolog identified by a search of the EST databases, FIZZ3 [also known as resistin or adipocytespecific secretory factor (ADSF)], was identical with the fat specific homolog that we had cloned. Steppan et al. (4) have subsequently proposed that resistin is elevated in type II diabetes and suggested that it is a potential link between obesity and insulin resistance. They report that the injection of recombinant resistin into mice results in reduced glucose tolerance and insulin action, whereas neutralization with antiresistin antibodies improves insulin action. Here, we will refer to the fat-specific protein, known as FIZZ3/resistin/ adipocyte-specific secretory factor as resistin and the homologs FIZZ1/RELM and FIZZ2/RELM as RELM and RELM, respectively. Very little is known about the potential function of resistin or its homologs. The conserved cysteine spacing and small size (between 8 and 12 kda) of these proteins suggested that they comprise a new family of cytokines. Indeed, RELM is up-regulated in the lung during inflammation (2) and the expression pattern of RELM is similar 1920

2 Rajala et al. Resistin and RELM in Adipose Tissue Mol Endocrinol, August 2002, 16(8): to that of other proteins that are believed to help protect the intestine against the constant barrage of inflammatory stimuli (5 7). Chronically elevated levels of acute phase reactants and inflammatory cytokines have been shown to be associated with diabetes and metabolic syndrome X (8). Adipose tissue has been shown to be an important source of a number of proinflammatory cytokines and acute phase reactants when exposed to the appropriate stimuli. Specifically, adipocytes are capable of expressing and secreting TNF, while remaining highly responsive to increased levels of TNF and lipopolysaccharide (LPS) (9). Significant levels of some of these acute phase reactant proteins, such as 1-acid glycoprotein and the lipocalin 24p3 can be observed under basal conditions as well. We have recently provided evidence that adipose tissue may indeed be a significant source for at least some of the circulating inflammatory proteins that are elevated in type II diabetes (10). In light of the responsiveness of adipose tissue to proinflammatory stimuli, and the up-regulation of RELM present in lung in response to chemically induced inflammation, we wanted to test whether pro inflammatory activity and/or transcriptional control by inflammatory signals could be observed for resistin and its closely related homolog, RELM. Here, we show that resistin and RELM are not induced by inflammatory stimuli in adipose tissue either in vivo or in vitro. RELM expression cannot be detected in either preadipocytes or adipocytes but is limited to the surrounding stromal vascular cells found in adipose tissue. However, despite expression in different cell types, regulation of RELM in adipose tissue and resistin in adipocytes is coordinated. Surprisingly, RELM expression in the lung is not regulated in the same way as it is in adipose tissue. This suggests that resistin and RELM may respond to similar stimuli in adipose tissue or that the expression of RELM may be influenced by the locally high concentrations of resistin. Irregardless, the coordinate expression of RELM in adipose tissue and high sequence homology with resistin may suggest at least partially overlapping functions. We also address the potential role for resistin as a factor that links obesity with insulin resistance. We provide data that resistin mrna and protein levels are suppressed in ob/ob and db/db mice. Furthermore, in agreement with Haugen et al. (11), we show that insulin may actually suppress expression of resistin mrna and protein in vivo and in vitro. Finally, we illustrate that acute hyperglycemia under euinsulinemic conditions acutely induces resistin and RELM mrna. A subtractive adipocyte library enriched for adipocyte specific transcripts was generated and screened in duplicate using the complex-subtracted and reversesubtracted pools as probes. As a result of this screen, we identified a transcript that is induced during adipocyte differentiation and is exclusively expressed in adipose tissue (Fig. 1). The open reading frame (ORF) encoded a protein of 10 kda with a predicted signal sequence. Pulse-chase experiments of a transient transfection of the entire predicted ORF in HEK 293T cells resulted in the production of a 10-kDa protein that was secreted into the media (data not shown). These initial experiments revealed that under nonreducing conditions, resistin forms a disulfide-bonded dimer that runs at approximately 20 kda on SDS-PAGE, an observation that was also reported in Ref. 12. Timed pulse-chase experiments were used to follow the biogenesis of resistin in fully differentiated 3T3-L1 adipocytes (Fig. 2). Under nonreducing conditions, resistin appears as a diffuse band early during biogenesis reflecting heterogeneous disulfide bond formation. The time required for the proper rearrangement of intramolecular bonds suggests that the monomer is at least partially folded before dimer formation. The rather slow overall kinetics of secretion (t 1/2 of 5 h) could not be further stimulated by inclusion of 160 nm insulin in the chase mix (data not shown). Resistin Is Neither Transcriptionally Nor Translationally Up-Regulated by Proinflammatory Stimuli To investigate whether resistin was transcriptionally regulated by proinflammatory stimuli, d 8 3T3-L1 adipocytes were treated with LPS, TNF, or IL-6. Neither 4-h (not shown) nor overnight treatment of 3T3-L1 adipocytes with LPS, TNF, or IL-6 leads to an increase of resistin transcription, whereas both LPS and TNF caused a marked increase in expression of the acute phase reactant serum amyloid A3 (SAA3) as previously reported (Fig. 3A). Western blot analysis of media from treated adipocytes also showed an increase in SAA3 protein, but not resistin protein levels (Fig. 3B). Fasshauer et al. (13) reported previously that RESULTS Identification of an Adipose Tissue-Specific Secretory Protein Fig. 1. Tissue Northern Blot Analysis Total RNA from the indicated tissues were harvested from a male FVB mouse as indicated in Materials and Methods and analyzed by Northern blot analysis with a probe comprising the open reading frame of mouse resistin.

3 1922 Mol Endocrinol, August 2002, 16(8): Rajala et al. Resistin and RELM in Adipose Tissue Fig. 2. Pulse/Chase Analysis of Resistin Secretion in 3T3-L1 Adipocytes Day 8 3T3-L1 adipocytes were pulse-labeled for 10 min in the presence of 35 S-methionine and cysteine. The labeling medium was then exchanged with medium containing excess unlabeled methionine and cysteine and de novo protein synthesis was stopped by addition of cycloheximide. Tissue culture supernatants were harvested and cells were lysed at the indicated time points. Both supernatants (bottom panels) and cellular lysates (top panels) were immunoprecipitated with antiresistin antibodies. To monitor the intracellular maturation of nascent resistin, immunoprecipitates were analyzed by SDS-PAGE under either reducing (left panels) or nonreducing conditions (right panels). It is interesting that the secretion of resistin from 3T3-L1 adipocytes under basal conditions is very slow, with detectable levels in the supernatant appearing only after 2 3 h. TNF represses resistin levels in 3T3-L1 adipocytes. Although we also observe a slight reduction ( 20%), this repression is not quite as dramatic as the one reported by Fasshauer. Importantly though, in neither case is an increase of resistin levels observed. Finally, addition of rosiglitazone for 12 h had no affect on resistin expression, whereas it did reduce the induction of SAA3 in the presence of TNF and LPS. Because in vitro experiments do not always accurately mimic events as they occur in vivo, we wanted to examine the transcriptional levels of resistin and RELM from adipose tissue 24 h post injection of LPS. The results reveal a down-regulation of both transcripts in adipose tissue in LPS-treated mice as compared with control mice (Fig. 3C). As expected, no transcript can be detected for resistin in lung tissue. However, RELM can readily be detected but is essentially unaffected by the presence of LPS. Strong proinflammatory stimuli that are able to induce an inflammatory response in adipose tissue were not only unable to induce resistin in vitro, but suppressed expression of resistin and RELM in vivo (Fig. 3C). It is therefore unlikely that resistin or RELM function as typical inflammatory cytokines. Resistin and RELM Are Both Expressed in Adipose Tissue, but Originate in Different Cell Types We wanted to determine whether RELM expression seen in adipose tissue by Northern blot analysis originates from adipocytes or from some other cell type within the fat pad. To address this, adipocytes were dissociated from the stromal vascular population of cells by treating adipose tissue with collagenase, straining for a single cell population, and separating the intact stromal vascular fraction and lipid-laden adipocytes by centrifugation. Northern blot analysis of total RNA collected from both populations show that resistin expression is limited to adipocytes, whereas RELM is exclusively expressed in the stromal vascular fraction (Fig. 4A). Consistent with these observations, Northern blot analysis of d 0 and d 8 3T3-L1 adipocytes show that resistin is expressed only in the mature adipocyte, whereas RELM is neither expressed in 3T3-L1 preadipocytes nor in mature adipocytes (Fig. 4B). A Comparison of Resistin and RELM Regulation Although adipocytes are not a source of RELM, adipose tissue Northern blots reveal significant RELM transcript levels. The sequence similarity of RELM to resistin, combined with the fact that both of these proteins are expressed in adipose tissue leads to the possibility that the two proteins may have partially redundant functions. To address this issue, we wanted to determine whether the regulation of RELM is similar to that of resistin despite different cellular distribution of expression. We used RELM to probe the same adipose tissue Northerns already studied for resistin. Interestingly, RELM mrna levels in the adipose tissue of LPS-treated mice displayed a similar reduction as resistin (Fig. 3C). This suggests that there are similar regulatory mechanisms in place for both proteins, at least under the experimental conditions studied so far. Resistin and RELM Regulation in Diabetic Mouse Models To determine whether resistin and RELM transcription were altered in diabetic mouse models, we examined the ob/ob and db/db murine models of type II diabetes.

4 Rajala et al. Resistin and RELM in Adipose Tissue Mol Endocrinol, August 2002, 16(8): Fig. 3. Resistin Is Not Induced by Proinflammatory Stimuli A, Northern blot analysis of d 8 3T3-L1 adipocytes treated with TNF, IL-6, or LPS for 16 h. Total RNA was run and blots were probed for either resistin, SAA3, or control. SAA3 has been shown previously to be markedly up-regulated in response to LPS or TNF. Resistin transcript levels are not altered by the strong proinflammatory stimuli LPS or TNF. B, Western blot analysis of growth media after d 8 3T3-L1 adipocytes were exposed to proinflammatory stimuli. Again, SAA3 levels were induced with no significant change in resistin levels. However, in the presence of the PPAR agonist (10 M), SAA3 induction is impaired, but no change is seen for resistin secretion. C, In vivo analysis of resistin, RELM and -actin levels in adipose tissue 24 h post injection of LPS into mice. Northern blot was probed for the expression levels of resistin, RELM and -actin in adipose and lung tissue. In vivo, there is a significant reduction for both resistin mrna and RELM in adipose tissue (n 3 mice for each treatment), but not in lung. Unexpectedly, a down-regulation in both RELM and resistin mrna is seen in ob/ob adipose tissue as compared with wild-type control (Fig. 5A). Way et al. (14) recently reported similar down-regulated resistin mrna levels in several monogenic obesity mouse models (ob/ ob, db/db, tub/tub and FFA y ). Analysis of serum samples from db/db and db/ littermates revealed that circulating levels of resistin protein are reduced, in line with the reduced tissue mrna levels (Fig. 5B). Thus, it seems unlikely that the decreased insulin sensitivity observed in ob/ob and db/db mice can be accounted for by altered resistin levels. However, this does not rule out the possibility that increased resistin levels may be observed in other models of obesity or insulin resistance. Resistin and RELM Regulation under Normal and Early Onset Diabetic Conditions We next examined a cohort of wild-type male mice (C57Bl/6J CBA) that were approximately wk old to determine whether variations in resistin or RELM transcript levels were apparent. Approximately half of the animals were kept on a high fat breeding chow that led to elevated insulin and in some cases elevated glucose levels. The other half of the animals were fed a standard chow diet and sustained vastly normal insulin and glucose levels. Serum samples for measurement of glucose and insulin levels were collected immediately before the mice were killed. Bilateral epididymal fat pads were excised and protein isolated from one pad and mrna from the other. On the low fat diet, there was a very close relationship between resistin protein and resistin mrna within the fat pad (Fig. 6A). On the high fat diet, mrna and protein remain proportional but are less linear, indicating that additional metabolic parameters influence message or intracellular protein stability (Fig. 6B). Furthermore, mrna for resistin and RELM were compared under the two conditions. The transcripts for these two genes are quite closely coregulated under normal con-

5 1924 Mol Endocrinol, August 2002, 16(8): Rajala et al. Resistin and RELM in Adipose Tissue Fig. 4. Resistin, But Not RELM, Is Expressed in Adipocytes A, Northern blot analysis of adipocytes and stromal vascular cells within the same sample of adipose tissue separated by a flotation assay. Expression of resistin in adipose tissue is limited to mature adipocytes, whereas RELM is exclusively expressed in the stromal vascular cell fraction. B, Northern blot analysis of pre- and fully differentiated adipocytes for the presence of resistin and RELM shows that resistin is expressed only in adipocytes, whereas RELM is expressed in neither preadipocytes nor adipocytes. Fig. 5. Resistin Levels Are Reduced in ob/ob and db/db Mice A, Northern blot analysis of adipose tissue isolated from wt and ob/ob mice (male, age-matched with otherwise identical genetic background). The Northern blot was probed with resistin, RELM, and -actin (top to bottom). Resistin and RELM expression is reduced approximately 70% in the ob/ob mice compared with controls. B, Western blot analysis of serum reveals reduced resistin levels in db/db as compared with heterozygous littermates. ditions but tend to be more widely scattered under high fat diet conditions (Fig. 6, C and D). Although the ratio between resistin mrna and protein remained relatively constant, it is noteworthy that mice with higher insulin levels had lower levels of both resistin protein and mrna (Fig. 6E) in their epididymal fat pads. Resistin and RELM Regulation during Fasting Is Tissue Specific Kim et al. (15) reported a nutritional regulation of resistin in rats. Significantly reduced resistin mrna levels are seen in rats fasted for a period as short as 12 h with further reduction up to 48 h. However, the transcript levels rebound rapidly to basal, or above-basal levels within 12 h of refeeding. To expand our observations on the coregulation of resistin and RELM, we repeated a similar experiment in mice. Consistent with the previous observation in rats by Kim et al. (15), resistin mrna levels in mice drop within 12 h of food deprivation and continue to drop rapidly with further food restriction (Fig. 7). This reduction is most pronounced in perirenal fat, whereas sc and epididymal (not shown) depots are slower to react. Again, RELM levels are regulated similarly as resistin within the stromal vascular cells of adipose tissue. In addition, because RELM is also expressed in lung tissue at levels similar to those observed in adipose tissue, we wanted to determine whether RELM levels from the lungs of the fasted mice are regulated in a similar manner. Interestingly, there is no change in mrna levels in lung by food deprivation, indicating metabolic regulation of RELM specific to a metabolically responsive tissue. Acute Transcriptional Regulation of Resistin and RELM by Hyperglycemia Considering the changes in resistin and RELM expression under chronically elevated glucose and insulin levels, we decided to test whether resistin and RELM levels were acutely regulated by elevated glucose under basal levels of insulin. Hyperglycemia was induced in rats by clamping glucose independent of compensating increases in insulin. Specifically, insulin levels were kept near basal levels and glucose infused to sustain hyperglycemia (hyperglycemic euinsulinemic clamp) (Fig. 8). Adipose tissue from rats excised rapidly after the hyperglycemic clamp showed a significant increase in resistin mrna within the relatively short time-frame of the experiment (3 h) (Fig. 8). These

6 Rajala et al. Resistin and RELM in Adipose Tissue Mol Endocrinol, August 2002, 16(8): Fig. 6. Expression of Resistin and RELM in a Cohort of Wild-Type Mice A cohort of 20 F1 offspring (18 wk old) of a cross between C57Bl/6J CBA were fed a standard chow diet or a high fat diet and analyzed for their levels of resistin and RELM by Northern blot analysis, and for resistin levels in adipose tissue by Western blot analysis. -Actin was used for normalization of both Western and Northern blot analysis. All samples were measured twice independently. Resistin mrna levels were plotted against RELM mrna levels (A and B), against resistin protein levels (C and D), and against insulin levels (E). The values on x- and y-axes represent arbitrary units, except insulin (ng/ml). in vivo results suggest that elevated glucose levels, independent of insulin, are capable of mediating the acute induction of resistin in adipose tissue. Once again, resistin mrna and RELM transcripts are regulated in a highly similar fashion. DISCUSSION We have used a subtractive library screen to identify a novel adipose specific transcript (resistin) that is upregulated during adipogenesis. Database searches identified two homologous proteins, RELM and RELM, with unknown function. Northern blot analysis revealed that these homologs were also expressed in a tissue-specific manner, either in intestine (RELM )or in lung and adipose (RELM ). Resistin, RELM, and RELM comprise a new gene family of small secreted proteins that are approximately 70% identical and share a fully conserved unique cysteine distribution: CX 11 CX 8 CX 10 CXCXCX 9 CC. Sequence identity between the three homologs is approximately 70% within a species, whereas the sequence identity observed for any of the three with their orthologs is also

7 1926 Mol Endocrinol, August 2002, 16(8): Rajala et al. Resistin and RELM in Adipose Tissue Fig. 7. Resistin and RELM mrna Levels Are Reduced during Food Restriction Three different fat pads and lung tissue were excised and total RNA isolated from mice that were either fed (F), fasted 12 h (12), 24 h (24), 36 h (36), and then refed for 12 h (RF). Blots were probed for resistin, RELM, and -actin. Resistin and RELM mrna levels in adipose tissue are suppressed in food-restricted mice. The decrease is present as early as 12 h and continues to decline with longer food restriction, but rapidly and robustly rebounds to normal or above normal within 12 h of refeeding. Although the levels of RELM in adipose tissue decrease with food restriction, RELM levels remain unchanged in the lungs of the same mice. Consistent with previous Northern blots, resistin is not expressed in the lung. Fig. 8. Hyperglycemia Induces Resistin and RELM mrna Levels Hyperglycemic/euinsulinemic clamp studies were performed as indicated in Materials and Methods with glucose levels that were kept around 300 mg/dl, whereas the insulin levels were kept at basal levels. At the end of the 3 h study, total RNA from adipose depots was isolated and analyzed by Northern blot analysis. Acute hyperglycemia induces resistin and RELM mrna compared with saline infused animals. Note that rats have three splice variants of resistin. only 70%. This fact has made the assignment of orthologs based only on sequence similarity difficult. To date there are two human (resistin and RELM ) and two rat (resistin and RELM ) sequences identified. Unlike secretion of RELM into the lung and RELM into the intestine, secretion into a lumen in adipose tissue is not possible. This allows for the exciting possibility that adipose tissue secretory proteins are released into serum where they could potentially exert profound effects on systemic carbohydrate and lipid metabolism. Steppan et al. (4) reported that resistin is elevated in type II diabetes and proposed that it is potentially the link between obesity and insulin resistance. Injection of recombinant resistin into mice resulted in reduced glucose tolerance and insulin action, whereas neutralization with antiresistin antibodies improved insulin action. Additionally, resistin levels were elevated in the serum of ob/ob mice and down-regulated by PPAR agonists. These observations led to the working hypothesis that resistin may be a link between obesity and insulin resistance. How could resistin confer insulin resistance in target cells? There is increasing evidence that cor-

8 Rajala et al. Resistin and RELM in Adipose Tissue Mol Endocrinol, August 2002, 16(8): relates the diabetic phenotype with chronically elevated systemic levels of acute phase reactants and inflammatory cytokines. It has been suggested that these elevated levels may contribute to, or partially cause, some of the pathologies underlying diabetes. Pickup and colleagues (8, 16, 17) have described a correlation between increased levels of several acute-phase reactants and IL-6 with the metabolic syndrome X. We have recently demonstrated that adipose tissue expresses increased levels of serum amyloid A3 in the diabetic state and may be responsible for the elevated levels of this protein observed in diabetes (10). Furthermore, Kim et al. (18) have recently reported that mice lacking IKK, a critical upstream activator of NF B and mediator of inflammation, are resistant to diet-induced obesity. Treatment of animals with high levels of the antiinflammatory agent salicylate reverses hyperglycemia, hyperinsulinemia and dyslipidemia in obese rodents. Perreault and Marette (19) have shown that lack of inducible nitric oxide synthase, an enzyme that is stimulated by many proinflammatory cytokines, confers improved glucose tolerance and insulinsensitivity in a diet induced model of diabetes. Combined, this suggests a tight link between systemic inflammation and insulin resistance. Activation of proinflammatory signaling cascades can be linked to the desensitization of the insulin-signal transduction cascade. We hypothesized that resistin functioned as an inflammatory cytokine that could lead to insulin resistance through an inflammatory mechanism. Circumstantial evidence that at least RELM could function as an inflammatory cytokine was provided by Holcomb et al. (2), who have shown that RELM is massively induced in the lungs of mice treated with a chemical irritant to induce pulmonary inflammation. We have shown here that strong inflammatory stimuli used to induce an inflammatory response in adipocytes both in vivo and in vitro were unable to increase resistin mrna levels in vitro and actually downregulated both resistin and RELM mrna in vivo. Our data suggest that resistin and RELM are most likely not involved in the inflammatory response in adipose tissue. We found it interesting that RELM was downregulated in adipose tissue of LPS-treated mice when the pulmonary inflammation induced by the chemical irritant was able to induce levels of RELM in the lung. It is possible that paracrine signals or the intracellular signal transduction cascades in the cells responsible for RELM production may explain this differential response. In support of this, we have shown that RELM expression in adipose tissue responds to food deprivation, whereas expression levels are unaltered in the lung tissue of the same mice. This raises the question as to what cell type within adipose tissue is responsible for tissue-specific regulation. Although RELM is found in adipose tissue, its expression is localized to the stromal vascular population of cells and not the adipocytes. Due to the lack of expression of RELM in 3T3-L1 preadipocytes, it is unlikely that the expression of RELM in adipose tissue could be accounted for by preadipocytes. The exact cell type that expresses RELM in the stromal vascular population is a subject of ongoing studies. After the initial report of resistin, several groups have recently questioned its role as a potential mediator of insulin resistance (14, 20 22). Way et al. (14) have presented Northern blot analysis demonstrating reduced resistin mrna levels in monogenic mouse models of obesity including ob/ob, db/db, tub/tub, and FFA y when compared with their respective wildtype littermates. In agreement, our findings show a reduction in resistin mrna in ob/ob mice and a reduction in circulating resistin in db/db mice. In addition, in a population of wild-type mice on a high fat diet, we show that reduced resistin mrna and protein levels are associated with hyperinsulinemia. Consistent with this observation, Haugen et al. (11) have shown that resistin transcription can be repressed by physiological levels of insulin in vitro. Although elevated levels of resistin do not appear to be the causative factor for insulin resistance in the monogenic mouse models, it is possible that resistin plays an important role in insulin sensitivity under normal conditions. Resistin transcription is up-regulated in response to acute hyperglycemia. However, resistin levels are decreased in mice in the hyperinsulinemic state. The acute effects of resistin on insulin sensitivity and target organs are currently being addressed in clamp studies with pharmacological amounts of highly purified resistin. Ongoing studies in our laboratory focusing on the hormonal regulation of another fat cell-specific protein, Acrp30 (23), reveal a very complex regulatory pattern. It is very likely that resistin expression is governed by a number of additional factors beyond glucose and insulin levels. Although acute changes in resistin transcription can be demonstrated in response to hyperglycemia, secretion of mature resistin protein from 3T3-L1 adipocytes requires several hours. This suggests that there is no systemic need for an acute increase in resistin levels. However, we cannot exclude the possibility that an appropriate stimulus may enable a more rapid processing and release, or that the synthesis of resistin in cell culture does not translate directly into the normal physiological time course in vivo. We are currently working on the development of more sensitive assays that will allow us to accurately monitor serum resistin levels in response to various metabolic conditions. It will also be of interest to determine whether RELM is present in serum as well and to what extent its concentration compares with that of resistin. Only two genes have been reported so far for the resistin gene family in humans instead of the three

9 1928 Mol Endocrinol, August 2002, 16(8): Rajala et al. Resistin and RELM in Adipose Tissue genes reported for mouse. Even though it is premature to formally exclude the possibility of the existence of a third gene in the human genome, some authors question the relevance of this gene family in the context of the pathophysiology of diabetes in humans (21). The data presented in this paper demonstrate a very close coupling of resistin and RELM expression profiles in murine adipose tissue. With the similarity in expression and sequence homology, these two proteins could have partially overlapping functions. Such a possibility may explain why resistin currently remains the only identified homolog expressed in human adipose tissue. An appropriate bioassay with recombinant RELM and resistin will be needed to address this issue. Finally, the resistin gene family constitutes a completely novel protein family. Very little information can be gathered based on homology or analogy. Further insights will result from the identification of the resistin receptor and from structural studies on the ligands that may reveal clues on the mechanism of action that are not apparent based on the primary amino acid sequence. MATERIALS AND METHODS Materials DMEM was purchased from Cellgro, Inc. (Herndon, VA). Murine TNF and IL-6 was purchased from PharMingen (San Diego, CA). LPS (Escherichia coli) was purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA). Cell Culture 3T3-L1 murine fibroblasts were propagated and differentiated as previously described (24). Subtractive Library Screen Using the PCR-Select cdna Subtraction kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer s protocol, a subtractive library was created in which 3T3-L1 preadipocyte transcripts (d 0) were subtracted from mature 3T3-L1 adipocyte messages. The library was screened in duplicate using the complex-subtracted and reverse-subtracted pools as probes. Animals Male FVB or C57Bl/6J mice were bred in house and used as indicated. Also used were twenty approximately 18-wkold male mice that were the F1 offspring of a cross between C57Bl/6J CBA that were either fed a breeding chow (Picolab mouse diet 20/5058) or a standard lab chow and killed mid-day. Male Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were housed in individual cages and subjected to a standard light ( h)/dark ( ) cycle. All rats were 3 months of age ( 300 g, n 12) and were fed ad libitum using regular rat chow that consisted of 64% carbohydrate, 30% protein, and 6% fat with a physiological fuel value of 3.3 kcal/g chow. One week preceding the in vivo studies, rats were anesthetized with methoxyflurane, which allows fast recovery and normal food consumption after 1 d. A venous indwelling catheter was placed into the right jugular vein and extended to the level of the right atrium and an indwelling arterial catheter was inserted into the left carotid artery and advanced to the level of the aortic arch. The rats were allowed to recover until body weight was within 3% of the preoperative weight ( 4 6 d). These stably catheterized rats were studied after approximately 24 h of fasting while awake and unstressed. LPS Injection into Mice Six-month-old FVB mice were injected with 100 ng/g body weight LPS (n 2 for each condition). Twenty-four hours post injection, adipose tissue was isolated (10) and analyzed by Northern blot analysis. Hyperglycemic Clamp Studies Both somatostatin (1.5 g/kg min) and a 25% glucose solution (in PBS) were infused into the arterial catheter of lean and obese rats to prevent endogenous insulin secretion and raise the plasma glucose concentration acutely to approximately 18 mm. Plasma glucose concentration was maintained at that level throughout the study (3 h) using a variable infusion of glucose, periodically adjusted to maintain plasma glucose levels. All rats also received a primedcontinuous (15 40 Ci/min) infusion of HPLC-purified [3-3 H]glucose (NEN Life Science Products, Boston, MA) throughout the study to determine glucose uptake. Similarly, a bolus of [U- 14 C]2-deoxyglucose (20 Ci) was administered 30 min before the end of the studies. Plasma samples for determination of plasma [U- 14 C]2-deoxyglucose-specific activity were obtained at 5-min intervals during the remainder of the clamp studies. Blots were probed with either the entire ORF of rat resistin or a fragment of RELM that were generated by PCR of first strand cdna from rat white adipose tissue. At the end of the study, rats were killed by iv injection of 60 mg pentobarbital-sodium/kg. The abdomen was quickly opened and adipose tissue samples were freeze-clamped in situ with aluminum tongs precooled in liquid nitrogen. Total RNA was isolated and used for Northern blot analysis. All study protocols were reviewed and approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine. Flotation Assay Isolation were performed essentially as described in (25, 26). Fat pads were excised, minced, and collagenase treated (1 ml/mg tissue of 0.1% collagenase in 100 mm HEPES, 120 mm NaCl, 50 mm KCl, 5 mm glucose, 1 mm CaCl 2, 1.5% BSA) for 45 min at 37 C. Cells were passed through a 100 m cell strainer and centrifuged at 100 g for 10 min at 4 C. Floating adipocytes were separated from pelleted nonadipocytes and centrifuged twice in fresh buffer without collagenase. RNA was isolated from both the combined pellets and floating adipocyte fractions. Total RNA Isolation and Northern blot analysis Total RNA from tissue and cultured cells was isolated with Trizol (Life Technologies, Inc., Gaithersburg, MD) according to manufacturer s directions. Twenty micrograms of total RNA were used for Northern blot analysis as previously described (10). Blots were prehybridized in Ultrahyb (Ambion, Inc.) for 2hat42C.Denatured [ 32 P]-labeled DNA probes

10 Rajala et al. Resistin and RELM in Adipose Tissue Mol Endocrinol, August 2002, 16(8): were added ( cpm/ml) and blots were hybridized overnight at 42 C. The filters were washed in 2 saline sodium citrate/0.1% sodium dodecyl sulfate ( 2, 10 ) and 0.1 saline sodium citrate/0.1% sodium dodecyl sulfate ( 2, 5 ) at 42 C before autoradiography. Antibodies Polyclonal antibodies to mouse resistin were raised in rabbits (Covance, Inc., Denver, PA) and guinea pigs (Linco Research, Inc., St Louis, MO; catalog no. 4096). The monoclonal antibody against -actin (AC40; A4700) was purchased from Sigma. Immunoblotting Separation of proteins by SDS-PAGE, fluorography, and immunoblotting were performed as described previously (27). Primary and secondary antibodies were diluted in Tris-buffered saline with 0.1% Tween-20 and 1% BSA. Horseradish peroxidase-conjugated secondary antibodies were detected with enhanced chemiluminescence according to the manufacturer s instructions (NEN Life Science Products). Acknowledgments We thank Puneeth Iyengar, Utpal Pajvani, and Anders Berg for comments on the manuscript and Dr. Terry Combs for his assistance with the in vivo experiments. We would also like to thank Dr. Maureen Charron for valuable assistance with the diabetic mouse models. Received January 30, Accepted May 7, Address all correspondence and requests for reprints to: Dr. Phillip Scherer, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York scherer@aecom.yu.edu. This work was supported by a grant from Pfizer, Inc. (to P.E.S.), by NIH Medical Scientist Training Grant T32- GM07288 (to M.R.), by a fellowship from the Juvenile Diabetes Foundation ( ; to Y.L.), and by NIH Grants 1R01-DK55758 (to P.E.S.) and 1R01-AG18381 (to N.B.). REFERENCES 1. 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