Proc Natl Acad Sci U S A 109(15): 5874-5879

PMC: PMC3326476

PMID: 22451920

Dichotomous effects of VEGF-A on adipose tissue dysfunction

Generation of Transgenic Mice with AT-Specific Expression of VEGF-A.

Fig. S1A shows a schematic representation of the mouse model. The tetracycline responsive element (TRE)-driven VEGF-A transgenic model has been described previously (14). In our model, the TRE is regulated by the reverse tetracycline-dependent transcriptional activator (rtTA), whose expression is under the control of the AT specific adiponectin promoter (15). VEGF-A in the double transgenic mouse is induced in the presence of Dox. To induce VEGF-A within a physiological range, we titrated different doses of Dox as food admixtures from 600 mg/kg to 60 mg/kg. We avoided high doses of Dox (600 mg/kg) to circumvent pathological changes associated with supraphysiological local levels of VEGF.

By titrating the effective doses of Dox, we identified that lower levels of Dox (60 mg/kg) induced VEGF-A within a physiological range without causing edema formation. All subsequent experiments were performed at these lower doses. Expression of VEGF-A in a number of fat pads was assayed in the presence or absence of 60 mg/kg Dox treatment. In EWAT, the transgenic VEGF-A mRNA levels were induced in the Dox-treated double transgenic mice only, whereas VEGF-A levels were undetectable in mice carrying either one of the transgenes as well as double transgenic mice without Dox treatment (Fig. S1B). VEGF-A was predominantly induced in white adipose tissues (WATs) (Fig. S1C), whereas only moderate induction was seen in brown AT (BAT). No induction was observed in other tissues, such as the liver (Fig. S1C). Circulating VEGF-A levels were not significantly increased (Fig. S1D). Taken together, our system allows inducible expression of VEGF-A, with expression restricted to ATs.

Overexpression of VEGF-A Promotes Local Angiogenesis in WAT.

Confocal fluorescent imaging reveals that compared with the control group, a higher vessel density is seen in the subcutaneous WAT (SWAT) of VEGF-A Tg mice (Fig. 1A). This differential vessel density is not observed in other tissues, e.g., in BAT (Fig. 1A), confirming the specificity of the VEGF-A effect in WAT. We then examined the levels of endothelial cell marker CD31. mRNA levels of CD31 were significantly induced in SWAT (Fig. 1B). The primers for q-PCR are listed in Table S1. Immunohistochemical analysis further confirmed enhanced CD31 staining in transgenic mice (Fig. 1C). Analysis of SWAT with anti-VEGF R2 antibodies also revealed an increased signal in the VEGF Tg mice (Fig. 1D). More importantly, our analysis with antiphospho-specific VEGFR2 also reveals more signal intensity in the SWATs of Tg mice (Fig. 1D). Activation of VEGF-A receptor 2 is considered to be a critical mediator of VEGF-A triggered proangiogenic function (16).

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Fig. 1.

VEGF-A stimulates angiogenesis in WAT. (A) Functional blood vessels in SWAT and BAT visualized by tail-injected rhodamine tagged lectin-1 in VEGF-A Tg and control mice. Blood vessels are shown in red and the nuclei are in blue, visualized by DAPI staining. The images were captured with confocal microscope. (B) q-PCR analysis of CD31 in SWAT of VEGF-A Tg and control mice (n = 4 in controls; n = 5 in VEGF-A Tg). The difference was analyzed by Student's t test. *P < 0.05. (C) Immunohistochemical analysis with α-CD31 in SWAT of VEGF Tg or their littermate controls. (Scale bar, 50 μm.) (D) Immunohistochemical analysis with α-VEGF receptor 2 and α-phosphorylated VEGF receptor 2 in SWAT in both VEGF Tg and controls. (Scale bar, 50 μm.)

Overexpression of VEGF-A in AT Improves Glucose Tolerance and Insulin Sensitivity in HFD Challenged Mice.

VEGF-A Tg mice weighed slightly less than control mice as we monitored body weights beyond week 7 (Fig. S2A). The modest body weight difference is due to a difference in fat mass in the VEGF-A Tg mice (Fig. S2A). Importantly, there were no signs of vascular leakage or edema formation, because NMR measurements show no difference in interstitial fluid in the transgenic mice (Fig. S2A). Both white fat pads examined (EWAT and SWAT) showed an increased density in vascular perfusion that is apparent, even at the level of the whole tissue, as judged by the darker color of both pads (Fig. S2B). The fasting levels of blood glucose and insulin were lower in transgenic mice (Fig. S2D). Five weeks after the initial HFD exposure, there were not yet any body weight differences between the groups. The oral glucose tolerance tests (OGTTs) demonstrate that in response to the glucose challenge, glucose tolerance in the VEGF-A Tg mice is significantly improved (Fig. 2A). Insulin sensitivity, as measured by insulin tolerance tests (ITTs), was also improved in the VEGF-A Tg mice (Fig. S2C). These results indicate a high degree of metabolic resistance to a HFD challenge upon supplementation of VEGF-A.

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Fig. 2.

VEGF-A Tg mice exhibit an improved metabolic profile. (A) Circulating glucose levels measured during an OGTT (n = 5 per group) 5 wk after HFD feeding. The difference at each time point was analyzed by Student's t test. *P < 0.05; **P < 0.001. (B) Indirect calorimetry was performed in a CLAMS system by housing mice after HFD plus Dox feeding for 6 wk. VO2 and RER (VCO₂/VO₂) were calculated from the average results during a 24-h light and dark cycle. Absolute contributions of carbohydrate and lipid metabolism to total energy expenditure and accumulated food intake were measured (ACC). *P < 0.05; **P < 0.001.

Overexpression of VEGF-A in AT Promotes an Increase in Lipid Clearance and a Decrease in HFD-Induced Hepatic Steatosis.

A lipid tolerance test showed that VEGF-A Tg mice cleared an oral lipid challenge much more efficiently than the littermate controls (Fig. S3A). Furthermore, both the cholesterol and free fatty acid (FFA) content in the livers were significantly lower in the VEGF-A Tg mice. Liver triglyceride (TG) levels also show a trend toward a decrease (Fig. S3C). H&E staining indicates fewer and smaller lipid droplets in hepatocytes from VEGF-A transgenic mice (Fig. S3D). Systemically, the improvements in lipid metabolism resulted in lowered plasma FFAs (Fig. S3B). The improvements in lipid parameters are, at least in part, due to higher levels of lipoprotein lipase (LPL), because mRNA levels of LPL in both adipose tissue and in the heart increased significantly (Fig. S3E). Collectively, these data highlight that AT VEGF overexpression results in significant systemic improvements in lipid metabolism.

Overexpression of VEGF-A in AT Leads to an Increase in Energy Expenditure.

The energy expenditure in metabolic chambers data indicate that the rate of oxygen consumption (VO₂) was significantly increased in the VEGF Tg mice (Fig. 2B). They also had a significantly higher respiratory exchange rate (RER), indicating that a higher proportion of energy expended in the transgenics derives from glucose metabolism (Fig. 2B). However, the absolute rate of lipid oxidation is similar between VEGF Tg and their littermate controls, whereas the use of carbohydrate is increased in the VEGF Tg mice (Fig. 2B). VEGF-A overexpression leads therefore to an increase in energy expenditure, and the increase is primarily driven by an increase in carbohydrate metabolism. Moreover, VEGF Tg mice have an increase in food intake (Fig. 2B), further illustrating the higher energy turnover in light of the weight differences.

Overexpression of VEGF-A Decreases Adipocyte Size and Enhances a “Brown Adipose” Phenotype in WAT.

Histological examination revealed dramatic differences, including smaller-sized adipocytes in WATs of VEGF Tg mice (Fig. 3A and Fig. S4A). Furthermore, the reduction of lipid depots and a more multilocular appearance of the SWAT cells are characteristic features of BAT (Fig. 3A; and see Fig. S6B). Indeed, both qPCR and Western blotting revealed marked increases in PGC-1α and UCP-1 levels (Fig. 3 B and C and Fig. S4C), lending further support for a browning of WATs in the VEGF-A Tg mice (13, 17). We also observed that mRNA levels for PPARγ and ZFP423 were unaffected (Fig. S4B). In VEGF Tg mice, leptin levels in both AT and serum were significantly decreased (Fig. S5A). Even though there was no difference at the level of adiponectin mRNA (Fig. S5B), circulating adiponectin levels decreased slightly, whereas intracellular pools were increased (Fig. S5 C and D), suggestive of a reduction in adiponectin secretion.

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Fig. 3.

VEGF-A Tg mice have smaller white adipocytes with BAT-like properties. (A) H&E staining of SWAT of VEGF-A Tg mice after HFD plus Dox feeding for 8 wk. (Scale bars, 50 μm.) (B) q-PCR analysis of PGC-1α and UCP-1 in EWAT of VEGF-A Tg and their littermate controls. The readings are normalized to hypoxanthine phosphoribosyltransferase (HPRT). *P < 0.05; **P < 0.001. (C) Western blot analysis for both PGC-1α and UCP-1 in WAT of VEGF Tg and their littermate controls. Results were normalized with β-actin.

Overexpression of VEGF-A Suppresses Hypoxia and Fibrosis and Reduces Local Inflammation in WAT During HFD Exposure.

Immunohistochemical assessment of local hypoxia using a hypoxyprobe indicates that hypoxia in WATs is significantly reduced in transgenic mice (Fig. 4A). Consistent with the reduction of hypoxic conditions, there is a significant reduction in the levels of HIF1α associated with induced VEGF-A overexpression (Fig. 4B). The fibrotic collagens III and VI (Col3 and Col6) are reduced (Fig. 4B). Interestingly, matrix metalloprotease-1 (MMP-1), one of the key enzymes facilitating the digestion of collagens in AT, was significantly induced in VEGF Tg mice (Fig. 4B). In line with these observations, a trichrome stain of SWAT and EWAT indicates that the extracellular matrix (ECM) accumulation is significantly reduced in the transgenic animals (Fig. S6B).

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Fig. 4.

VEGF-A overexpression ameliorates hypoxia, fibrosis, and inflammatory responses in WAT induced by HFD. (A) Immunohistochemical staining by hypoprobe-1 in WAT of VEGF Tg mice and their littermate controls after HFD plus Dox treatment for 8 wk. The darker staining on the Left indicates more hypoxic environment in controls. (B) q-PCR analysis of HIF-1α and its target collagen genes in EWAT. The readings are normalized to HPRT. *P < 0.05; **P < 0.001. (C) q-PCR analysis of inflammatory cytokines IL6, TNFα, SAA3, and macrophage marker F4/80 in EWAT. The readings are normalized to HPRT. *P < 0.05; **P < 0.001. (D) Immunohistochemical staining of F4/80 in SWAT of VEGF-A Tg mice. Red arrows indicate the crowns formed by accumulation of macrophages surrounding the dysfunctional adipocytes.

We further observed significantly decreased expression of inflammatory factors, such as IL6, F4/80, TNFα, and SAA3 by qPCR in EWAT of VEGF-A Tg mice (Fig. 4C). In line with these transcriptional changes, the levels of the generic plasma inflammatory marker serum amyloid A (SAA) were significantly reduced in VEGF Tg mice (Fig. S6A). Immunohistochemical analysis of both SWAT and EWAT with an anti-F4/80 antibody further confirms a reduced frequency of “crown-like” structures surrounding adipocytes in the VEGF-A Tg mice (Fig. 4D and Fig. S6C). Collectively, we conclude that local overexpression of VEGF-A in AT prevents tissue dysfunction.

Angiogenesis-Inhibitor Treatment During Early Stages of HFD-Induced Weight Gain Aggravates Systemic Metabolic Health.

To investigate the “loss-of-function” effect of angiogenesis, we used anti-VEGF monoclonal antibody Mcr84. This monoclonal antibody binds mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 (18). We treated 6-wk-old wild-type mice with Mcr84 or control IgG just before and throughout a 6-wk time course of HFD feeding. Whereas food intake, weight gain (Fig. S7A), and dissected fat pad size did not differ between groups, we observed an impaired response to the HFD in the Mcr84-treated mice. The Mcr84-treated mice required higher insulin to control their glucose levels during an OGTT (Fig. 5B), even though a comparable rate of glucose clearance was maintained, along with a reduction in lipid clearance (Fig. 5C). As a reflection of the dysfunctional ATs, the circulating adiponectin levels were lower in Mcr84-treated mice (Fig. S7C). In an attempt to assess metabolic flexibility, we measured FFAs in the fed and fasted state. Control mice display the anticipated increase and decrease of FFAs during the fasted and fed state, respectively. However, this dynamic regulation was absent in the Mcr84-treated mice (Fig. 5D). As expected, Mcr84-treated WAT displays a reduction in the amount of fluorescently labeled lectin (Fig. 5A). We also observed that the mRNA levels of CD31 decreased in SWAT but not in livers (Fig. S7B), indicating that angiogenesis inhibition is primarily affecting fat tissues. Furthermore, there was an increase in macrophage infiltration in EWAT in the Mcr84-treated mice (Fig. S7D). Despite similar overall fat pad weight, we observed an increase in average adipocyte cell size in the Mcr84-treated mice (Fig. S7D).

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Fig. 5.

Blockade of VEGF-A binding to VEGFR2 by Mcr84 accelerates metabolic dysfunction induced by HFD. (A) Functional blood vessels in AT labeled by rhodamine-tagged lectin. The mice were fed with HFD for 6 wk before the experiment. Mcr84 or control IgG were injected i.p at the initial stage and continued twice per week for the whole HFD feeding process. Blood vessels are shown in red and the nucleus in blue, by DAPI staining. (B) Insulin levels for an OGTT in Mcr84- or control IgG-treated mice (n = 5 per group) after antibody treated for 6 wk. *P < 0.05. (C) Triglyceride levels for a lipid clearance test in Mcr84- or control IgG-treated mice (n = 5 per group). *P < 0.05. (D) Serum nonesterified fatty acid (NEFA) levels in Mcr84- or control IgG-treated mice (n = 5 per group) after antibody treated for 7 wk. *P < 0.05.

Angiogenesis-Inhibitor Treatment in Dysfunctional AT Minimizes Body-Weight Gain and Enhances Insulin Sensitivity in the Context of ob/ob Mice.

Our previous observations demonstrated the metabolically challenged state of WAT in adult ob/ob mice (10). We therefore selected 6-wk-old ob/ob mice as our model. During a 3-wk treatment regimen, Mcr84-treated ob/ob mice gained slightly less body weight than their control IgG-treated littermates (Fig. S8A). Furthermore, the Mcr84-treated ob/ob group displayed a trend toward improvement of glucose-tolerance compared with the control group (Fig. S9B). Upon examination of acute fasting serum parameters, circulating FFA levels were significantly lower in the Mcr84-treated ob/ob mice compared with control IgG-treated mice (Fig. S9C). Circulating glucose levels following an overnight fast were lower in Mcr84-treated ob/ob mice compared with controls (Fig. S9C). Furthermore, the mRNA expression levels of the macrophage marker F4/80 significantly decreased in the Mcr84-treated mice (Fig. S9D).

Consistent with these observations, immunofluorescence staining of EWAT shows that the anti–Mcr84-treated animals displayed a marked reduction in vascular density (Fig. S9A). Similarly, the mRNA levels of CD31were significantly down-regulated in EWAT of anti–Mcr84-treated ob/ob mice (Fig. S8B). We also found a dramatic increase in adipocyte death as judged by the number of perilipin-negative lipid droplets in the AT of Mcr84-treated mice compared with control (Fig. S9E).

Animals.

TRE–VEGF-A transgenic mice have been described previously (14). Mice in the original Friend leukemia virus B strain (FVB) background have been backcrossed to pure C57BL/6 mice (Jackson Laboratories) for eight generations to obtain a pure C57BL/6 background. ob/ob experiments were performed on mice on a pure FVB background. Adiponectin-rtTA transgenic mice were on a pure C57BL/6 background. These mice were crossed with TRE–VEGF-A mice to generate the AT-specific Dox-inducible VEGF-A transgenic mice. All experiments were conducted using littermate-controlled male mice and were started when they were 5 wk old. Mice were housed in cages with 12-h dark–light cycle with free access to water and regular chow. All animal experiments were performed with the approval of the institutional animal care and use committee of University of Texas Southwestern Medical Center at Dallas.

Mcr84 (Anti-VEGF) Treatment.

The VEGF-specific antibody Mcr84 had been described previously (18). For the treatment of wild-type mice, 6-wk-old male C57B/6 wild-type mice were injected intraperitoneally (i.p.) with Mcr84 or isotype control IgGs (0.2 mg per mouse) twice per week. The injection began just before the introduction of HFD and continued for 6 wk. Food intake and body weights were monitored throughout the entire time course. An oral glucose and lipid tolerance test were performed after 4 and 5 wk on HFD, respectively. At week 6, the mice were fasted for 3 h and anesthetized with isoflurane. Tissues and sera were collected for analysis. Similarly, for the experiments in ob/ob mice, 6-wk-old male ob/ob mice were injected i.p. with either control IgG or mcr84 twice per week for 3 wk.

Statistical Analysis.

All results are given as means ± SEM. Differences between two groups were examined for statistical significance with Student's t test. Significance was accepted at a P value of <0.05.

Additional Materials and Methods.

Please refer to SI Materials and Methods.

Supplementary Material

Supporting Information:

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^{Department of Internal Medicine, Touchstone Diabetes Center,}

^{Department of Cell Biology, and}

^{Hamon Center for Therapeutic Oncology and Division of Surgical Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390;}

^{Department of Pediatrics, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil; and}

^{Department of Developmental and Molecular Biology, Center of Reproductive Biology and Women's Health, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY, 10461}

^{To whom correspondence should be addressed. E-mail:}ude.nretsewhtuostu@rerehcs.ppilihp.

Edited^* by Roger H. Unger, Touchstone Center for Diabetes Research, Dallas, TX, and approved February 29, 2012 (received for review January 10, 2012)

Author contributions: K.S., I.W.A., C.M.K., and P.E.S. designed research; K.S., I.W.A., C.M.K., and A.C.B. performed research; Z.V.W., J.W.P., and R.A.B. contributed new reagents/analytic tools; Z.V.W. generated the mouse model; K.S., I.W.A., C.M.K., and P.E.S. analyzed data; and K.S., I.W.A., C.M.K., and P.E.S. wrote the paper.

^{I.W.A. and C.M.K. contributed equally to this work.}

Edited^* by Roger H. Unger, Touchstone Center for Diabetes Research, Dallas, TX, and approved February 29, 2012 (received for review January 10, 2012)

Abstract

Obese fat pads are frequently undervascularized and hypoxic, leading to increased fibrosis, inflammation, and ultimately insulin resistance. We hypothesized that VEGF-A–induced stimulation of angiogenesis enables sustained and sufficient oxygen and nutrient exchange during fat mass expansion, thereby improving adipose tissue function. Using a doxycycline (Dox)-inducible adipocyte-specific VEGF-A overexpression model, we demonstrate that the local up-regulation of VEGF-A in adipocytes improves vascularization and causes a “browning” of white adipose tissue (AT), with massive up-regulation of UCP1 and PGC1α. This is associated with an increase in energy expenditure and resistance to high fat diet-mediated metabolic insults. Similarly, inhibition of VEGF-A–induced activation of VEGFR2 during the early phase of high fat diet-induced weight gain, causes aggravated systemic insulin resistance. However, the same VEGF-A–VEGFR2 blockade in ob/ob mice leads to a reduced body-weight gain, an improvement in insulin sensitivity, a decrease in inflammatory factors, and increased incidence of adipocyte death. The consequences of modulation of angiogenic activity are therefore context dependent. Proangiogenic activity during adipose tissue expansion is beneficial, associated with potent protective effects on metabolism, whereas antiangiogenic action in the context of preexisting adipose tissue dysfunction leads to improvements in metabolism, an effect likely mediated by the ablation of dysfunctional proinflammatory adipocytes.

Keywords: neovascularization, VEGF receptor 2, hypoxia, obesity

Abstract

Adipose tissue (AT) has unique plasticity, illustrated by its ability for rapid and dynamic expansion or reduction in periods of excess energy exposure or demand to ensure proper systemic energy homeostasis. AT expansion includes both hypertrophic and hyperplastic growth (1, 2). During the progression to chronic obesity, AT depots undergo profound pathological changes (3), such as enhanced oxidative damage, ER stress, local hypoxia, fibrosis, as well as immune cell infiltration and inflammation; many of these changes ultimately promote the development of insulin resistance (3).

However, not all AT expansion is associated with pathological changes. A subgroup of individuals that we refer to as “metabolically healthy obese” manage to expand their AT mass without the associated pathological consequences. The concept of “healthy AT expansion” suggests that fat pads differ largely with respect to how well they cope with the local tissue growth (3). In contrast to the pathological expansion widely seen upon weight gain, a healthy expansion consists of an enlargement of a given fat pad through recruitment of new adipocytes, along with the adequate development of the vasculature, minimal associated fibrosis, and the lack of hypoxia and inflammation (3).

AT is a highly vascularized tissue. Almost all adipocytes are surrounded by capillaries (4). A functional vascular system is critical for AT expansion. The macro- and microvasculature in AT supplies oxygen, nutrients, hormones, and growth factors, supporting expansion and homeostasis (4). The vasculature is also critical for the effective local removal of free fatty acids during fasting. Thus, angiogenesis has been considered to be a rate-limiting step for fat tissue expansion (3). Importantly, several crucial angiogenic factors, such as leptin, adiponectin, HGF-1, angiopoeitin-2, and VEGF-A are secreted by adipocytes, suggesting an autoregulatory function for angiogenesis in AT (4). Among them, VEGF-A is the only bona fide endothelial cell growth factor. VEGF-A accounts for most of the proangiogenic activity in AT (5, 6). VEGF-A binds to two tyrosine kinase receptors, VEGF receptors 1 (R1) and 2 (R2), prompting homo- and heterodimerization and becomes activated through transphosphorylation. VEGFR2 mediates most of the known cellular responses to VEGF. The function of VEGFR1 is less defined (7). VEGF-A levels in AT are regulated by exercise, hypoxia, insulin, a subset of cytokines, and several growth factors (8, 9). However, adipocytes frequently fail to mount a proper response to local hypoxia and do not produce sufficiently high levels of VEGF (10).

To date, a number of reports demonstrate that the disruption of neovascularization in AT prevents the development of obesity (11, 12). However, despite these efforts, much remains to be learned about targeting established blood vessels in the metabolically challenged AT (4, 11) and the role of angiogenesis and its regulation in healthy expanding fat tissue. This is especially true for the early stages of tissue expansion. Even though the literature has primarily focused on the inhibition of angiogenesis at later stages of metabolic dysfunction, we hypothesized that enhanced neovascularization in expanding fat pads should have beneficial effects. To test this hypothesis, we used a doxycycline (Dox)-inducible mouse model that allows us to overexpress VEGF-A uniquely in white adipose tissue at early stages of a high fat diet (HFD) challenge. We find that angiogenesis facilitates healthy fat pad expansion, reflected by smaller average size of adipocytes, absence of hypoxia, minimal fibrosis, and essentially none of the hallmarks of inflammation characteristic of dysfunctional AT. The mice remain metabolically fit on a HFD, with improved insulin sensitivity and increased energy expenditure. More importantly, VEGF-A also up-regulates UCP-1 and PGC1α levels, prompting white adipose tissue to assume a phenotype resembling more closely brown adipose tissue (13). In contrast, when we blocked functional blood vessel expansion by using the anti-VEGF antibody (Mcr84) that specifically inhibits binding of VEGF-A to VEGF receptor 2 at the initial stages of HFD feeding, we found that the mice showed a significant metabolic impairment and reduced insulin sensitivity. However, using the neutralizing antibody in ob/ob mice with preexisting metabolic dysfunction resulted in the opposite effect: the blockade decreased local inflammation in epididymal white adipose tissue (EWAT), improved insulin sensitivity, and overall metabolic health. Taken together, these results highlight the complexity of interfering with angiogenesis in adipose tissue.

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Acknowledgments

We thank members in the P.E.S. laboratory, especially Dr. Nils Halberg, for discussion and technical help; the pathology core facility at University of Texas (UT) Southwestern for help with histology; Dr. Bob Hammer and the transgenic core facility at UT Southwestern for generating the adiponectin-rtTA transgenic mice; the Metabolic Core Unit at UT Southwestern for phenotyping efforts; and Steven Spurgin for help with manuscript preparation. This work was supported by National Institutes of Health Grants R01-DK55758, RC1DK086629, and P01DK088761 (to P.E.S.).

Acknowledgments

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1200447109/-/DCSupplemental.

Footnotes

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