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Departments of Cell Biology (T.P.C., U.B.P., A.H.B., Y.L., A.R.N., M.W.R., L.C., Y.Y.D., P.E.S.), Physiology and Biophysics (L.A.J.), Pathology (R.G.R.), Neuroscience (A.H.), Medicine (S.O., L.R.), and Molecular Pharmacology (S.O., L.R.); Institute for Animal Studies (R.G.R.); Department of Medicine, Division of Endocrinology, Diabetes Research and Training Center (S.O., L.R., P.E.S.), Albert Einstein College of Medicine, Bronx, New York 10461; Department of Anatomy and Physiology, Laval Hospital Research Center, School of Medicine, Laval University (M.La., Y.Y.D.), Québec, Canada; National Hormone and Peptide Program, Harbor-University of California-Los Angeles Medical Center (A.F.P.), Torrance, California 90509; Institute for Virology, Technical University Dresden (D.L.), 01307 Dresden, Germany; and Department of Medicine, University of Wales College of Medicine (G.R.C.B., M.Lu.), Heath Park, Cardiff, United Kingdom CF14 4XN
Address all correspondence and requests for reprints to: Dr. Philipp E. 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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2, caveolin-1, and mitochondrial markers in white adipose tissue. These studies strongly suggest that increasing endogenous adiponectin levels has direct effects on insulin sensitivity and may induce similar physiological responses as prolonged treatment with peroxisomal proliferator-activated receptor agonists. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many studies have found a correlation between insulin sensitivity and elevated adiponectin levels in both humans and experimental animals. For example, type II diabetic patients have lower adiponectin levels than nondiabetic controls, and chronic calorie restriction regimens lead to elevated adiponectin (5). Furthermore, in rhesus monkeys predisposed to type II diabetes, the levels of circulating adiponectin decrease before the onset of hyperglycemia (6).
Pharmacological studies have shown that recombinant adiponectin suppresses endogenous glucose production by raising hepatic insulin sensitivity, an effect also seen in primary hepatocyte cultures (4, 7). Glucose-lowering effects in vivo have also been seen with the globular domain of adiponectin (8).
Two reports recently described mouse models with a disruption of the adiponectin locus (9, 10). Mice lacking adiponectin exhibit earlier onset of diet-induced insulin resistance. Even on a normal laboratory chow diet, both adiponectin -/- and -/+ mice display moderate insulin resistance and impaired free fatty acid clearance. However, the effects of chronically elevated serum adiponectin have not been explored to date due to the limited supply of recombinant full-length material.
These observations suggest that low plasma adiponectin contributes to the pathogenesis of insulin resistance and type 2 diabetes. Therefore, elevating adiponectin values can be viewed as a potentially therapeutic objective. Furthermore, transgenic (Tg) methods using conventional strategies of overexpression have also failed to yield a Tg mouse with endogenously elevated adiponectin. Yamauchi et al. (11) expressed a truncated analog of adiponectin in Tg mice that only contains the globular domain. However, this fragment clearly lacks the structural integrity of circulating adiponectin, and it is thus unlikely that it shares the same physiological properties as the native full-length oligomeric complex. Chronic exposure to a class of peroxisomal proliferator-activated receptor (PPAR) ligands known as thiazolidinediones can also increase serum adiponectin levels (12), but this class of drugs clearly induces pleiotropic effects in multiple tissues.
We discovered that the expression of adiponectin lacking a portion of the collagenous domain has a dominant and specific effect on the secretion of native adiponectin. This mechanism was studied in vitro and was used to generate a Tg mouse that exhibited a chronic 3-fold induction of adiponectin in serum. This mouse model is unique in that it displays elevated levels of the native oligomeric adiponectin complexes and preserves the effects seen with sexual maturation and sexual differentiation. Adiponectin in this model is therefore under the same developmental control as adiponectin in wild-type (Wt) mice, except that the basal set-point is approximately 3 times higher in the former. Not only do these mice provide insight into the biogenesis of this fat-specific secretory protein, but they also demonstrate a significant improvement in insulin sensitivity that can be achieved through a relatively modest elevation of the serum levels of this protein.
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Materials and Methods |
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Generation of adiponectin Tg mice
The open reading frame encoding murine adiponectin was subcloned into the multiple cloning site (MCS) of pBluescript. The region encoding amino acids 57–95, containing 13 collagen repeats, was deleted using site-directed mutagenesis. The 5.4-kb aP2 enhancer/promoter sequence (provided by Bruce M. Spiegelman, Dana-Farber Cancer Institute, Boston, MA) was introduced into the MCS region upstream of the cDNA. In addition, the simian virus 40 splice and polyadenylase sequences were introduced into the MCS region downstream of the cDNA. For expression of the full-length protein, the complete open reading frame was inserted into the MCS of pCB7 (gift from J. Casanova, Massachusetts General Hospital, Boston, MA). This vector provides expression from a CMV promoter and contains a human GH terminator sequence. The entire cassettes were excised from both vectors and purified for pronuclear injection into fertilized one-cell murine zygotes from FVB mice (Taconic Farms, Germantown, NY). The embryos were implanted in CD-1 mice (Charles River Breeding Laboratories, Wilmington, MA) at the Transgenic Mouse Facility of the Albert Einstein College of Medicine. Transgenic mice were identified by slot-blot analysis using genomic DNA isolated from the tip of the tail. A fragment containing the simian virus 40 region was radiolabeled and used for hybridization by slot-blot analysis.
Laboratory animals
Mice were housed in groups of two to five in filter-top cages and were given free access to water and Teklad rodent diet no. 8604 (Harlan Teklad, Indianapolis, IN). The colony was maintained in a pathogen-free Association for the Accreditation of Laboratory Animal Care-accredited facility at the Albert Einstein College of Medicine under controlled environment settings (22–25 C; 40–50% humidity; 12-h light, 12-h dark cycle with lights on from 0600–1800). The transgene was propagated in a pure FVB background by mating Tg males with Wt females.
Total RNA isolation, RT-PCR analysis, and Northern blot analysis
Tissue was dissected immediately after sacrifice, snap-frozen in liquid nitrogen, and stored at -80 C. Total RNA was isolated from frozen tissue (-80 C) with TRIzol (Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol. cDNA was prepared from 4 µg RNA using Superscript II, and random hexamers (Invitrogen, Carlsbad, CA). The PCR products were analyzed by gel electrophoresis on 1% agarose and scanned under UV light. Northern blot analysis was performed as described previously (16).
Pulse-chase analysis of adiponectin and resistin secretion in 3T3-L1 adipocytes
Fully differentiated d 8 3T3-L1 adipocytes were pulse-labeled for 10 min in the presence of [35S]methionine and cysteine (NEN Life Science Products, Boston, MA). The labeling medium was then exchanged and washed four times with medium containing excess unlabeled methionine and cysteine, and the cells were kept in that medium supplemented with 10% fetal calf serum for up to 24 h. Tissue culture supernatants were harvested, and cells were lysed at the indicated time points. Both supernatants and cellular lysates were immunoprecipitated with antiresistin and antiadiponectin antibodies and analyzed by SDS-PAGE.
Measurement of specific protein levels in tissue by Western blot
Protein was extracted by immersing frozen tissue in cold buffer [20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM KCl, and 1 mM phenylmethylsulfonylfluoride] and sonicating for 20 sec. The homogenate was centrifuged at 4 C to separate lipids, lysate, and insoluble material. Lysate infranatant was removed and mixed with sodium dodecyl sulfate to a final concentration of 2% (w/v) and heated to 95 C. After 5 min, lysates were cooled to room temperature and centrifuged again. The supernatant was assayed for protein concentration by bicinchoninic acid (protein assay kit, Pierce Chemical Co., Rockford, IL), and 50 µg were assayed for adiponectin, caveolin-1, or GDP dissociation inhibitor (GDI) content by Western blot as previously described. For protein extractions from liver, the tissue was homogenized in a buffer containing 1% Triton X-100, 60 mM octyl-glucoside, 150 mM NaCl, 20 mM Tris (pH 8.0), 2 mM EDTA, 50 mM sodium fluoride, 30 mM sodium pyrophosphate, 100 mM sodium orthovanadate, and 1 mM phenylmethylsulfonylfluoride and sonicated for 20 sec. The resulting homogenate was centrifuged at 4 C, and the supernatant was assayed for protein content by bicinchoninic acid and processed for Western blotting as described above. The antibodies against GDI were a gift from Dr. Perry Bickel (Washington University, St. Louis, MO).
Measurement of adiponectin levels in plasma by Western blot analysis
Blood samples were obtained from the tail vain of unrestrained conscious animals using heparinized capillary tubes (Fisher Scientific, Fairlawn, NJ). Plasma was stored at -20 C for future measurement. Adiponectin in plasma (3 µl) was measured by Western blot analysis. After SDS-PAGE (12%), proteins were transferred to BA83 nitrocellulose (Schleicher & Schuell, Inc., Keene, NH). Blots were exposed to 125I-labeled rabbit antibody against murine adiponectin and analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant 1.2 software. Each gel contained four standards of purified mouse adiponectin at four different concentrations to ensure linearity and reproducibility of the signal. Inter- and intraassay variations for adiponectin were less than 10%, and the limit of detection was approximately 5 ng adiponectin (12).
Velocity sedimentation for size separation of adiponectin complexes
Sucrose gradients [5–20% in 10 mM HEPES (pH 8) and 125 mM NaCl] were poured stepwise (5%, 10%, 15%, and 20%) in 2-ml, thin-walled, ultracentrifuge tubes (BD Biosciences, Mountain View, CA) and allowed to equilibrate overnight at 4 C. After layering of the plasma sample on top [diluted 1:10 with 10 mM HEPES (pH 8) and 125 mM NaCl in the case of serum], the gradients were spun at 55,000 rpm for 4 h at 4 C in a TLS55 rotor in a TL-100 table-top ultracentrifuge (Sorvall, Kendro Laboratory Products, Newton, CT). Gradient fractions (150 µl) were retrieved sequentially from the top of the gradient and analyzed by quantitative Western blot analysis. This protocol was described in more detail previously (17).
Percent fat mass
The percent fat mass was determined on the basis of the low partition coefficient of H2O. A known amount of 3H2O was injected into the ip cavity and allowed to equilibrate over 8 h. The distribution of tracer in the aqueous compartment was used to calculate the contribution of the lipid stores to the total body weight. The calculation was made under the assumption that 7.5% of the aqueous volume is occupied by nonlipids that nevertheless exclude the tracer.
Indirect calorimetry
Animals undergoing indirect calorimetry were acclimated to the respiratory chambers for 1 d before the gas exchange measurements. Mice were individually housed in the calorimeter cages, and data on gas exchanges, activity, and food intake were collected for 2–3 d. Indirect calorimetry was performed with a computer-controlled Oxymax open circuit calorimetry system (Columbus Instruments, Columbus, OH). Our instrument comprises 4 respiratory chambers with a stainless steel, elevated, wire floor. Each chamber is equipped with water bottle, food tray connected to a balance, and activity monitor. Oxygen consumption and carbon dioxide production were measured for each mouse at 6-min intervals. Outdoor air reference values were measured after every 10 measurements. Instrument settings were: gas flow rate, 0.5 liter/min; settle time, 240 sec; and measure time, 60 sec. Gas sensors were calibrated daily with primary gas standards containing known concentrations of O2, CO2, N2 (Tech Air, Danbury, CT). A mass flow meter was used to measure air flow. A limited diffusion metal-air battery was used as an oxygen sensor. CO2 was measured with a spectrophotometric sensor. The respiratory quotient was calculated as the ratio of CO2 production over O2 consumption. The energy expenditure (EE) was calculated as follows: EE = (3.815 + 1.232 x VCO2/VO2) x VO2, where VCO2 is the expired CO2 volume (ml/kg·h), and VO2 is the expired O2 volume (ml/kg·h).
Magnetic resonance imaging
All images were obtained using a 9.4-Tesla imaging system. Mice were first anesthetized by ip injection with a ketamine/xylazine mixture (0.1 ml/20 g body weight). To quantitatively assess whole body fat and water, each mouse was subjected to a 16-scan pulse-acquire sequence in a 40-mm 1H coil, and spectra, including the water and fat peaks, were obtained and analyzed. For imaging, several datasets of eight slices of 2-mm thickness spanning the whole body were obtained. Imaging was conducted using a 35-mm 1H coil and a routine spinecho pulse sequence (18-msec echo time, 600-msec repetition time, and four-signal averages per scan).
Light and transmission electron microscopy
Adipose tissue was fixed with 2.5% glutaraldehyde/1.2% acrolein in sym-collidine buffer, postfixed with 1% osmium tetroxide, followed by 1% uranyl acetate, dehydrated through a graded series of ethanol concentrations, and embedded in LX112 resin (LADD Research Industries, Burlington VT). One-micron-thick sections were cut on an Ultracut UCT (Reichert, Analytical Instruments, Buffalo, NY), stained with 1% toluidine blue in 1% sodium borate, and viewed on an Axiophot light microscope (Carl Zeiss, Inc., New York, NY). Ultrathin sections were cut on a Reichert Ultracut UCT, stained with uranyl acetate, followed by lead citrate, and viewed on a 1200EX transmission electron microscope (JEOL, Inc., Peabody, MA) at 80 kV. For light microscopy of the orbital fat, the entire skull was formalin-fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin. For electron microscopy, adipose tissue was dissected from the intraconal region of the orbit and frozen under high pressure using an EMpact High Pressure Freezer (Leica Microsystems, Solms, Austria). The frozen samples were transferred to a Leica EM AFS Freeze Substitution Unit, freeze-substituted in 1% osmium tetroxide in acetone, brought from -90 C to room temperature over 2–3 d, rinsed in acetone, and embedded in LX112 epoxy resin (LADD Research Industries). Ultra thin sections of 70–80 nm were cut on a Reichart Ultracut UCT, stained with uranyl acetate, followed by lead citrate, and viewed on a JEOL 1200EX transmission electron microscope at 80 kV. Photographs were taken with a Zeiss digital imaging system.
Triglyceride clearance
Mice were weighed at 1000 h and were given 15 µl olive oil/g body weight by gastric gavage. Approximately 20 µl blood were collected at 0, 2, 4, and 12 h and assayed for triglycerides (TG; Sigma-Aldrich Corp., St. Louis, MO) and nonesterified free fatty acids (WAKO Diagnostic Products, Richmond, VA). The mice were denied access to food during the course of the study.
Measurement of tissue lipoprotein lipase (LPL) activity
Enzyme activity in adipose tissue was performed as previously described (18). Briefly, tissue homogenates were incubated with a substrate mixture containing [carboxyl-14C]triolein, and nonesterified fatty acids (NEFA) released by LPL were separated and counted. LPL activity was expressed as microunits (1 mU = 1 µmol NEFA released/h of incubation at 28 C). Data are expressed as LPL activity per gram of tissue. The interassay coefficient of variation (4.1%) was using bovine skim milk as a standard source of LPL.
Measurement of glucose flux
Adult (7- to 10-month-old) male Tg mice and their Wt littermates (30–35 g) were anesthetized with chloral hydrate (400 mg/kg body weight, ip) and catheterized through the right internal jugular vein. The venous catheter was used for infusion while blood samples were collected from the tail vein. Each animal was monitored for food intake and weight gain for 3–4 d after surgery to ensure complete recovery. Euglycemic insulin (1 or 4 mU/kg·min) clamps were performed in conscious catheterized mice as previously described (7). Food was removed for 5 h before beginning the in vivo studies. A solution of glucose (10%) was infused at a variable rate as required to maintain euglycemia (6 mM). Mice received a constant infusion of HPLC-purified [3H]glucose (0.1 mCi/min; NEN Life Science Products, Boston, MA) or insulin (1 or 4 mU/min·kg body weight). Thereafter, plasma samples were collected to determine glucose levels as well as [3H]glucose specific activity (at 40, 50, 60, 70, 80, and 90 min). Consecutive samples were used for assessment of plasma insulin levels. Steady state conditions for both plasma glucose concentration and specific activity were achieved by 40 min in these studies. In the final stage, mice were anesthetized (60 mg pentobarbital/kg body weight, iv), the abdomen was quickly opened, and the liver was freeze-clamped in situ with aluminum tongs cooled in liquid nitrogen. The time between the injection of anesthesia and the freeze-clamping of tissue samples never exceeded 60 sec. Tissue samples were stored at -80 C for further analysis. The euglycemic clamp protocol was approved by the institutional animal care and use committee of the Albert Einstein College of Medicine.
High fat feeding and oral glucose tolerance tests
A cohort of mice was maintained on a high fat diet from weaning at 3 wk to 6 months of age (59% of calories derived from fat; D12492 Research Diets, Harlan Teklad, Madison, WI). Fasted mice (5 h) were given an oral glucose load (2.5 mg glucose/g body weight using a solution of 10% glucose in physiological saline). Blood was collected at 0, 30, 60, and 120 min and used for glucose (Sigma-Aldrich Corp.) and insulin (Linco Research, St. Charles, MO) measurements. Access to food was denied during the course of the study.
Statistical analysis
Results are shown as the mean ± SEM. Statistical analysis was performed by unpaired t test (nonparametric), assuming unequal variance unless otherwise indicated. Significance was accepted at P < 0.05.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The strategy was initially developed in tissue culture to elucidate the biogenesis of the adiponectin complex.
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Gly-adiponectin) interferes with the folding of individual trimer units.
The effects on adiponectin secretion can be inverted based on the expression of full-length to Gly-adiponectin. Low Gly-adiponectin expression increases the secretion of full-length adiponectin, whereas high levels lower the secretion of full-length adiponectin. This was shown in a dose-dependent induction of the mutant protein in 3T3-L1 adipocytes by Western blot analysis under reducing conditions (Fig. 1B
). Note that a greater exposure period was needed to detect the intracellular expression of Gly-adiponectin compared with full-length adiponectin, and that Gly-adiponectin is not secreted under any circumstances. This effect can be exclusively achieved at the level of secretion, as endogenous adiponectin mRNA levels are not altered by Gly-adiponectin (data not shown). This is consistent with our previous observations that point to a critical posttranscriptional component in the relationship of sexual dimorphism to adiponectin levels (20). This suggested that a pool of endogenous adiponectin within the secretory pathway of adipocytes could be effectively released through low expression of Gly-adiponectin.
More detailed analysis of the secretion kinetics for adiponectin using a pulse-chase study revealed that near-maximal levels of secretion could be achieved within 3 h after initiation of the chase. The remaining material was retained intracellularly and slowly degraded (Fig. 1C). This is in stark contrast to the secretion kinetics of resistin, another adipocyte-specific secretory protein that could be almost quantitatively secreted under the same conditions. The inability of the remaining intracellular adiponectin pool to be released is not a reflection of a high level of misfolding, as a number of treatments (e.g. treatment with ß3 agonists or reducing agents) can effectively tap into this intracellular pool and release it (Berg, A., and U. Pajvani, unpublished observations). This suggests that under normal conditions, a futile cycle is in place that is characterized by high level production of adiponectin, followed by degradation of nearly 50% of the newly synthesized material. Degradation most likely involves the cytosolic proteasomal complex, because treatment of cells with MG132, a proteasomal inhibitor, leads to a significant accumulation of intracellular adiponectin, as shown by Western blot analysis in 3T3-L1 cells (Fig. 1D). In the absence of Gly-adiponectin, normal basal levels of full-length adiponectin are secreted. Upon low level induction of Gly-adiponectin, a small amount of mixed trimers is formed, blocking the futile degradation cycle of the Wt protein. This significantly increases the secretion of full-length adiponectin. At higher levels of the mutant protein, the deletion construct effectively integrates into all trimers formed, thereby quantitatively blocking the secretion of almost all of the full-length adiponectin. The net result of transgene expression is therefore a more efficient secretion of endogenous Wt adiponectin complexes.
Based on our observations in tissue culture, we expressed Gly-adiponectin under the control of a fat cell-specific aP2 promoter in mice. Tg mice with Wt adiponectin under control of the more ubiquitously expressed CMV promoter were also generated in parallel. The results from three and four founder lines are shown for both constructs (Fig. 2B). In the case of CMV-adiponectin, a much larger number of founders was analyzed (>30), but they are not shown. RT-PCR analysis of gonadal fat with transgene-specific primers showed three lines expressed Gly-adiponectin, but only line 2 had increased circulating levels in serum (Fig. 2C). Because Gly-adiponectin females are infertile, the line was propagated through Tg males and Wt females that yielded a Tg birth rate of 50%.
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Gly-adiponectin and line 3 of CMV-adiponectin mice developed an unusual phenotype at midlife that included expansion of the interscapular region and bilateral exophthalmia (Fig. 2D). An increase in serum adiponectin correlated with the appearance of this phenotype. The expression of either aP2-Gly-adiponectin or CMV-adiponectin without an increase in serum adiponectin did not produce expansion of the interscapular region and exophthalmia in old age (24–30 months).
Line 2 from aP2-Gly-adiponectin was chosen for further analysis because it showed the greatest elevation of serum adiponectin. A survey of transgene expression by RT-PCR in various fat pads and other tissues (Fig. 3A) revealed that Gly-adiponectin expression under the aP2 promoter overlapped endogenous adiponectin expression and is thus limited to adipose tissue. Adiponectin levels in various fat pads (interscapular, inguinal, mammary, and epididymal) were elevated in both male and female Tg mice. Interestingly, perimetrial (uterine) fat differed from all other fat pads examined in that a reduction of adiponectin was observed in the Tg animals (Fig. 3B). As the clearance of adiponectin was not markedly different in Wt and mutant mice (data not shown), the net increase in serum in Tg mice was primarily due to increased secretion of the protein from a range of adipose pads.
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Gly-adiponectin allowed endogenous adiponectin to escape degradation, the early rise in serum levels and the sexual dimorphism were both preserved in Gly-adiponectin mice at an overall higher level. Both male and female Gly-adiponectin mice exhibited serum adiponectin levels 3-fold above those in their age- and sex-matched Wt littermates (Fig. 4A).
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). This strongly suggests that the complexes generated in Tg mice fold properly and resemble Wt protein in their oligomeric make-up.
Male Tg mice displayed similar body weights as their Wt littermates up to 1 yr (Fig. 4C). Tg females, however, started to weigh significantly more than their Wt littermates around 16 wk of age and continued to be heavier until 1 yr of age when they displayed a small, but significant, 15% increase in overall body mass (Fig. 4C).
Chronic elevation of adiponectin does not affect food intake, but leads to an increase in adiposity in females
At 6–8 months of age, we determined the percent body fat content in males and females. There was a significant increase in percent total body fat by that age in the females, which reached approximately 30% of their total body weight, in contrast to 18% in their Wt siblings (Fig. 5A). The males did not display any significant differences at that age (data not shown). These differences in the females could not be explained by a difference in food intake within the accuracy with which these experiments can be performed (Fig. 5B). We were also unable to observe any changes with respect to the average 24-h energy expenditure (Fig. 5C), nor were there differences with respect to the respiratory quotient in either the resting or the active state (Fig. 5D).
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) that was particularly striking in a cross-sectional view (Fig. 6B). Magnetic resonance imaging strongly suggested that the expansion consists primarily of lipid mass (Fig. 6B). A histological analysis at the light microscopic (Fig. 6C) and electron microscopic (Fig. 6D) levels clearly identified the expanding tissue mass as adipose tissue. Interscapular brown adipose tissue (BAT) could be identified in both Wt and Tg mice on the basis of its darker brown appearance. Rather than the classical multilobular appearance of the lipid droplets as they appeared in Wt mice, the Tg BAT had a more unilocular appearance.
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). High resolution magnetic resonance imaging (Fig. 7B) and histological analysis (Fig. 7C) clearly indicated the intraconal fat pad as the primary cause of the proptosis. There was no indication of immune infiltration or inflammatory activity within the muscle fibers, but individual orbital muscles separated into multiple fibers by the expansion of adipose tissue. At the orbital apex, the tendinous ring insertion of the ocular muscles dilated as fat prolapsed. Chronic exposure keratopathy due to proptosis ultimately ulcerated the entire cornea and resulted in globe perforation with hemorrhagic ulcerated plaque clinging to the sclera beneath, followed by complete globe shrinkage and fibrosis (phthisis). The intraconal fat further expanded between the straps of ocular muscles. The proptosis worsened progressively and mice were usually euthanized by 15 months of age.
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). The primary source of expanding fat in Tg mice therefore appears to originate from BAT-like tissue, as judged by low level UCP1 expression even in Wt fat (Fig. 7E). This adipose mass expanded laterally, eventually wrapping around the entire neck. With age, the adipose tissue stretched even further and included regions of the head, including the submandibular fat pad (see three-dimensional nuclear magnetic resonance movie of Wt and Tg published as supplemental data on The Endocrine Society’s Journals Online web site, http://endo.endojournals.org). Macroscopic appearance and weight were comparable between Wt and Tg in all other adipose pads examined. The expansion of the interscapular and orbital adipose tissues was significantly delayed in males.
Chronic elevation of adiponectin affects PRL levels
Serum levels of adiponectin are under complex hormonal control (20). To test whether chronic adiponectin overexpression feeds back on any factors known to affect adiponectin levels under normal conditions, we determined serum levels of a number of hormones and metabolites. Table 2 shows that levels of TNF
and glucocorticoids were normal; the pituitary hormones GH and TSH were also comparable in Wt and Tg. Surprisingly, despite overall increased fat mass in Tg females, leptin levels were comparable as well. In contrast, Tg males and females displayed a 2-fold increase in PRL levels. PRL exerts a strong negative influence on serum adiponectin levels in Wt mice (20). The chronic elevation of PRL levels in Tg animals may therefore reflect a compensatory mechanism to lower the elevated concentration of adiponectin in serum.
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, A and B) levels before the gavage were vastly overlapping. However, subsequent to the gavage, plasma levels of TG and NEFAs were consistently lower in the Tg animals, never approaching the peak levels found in Wt females. These observations suggest that chronically increased levels of adiponectin in females have a positive impact on TG clearance and are consistent with acute pharmacological effects that lead to a decrease in plasma TG upon injection of recombinant adiponectin. The more modest increases in serum adiponectin levels in males are not sufficient to cause any changes in lipid clearance, because the kinetics of TG and NEFA disposal are vastly overlapping between Wt and Tg males. A partial explanation for this effect is revealed upon analysis of adipose tissue lipoprotein lipase activity (Fig. 8C). The Tg females displayed significantly elevated LPL activity per gram of tissue in the fed state in a number of different fat pads. Similar trends were observed in the fasted state (not shown). Consistent with the improved clearance of TG that could only be observed in females, the increased lipoprotein lipase activity was observed in females only. Additional metabolic parameters were analyzed in these mice, but did not reveal any significant differences (Table 3).
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activation, we tested whether we would detect increased PPAR2 levels in Tg mice. At least in a subset of white fat pads (Fig. 9B shows abdominal fat as a representative example), PPAR2 mRNA levels were markedly increased, particularly in females. Furthermore, resistin mRNA levels were slightly induced compared with control levels, and a very significant induction of mRNA levels of several mitochondrial markers (the ADP/ATP translocator and the dicarboxylate transporter are shown) was detected.
Euglycemic insulin clamps reveal dramatically improved hepatic insulin action in Tg adiponectin mice
To determine whether chronic increases in circulating levels of adiponectin improve glucose tolerance by increasing glucose uptake, decreasing glucose production, or both, we used the insulin clamp technique on a cohort of 7- to 10-month old male and female mice that were fed a standard laboratory chow diet. Insulin infusion rates were selected to raise plasma insulin levels similarly in all groups, and a variable infusion of a 10% glucose solution was started and periodically adjusted to maintain the plasma glucose concentration at approximately 5 mM for the rest of the study. Similar to the acute administration of recombinant adiponectin, the rate of disappearance of glucose was not altered in Tg mice, indicating that whole body glucose uptake was not affected. However, with either low (1 mU/kg·min) or high (4 mU/kg·min) insulin, glucose production in Tg females was lower than that in Wt mice, indicating greater insulin sensitivity in the Tg animals (Fig. 10, A and B). This effect became even more striking with hyperinsulinemia, which demonstrated a marked decrease in the rates of glucose production in Tg animals compared with Wt mice in both females and males (Fig. 10B). Therefore, both male and female Tg mice exhibit increased hepatic insulin sensitivity. The relevant metabolic parameters for these experiments are indicated in Table 4. From that table it is apparent that the Tg mice displayed a marked decrease in postabsorptive plasma insulin concentrations compared with Wt mice. Although female Tg mice do show initial signs of increased interscapular fat mass at this age, it is important to note that the males do not display any detectable differences in fat distribution between Wt and Tg animals, emphasizing that the improvement in hepatic insulin sensitivity is not a consequence of the interscapular fat hyperproliferation.
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), consistent with our own observations in primary hepatocytes upon treatment with adiponectin (not shown) and in vivo results by other groups (23), further corroborating that elevated adiponectin levels will lead to activation of AMPK.
Elevated circulating adiponectin improves oral glucose tolerance
Our previous studies suggested that pharmacological levels of recombinant adiponectin dramatically improve hepatic insulin sensitivity in Wt and type I and type II diabetic mice. To further test whether the constitutively elevated adiponectin levels in our Tg mice have an impact on insulin sensitivity, we subjected a cohort of high fat-fed Wt and Tg males to an oral glucose tolerance test. The presence of the transgene did not prevent weight gain during a high fat diet for 6 months. A modest increase in body weight was observed in female Tg compared with their Wt littermates, similar to chow-fed animals (Fig. 11A). The Tg females responded with a significantly improved glucose excursion during an oral glucose tolerance test, indicating improved insulin sensitivity compared with Wt littermates (Fig. 11B). Although glucose disposal was comparable between Wt and Tg, the males not only displayed reduced insulin levels in the basal state, but also achieved normal glucose disposal with significantly lower insulin levels. Female Tg demonstrated improved glucose clearance compared with Wt littermates and required less insulin to achieve these improved clearance kinetics (Fig. 11C). This indicates that the Tg animals are vastly resistant to diet-induced decreases in insulin sensitivity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epidemiological studies in humans and animal models describe a close association between insulin sensitivity and adiponectin levels within a 2- to 3-fold range from baseline. Consistent with the effects of a pharmacological 2- to 3-fold elevation of adiponectin after the injection or infusion of recombinant adiponectin (4, 7), congenital elevation of adiponectin led to improved hepatic insulin sensitivity. Female Tg mice exhibit a significant increase in the rate of oral lipid clearance, consistent with the phenotype reported for mice carrying a genetic deletion of the adiponectin locus, which display delayed clearance of free fatty acids after injection of a TG emulsion. A recent study by Matsuzawa and colleagues (9) in adiponectin-/- mice focuses on a mechanism involving free fatty acid uptake and oxidation by muscle in line with original suggestions by Lodish and colleagues (8). Although this represents a potential mechanism, we are also intrigued by the effects of adiponectin overexpression on serum TG clearance. It will be interesting to determine whether adiponectin has additional effects on lipolysis or lipoprotein clearance by the liver, perhaps via the same mechanisms as those responsible for the liver glucose effects. Additionally, we reported (22) that mice lacking caveolin-1 display dramatically reduced adiponectin levels coupled with severely elevated TG and free fatty acid levels, especially in the postprandial state. In light of the findings presented here, this suggests that the postprandial hypertriglyceridemia may be functionally related to the reduced adiponectin levels in the caveolin-1-/- mice. It is intriguing that the lack of caveolin-1 triggers reduced adiponectin levels in serum, whereas the Tg up-regulation of adiponectin in Wt mice causes increased adipose caveolin-1 expression. The dramatically increased lipoprotein lipase activity that we observe in female adipose tissues analyzed could contribute to increased clearance of a postprandial TG load. Similar increases in LPL activity have recently been reported in PPAR agonist-treated rats and linked to improvements in postprandial TG clearance (18).
Humans and mice display a sexual dimorphism for circulating adiponectin values, with female levels increased compared with those in males. This sexual dimorphism is preserved in Tg mice, with the transgene effectively amplifying the host’s endogenous levels. Due to overexpression, Tg male mice have levels of adiponectin approximately equal to those in Wt females. The serum concentrations found in Tg males are therefore physiological and readily found in Wt mice, albeit in a different (female) hormonal milieu. Female Tg have levels approximately 3-fold above those in Wt females. Relatively small changes in serum adiponectin concentrations can have a significant effect on insulin sensitivity. An example is seen in heterozygous mice (+/-) with only one functional allele for adiponectin. These mice have approximately 40% of the Wt adiponectin serum levels, but they have a significantly reduced insulin sensitivity (10). Wt females with higher circulating adiponectin levels are more insulin sensitive than Wt males (20). Acute injections that alter circulating levels by as little as 2-fold have a profound impact on insulin sensitivity (4, 7).
Finally, a number of epidemiological studies in humans that draw strong positive correlations between insulin sensitivity and adiponectin levels focus on values that vary by less than 2- to 3-fold from baseline. Along with the sexually dimorphic expression pattern, we observed various sexually dimorphic phenotypes in a number of different areas. By midlife, the female Tg mouse exhibits a moderate increase in body weight due to a dramatic increase in interscapular adipose mass. The expansion of interscapular and orbital adipose tissues is also apparent in males older than 20 months. We previously showed that estrogen represses serum adiponectin levels (20) and that ovariectomy results in a significant increase in adiponectin levels in Wt mice. Interestingly, consistent with our hypothesis that the expansion of interscapular and orbital adipose pads is a direct consequence of increased serum adiponectin levels, ovariectomy accelerated the remodeling of fat tissue in the Tg by 3–4 months (data not shown).
Based on the reports by Fruebis et al. (8) that increased levels of the globular domain of adiponectin lead to increased ß-oxidation in muscle, we suspected that overexpression of adiponectin might lead to a lean phenotype. This is clearly not the case. There is overall an increase in fat mass in females and relatively normal levels in males. Indirect calorimetry confirmed that there is no evidence for an increased metabolic rate in these mice. Importantly, the studies by Fruebis et al. (8) focused primarily on the effects of the globular domain, whose pharmacological activities become increasingly more established. However, this globular domain, a putative proteolytic fragment, is difficult to visualize in vivo, and it is not clear at this stage whether this fragment is a bona fide processing intermediate or represents a pharmacological antagonist only. We have also not been able to detect any specific adiponectin degradation products in the Tg mice with either amino- or with carboxyl-terminal-specific antibodies. We cannot exclude the possibility that there is a rate-limiting step along the activation pathway that we do not overcome by mere overexpression of the full-length product. Nevertheless, the overproduced material in Tg mice is, by all possible criteria established to date, identical to endogenous adiponectin observed under normal conditions. Adiponectin in serum of Tg mice forms complexes of similar molecular weight as adiponectin in Wt mice, with discrete trimer-dimers and a high molecular weight complex. However, based on the data presented, it is clear that overexpression of full-length adiponectin is sufficient to cause marked improvement of insulin sensitivity in liver. Yamauchi et al. (11) recently reported overexpression of the globular adiponectin fragment from a liver-specific promoter. When analyzed in a leptin-deficient ob/ob background, these mice displayed increased expression of enzymes involved in ß oxidation and energy dissipation (such as uncoupling proteins), but did not display decreased adipose mass due to a compensatory increase in food intake. Even though these effects of the globular domain of adiponectin are well established, data from a number of different laboratories indicate the fundamentally different nature of this globular domain compared with the full-length protein found in vivo.
Consistent with a recent report by Kadowaki and colleagues (23) demonstrating activation of AMPK by full-length adiponectin in the liver, we observed increased activation of AMPK in our Tg mice at the end of the clamp studies. This further corroborates the functional link between increased adiponectin levels in serum and AMPK activation in the liver.
Yokota et al. (24) reported that bacterially produced recombinant adiponectin blocked fat cell formation in long-term bone marrow cultures and inhibited the differentiation of cloned stromal preadipocytes. We have no evidence for an inhibitory activity on differentiation based on the expression of other differentiation-related marker proteins, such as resistin (3, 16), whose expression is, in fact, slightly elevated in fad pads from Tg animals. On the contrary, increased levels of adiponectin cause expansion of interscapular and orbital pads. The hyperproliferative response of the interscapular adipose tissue is not unique and has been reported previously. Tg overexpression of serum response element-binding protein-1c (SREBP-1c) in adipose tissue leads to development of a large fat mass in the interscapular region consisting of an enlarged, pale, bilobed fat pad highly similar in our mice (25, 26). These mice, in contrast to ours, are severely insulin resistant due to a generalized lipodystrophy, lack of leptin, and the presence of fatty livers.
Acute treatment of mice with thiazolidinediones has selective proliferative effects on interscapular brown adipocytes as well and increases UCP1 expression (27), eventually resulting in a very significant increase in adipose tissue in the interscapular region. These observations were recently also reported in both rats and monkeys treated with the potent PPAR agonist darglitazone that resulted in hyperproliferation of a number of fat pads, in particular the dorsal thoracic and the interscapular brown adipose pads (28). It is noteworthy that thiazolidinediones also trigger a significant and sustained increase in circulating adiponectin levels, similar in magnitude to the increases achieved in our Tg model (12). Additional similarities to chronic PPAR activation are found at the level of adipose tissue LPL induction that can be observed upon PPAR agonist treatment (18) as well as in mice chronically overexpressing adiponectin, as demonstrated here. Interestingly, Wilson-Fritch and colleagues (29) recently reported that treatment of 3T3-L1 adipocytes with the PPAR agonist rosiglitazone leads to a dramatic up-regulation of a host of nuclear-encoded mitochondrial proteins, consistent with other observations made in WAT of rats and dogs that describe significant effects of rosiglitazone treatment on mitochondrial number and morphology (30). These increased levels of mitochondrial proteins may explain in part the ability of PPAR agonists to increase ß oxidation, which results in an increased clearance of free fatty acids that, in turn, can improve insulin sensitivity (31). Finally, even in humans there is an apparent propensity for hyperproliferation of a similar fat pad in the context of human immunodeficiency virus-mediated lipodystrophies, particularly in individuals undergoing highly aggressive antiretroviral therapy (HAART) (32, 33). There is, therefore, an increased propensity for the adipose tissue located in the interscapular region to undergo proliferation under a number of different conditions. Mechanistically, however, we do not know the link between serum response element-binding protein-1c overexpression in adipose tissue, thiazolidinedione-increased serum adiponectin levels, and human immunodeficiency virus protease inhibitors.
Orbital fat proliferation is a unique phenomenon among animal models. Only one other mouse model, the TSH receptor-induced model of Graves’ ophthalmopathy, has displayed a similar phenotype, albeit on a much less dramatic scale (34). In this respect, our Tg mice recapitulate some phenotypic changes associated with Graves’ exophthalmia. However, the Tg mice do not show evidence of hyperthyroidism or TSH receptor autoantibodies. It is therefore unlikely that the underlying causes of Graves’ disease are directly linked to adiponectin overexpression. It is noteworthy that in a small cohort of Graves’ patients displaying exophthalmos, we found serum adiponectin levels elevated by a factor of 2–3 above average levels and are currently testing whether the increased serum adiponectin is due to the hyperthyroidism per se (Combs, T. P., G. Baker, E. Ludgate, and P. E. Scherer, unpublished observations). Nevertheless, this suggests that the hypertrophy of the orbital fat pad observed in a subset of Graves’ patients may be directly linked to the elevation of serum adiponectin, which, in turn, is secondary to a hormonal change induced by the autoimmune reaction. Consistent with that, a recent case has been reported in which a type 2 diabetic patient, upon initiation of treatment with the PPAR agonist pioglitazone, experienced exacerbation of his thyroid eye disease, which had been stable and inactive for more then 2 yr. This patient displayed a massive expansion of the orbital fat pad (35).
This is the first time that orbital proliferation has been observed in mice to such a significant extent. Therefore, this mouse model offers a tool to study noninflammatory fat proliferation as often seen in Graves’ ophthalmopathy.
It is surprising that the female Tg mice display such dramatic improvements in insulin sensitivity despite their increased adipose mass. Again, this phenomenon resembles the effects of thiazolidinedione treatment, which is known to lead to increases in serum adiponectin levels, insulin sensitivity, fat cell differentiation, and fat mass. Importantly, though, we ruled out the possibility that the improvement in insulin sensitivity is a secondary effect of the interscapular hyperproliferative fat pad, because we observed similarly striking improvements in insulin sensitivity in young male mice that showed no evidence of adipose depot redistribution.
In a very recent paper, Yamauchi and colleagues (36) reported the cloning of two receptors for adiponectin. Interestingly, one of these receptors shows preferential binding to the full-length native version of adiponectin and is predominantly expressed in the liver. The other receptor displays preferential binding to the globular trimeric version of adiponectin and shows a more ubiquitous tissue distribution, with strong expression in muscle. This is fully consistent with previous reports that show a differential effect of the two ligands on liver and muscle, respectively. It will be interesting to determine whether these receptors play any role in the differential adipose tissue distribution observed in the Tg mouse model presented here.
In summary, this is the first mouse model in which adiponectin levels are chronically elevated. This elevation is within physiological levels and accurately recapitulates the changes in endogenous adiponectin levels during maturation of the animal. These elevated serum adiponectin levels dramatically improve insulin sensitivity and at a later age lead to the hypertrophy of select fat pads. It is not clear whether this hypertrophy is the consequence of an autocrine effect of adiponectin or an indirect effect of improved systemic insulin sensitivity or is due to a hormonal change secondary to adiponectin overexpression. Consistent with our acute pharmacological studies, chronic elevation of full-length adiponectin in serum had a positive effect on insulin sensitivity, primarily in the liver. As several groups have implicated adiponectin as a possible mediator of thiazolidinedione action (12, 37, 38, 39), it is striking that chronic elevation of adiponectin by means of Tg overexpression causes effects that are vastly overlapping with those of chronic treatment with thiazolidinediones. This further strengthens the relationship between the effects of thiazolidinedione treatment and the induction of adiponectin as a prerequisite step to achieve clinical improvement in insulin sensitivity.
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Abbreviations: AMPK, 5'-AMP-activated protein kinase; BAT, brown adipose tissue; CMV, cytomegalovirus; GDI, GDP dissociation inhibitor; LPL, lipoprotein lipase; MCS, multiple cloning site; NEF
, Female Wt mice; 
, female Tg littermates. The levels are indicated as arbitrary densitometric units. C, Transgene expression assessed by nonquantitative RT-PCR using primers that recognize the endogenous Wt adiponectin and the 
) and their Wt littermates (
) measured in a longitudinal 1-yr study by Western blot analysis. B, Size distribution of circulating adiponectin in 4- to 6-month-old female (left) and male (right) Tg mice (
). Gas exchanges were measured in mice individually housed in metabolic chambers, whereas activity was monitored with an infrared light movement sensor. Resting values for energy expenditure were calculated from measurements obtained during times when activity sensors recorded less than 20 events in a 20-min interval. Gas exchanges values were the mean of measurements made every 20 min for a 24-h period after a 24- to 48-h acclimation period. Each group represents 8–10 mice. *, P < 0.05, active is significantly higher than resting.
