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Endocrinology Vol. 143, No. 4 1502-1511
Copyright © 2002 by The Endocrine Society

INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS

Exclusive Action of Transmembrane TNF{alpha} in Adipose Tissue Leads to Reduced Adipose Mass and Local But Not Systemic Insulin Resistance

Haiyan Xu, Jiro Hirosumi, K. Teoman Uysal, A. Deniz Guler and Gökhan S. Hotamisligil

Harvard School of Public Health, Division of Biological Sciences and Department of Nutrition, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Gökhan S. Hotamisligil, M.D., Ph.D., Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115. E-mail: . ghotamis{at}hsph.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aberrant TNF{alpha} expression in adipocytes is a molecular mechanism by which insulin action is modulated in adipose tissue. While this might be a compensatory response to limit adipose expansion, neither the mechanisms underlying this local effect nor its systemic biological consequences have been studied. It is also not clear whether TNF{alpha}-induced insulin resistance in adipocyte alone is responsible for systemic insulin resistance in the absence of obesity. In a transgenic mouse model deficient in endogenous TNF{alpha}, we demonstrate that specific expression of the transmembrane TNF{alpha} (mTNF{alpha}) in adipocytes leads to decreased whole body adipose mass, and local, but not systemic insulin resistance. These data demonstrate that exclusive action of TNF{alpha} in adipose tissue strongly inhibits insulin action at this site and leads to reduced adiposity in mice. However, this isolated adipocyte insulin resistance in the context of reduced fat mass and/or the absence of obesity is insufficient to alter systemic glucose homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAJOR ROLE of white adipose tissue (WAT) during evolution is to store excessive energy in the form of triglyceride (TG) under conditions of positive energy balance and release them via lipolysis during periods of energy shortage or fasting (1) Fat mass is highly variable in mammals, and this almost unprecedented short- and long-term plasticity is determined by both genetic and environmental factors. While the short-term changes in adiposity are critical in metabolic control, long-term disruption of the delicate balance controlling fat deposition results in either insufficient (such as lipodystrophies) or excessive fat mass (such as obesity). Change in adipose mass has a critical impact on systemic insulin action, which is a critical regulator of glucose and lipid metabolism in mammals (2, 3, 4, 5). In both animal models and in humans, it has been firmly established that excess adiposity is associated with insulin resistance and is one of the major risk factors for type 2 diabetes (6). Consequently, weight loss is among the most efficient therapeutic remedies to alleviate insulin resistance (7, 8). Interestingly, excessive loss of adiposity also leads to severe impairments of systemic glucose metabolism and insulin sensitivity (9, 10).

It has been proposed that adipocyte insulin resistance might act as a limiting factor in adipogenesis owing to insulin’s strong positive effects on lipogenesis and adipocyte differentiation (11, 12, 13). This latter hypothesis is supported by the inverse relation between the severity of insulin resistance and the extent of obesity in humans (14). In addition, the use of insulin-sensitizing agents for treatment of type 2 diabetes is associated with weight gain, irrespective of the mechanism of action of these drugs. According to this model, isolated adipocyte insulin resistance might even be beneficial by limiting excess obesity. However, most of these questions remain unanswered because the molecular mechanism(s) by which insulin action is locally modified in adipocytes are not known. Furthermore, the impact of adipocytes on systemic insulin resistance is not well understood, primarily due to lack of appropriate experimental models.

In the obese state, the adipose expression of TNF{alpha} is elevated in many experimental rodent obesity models (15) as well as in obese humans (16, 17). Because TNF{alpha} is capable of inducing a catabolic state, the increased production of TNF{alpha} in adipocytes has been proposed to be a candidate mechanism used by the organism to induce local insulin resistance and limit further development of the fat mass (11, 12, 13). In line with this, TNF{alpha} interferes with adipocyte differentiation (18, 19, 20, 21, 22) and profoundly impacts insulin-responsive cells and tissues (23). Removal of TNF{alpha} activity by biopharmaceutical agents (15, 24, 25) or by targeted genetics (26, 27, 28) results in increased responsiveness to insulin. The more challenging aspects of this model are related to the underlying mechanisms to spatially control TNF{alpha} action at specific sites such as adipocytes and examine whether such a restricted activity would have local and/or systemic metabolic consequences. In this study, we have attempted to address some of these issues by generating a mouse model where a nonsecretable form of TNF{alpha} (mTNF{Delta}1-9K11E) is transgenically expressed in an adipocyte-specific manner in animals lacking both copies of the endogenous TNF{alpha} gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant murine soluble TNF{alpha} and ELISA kits for murine and human TNF{alpha} were purchased from Genzyme (Cambridge, MA). The biotinylated polyclonal goat antimurine TNF{alpha} antibody was purchased from R&D Systems (Minneapolis, MN). The polyclonal rabbit antihuman insulin receptor and insulin receptor substrate-1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The polyclonal rabbit antimouse adiponectin/ACRP30 was a gift from Dr. Philip Scherer (Albert Einstein College of Medicine, New York, NY).

Generation of the transgenic mice
The 5.4-kb aP2 gene enhancer/promoter was used to drive the target gene (mTNF{Delta}1–9K11E) expression specifically in adipose tissue. The plasmid was constructed as follows: a 1.65-kb XbaI/SfiI fragment containing the SV40 splice and Poly (A) sites was cloned into the SmaI site of pBluescript SKII+ after Klenow fill-in and denoted as pBS-SV40. A 0.76-kb BglII/BamHI fragment containing the coding sequence of mTNF{Delta}1–9K11E was cloned into the BamHI site of pBS-SV40 in sense orientation and named as pBS-TNF-SV40. Finally, the 5.4-kb aP2 gene enhancer/promoter was isolated as a NotI fragment and cloned into NotI site of pBS-TNF-SV40 in correct orientation. For microinjection, the final construct pBS-aP2-TNF-SV40 was digested with KpnI, and the 10-kb fragment containing the aP2 enhancer/promoter, the noncleavable transmembrane TNF{alpha} SV40 splice and Poly (A) sites were isolated free of vector sequence. The purified and linearized expression vector was injected into fertilized eggs derived from TNF{alpha}-/- C57BL/6 mice. Out of 19 offspring, 7 (36.8%) had integrated the transgene as determined by PCR and southern blot analysis. The primers used to genotyping were as the follows: 5' primer, 5'-GAAGTTCCCAAATGGCCTCC-3'; and 3' primer, 5'-GGATCCAGAGTAAAGGGG TCAGAGTG-3'. These founder mice were bred with nontransgenic TNF{alpha}-/- littermates. Heterozygous F1 males were intercrossed with nontransgenic TNF{alpha}-/- female littermates. The entire progeny were genotyped by genomic southern blot analysis.

Biochemical assays and insulin and glucose tolerance tests
Tolerance tests were performed on 20-wk-old male mice after 6 h daytime food withdrawal. Insulin and glucose solutions were injected into peritoneal cavity at the dose of 0.5 U/kg and 10 ml/kg (1 M solution), respectively. Blood was collected via tail vein at different time points and glucose levels were measured by the use of a glucometer (Precision, Willow Grove, PA). Plasma insulin, leptin and FFA levels were measured with the rat insulin RIA (Linco Research, Inc., St. Charles, MO), mouse leptin RIA (Linco Research, Inc.) and the NEFA-C assay (Wako Chemicals USA Inc., Richmond, VA), respectively. TG, glycerol, and cholesterol levels were measured with the corresponding commercially available assay systems (Sigma, St. Louis, MO).

Glucose uptake in isolated adipocytes
Epididymal white fat pads were excised, weighed, and rinsed in isolation buffer (120 mM NaCl, 0.5 mM KCl, 1.2 mM KH2PO4, 0.6 mM MgSO4·7H2O and 0.9 mM CaCl2·6H2O, 20 mM HEPES, 200 nM adenosine, and 2.5% BSA). Fat pads were then cut into small pieces in isolation buffer supplemented with 1 mg/ml type I collegenase (Worthington Biochemical Corp., Lakewood, NJ) and digested at 37 C in shaking water bath (Precision) at 100 rpm per minute for 45 min. Then, digested tissues were filtered through 100 µM mesh (TETKO Inc., Briarcliff Manor, NY) to get single cell suspension and cells were rinsed twice with isolation buffer. For glucose uptake, 100 µl cell suspension was used in a 1.5-ml tube together with 350 µl buffer with or without insulin (basal uptake) for a 30-min incubation at 37 C with gentle shaking. Nonspecific uptake was determined by incubating cells with 50 µM cytochalsin B (Sigma), a glucose transporter inhibitor. Then, 50 µl isolation buffer containing 0.5 µCi 14C-deoxy-glucose (NEN Life Science Products, Boston, MA) was added to all tubes and incubated at 37 C for 45 min with gentle shaking. The uptake was terminated by adding 500 µl di-isononyl ester (Fluka, Milwaukee, WI). After gentle mixing, the tubes were spun at 1,000 rpm for 2 min and cells on the top layer were transferred to scintillation vials for counting.

Southern blot analysis
Genomic DNA samples were extracted from mouse tails and digested with BamHI. Samples were loaded on 1% agarose gel along with molecular weight markers and run at 20 V overnight. DNA samples were then depurinated in 0.25 M HCl for 30 min, denatured in 1.5 M NaCl/0.5 M NaOH for 30 min, and neutralized in 1.5 M NaCl/0.5 M Tris·Cl (pH 7.0) for 30 min. After neutralization, DNAs were transferred to a biotran membrane (ICN, Costa Mesa, CA), UV cross-linked and baked at 80 C for 1 h. Hybridization with {alpha}32P-deoxy-CTP (NEN Life Science Products) labeled cDNA probes and subsequent washings were done as described previously (22).

Northern blot analysis
RNA samples were extracted as described (15). Following denaturation, RNAs were loaded on a 1% agarose gel containing 3% formaldehyde. After electrophoresis, RNAs were transferred to a biotran membrane (ICN Biomedicals), UV cross-linked and baked at 80 C for 1 h. Hybridization with {alpha}32P-deoxy-CTP (NEN Life Science Products) labeled cDNA probes and subsequent washings were done as described previously (22). Pictures of ethidium bromide-stained gels were shown for loading adjustment.

Immunoprecipitation and immunoblotting
To immunoprecipitate membrane-associated TNF{alpha} from adipose tissue, two transgenic and two control mice were used for plasma membrane preparations. The monoclonal hamster antimurine TNF{alpha} antibody was used for immunoprecipitation (a gift from Dr. Robert D. Schreiber, Washington University, St. Louis, MO). Four micrograms of antibody were used for 1 mg membrane material. The tissue lysates were first cleared with 50 µl protein A (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) beads for 1 h at 4 C and then incubated with the immunoprecipitating antibody for 2 h at 4 C. Immunocomplexes were then collected by incubation with 50 µl protein A beads for 1 h at 4 C. Immunoblots were performed by using the biotinylated polyclonal goat antimurine TNF{alpha} antibody (R&D Systems Inc.) at a concentration of 0.2 µg/ml. ACRP30/adiponectin immunoblots were done as described (29), using 0.5-µl aliquots of serum and 1:100 dilution of the antibody polyclonal rabbit antimouse ACRP30/adiponectin antibody.

Food intake and feces lipid analysis
Mice were individually caged (10 mice per group) and fed with 50 g of transgenic dough diet. The dough was replaced once every week and weighted daily for 2 wk. The data were presented as an average daily intake from a 2-wk study. The feces were collected weekly for individual mouse. For lipid analysis, equal amount of feces from control and transgenic mice were dissolved in alcoholic KOH (2 parts ethonal:1 part 30% KOH) at 60 C overnight. The above solution was mixed well and 0.5 ml was removed to a microfuge tube, 0.54 ml of 1 M MgCl2 was added. The content was mixed and then let sit on ice for 10 min. Microfuge was spun at top speed and supernatant was saved for glycerol assay.

In vivo insulin-stimulated IR phosphorylation
After an overnight fast, 20-wk-old male mice were anesthetized by ip administration of xylasine (10 mg/kg) and ketamine (100 mg/kg). The abdominal cavity was opened and 500 mIU per kg insulin (Eli Lilly \|[amp ]\| Co., Indianapolis, IN) or an equal volume of PBS solution was administered through the portal vein. Liver and epididymal fat pads were collected 120 sec after the injection and immediately frozen in liquid nitrogen. Protein extracts from the tissue samples were then prepared for detecting IR phosphorylation as described previously (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of transgenic mice with exclusive mTNF{alpha} expression in adipocytes
We generated a transgenic mouse model to examine the action of mTNF{alpha} exclusively in the adipocyte and test whether it is sufficient to interfere with adipogenesis and adipose tissue insulin action in vivo. To achieve adipose-tissue specific expression of transmembrane TNF{alpha}, we constructed a transgenic expression vector driven by the 5.4-kb aP2 gene enhancer/promoter (Fig. 1AGo) (30). The transgene encodes a noncleavable TNF{alpha} variant, mTNF{Delta}1-9K11E (22), the actions of which should be limited to adipose tissue. This TNF{alpha} variant carries a 9-amino acid deletion at the site of cleavage recognized by the TNF{alpha} converting enzyme and a substitution of lysine at position 9 to glutamic acid. The mutant retains the actions of the wild-type protein as tested previously (22, 31, 32). Under normal circumstances, TNF{alpha} is localized to the cell surface as a type 2 transmembrane protein and cleaved at the cell surface to release the soluble cytokine. The mutant used in our work is also inserted to the plasma membrane the same way; however, since the cleavage site is altered it is not recognized by the protease, hence, not cleaved. The biologically active domain is present and it does interact with TNF receptors. This mutant has been characterized extensively in adipocytes (22, 32) and immune cells (31). The transgenic lines were created directly in the TNF{alpha}-/- mice to prevent interference from the endogenous TNF{alpha} expression at other sites. After microinjection, 19 viable offspring were obtained, and 7 of them had the transgene integrated into their genome. Genomic Southern blotting revealed that they had different copy numbers of the transgene (Fig. 1BGo). Four founder mice transmitted the transgene and were designated based on the order of production as lines 1, 2, 18, and 19. There was no obvious difference in behavior and fertility between transgenic and control mice. Experiments were first performed with the F1 generation of heterozygous line 1 mice and results were confirmed in the F2 generation of transgenic lines 1, 18, and 19 (8, 12, and 15 copies of the transgene).



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  		Figure 1. Establishment of a transgenic mouse model that expresses mTNF{alpha} specifically in adipose tissue. A, Construction of the transgenic expression vector. The mTNF{Delta}1–9K11E cDNA encoding a noncleavable transmembrane TNF{alpha} protein and the 5.4-kb mouse aP2 gene promoter/enhancer were chosen to control adipose tissue-specific expression of the transgene. B, Genomic Southern blot analysis of tail DNA from transgenic founder mice. Genomic DNA was digested with BamHI, which excised the 0.76-kb transgene, then probed with full length TNF{alpha} cDNA. The aP2 promoter/enhancer-driven construct is expressed in TNF{alpha}-deficient (TNF{alpha}-/-) background where two of the four exons (exons 1 and 2) have been replaced by a neo cassette. M, Male; F, female. Independent transgenic lines are named after the numbers shown above the corresponding lanes with asterisks. Four founders (line 1, line 2, line 18, and line 19) gave offspring. C, Adipose tissue-specific expression of a noncleavable transmembrane TNF{alpha} (mTNF{alpha}). Eleven different tissues and organs were collected and used for RNA extraction. The signal of mTNF{alpha} transgene was highly expressed in both white (WAT) and brown (BAT) adipose tissues from mTNF{Delta}1–9K11E-expressing transgenic mice. In contrast, no signal could be detected in WAT from the control (Ctr) mouse. D, Detection of adipose tissue mTNF{alpha} protein in membrane fractions prepared from the control and transgenic mice.

 
To determine whether the transgene was expressed properly in the targeted tissues, Northern blot analysis was performed to examine the tissue distribution of the transgenic mRNA encoding mTNF{Delta}1–9K11E. As shown in Fig. 1CGo, the transgene was highly expressed in both WAT and BAT. Low levels of expression were also detected in testes and pancreas, which might reflect either leaky activity of the promoter or contaminating adipocytes at these sites. To determine whether the protein product was also properly translated and transported to the cell surface, WAT was fractionated and the fraction containing plasma membranes was used to examine the existence of mTNF{Delta}1–9K11E protein by immunoblotting. These experiments demonstrated the presence of the protein product of the transgene specifically in transgenic but not control animals (Fig. 1DGo). The glycosylated forms, which are often observed with wild-type endogenous TNF{alpha}, were not detected because mTNF{Delta}1–9K11E lacks the glycosylation site that give rise to these species. We also tested whether there was any unanticipated processing of the transgenic product in adipose tissue and consequent release into circulation in vivo at the baseline conditions and upon administration of lipopolysaccharide. Analysis of blood samples by a TNF{alpha}-specific ELISA did not reveal any detectable level of circulating TNF{alpha} in transgenic animals even upon 3 mg/kg lipopolysaccharide stimulation for 3 h, where high levels of circulating TNF{alpha} was readily detectable in the wild-type animals with intact endogenous TNF{alpha} gene (data not shown). These data indicate that the transgene was properly transcribed, translated, translocated, and retained at the desired site and there was no detectable secreted TNF{alpha} in these transgenic animals.

Phenotypic characterization of mTNF{Delta}1–9K11E transgenic mice
At birth, the mTNF{Delta}1–9K11E transgenic mice appeared normal and could not be distinguished from controls (TNF{alpha}-deficient littermates) by their appearance. The body weight was monitored from 4 wk of age, at which time no significant differences were apparent between the genotypes. At 8 wk, the body weights of the transgenic mice were lower compared with their littermates. This difference became more significant in the subsequent measurements and by 20 wk, there was a 15% reduction in the body weight of all three transgenic mice lines compared with controls (Fig. 2AGo). A general decrease in adipose mass was readily observable upon gross dissection of the mTNF{Delta}1–9K11E transgenic mice (Fig. 2Go, C–F) and both white (epididymal) and brown fat pads (interscapular) were reduced in mass compared with those from control animals (Fig. 2BGo). No difference was evident in the weights of liver, spleen, and heart between the transgenic and control mice indicating that the reduction was specific to fat depots. Histological analysis of fixed adipose tissue showed that transgenic animals had smaller fat cells compared with control mice (Fig. 3Go, A and B). The average adipocyte size in the transgenic animals was reduced by 20% compared with the wild-type controls. Although reduction in adiposity is commonly associated with excess lipid accumulation in the liver, sections prepared from the mTNF{Delta}1–9K11 transgenic mice did not reveal any lipid accumulation (Fig. 3Go, C and D).



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  		Figure 2. The transgenic mice have reduced body weight, smaller white and brown fat pads. A, Body weight recordings of transgenic vs. control mice in three independent lines. Body weights were measured every 4 wk in fed state. B, Weight measurements of WAT, BAT, and liver. Tissue and organs were collected from 20-wk-old male mice. C, Dorsal views of the transgenic (left) and control (right) mice at an age of 20 wk. D, Ventral views of the transgenic (left) and control (right) mice. E, Display of excised WATs of the transgenic (left) and control (right) mice. F, Display of excised BAT of the transgenic (left) and control (right) mice. The asterisk indicates statistical significance with P < 0.05.

 


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  		Figure 3. A and B, Hematoxylin and eosin-stained adipose tissue sections from mTNF{alpha} transgenic and control mice, respectively. C and D, Hematoxylin and eosin-stained liver tissue sections from mTNF transgenic and control mice, respectively. E–H, Comparison of total body composition, intestinal lipid absorbtion, daily food intake and core body temperature between control and transgenic mice. The data shown are obtained from line 1 and reproduced in part in the remaining lines. The differences of food intake and core body temperature showed similar trends in the additional lines but did not reach statistical significance. The asterisk indicates statistical significance with P < 0.05.

 
Total body composition analysis also revealed a significant reduction in lipid content of transgenic mice compared with controls (Fig. 3EGo, 0.0291 vs. 0.0360 mM/g glycerol/body weight, P < 0.05). To determine the potential alterations that might contribute to loss of adipose mass, we studied intestinal lipid absorption in control and transgenic mice, but did not observe any differences between genotypes (Fig. 3FGo). There was a very small decrease in daily food intake (O.4 g/d) in the transgenic mice compared with controls (Fig. 3GGo). Interestingly, there was also a small increase (0.42 C) in core body temperature in the transgenic animals (Fig. 3HGo). The steady-state lipid metabolism in the transgenic mice was normal with similar plasma TG, cholesterol, FFA, and glycerol levels in both groups (data not shown)

The leptin level in circulation is strongly related to total fat mass. In further support of reduced adiposity, adipose tissue leptin mRNA (Fig. 4AGo) and circulating leptin protein (Fig. 4BGo) levels were significantly reduced in the transgenic animals. Interestingly, despite reduced overall adiposity and altered adipocyte size, the expression of mTNF{Delta}1–9K11 under the control of aP2 promoter did not result in alterations of several other adipogenic markers including adipsin, aP2 and ACRP30/AdipoQ (Fig. 4AGo). These data indicated that adipocyte de-differentiation did not occur under the experimental conditions used in this study despite reduced overall adiposity. Instead, mTNF{Delta}1–9K11E affected gene expression in a highly selective manner without a general suppression of adipocyte differentiation.



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  		Figure 4. Adipocyte gene expression and plasma leptin levels in transgenic and control mice. A, Expression of adipsin, aP2, and leptin in WAT and BATs of mTNF{alpha} transgenic mice and littermate controls. B, Circulating leptin levels in transgenic and control mice. The data shown are obtained from line 1 and reproduced in the remaining lines.

 
Local insulin action in adipocytes
To investigate whether mTNF{alpha} has any effect on insulin sensitivity in adipose tissue, glucose uptake experiments were performed with isolated primary adipocytes from epididymal fat pad of transgenic mice as well as controls. Upon insulin stimulation, glucose uptake levels were increased 2.0- to 3.9-fold over baseline in transgenic and control animals, respectively (Fig. 5AGo). These data demonstrated that the insulin-stimulated glucose transport capacity in mTNF{Delta}1–9K11E-expressing primary adipocytes was significantly reduced compared with that in control cells. This reduction was apparent at both saturating and submaximal doses of insulin. Hence, local TNF{alpha} action in fat cells was associated with local insulin resistance.



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  		Figure 5. Glucose uptake in adipocytes. A, Insulin-stimulated glucose uptake in primary adipocytes isolated from epididymal white fat pad of male transgenic (n = 7) and control mice (n = 7) at the age of 20 wk. 14C-deoxy-glucose uptake in adipocytes of both genotypes was measured in triplicate following stimulation with 10 ng/ml insulin. Insulin-stimulated glucose uptake was expressed as percent over basal level. B, Expression of glucose transporters, resistin, and adiponectin in WAT and BATs of mTNF{alpha} transgenic mice and littermate controls. C, Serum levels of ACRP30/adiponectin in transgenic and control mice. The data shown are obtained from line 1.

 
We next examined whether the decrease in glucose transport capacity was related to the expression of adipose tissue glucose transporters. The two glucose transporter isotypes that are found in adipose tissue, Glut1 and Glut4, were expressed at comparable levels in control and transgenic mice WAT. There was, however, a very small (15%) reduction observed in the expression of these genes in BAT. Taken together, it is unlikely that the quantitative regulation of glucose transporters can account for the decrease in insulin action in adipose tissue. We also measured the expression of resistin because this gene was recently proposed as an adipose tissue-derived mediator of insulin resistance that can interfere with insulin-stimulated glucose transport in adipocytes (33). There was no difference between genotypes in the adipose tissue resistin expression (Fig. 5BGo). Finally, we determined both mRNA and serum protein levels for adiponectin/ACRP30, a candidate effector that reduces adiposity and increases insulin sensitivity (29, 34, 35). These experiments demonstrated a 2-fold increase in the serum levels of adiponectin/ACRP30 in the transgenic animals compared with the controls (Fig. 5CGo).

Experiments were also performed to examine whether mTNF{Delta}1–9K11E expression in adipocytes interfered with insulin signaling at this site in vivo, thereby reducing glucose transport. To do this, insulin was administered via the portal vein and after 2 min epididymal fat pads and livers were collected for the preparation of protein extracts (26). Insulin receptors were immunoprecipitated with a polyclonal antibody and tyrosine phosphorylation levels were examined by immunoblotting with a monoclonal antibody against phosphotyrosine (26). As shown in Fig. 6AGo, insulin-stimulated phosphorylation of the insulin receptor ß-chain was readily detectable in controls. The extent of this phosphorylation was reduced by 70% in the adipose tissue of transgenic mice compared with that in controls (Fig. 6Go, A and B). In contrast, no obvious difference in IR phosphorylation was detected in liver lysates (data not shown). These data indicate that mTNF{Delta}1–9K11E directly suppresses insulin receptor signaling in adipose tissue in vivo and induces strong local insulin resistance in adipocytes of the transgenic mice.



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  		Figure 6. Insulin receptor signaling in adipocytes. A, Insulin receptors (IR) were immunoprecipitated from 1 mg of adipose tissue total protein lysates prepared from mTNF{alpha} transgenic (n = 6) and control (n = 6) animals with or without insulin stimulation. Immunoblotting was performed to show IR phosphorylation and total protein levels. B, Quantitation of experiments performed in A. Ins, Insulin. The data shown are obtained from line 1.

 
Systemic insulin action in vivo
A critical question in general is whether the localized suppression of insulin action via the TNF{alpha} transgene in adipose tissue could be sufficient to alter systemic glucose metabolism. This issue became particularly interesting because the transgenic animals exhibited a reduction in adiposity and an increase in circulating adiponectin/ACRP30 level, both of which would enhance insulin action systemically whereas their adipocytes were highly insulin resistant locally. To investigate the effects of adipose tissue-restricted TNF{alpha} action on whole body glucose metabolism and insulin sensitivity, steady-state plasma glucose and insulin levels were determined in transgenic animals and controls. At 20 wk of age, both steady-state and fasting plasma insulin levels in the transgenic mice were significantly lower than that in control mice (Fig. 7AGo). The data shown is obtained from F1 generation of line 1 and also reproduced in the F2 generations of both lines 1 and 19 (data not shown) at 16 wk of age. This time course coincides with the reduction of body weight of these animals. Although there was also a consistent trend for lower plasma glucose levels starting at 12 wk in both lines of transgenic mice, these differences did not approach statistical significance (Fig. 7BGo). These data suggested that the mTNF{Delta}1–9K11E transgenic mice might not have developed systemic insulin resistance. To further address this and explore systemic insulin action and glucose metabolism, both ip insulin and glucose tolerance tests were performed. In insulin tolerance tests, the hypoglycemic response to insulin was more pronounced in the mTNF{Delta}1–9K11E transgenic mice line 1 throughout the experiments compared with nontransgenic controls (Fig. 7CGo). Similarly, the ip glucose tolerance test also demonstrated a lower degree of hyperglycemia in these transgenic mice compared with controls (Fig. 7DGo). There was no significant difference in serum insulin levels between genotypes at 0- and 30-min samples collected during the glucose tolerance tests. These data demonstrated that the impaired insulin action in adipocytes and adipose tissue in the mTNF{alpha} transgenic animals did not have a systemic impact. Surprisingly, one of the transgenic lines exhibited even improved systemic insulin sensitivity, presumably as a result of the dominant effects of reduced overall adiposity, which was quite significant. The two additional transgenic lines also exhibited similar trends, although the differences in ITT and GTT did not reach statistical significance (data not shown).



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  		Figure 7. Systemic insulin and glucose homeostasis in transgenic and control mice. Steady-state and fasting plasma insulin (A) and glucose (B) levels of 20-wk-old line 1 F1 generation male mice. n = 7 for transgenic and n = 11 for controls. Insulin (C) and glucose (D) tolerance test (ITT and GTT) performed in 20-wk-old male mice. After insulin or glucose administration, blood was collected via tail vein at several time points and glucose levels were measured by the use of Glucometer (n = 7 for transgenic and 11 for controls). The asterisk indicates statistical significance in ANOVA repeated measures with P < 0.05. The data shown are from line 1. There was no significant difference in insulin levels between genotypes at 0 and 30 min samples collected during the glucose tolerance tests (0.6 ± 0.04 and 1.44 ± 0.1 in controls and 0.5 ± 0.03 and 1.58 ± 0.1 in transgenic mice at t = 0 and t = 30 min). The glucose disposal curves in the other two transgenic lines were also not statistically significantly different.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Adipose tissue exhibits tremendous plasticity of mass based on the metabolic status of the organism. It has been proposed that fluctuations in adipose mass are actively controlled by mechanisms endogenous to adipocytes, including, but not limited to, the sensitivity to the actions of insulin. In this study, we demonstrate in a transgenic mouse model expressing a noncleavable form of TNF{alpha} in adipose tissue that exclusive action of this cytokine in adipocytes led only to local insulin resistance in adipose tissue and reduced adipose mass. These results demonstrate that regulated processing of TNF{alpha} could be a potential mechanism to limit adiposity.

Obesity is the only known condition where TNF{alpha} is expressed at elevated levels in adipocytes (15, 23). Substantial data have accumulated to demonstrate the role of TNF{alpha} in inducing insulin resistance through a cross-talk between TNF and insulin receptor signaling pathways (36, 37, 38, 39, 40). In fact, this action of TNF{alpha} in adipose tissue is localized perhaps in an autocrine/paracrine fashion to combat further adiposity. While studies in isolated human adipocytes (41) and disproportionately low circulating levels of TNF{alpha} protein in obese animal models and humans (23) have supported such a hypothesis, no mechanism has been identified to account for the spatial control of TNF{alpha} action and its biological consequences. Recently, we demonstrated that adipocytes are defective in TNF{alpha} processing in vitro and in vivo and hence retain excess amount of this molecule on the cell surface. In line with this observation, significant elevations were evident in transmembrane TNF{alpha} levels in adipose tissues obtained from several different experimental mouse obesity models, as well as obese humans where adipose TNF{alpha} production is abnormally elevated (42). Here, we transgenically reconstituted TNF{alpha} expression in the TNF{alpha}-/- mice with a noncleavable form of TNF{alpha} expressed under the control of the adipocyte specific aP2 promoter/enhancer. The exclusive presence of transmembrane TNF{alpha} in adipose tissue resulted in a lean phenotype in transgenic mice, indicating that localized TNF{alpha} action could limit adiposity. However, whether this would also be the case in the presence of caloric imbalance and obesity remains to be seen.

The decrease in adiposity in these transgenic mice was not severe. The total body composition analysis revealed a 10% reduction in adiposity. Unlike the transgenic mouse models of lipodystrophy generated by the expression of SREBP-1c and A-ZIP/F under the control of aP2 promoter/enhancer, there were no pathological changes in other tissues and organs in this model. The morphology of adipose tissue, however, was altered in the transgenic animals and the adipocyte size was significantly reduced. If no other aspect of energy metabolism has been altered in these animals, the 10% decrease in adipose mass could have a strong impact on systemic lipid metabolism and lead to a potential increase in the lipid content of the liver. Interestingly, none of these is evident in the aP2-mTNF{alpha} transgenic mice. Under gross examination, livers appeared normal. There were no changes in color, and liver lipid content was indistinguishable from the nontransgenic controls. Similarly, no alterations were observed in the plasma lipid profiles.

These observations might have several potentially important implications. First, the target for TNF{alpha} in mediating its in vivo effects on lipid metabolism has been a subject of discussion where both adipocytes and liver have been implicated (43, 44, 45, 46). Our data demonstrates that isolated TNF{alpha} action on the adipocyte is insufficient to induce systemic dyslipidemia and hence, the role of liver should be dominant with respect to this particular effect (46). Second, it is possible that TNF-induced alterations in adipose tissue have secondary effects on other systems, for example through the regulation of adipocyte communication with other metabolically significant sites to establish metabolic equilibrium. In this regard, we determined fecal lipid content, food intake, and body temperature. There was no evidence for intestinal lipid loss in the transgenic animals. There was a small decrease in the daily food intake and a small increase in body core temperature in mTNF{alpha} transgenic mice compared with nontransgenic controls. Although, the mechanisms underlying these effects of mTNF{alpha} are not yet clear, reduced food intake and increased energy expenditure are the likely underlying causes contributing to the modest reduction in fat mass. Interestingly, we observed higher serum levels of adiponectin/ACRP30 protein. Because this protein can influence muscle to increase fatty acid oxidation and liver to enhance insulin action (29, 34), it is possible that its elevated levels could be critical in the reduced adiposity and increased insulin sensitivity seen in the transgenic animals.

While the transgenic models of lipodystrophy exhibit systemic insulin resistance (9, 10, 47), those with mild reductions in adiposity display enhanced systemic insulin sensitivity (48, 49). A striking recent example for the latter is the unanticipated increase in systemic insulin action in mice heterozygous for a null allele in the PPAR{gamma} gene (49, 50). Interestingly, these mice also had reduced adiposity and decrease in the size of the adipocytes, both of which support enhanced insulin action, similar to what we have observed in the transgenic model presented here. PPAR{gamma} is primarily expressed in adipose tissue, and loss of function is expected to result in reduced insulin sensitivity. Unfortunately, insulin action in the adipocytes has not yet been studied in this model. Even more interesting and revealing is the phenotype of mice with an adipocyte-specific deletion of the insulin receptor gene. These mice also have reduced adipose mass and despite complete absence of insulin action in adipocytes, do not become diabetic (51). Similarly, in this study, we did not observe any sign of systemic insulin resistance in the mTNF{alpha} transgenic mice. On the contrary, transgenic mice were slightly more sensitive to insulin, suggesting that the lean state of these animals might be the predominant factor for determining systemic insulin sensitivity. Taken together, these data indicate that modestly reduced adipocyte volume and adiposity, whether it is the direct or indirect consequence of adipocyte insulin resistance, appear to dominate systemic insulin action. It is even possible that adipocyte insulin resistance itself is a defense against the expansion of adipose mass. In instances where adipose mass is not reduced, such as the case in the adipose specific Glut4-deficient mice, adipocytes could alter systemic glucose metabolism (52).

The transgenic mouse model used in this study was created in the TNF{alpha}-deficient background to investigate mTNF{alpha} action exclusively in adipose tissue. Although it has been established that the absence of TNF{alpha} ligand or receptors partially improve insulin sensitivity in the context of obesity (26, 27, 28), reconstitution of TNF{alpha} action only in adipose tissue in lean mice led to local but not systemic insulin resistance (this study). These data indicate that TNF{alpha}-induced insulin resistance in adipocytes specifically or adipocyte insulin resistance in general may not be sufficient to induce systemic insulin resistance. If this is the case, it is likely that TNF{alpha} action in other target tissues (such as liver, muscle and pancreas) and/or combination of TNF{alpha} action in more than one site is responsible for its impact on insulin action systemically. However, it is also likely that, in the context of obesity, local reconstitution of TNF{alpha} action in adipocytes would exacerbate insulin resistance in these transgenic mice. Induction of obesity in the transgenic mice described here as well as transgenic mice in the wild-type genetic background should address these questions and these studies are underway. We do also recognize the possibility that our transgenic reconstitution might not have faithfully represented the dose, timing, and the dynamics of TNF{alpha} production necessary for its full action and that the results observed are a function of this shortcoming. These questions notwithstanding, the data presented here provides a model where local actions of this molecule is conferred by the transmembrane form of TNF{alpha} and result in decreased adiposity and reduced local, but not systemic, insulin sensitivity.

These findings might have important implications in modeling and targeting TNF{alpha} biology in obesity as well as dissecting its actions in adipocytes vs. other sites in vivo. For example, if adipose tissue-restricted TNF{alpha} action alone is insufficient in conferring insulin resistance or results in distinct phenotypes, then strategies aimed at complete blocking of TNF{alpha} cleavage with metalloproteinase inhibitors might provide important tools for alternative therapeutic purposes. So far, one report has shown that KB-R7785, a novel matrix metalloproteinase inhibitor, exerts its antidiabetic effect by inhibiting TNF{alpha} processing (53). Additional studies will be necessary to further address these critical questions.


   Acknowledgments
 
We thank Guo Tan, Gökhan Dalgin, and Drs. Qiang Tong and Cem Gorgun for their advice and help.


   Footnotes
 
This work was supported by grants from American Diabetes Association Pew Foundation and National Institutes of Health (to G.S.H.).

Abbreviations: BAT, Brown adipose tissue; mTNF{alpha}, transmembrane form of TNF{alpha}; mTNF{Delta}1-9K11E, nonsecretable form of TNF{alpha} mutant; TG, triglyceride; WAT, white adipose tissue.

Received August 28, 2001.

Accepted for publication December 10, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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