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in Adipose Tissue
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 10461
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 10461. E-mail: scherer{at}aecom.yu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 up-regulated 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, FIZZ2 (also known as RELMß), 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/RELMß 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 adipocyte-specific 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/RELMß as RELM and RELMß, 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 RELMß is similar to that of other proteins that are believed to help protect the intestine against the constant barrage of inflammatory stimuli (5, 6, 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 proinflammatory 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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 .
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). 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 (t1/2 of >5 h) could not be further stimulated by inclusion of 160 nM insulin in the chase mix (data not shown).
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, 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 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.
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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).
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Regulation, 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. 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 FFAy). 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.
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Regulation under Normal and Early Onset Diabetic Conditions 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 conditions 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.
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Regulation during Fasting Is Tissue Specific, 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.
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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 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and RELMß, with unknown function. Northern blot analysis revealed that these homologs were also expressed in a tissue-specific manner, either in intestine (RELMß) or in lung and adipose (RELM).
Resistin, RELM, and RELMß comprise a new gene family of small secreted proteins that are approximately 70% identical and share a fully conserved unique cysteine distribution: CX11CX8CX10CXCXCX9CC. 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 only 70%. This fact has made the assignment of orthologs based only on sequence similarity difficult. To date there are two human (resistin and RELMß) and two rat (resistin and RELM) sequences identified.
Unlike secretion of RELM into the lung and RELMß 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 correlates 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 IKKß, 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 insulin-sensitivity 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 down-regulated 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 down-regulated 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, 21, 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 FFAy when compared with their respective wild-type 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 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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-wk-old male mice that were the F1 offspring of a cross between C57Bl/6J x 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 (0600–1800 h)/dark (1800–0600) 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 µg/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 primed-continuous (15–40 µCi/min) infusion of HPLC-purified [3-3H]glucose (NEN Life Science Products, Boston, MA) throughout the study to determine glucose uptake. Similarly, a bolus of [U-14C]2-deoxyglucose (20 µCi) was administered 30 min before the end of the studies. Plasma samples for determination of plasma [U-14C]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 CaCl2, 1.5% BSA) for 45 min at 37 C. Cells were passed through a 100 µm cell strainer and centrifuged at 100 x 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 2 h at 42 C. Denatured [32P]-labeled DNA probes were added (2 x 106 cpm/ml) and blots were hybridized overnight at 42 C. The filters were washed in 2x saline sodium citrate/0.1% sodium dodecyl sulfate (x2, 10') and 0.1x saline sodium citrate/0.1% sodium dodecyl sulfate (x2, 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 ß-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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: EST, Expressed sequence tag; FIZZ, found in inflammatory zone; LPS, lipopolysaccharide; ORF, open reading frame; RELM, resistin-like molecule; SAA3, serum amyloid A3.
Received for publication January 30, 2002. Accepted for publication May 7, 2002.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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- and ß-defensins mRNA in gastrointestinal epithelia. Eur J Clin Invest 30:695–701[CrossRef][Medline]
is a negative regulator of resistin gene expression and secretion in 3T3-L1 adipocytes. Biochem Biophys Res Commun 288:1027–1031[CrossRef][Medline]
agonists. J Biol Chem 276:25651–25653 action in humans. Diabetes 50:2199–202: direct role in obesity-linked insulin resistance. Science 259:87–91This article has been cited by other articles:
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|
 |
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  E. Szalowska, M. G. L. Elferink, A. Hoek, G. M. M. Groothuis, and R. J. Vonk Resistin Is More Abundant in Liver Than Adipose Tissue and Is Not Up-Regulated by Lipopolysaccharide J. Clin. Endocrinol. Metab., August 1, 2009; 94(8): 3051 - 3057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Jorgensen, J. Honeyman, J. S. Oakhill, D. Fazakerley, J. Stockli, B. E. Kemp, and G. R. Steinberg Oligomeric resistin impairs insulin and AICAR-stimulated glucose uptake in mouse skeletal muscle by inhibiting GLUT4 translocation Am J Physiol Endocrinol Metab, July 1, 2009; 297(1): E57 - E66. [Abstract] [Full Text] [PDF]
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|
|
|
 |
|
 T. Tomaru, D. J. Steger, M. I. Lefterova, M. Schupp, and M. A. Lazar Adipocyte-specific Expression of Murine Resistin Is Mediated by Synergism between Peroxisome Proliferator-activated Receptor {gamma} and CCAAT/Enhancer-binding Proteins J. Biol. Chem., March 6, 2009; 284(10): 6116 - 6125. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. I. Beier, L. Guo, C. von Montfort, J. P. Kaiser, S. Joshi-Barve, and G. E. Arteel New Role of Resistin in Lipopolysaccharide-Induced Liver Damage in Mice J. Pharmacol. Exp. Ther., June 1, 2008; 325(3): 801 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Wendel, A. Purushotham, L.-F. Liu, and M. A. Belury Conjugated linoleic acid fails to worsen insulin resistance but induces hepatic steatosis in the presence of leptin in ob/ob mice J. Lipid Res., January 1, 2008; 49(1): 98 - 106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. V. Wang, T. D. Schraw, J.-Y. Kim, T. Khan, M. W. Rajala, A. Follenzi, and P. E. Scherer Secretion of the Adipocyte-Specific Secretory Protein Adiponectin Critically Depends on Thiol-Mediated Protein Retention Mol. Cell. Biol., May 15, 2007; 27(10): 3716 - 3731. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 N. Pischon, N. Heng, J.-P. Bernimoulin, B.-M. Kleber, S.N. Willich, and T. Pischon Obesity, Inflammation, and Periodontal Disease Journal of Dental Research, May 1, 2007; 86(5): 400 - 409. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 M. E. Trujillo and P. E. Scherer Adipose Tissue-Derived Factors: Impact on Health and Disease Endocr. Rev., December 1, 2006; 27(7): 762 - 778. [Abstract] [Full Text] [PDF]
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|
|
M. J. Bahr, J. Ockenga, K. H. W. Boker, M. P. Manns, and U. J. F. Tietge Elevated resistin levels in cirrhosis are associated with the proinflammatory state and altered hepatic glucose metabolism but not with insulin resistance Am J Physiol Endocrinol Metab, August 1, 2006; 291(2): E199 - E206. [Abstract] [Full Text] [PDF]
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|
 |
|
 P. E. Scherer Adipose Tissue: From Lipid Storage Compartment to Endocrine Organ Diabetes, June 1, 2006; 55(6): 1537 - 1545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. E Krassas, N. Pontikides, K. Loustis, G. Koliakos, T. Constantinidis, and D. Panidis Resistin levels in hyperthyroid patients before and after restoration of thyroid function: relationship with body weight and body composition Eur. J. Endocrinol., August 1, 2005; 153(2): 217 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bokarewa, I. Nagaev, L. Dahlberg, U. Smith, and A. Tarkowski Resistin, an Adipokine with Potent Proinflammatory Properties J. Immunol., May 1, 2005; 174(9): 5789 - 5795. [Abstract] [Full Text] [PDF]
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J. H. Lee, J. W. Bullen Jr., V. L. Stoyneva, and C. S. Mantzoros Circulating resistin in lean, obese, and insulin-resistant mouse models: lack of association with insulinemia and glycemia Am J Physiol Endocrinol Metab, March 1, 2005; 288(3): E625 - E632. [Abstract] [Full Text] [PDF]
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Y. Lin, A. H. Berg, P. Iyengar, T. K. T. Lam, A. Giacca, T. P. Combs, M. W. Rajala, X. Du, B. Rollman, W. Li, et al. The Hyperglycemia-induced Inflammatory Response in Adipocytes: THE ROLE OF REACTIVE OXYGEN SPECIES J. Biol. Chem., February 11, 2005; 280(6): 4617 - 4626. [Abstract] [Full Text] [PDF]
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 K. F. Wagner, A.-K. Hellberg, S. Balenger, R. Depping, J. Dodd-O, R. A. Johns, and D. Li Hypoxia-Induced Mitogenic Factor Has Antiapoptotic Action and Is Upregulated in the Developing Lung: Coexpression with Hypoxia-Inducible Factor-2{alpha} Am. J. Respir. Cell Mol. Biol., September 1, 2004; 31(3): 276 - 282. [Abstract] [Full Text] [PDF]
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T. Liu, H. Jin, M. Ullenbruch, B. Hu, N. Hashimoto, B. Moore, A. McKenzie, N. W. Lukacs, and S. H. Phan Regulation of Found in Inflammatory Zone 1 Expression in Bleomycin-Induced Lung Fibrosis: Role of IL-4/IL-13 and Mediation via STAT-6 J. Immunol., September 1, 2004; 173(5): 3425 - 3431. [Abstract] [Full Text] [PDF]
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 U. Meier and A. M. Gressner Endocrine Regulation of Energy Metabolism: Review of Pathobiochemical and Clinical Chemical Aspects of Leptin, Ghrelin, Adiponectin, and Resistin Clin. Chem., September 1, 2004; 50(9): 1511 - 1525. [Abstract] [Full Text] [PDF]
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 S. D. Patel, M. W. Rajala, L. Rossetti, P. E. Scherer, and L. Shapiro Disulfide-Dependent Multimeric Assembly of Resistin Family Hormones Science, May 21, 2004; 304(5674): 1154 - 1158. [Abstract] [Full Text] [PDF]
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 C. Asensio, P. Cettour-Rose, C. Theander-Carrillo, F. Rohner-Jeanrenaud, and P. Muzzin Changes in Glycemia by Leptin Administration or High- Fat Feeding in Rodent Models of Obesity/Type 2 Diabetes Suggest a Link between Resistin Expression and Control of Glucose Homeostasis Endocrinology, May 1, 2004; 145(5): 2206 - 2213. [Abstract] [Full Text] [PDF]
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F. Felipe, M. L. Bonet, J. Ribot, and A. Palou Modulation of Resistin Expression by Retinoic Acid and Vitamin A Status Diabetes, April 1, 2004; 53(4): 882 - 889. [Abstract] [Full Text] [PDF]
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T. P. Combs, U. B. Pajvani, A. H. Berg, Y. Lin, L. A. Jelicks, M. Laplante, A. R. Nawrocki, M. W. Rajala, Albert. F. Parlow, L. Cheeseboro, et al. A Transgenic Mouse with a Deletion in the Collagenous Domain of Adiponectin Displays Elevated Circulating Adiponectin and Improved Insulin Sensitivity Endocrinology, January 1, 2004; 145(1): 367 - 383. [Abstract] [Full Text] [PDF]
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P. G. McTernan, F. M. Fisher, G. Valsamakis, R. Chetty, A. Harte, C. L. McTernan, P. M. S. Clark, S. A. Smith, A. H. Barnett, and S. Kumar Resistin and Type 2 Diabetes: Regulation of Resistin Expression by Insulin and Rosiglitazone and the Effects of Recombinant Resistin on Lipid and Glucose Metabolism in Human Differentiated Adipocytes J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6098 - 6106. [Abstract] [Full Text] [PDF]
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J. H. Lee, J. L. Chan, N. Yiannakouris, M. Kontogianni, E. Estrada, R. Seip, C. Orlova, and C. S. Mantzoros Circulating Resistin Levels Are Not Associated with Obesity or Insulin Resistance in Humans and Are Not Regulated by Fasting or Leptin Administration: Cross-Sectional and Interventional Studies in Normal, Insulin-Resistant, and Diabetic Subjects J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4848 - 4856. [Abstract] [Full Text] [PDF]
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S. R. Smith, F. Bai, C. Charbonneau, L. Janderova, and G. Argyropoulos A Promoter Genotype and Oxidative Stress Potentially Link Resistin to Human Insulin Resistance Diabetes, July 1, 2003; 52(7): 1611 - 1618. [Abstract] [Full Text] [PDF]
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 X. Teng, D. Li, H. C. Champion, and R. A. Johns FIZZ1/RELM{alpha}, a Novel Hypoxia-Induced Mitogenic Factor in Lung With Vasoconstrictive and Angiogenic Properties Circ. Res., May 30, 2003; 92(10): 1065 - 1067. [Abstract] [Full Text] [PDF]
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