Gsα deficiency in adipose tissue improves glucose metabolism and insulin sensitivity without an effect on body weight Yong-Qi Lia, Yogendra B. Shresthaa, Min Chena, Tatyana Chanturiyab, Oksana Gavrilovab, and Lee S. Weinsteina,1 a Metabolic Diseases Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; and bMouse Metabolism Core Laboratory, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Edited by Gerald I. Shulman, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved December 2, 2015 (received for review August 27, 2015)
Gsα, the G protein that transduces receptor-stimulated cAMP generation, mediates sympathetic nervous system stimulation of brown adipose tissue (BAT) thermogenesis and browning of white adipose tissue (WAT), which are both potential targets for treating obesity, as well as lipolysis. We generated a mouse line with Gsα deficiency in mature BAT and WAT adipocytes (Ad-GsKO). Ad-GsKO mice had impaired BAT function, absent browning of WAT, and reduced lipolysis, and were therefore cold-intolerant. Despite the presence of these abnormalities, Ad-GsKO mice maintained normal energy balance on both standard and high-fat diets, associated with decreases in both lipolysis and lipid synthesis. In addition, Ad-GsKO mice maintained at thermoneutrality on a standard diet also had normal energy balance. Ad-GsKO mice had improved insulin sensitivity and glucose metabolism, possibly secondary to the effects of reduced lipolysis and lower circulating fatty acid binding protein 4 levels. Gsα signaling in adipose tissues may therefore affect whole-body glucose metabolism in the absence of an effect on body weight.
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G proteins adipocyte insulin sensitivity lipid metabolism brown adipose tissue
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he sympathetic nervous system (SNS) regulates energy homeostasis and adiposity through several mechanisms, including activation of nonshivering thermogenesis in brown adipose tissue (BAT), browning (formation of BAT-like “beige” cells) of white adipose tissue (WAT), and stimulation of lipolysis. Although these processes have been shown to be potential targets in treating obesity and diabetes (1–3), ablation of sympathetic nerves (4–6) or their main effectors (norepinephrine and epinephrine) (7) does not result in obesity or insulin resistance. Although mice lacking β adrenergic receptors (β-less mice) do develop obesity (8), it is likely that this effect is not due only to loss of β-adrenergic signaling in adipose tissue. The main mediator of SNS function in adipose tissues is Gsα (9, 10), a ubiquitously expressed G protein α-subunit that in adipose tissue couples adrenergic and other receptors, such as the adenosine A2A receptor (11), to the generation of intracellular cAMP. We have previously generated adipose-specific Gsα knockout mice (FGsKO) using fatty acid binding protein 4 (FABP4) (aP2)-cre and showed these mice to have significant early mortality and a severely lean phenotype (12). However, the usefulness of this model to examine the role of Gsα in mature adipocytes is limited due to both the lack of specificity of FABP4-cre expression in adipose tissue and the presence of a severe defect in adipogenesis due to expression of FABP4, and therefore loss of Gsα, during an early step in adipocyte differentiation. Adiponectin is a mature adipocyte marker expressed late in adipocyte differentiation (13). The more recent availability of adiponectin-cre mouse lines (14, 15) has enabled us to generate adipose-specific Gsα knockout mice (Ad-GsKO) in which Gsα deletion is restricted to mature adipocytes. Despite having loss of BAT function or browning of WAT, Ad-GsKO mice failed to develop obesity on either standard chow or a high-fat diet (HFD). 446–451 | PNAS | January 12, 2016 | vol. 113 | no. 2
Moreover, Ad-GsKO mice had improved glucose tolerance and insulin sensitivity associated with a significant reduction of circulating FABP4. Our results show that thermogenesis in BAT and in beige adipocytes is not required for normal weight maintenance and that Gsα signaling in adipose tissue has an effect on wholebody glucose metabolism independent of adiposity. Results Ad-GsKO Mice Have Normal Energy Balance. We generated mice with adipose tissue-specific Gsα deficiency (Ad-GsKO: adiponectin-cre+, Gsαfl/fl). Loss of Gsα expression in WAT and BAT adipocytes was confirmed by immunoblotting (Fig. 1A), whereas Gsα expression was unaffected in other tissues (brain, liver, or kidney; Fig. S1). The number of mutants alive at weaning was consistent with expected Mendelian ratios and no premature death was observed up until age 4 mo. Both male and female Ad-GsKO mice had normal body weight and composition on both standard diet and HFD (Fig. 1 B–E and Fig. S2 A and B), as well as normal body length (Fig. 1F). Interscapular BAT weight was significantly increased in Ad-GsKO mice on standard diet (Fig. 1G), consistent with this tissue’s being metabolically inactive and having enlarged adipocytes (Fig. 2 and discussed below). However, there were no differences in the weights of inguinal (Ing), retroperitoneal, or epididymal (Epi) WAT pads (Fig. 1G), and adipocytes in Epi WAT of Ad-GsKO mice on standard diet showed a normal mean diameter and size distribution (Fig. 1 H and I). Kidney, heart, and liver weights were also unaffected (Fig. 1G). Consistent with no change in adiposity, serum leptin levels were similar in
Significance The G protein Gsα mediates the activation of lipolysis in white and brown adipose tissue (WAT and BAT) and thermogenesis in BAT by sympathetic nerves. In this study we show that Gsα deficiency in BAT and WAT leads to cold intolerance and inactive BAT in mice, as expected, but does not alter body weight or energy balance, associated with a reduction in both lipolysis and lipogenesis. Despite having similar adiposity, these mice had improved insulin sensitivity and glucose metabolism, possibly secondary to reduced release of free fatty acids and fatty acid binding protein 4 from adipose tissue. These findings show that Gsα in adipocytes is not required to maintain energy balance but still has an important effect on glucose metabolism. Author contributions: Y.-Q.L., Y.B.S., M.C., O.G., and L.S.W. designed research; Y.-Q.L., Y.B.S., M.C., T.C., and O.G. performed research; Y.-Q.L., Y.B.S., M.C., T.C., and O.G. analyzed data; and Y.-Q.L. and L.S.W. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1517142113/-/DCSupplemental.
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Fig. 1. Energy balance is unaffected in Ad-GsKO mice. (A) Immunoblots of adipocytes isolated from interscapular BAT and Epi WAT of control and Ad-GsKO mice with Gsα and β-actin antibodies. (B and C) Body weight curves of male Ad-GsKO and control mice fed a standard diet (B) or HFD (C). HFD was started at 9 wk of age. (D and E) Total, fat, and lean mass in 4- to 5-mo-old male mice maintained on standard diet (D) or HFD (E). (F and G) Body length (F) and organ weights (G) (expressed as percent body weight) of 4- to 5-mo-old Ad-GsKO and control mice on standard diet. BAT, interscapular BAT; Retro, retroperitoneal WAT. (H and I) Mean adipocyte diameter (H) and adipocyte size (I) distribution in Epi WAT from 4-mo-old male Ad-GsKO and control mice on standard diet. (J) Food intake of 4-mo-old male Ad-GsKO mice and controls. (K and L) Resting and total energy expenditure rate (K) and ambulatory (Amb) and total activity levels (L) measured over 24 h at 24 °C and 30 °C in 4-mo-old male mice on standard diet (n = 6–8 per group). (M) Respiratory exchange ratios (vCO2/vO2) measured over three consecutive days from 1200 to 1800 hours in 4-mo-old male mice on standard diet. Data are mean ± SEM, n = 5–8 per group. *P < 0.05 vs. controls.
to 1800 hours at a time when mice do not generally eat tended to be higher in Ad-GsKO mice (P = 0.08) (Fig. 1M), suggesting that Ad-GsKO mice probably metabolize a lower ratio of lipid to carbohydrate during this time period.
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control and Ad-GsKO mice (Table 1). Food intake (Fig. 1J), energy expenditure (Fig. 1K), and activity levels (Fig. 1L) were all unaffected in Ad-GsKO mice maintained on standard diet. Respiratory exchange ratios (RERs, vCO2/vO2) measured from 1200
Fig. 2. Gsα is required for BAT thermogenesis and browning of WAT. (A) Histology of interscapular BAT (above) and Ing WAT (below) from a 5-mo-old male control and an Ad-GsKO mouse maintained on a standard diet (H & E). Areas featuring beiging in the Ing WAT of control mice only comprise less than 1/20 of the total random selected areas examined. (Scale bar, 100 μm.) (B) Ucp1 and Serpina3k mRNA expression in BAT from 3- to 4-mo-old male control and Ad-GsKO mice (n = 3 per group). (C) Rectal temperature in 3- to 4-mo-old male control and Ad-GsKO mice maintained on standard diet at room temperature (time 0) and hourly after being placed at 4 °C (n = 7 per group). (D) Ucp1 mRNA levels in BAT of male control and Ad-GsKO mice at baseline (24 °C) and after 3 h at 4 °C (Cold) (n = 3 per group). (E) O2 consumption in 4-mo-old female control and Ad-GsKO mice on a standard diet measured at 30 °C before and after administration of the β3-adrenergic receptor agonist CL316243 (0.01 mg/kg i.p.; n = 5–6 per group). (F) Respiratory exchange ratios in control and Ad-GsKO mice before and after CL316243 administration. Differences before and after treatment were significantly different between groups. Data are mean ± SEM *P < 0.05 or ***P < 0.001 vs. controls; #P < 0.05 vs. room temperature or baseline.
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Table 1. Serum chemistries in control and Ad-GsKO mice Metabolite/factor Random Glucose, mg/dL Insulin, ng/mL FFAs, mM TGs, mg/dL Cholesterol, mg/dL Leptin, ng/mL FABP4, ng/mL FGF21, pg/mL RBP4, μg/mL AdipoQ, μg/mL T4, μg/dL T3, ng/dL Starved Glucose, mg/dL Insulin, ng/mL
Control 90 0.58 0.39 139 91 11 223 100 29 16.8 3.46 90.0
± ± ± ± ± ± ± ± ± ± ± ±
6 0.10 0.05 6 8 3 41 11 1 1.8 0.18 2.7
48 ± 2 0.49 ± 0.06
Ad-GsKO 89 0.37 0.23 104 95 10 121 92 27 11.0 3.73 92.1
± ± ± ± ± ± ± ± ± ± ± ±
4 0.05 0.04* 13* 11 2 19* 8 1 1.4* 0.24 4.2
48 ± 2 0.34 ± 0.01*
Data are mean ± SEM n = 6–8 per group. Blood was collected from 12- to 16-wk-old female mice. *P < 0.05 vs. controls. AdipoQ, adiponectin.
We also observed no differences in body weight, body composition, food intake, or energy expenditure when mice were maintained at thermoneutral temperature (30 °C) for 8 wk (Fig. S3). This is in contrast to Ucp1 knockout mice, which were shown to have increased weight gain and metabolic efficiency when maintained at thermoneutrality (2). Gsα Is Required for BAT Thermogenesis and Browning of WAT. Consistent with their increased weight (Fig. 1G), interscapular BAT pads from Ad-GsKO mice were grossly enlarged and pale in comparison with BAT from controls. Histology revealed that BAT from Ad-GsKO mice contained enlarged adipocytes with unilocular lipid droplets (Fig. 2A), which were morphologically similar to WAT adipocytes. Consistent with this, Ucp1 mRNA expression was decreased by ∼92% in BAT from Ad-GsKO, whereas expression of Serpina3k, a relatively WAT-specific marker (16), was increased >10-fold (Fig. 2B). Next, we investigated the impact of Gsα deficiency on the process of browning of WAT, a feature normally most prominent in s.c. WAT. Histological examination of s.c. Ing WAT from control mice raised at 22 °C showed areas of browning (smaller adipocytes with multiple lipid droplets), whereas browning was absent in Ing WAT from Ad-GsKO mice (Fig. 2A). Consistent with this, Ucp1 mRNA could be detected in control Ing WAT by RT-PCR (Cт: 30.0 ± 1.8) but was undetectable in Ad-GsKO Ing WAT (Cт > 40). BAT and beige adipose tissue are known to be required for mice to maintain normal body temperature (Tb) in a cold environment. In contrast to controls, Ad-GsKO mice were unable to maintain their Tb when placed at 4 °C (Fig. 2C), consistent with the presence of inactive BAT and absence of beige fat. Induction of BAT Ucp1 mRNA in the cold was almost completely absent (Fig. 2D), which would be expected because the absence of Gsα would make BAT resistant to SNS stimulation. The ability of a β3-adrenergic receptor agonist (CL31624), which is relatively specific for adipose tissue, to stimulate whole-animal energy expenditure (O2 consumption rate) was also markedly impaired in Ad-GsKO mice, although not completely lost (Fig. 2E), a result very similar to that observed in Ucp1 knockout mice (17). Compared with controls, mutants also showed a lower reduction in the RER (vCO2/vO2) in response to CL31624 (Fig. 2F), indicating a lower stimulation of fatty acid metabolism in response to the drug. Interestingly, Ad-GsKO mice had a slightly higher baseline Tb at 22 °C that was statistically significant during the light phase 448 | www.pnas.org/cgi/doi/10.1073/pnas.1517142113
(Table S1). There were no associated differences in activity levels during either the light or dark phase (Table S1), and quadratic fit of activity vs. Tb showed that Tb was higher in the dark phase independent of the activity level (Fig. S4A). When mice were placed in a temperature gradient during the light phase we observed no difference in temperature preference between Ad-GsKO mice and controls (Fig. S4 B and C), suggesting that the mutants prefer their slightly higher Tb and did not perceive this as hyperthermia (18). Although the mechanism responsible for this small shift in Tb is unclear, our results suggest that mice can chronically defend against hypothermia at a temperature moderately below thermoneutrality by BAT/beige fat-independent mechanisms. Our results are consistent with the small increase in Tb (0.1–0.3 °C) that has also been observed in Ucp1 knockout mice maintained at 20 °C (19). In summary, Ad-GsKO mice raised at 22 °C showed many metabolic and thermogenic features in common with Ucp1 knockout mice (normal energy balance on standard diet and HFD, similar loss of metabolic response to a β3 adrenergic agonist, small increase in baseline Tb, and inability to defend Tb acutely at 4 °C) (17, 19). These changes in temperature in Ad-GsKO mice were not associated with any changes in circulating levels of thyroid hormones [triiodothyronine (T3) and thyroxine (T4); Table 1]. Both Adipose Tissue Lipolysis and Lipogenesis Are Reduced in Ad-GsKO Mice. Ad-GsKO mice had significant down-regulation of multiple
genes in BAT required for lipolysis, including those encoding perilipin 1 (plin1), hormone-sensitive lipase (lipe), and adipose triglyceride lipase (ATGL, pnpla2) and its coactivator CGI-58 (abhd5) (20) (Fig. 3A). Similar reductions of plin1, lipe, and pnpla2 mRNA, but not abhd5, mRNA were also observed in Ad-GsKO Ing WAT (Fig. 3B). Consistent with markedly reduced expression of genes related to lipolysis, Ad-GsKO mice had reduced levels of serum free fatty acids (FFA; Fig. 3E and Table 1) and glycerol (Fig. 3F) at baseline, which probably reflect reduced lipolysis but could also be due to changes in FFA and triglyceride (TG) turnover. In contrast to controls, Ad-GsKO mice showed minimal induction of either plasma FFA or glycerol in response to CL31624 (Fig. 3 E and F). This latter observation is likely secondary to both loss of β-adrenergic responsiveness in adipose tissue due to Gsα deficiency and marked reduction in expression of lipolysis-related genes and is consistent with the impaired induction of energy expenditure and reduced lowering of RER in response to CL31624 that we also observed in these mice (Fig. 2 E and F), although direct measures of lipolytic protein and phosphorylation levels were not examined. We also observed markedly reduced expression of genes required for glucose uptake and fatty-acid synthesis, including genes encoding glucose transporter 4 (Glut4), pyruvate carboxylase (Pcx), malic enzyme 1 (Me1), acyl-CoA synthetase longchain family member 1 (Ascl1), fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1), and carbohydrate response element binding protein (Chrebp) in BAT and Ing WAT of AdGsKO mice (Fig. 3 C and D). There was no change in expression of malic enzyme 2 (Me2) in BAT or Ing WAT of Ad-GsKO mice (Fig. 3 C and D). Consistent with these gene expression results, 2-deoxyglucose (DG) uptake in BAT after i.p. administration of insulin in vivo was significantly reduced in BAT (Fig. 4E) and tended to be lower in Ing and Epi WAT (Fig. 4 F and G) of AdGsKO mice. In addition, Ad-GsKO mice had reduced rates of incorporation of glucose into lipid in BAT and epididymal WAT in vitro (Fig. 3F). Ad-GsKO also had reduced expression of genes involved in fatty acid uptake [e.g., lipoprotein lipase (Lpl), fatty acid translocase (Cd36)] and esterification [e.g., phosphoenolpyruvate carboxykinase (Pepck), diacylglycerol acyltransferase 1 (Dgat1), and mitochondrial glycerol-3-phosphate acyltransferase (Gpam)] in both BAT (Fig. S5A) and Ing WAT (Fig. S5B). Pepck, a gene involved in glycerol-3-phosphate production, was also significantly Li et al.
Fig. 3. Reduced lipolysis and lipogenesis in adipose tissues of Ad-GsKO mice. (A and B) mRNA expression of lipolysis-related genes in BAT (A) and Ing WAT (B) from 3- to 4-mo-old male control and Ad-GsKO mice maintained on standard diet (n = 3–5 per group). Abhd5, abhydrolase domain containing 5 (CGI-58); Lipe, hormone-sensitive lipase; Plin1, perilipin 1; Pnpla2, adipose triglyceride lipase. (C and D) mRNA expression of glucose uptake and de novo lipogenesis related genes in interscapular BAT (C) and Ing WAT (D) from 3- to 4-mo-old male control and Ad-GsKO mice maintained on standard diet (n = 3–5 per group). Ascl1, acyl-CoA synthetase long-chain family member 1; Chrebp, carbohydrate responsive element binding protein; Fasn, fatty acid synthase; Glut4, glucose transporter 4; Me1 and Me2, malic enzymes 1 and 2; Pcx, pyruvate carboxylase; Scd1, stearoyl-CoA desaturase 1. (E and F) Plasma FFA (E) and glycerol (F) levels in 8-mo-old male control and Ad-GsKO mice after i.p. injection of saline or CL316243 (CL; n = 5–7 per group). (G) D-[14C(U)]-glucose incorporation into lipid in BAT and Epi WAT isolated from 4-mo-old male control and Ad-GsKO mice (n = 3–5 per group). Results are normalized to tissue weight and expressed as percent of control. Data are mean ± SEM *P < 0.05 vs. controls; #P < 0.05 vs. baseline.
Despite a reduction in adipose tissue lipid metabolism, AdGsKO mice were able to maintain a normal rate of whole-body fatty acid oxidation in vivo (Fig. S6A). To what extent increased metabolism in skeletal muscle prevented the development of obesity in Ad-GsKO mice is not fully clear. It is interesting to note that in these animals gastrocnemius muscle had increased insulin-stimulated glucose uptake (Fig. 4 F and G) and soleus muscle had increased expression of the peroxisome proliferatoractivated receptor-γ coactivator-1α (PGC-1α) and cytochrome c genes, although no increase in expression of the mitochondrial genes Tfam or Nrf1 was observed (Fig. S6B). We also did not observe an increase in expression of the gene encoding sarcolipin, a protein implicated in nonshivering thermogenesis in skeletal muscle (21), in soleus muscle of Ad-GsKO mice. Rather, its expression was decreased (Fig. S6B).
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down-regulated in Epi WAT in both fed and fasted Ad-GsKO mice (Fig. S5C). Consistent with these changes in gene expression, we showed that glucose incorporation into TGs and fatty acids (both free and associated with TG) were markedly reduced in explanted BAT and Ing and Epi WAT (Fig. S5D). The effect of insulin on glucose incorporation was not examined. Incorporation of extracellular oleic acid to TG in explanted BAT and Epi WAT of Ad-GsKO mice were also significantly reduced (Fig. S5E), which could be due to either reduced lipid uptake, esterification, or both. Overall our results suggest that glucose and lipid uptake, fatty acid synthesis, and esterification may all be reduced in adipose tissue of Ad-GsKO mice. At least for WAT, normal adipocyte cell size and lipid content may be the result of similar reductions in lipolysis and lipid synthesis, which counterbalance their opposing effects on lipid storage.
Fig. 4. Improved glucose metabolism and insulin sensitivity in Ad-GsKO mice. (A and B) Results of glucose tolerance tests on 4- to 5-mo-old male control and Ad-GsKO mice maintained on standard diet (A) or after 8 wk of HFD (B). (C and D) Results of insulin tolerance tests on 4- to 5-mo-old control and Ad-GsKO mice maintained on standard diet (C) or 8 wk of HFD (D) (n = 5–8 per group). (E–G) In vivo 2-DG uptake in interscapular BAT (E), Epi WAT (F), Ing WAT (G), gastrocnemius muscle (H), and cardiac muscle (I) of 2-mo-old male control and Ad-GsKO mice after administration of insulin (Humulin, 0.75 mU/kg i.p.; n = 3–5 per group). (J) Blood glucose levels in 4-mo-old male control and Ad-GsKO mice before and after administration of pyruvate (2 g/kg i.p.; n = 5–8 per group). (K and L) Liver (K) and soleus muscle (L) TG content in 4-mo-old male control and Ad-GsKO mice (n = 6 per group). (M) mRNA expression of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in Epi WAT from 3-mo-old male control and Ad-GsKO mice (n = 6 per group). Area under curves for glucose, insulin, and pyruvate tolerance tests were all significantly different between control and Ad-GsKO mice. Data are mean ± SEM *P < 0.05 vs. controls.
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Gsα Deficiency in Fat Tissues Improved Glucose Metabolism and Insulin Sensitivity. Ad-GsKO mice had normal fed and starved
serum glucose levels, whereas serum insulin levels were significantly lower in the starved state and tended to be lower in the fed state (Table 1). Glucose and insulin tolerance tests showed AdGsKO mice to have significantly improved glucose tolerance and insulin sensitivity on both standard diet and HFD (Fig. 4 A–D and Fig. S2C). Consistent with this finding, insulin-stimulated 2-DG uptake was significantly increased in vivo in both skeletal (gastrocnemius) and cardiac muscle in Ad-GsKO mice (Fig. 4 H and I). In addition, Ad-GsKO mice showed a smaller rise in serum glucose after administration of pyruvate, consistent with reduced hepatic gluconeogenesis, although this difference could reflect increased pyruvate oxidation in liver to compensate for reduced energy substrate availability (Fig. 4J). Glycogen levels (measured at 1300 hours) were reduced by 15% in livers of AdGsKO mice whereas they were unchanged in soleus muscle (Fig. S7 A and B). AMP kinase phosphorylation, a downstream marker of insulin signaling, was unaffected in liver but increased by 41% in soleus muscle of Ad-GsKO mice (Fig. S7 C–F). Improved insulin sensitivity in Ad-GsKO mice may be partially explained by improved lipid status, because Ad-GsKO mice had lower serum FFA and TG levels, as well as liver and muscle TG levels (Fig. 4 K and L). Expression of genes involved in lipid synthesis were either unchanged or reduced (e.g., Pcx, Chrebp, Cd36, and Gpam) in livers from Ad-GsKO mice (Fig. S8A) and liver TG secretion rates were significantly reduced in Ad-GsKO mice (Fig. S8B). We also examined several circulating factors that have been shown to affect glucose metabolism and insulin sensitivity. Serum retinol binding protein 4 (RBP4) and fibroblast growth factor 21 (FGF21) levels were unaffected (Table 1). Serum adiponectin levels were reduced in Ad-GsKO mice, consistent with our prior results showing that adiponectin expression in adipose tissue is induced by stimulation with a β3 agonist (22). However, reduced serum adiponectin levels are associated with impaired, rather than improved, peripheral glucose metabolism. Serum levels of FABP4, an adipokine that is markedly elevated in obese/diabetic patients and that stimulates hepatic gluconeogenesis (23–25), were markedly reduced by almost 50% in Ad-GsKO mice (Table 1), making this a possible mechanism contributing to the improved glucose metabolism present in Ad-GsKO mice. There were no differences in expression of the inflammatory genes interleukin-6 and tumor necrosis factor-α in Epi fat from Ad-GsKO and control mice (Fig. 4M), making it unlikely that differences in adipose tissue inflammation are contributing to the observed differences in glucose metabolism. Discussion In this paper we generated mice with loss of Gsα signaling (and therefore resistance to activation by SNS and other hormones) in mature BAT and WAT adipocytes and examined the effects on temperature maintenance, adipocyte metabolism, energy balance, and glucose homeostasis. Our results provide important insights into on how these physiological parameters are regulated by SNS activation of adipose tissue. As expected, Ad-GsKO mice showed marked reductions in BAT activation, cold tolerance, cold-induced Ucp1 up-regulation, and browning of WAT. This result is expected, given the known role of SNS activation of BAT and WAT in these processes, and is consistent with the cold intolerance observed in both our prior adiposespecific Gsα knockout model (10) and Ucp1 knockout mice (17). Similar to Ucp1 knockout mice (17), we observed a small increase in Tb at room temperature in Ad-GsKO mice. Presumably this reflects increased thermogenesis in other tissue(s), although the mechanisms responsible for these observations are unclear. Although it has been shown that loss of BAT function can be compensated for by increased browning of WAT (26), this 450 | www.pnas.org/cgi/doi/10.1073/pnas.1517142113
is not an explanation in our model because WAT browning was absent. The observed absence of a detectable change in energy expenditure despite a small increase in Tb may reflect compensation by the small, though not significant, reduction in activity levels or may reflect the limitations of indirect calorimetry to detect small differences in energy expenditure. Despite loss of BAT and WAT activation by SNS, Ad-GsKO mice had normal body weight and adiposity on both standard diet and HFD. One potential explanation for this observation is that loss of Gsα in adipocytes led not only to reduced lipolysis, a process known to be activated by Gsα and cAMP, but also to a similar reduction in expression of genes critical for glucose and lipid uptake, fatty acid synthesis, and esterification in both BAT and WAT, associated with reduced incorporation of glucose and fatty acids into TG. This finding is consistent with a recent study showing that β3-adrenergic stimulation increased both lipolysis and de novo lipogenesis in BAT and WAT and that induction of de novo lipogenesis may be coupled to lipolysis (27). Another study showed that although norepinephrine acutely inhibited lipogenesis in cultured BAT cells, longer treatments led to stimulation of lipogenesis (28). It is interesting to note that mutations of lipolytic genes encoding ATGL and hormone-sensitive lipase are associated with only small changes in adiposity and reduced lipogenesis in adipose tissue, suggesting that lipid anabolic and catabolic pathways may be coupled (29, 30). It is also possible that the reduced lipogenesis observed in Ad-GsKO mice may reflect fundamental changes in the adipogenic program, because a profound effect of Gsα on adipogenesis was observed in our prior FGsKO mouse model in which Gsα deletion occurred at an earlier stage of adipogenesis (12). Further studies will be required to understand the mechanisms by which Gsα seems to affect both lipolysis and lipogenesis in the same direction. The role of SNS-induced BAT activation and browning of WAT in diet-induced thermogenesis has great appeal and stimulation of these responses has been proposed as a potential treatment for obesity (1, 2, 8, 9). However, whether BAT function is necessary for the maintenance of normal body weight is still not definitively answered. Ablation of sympathetic nerves (4–6) or their neurotransmitters (norepinephrine and epinephrine) (7) does not result in obesity, even when BAT is functionally inactive. Similarly, Ucp1 knockout mice fed on either a standard chow or HFD do not develop obesity relative to littermate controls at room temperature (17). Although Ucp1 knockout mice showed increased gain in fat mass and diet-induced thermogenesis when maintained at thermoneutrality and this was ascribed to loss of diet-induced thermogenesis (2), we observed no changes in fat mass or energy expenditure in Ad-GsKO maintained at thermoneutrality. The absence of greater adiposity of Ad-GsKO mice on either standard diet or HFD provides further direct evidence that activation of BAT or browning of WAT is not required for maintenance of normal energy balance. Another striking finding in Ad-GsKO mice is significant improvement in insulin sensitivity and peripheral glucose metabolism despite the fact that BAT is inactive and the absence of improvement in whole-body adiposity. This effect of glucose metabolism is probably independent of changes in Ucp1 expression, because Ucp1 knockout mice on standard diet were shown to have no differences in glucose tolerance or insulin levels (31). We observed no differences in circulating FGF21 or RBP4 levels to help explain this effect, and circulating levels of adiponectin, a factor associated with improved glucose metabolism, were actually decreased in Ad-GsKO mice. In addition, we found no evidence for differences in WAT inflammation that might contribute to alterations in glucose metabolism. One potential mechanism contributing to improved insulin sensitivity and glucose metabolism in Ad-GsKO mice is reduced lipolysis in adipose tissue resulting in lower circulating FFA levels. It is well established that FFAs, the major product of lipolysis, cause deleterious effects on insulin Li et al.
sensitive organs (32, 33) and that genetic or pharmacologic inhibition of adipose tissue lipolysis in mice improves glucose metabolism and insulin sensitivity (29, 34). Moreover, high lipolysis rate is associated with low insulin sensitivity in humans (34). The lower TG content in liver present in Ad-GsKO may also contribute to improved glucose metabolism in these mice. FABP4 is a recently identified adipokine released from adipocyte that leads to impaired glucose metabolism via increased hepatic glucose production and perhaps other mechanisms as well, although alternative mechanisms have not been directly examined (23, 25). FABP4 release from adipocytes is stimulated by β-adrenergic receptor agonists that activate Gsα, and circulating FABP4 levels have been shown to be markedly elevated in obese/diabetic patients (23, 24). Conversely, reduced plasma FABP4 levels are associated with improved glucose metabolism and insulin sensitivity (23, 35). Therefore, inhibiting Gsα signaling pathway in adipocytes may improve insulin sensitivity and glucose metabolism via reduced lipolysis, release of FABP4, and perhaps other additional mechanisms. In conclusion, although the level of functional BAT is inversely correlated with adiposity in humans (36, 37) and positively
correlated with improved insulin sensitivity and glucose metabolism in mice and humans (38–40), our results suggest that reduced BAT function alone may be insufficient for the development of obesity or insulin resistance. Moreover, specific inhibition of adipose tissue Gsα signaling seems to dramatically affect whole-body glucose metabolism in the absence of an effect on body weight.
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24. Tso AW, et al. (2007) Serum adipocyte fatty acid binding protein as a new biomarker predicting the development of type 2 diabetes: A 10-year prospective study in a Chinese cohort. Diabetes Care 30(10):2667–2672. 25. Kralisch S, et al. (2014) Adipocyte fatty acid-binding protein is released from adipocytes by a non-conventional mechanism. Int J Obes 38(9):1251–1254. 26. Schulz TJ, et al. (2013) Brown-fat paucity due to impaired BMP signalling induces compensatory browning of white fat. Nature 495(7441):379–383. 27. Mottillo EP, et al. (2014) Coupling of lipolysis and de novo lipogenesis in brown, beige, and white adipose tissues during chronic β3-adrenergic receptor activation. J Lipid Res 55(11):2276–2286. 28. Bianco AC, Carvalho SD, Carvalho CR, Rabelo R, Moriscot AS (1998) Thyroxine 5′deiodination mediates norepinephrine-induced lipogenesis in dispersed brown adipocytes. Endocrinology 139(2):571–578. 29. Schreiber R, et al. (2015) Hypophagia and metabolic adaptations in mice with defective ATGL-mediated lipolysis cause resistance to HFD-induced obesity. Proc Natl Acad Sci USA 112(45):13850–13855. 30. Zimmermann R, et al. (2003) Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue. J Lipid Res 44(11):2089–2099. 31. Kontani Y, et al. (2005) UCP1 deficiency increases susceptibility to diet-induced obesity with age. Aging Cell 4(3):147–155. 32. Boden G (2011) Obesity, insulin resistance and free fatty acids. Curr Opin Endocrinol Diabetes Obes 18(2):139–143. 33. Samuel VT, Petersen KF, Shulman GI (2010) Lipid-induced insulin resistance: unravelling the mechanism. Lancet 375(9733):2267–2277. 34. Girousse A, et al. (2013) Partial inhibition of adipose tissue lipolysis improves glucose metabolism and insulin sensitivity without alteration of fat mass. PLoS Biol 11(2): e1001485. 35. Furuhashi M, et al. (2007) Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2. Nature 447(7147):959–965. 36. Cypess AM, et al. (2009) Identification and importance of brown adipose tissue in adult humans. N Engl J Med 360(15):1509–1517. 37. van Marken Lichtenbelt WD, et al. (2009) Cold-activated brown adipose tissue in healthy men. N Engl J Med 360(15):1500–1508. 38. Lee P, et al. (2014) Temperature-acclimated brown adipose tissue modulates insulin sensitivity in humans. Diabetes 63(11):3686–3698. 39. Stanford KI, et al. (2013) Brown adipose tissue regulates glucose homeostasis and insulin sensitivity. J Clin Invest 123(1):215–223. 40. Chondronikola M, et al. (2014) Brown adipose tissue improves whole-body glucose homeostasis and insulin sensitivity in humans. Diabetes 63(12):4089–4099. 41. Chen M, et al. (2005) Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific Gsα deficiency. J Clin Invest 115(11):3217–3227. 42. Kim JK, Gavrilova O, Chen Y, Reitman ML, Shulman GI (2000) Mechanism of insulin resistance in A-ZIP/F-1 fatless mice. J Biol Chem 275(12):8456–8460. 43. Gautam D, et al. (2006) Beneficial metabolic effects of M3 muscarinic acetylcholine receptor deficiency. Cell Metab 4(5):363–375. 44. Ravussin Y, Gutman R, LeDuc CA, Leibel RL (2013) Estimating energy expenditure in mice using an energy balance technique. Int J Obes 37(3):399–403. 45. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1):497–509. 46. Chen H, et al. (1997) Protein-tyrosine phosphatases PTP1B and syp are modulators of insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. J Biol Chem 272(12):8026–8031.
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Materials and Methods All studies were approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee. Gsα exon 1-floxed mice (E1fl/fl) (41) were mated with adiponectin-cre mice (15) to generate Ad-GsKO mice (adiponectin-cre+, E1fl/fl). Animals were maintained on a 12-h light/dark cycle (6:00 AM/6:00 PM) and standard pellet diet (NIH-07, 5% fat by weight) unless indicated. HFD (Bio-Serv) consisted of 59.4 kcal% fat, 16.2 kcal% protein, and 24.5 kcal% carbohydrate. All data are expressed as mean ± SEM and analyzed by Student’s t test with differences considered significant at P < 0.05. Detailed methods are described in SI Materials and Methods.
PNAS | January 12, 2016 | vol. 113 | no. 2 | 451
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ACKNOWLEDGMENTS. We thank M. Reitman and A. Cypess for suggestions and technical assistance. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.
Edited by Gerald I. Shulman, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved December 2, 2015 (received for review August 27, 2015)
Gsα, the G protein that transduces receptor-stimulated cAMP generation, mediates sympathetic nervous system stimulation of brown adipose tissue (BAT) thermogenesis and browning of white adipose tissue (WAT), which are both potential targets for treating obesity, as well as lipolysis. We generated a mouse line with Gsα deficiency in mature BAT and WAT adipocytes (Ad-GsKO). Ad-GsKO mice had impaired BAT function, absent browning of WAT, and reduced lipolysis, and were therefore cold-intolerant. Despite the presence of these abnormalities, Ad-GsKO mice maintained normal energy balance on both standard and high-fat diets, associated with decreases in both lipolysis and lipid synthesis. In addition, Ad-GsKO mice maintained at thermoneutrality on a standard diet also had normal energy balance. Ad-GsKO mice had improved insulin sensitivity and glucose metabolism, possibly secondary to the effects of reduced lipolysis and lower circulating fatty acid binding protein 4 levels. Gsα signaling in adipose tissues may therefore affect whole-body glucose metabolism in the absence of an effect on body weight.
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G proteins adipocyte insulin sensitivity lipid metabolism brown adipose tissue
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he sympathetic nervous system (SNS) regulates energy homeostasis and adiposity through several mechanisms, including activation of nonshivering thermogenesis in brown adipose tissue (BAT), browning (formation of BAT-like “beige” cells) of white adipose tissue (WAT), and stimulation of lipolysis. Although these processes have been shown to be potential targets in treating obesity and diabetes (1–3), ablation of sympathetic nerves (4–6) or their main effectors (norepinephrine and epinephrine) (7) does not result in obesity or insulin resistance. Although mice lacking β adrenergic receptors (β-less mice) do develop obesity (8), it is likely that this effect is not due only to loss of β-adrenergic signaling in adipose tissue. The main mediator of SNS function in adipose tissues is Gsα (9, 10), a ubiquitously expressed G protein α-subunit that in adipose tissue couples adrenergic and other receptors, such as the adenosine A2A receptor (11), to the generation of intracellular cAMP. We have previously generated adipose-specific Gsα knockout mice (FGsKO) using fatty acid binding protein 4 (FABP4) (aP2)-cre and showed these mice to have significant early mortality and a severely lean phenotype (12). However, the usefulness of this model to examine the role of Gsα in mature adipocytes is limited due to both the lack of specificity of FABP4-cre expression in adipose tissue and the presence of a severe defect in adipogenesis due to expression of FABP4, and therefore loss of Gsα, during an early step in adipocyte differentiation. Adiponectin is a mature adipocyte marker expressed late in adipocyte differentiation (13). The more recent availability of adiponectin-cre mouse lines (14, 15) has enabled us to generate adipose-specific Gsα knockout mice (Ad-GsKO) in which Gsα deletion is restricted to mature adipocytes. Despite having loss of BAT function or browning of WAT, Ad-GsKO mice failed to develop obesity on either standard chow or a high-fat diet (HFD). 446–451 | PNAS | January 12, 2016 | vol. 113 | no. 2
Moreover, Ad-GsKO mice had improved glucose tolerance and insulin sensitivity associated with a significant reduction of circulating FABP4. Our results show that thermogenesis in BAT and in beige adipocytes is not required for normal weight maintenance and that Gsα signaling in adipose tissue has an effect on wholebody glucose metabolism independent of adiposity. Results Ad-GsKO Mice Have Normal Energy Balance. We generated mice with adipose tissue-specific Gsα deficiency (Ad-GsKO: adiponectin-cre+, Gsαfl/fl). Loss of Gsα expression in WAT and BAT adipocytes was confirmed by immunoblotting (Fig. 1A), whereas Gsα expression was unaffected in other tissues (brain, liver, or kidney; Fig. S1). The number of mutants alive at weaning was consistent with expected Mendelian ratios and no premature death was observed up until age 4 mo. Both male and female Ad-GsKO mice had normal body weight and composition on both standard diet and HFD (Fig. 1 B–E and Fig. S2 A and B), as well as normal body length (Fig. 1F). Interscapular BAT weight was significantly increased in Ad-GsKO mice on standard diet (Fig. 1G), consistent with this tissue’s being metabolically inactive and having enlarged adipocytes (Fig. 2 and discussed below). However, there were no differences in the weights of inguinal (Ing), retroperitoneal, or epididymal (Epi) WAT pads (Fig. 1G), and adipocytes in Epi WAT of Ad-GsKO mice on standard diet showed a normal mean diameter and size distribution (Fig. 1 H and I). Kidney, heart, and liver weights were also unaffected (Fig. 1G). Consistent with no change in adiposity, serum leptin levels were similar in
Significance The G protein Gsα mediates the activation of lipolysis in white and brown adipose tissue (WAT and BAT) and thermogenesis in BAT by sympathetic nerves. In this study we show that Gsα deficiency in BAT and WAT leads to cold intolerance and inactive BAT in mice, as expected, but does not alter body weight or energy balance, associated with a reduction in both lipolysis and lipogenesis. Despite having similar adiposity, these mice had improved insulin sensitivity and glucose metabolism, possibly secondary to reduced release of free fatty acids and fatty acid binding protein 4 from adipose tissue. These findings show that Gsα in adipocytes is not required to maintain energy balance but still has an important effect on glucose metabolism. Author contributions: Y.-Q.L., Y.B.S., M.C., O.G., and L.S.W. designed research; Y.-Q.L., Y.B.S., M.C., T.C., and O.G. performed research; Y.-Q.L., Y.B.S., M.C., T.C., and O.G. analyzed data; and Y.-Q.L. and L.S.W. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1517142113/-/DCSupplemental.
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Fig. 1. Energy balance is unaffected in Ad-GsKO mice. (A) Immunoblots of adipocytes isolated from interscapular BAT and Epi WAT of control and Ad-GsKO mice with Gsα and β-actin antibodies. (B and C) Body weight curves of male Ad-GsKO and control mice fed a standard diet (B) or HFD (C). HFD was started at 9 wk of age. (D and E) Total, fat, and lean mass in 4- to 5-mo-old male mice maintained on standard diet (D) or HFD (E). (F and G) Body length (F) and organ weights (G) (expressed as percent body weight) of 4- to 5-mo-old Ad-GsKO and control mice on standard diet. BAT, interscapular BAT; Retro, retroperitoneal WAT. (H and I) Mean adipocyte diameter (H) and adipocyte size (I) distribution in Epi WAT from 4-mo-old male Ad-GsKO and control mice on standard diet. (J) Food intake of 4-mo-old male Ad-GsKO mice and controls. (K and L) Resting and total energy expenditure rate (K) and ambulatory (Amb) and total activity levels (L) measured over 24 h at 24 °C and 30 °C in 4-mo-old male mice on standard diet (n = 6–8 per group). (M) Respiratory exchange ratios (vCO2/vO2) measured over three consecutive days from 1200 to 1800 hours in 4-mo-old male mice on standard diet. Data are mean ± SEM, n = 5–8 per group. *P < 0.05 vs. controls.
to 1800 hours at a time when mice do not generally eat tended to be higher in Ad-GsKO mice (P = 0.08) (Fig. 1M), suggesting that Ad-GsKO mice probably metabolize a lower ratio of lipid to carbohydrate during this time period.
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control and Ad-GsKO mice (Table 1). Food intake (Fig. 1J), energy expenditure (Fig. 1K), and activity levels (Fig. 1L) were all unaffected in Ad-GsKO mice maintained on standard diet. Respiratory exchange ratios (RERs, vCO2/vO2) measured from 1200
Fig. 2. Gsα is required for BAT thermogenesis and browning of WAT. (A) Histology of interscapular BAT (above) and Ing WAT (below) from a 5-mo-old male control and an Ad-GsKO mouse maintained on a standard diet (H & E). Areas featuring beiging in the Ing WAT of control mice only comprise less than 1/20 of the total random selected areas examined. (Scale bar, 100 μm.) (B) Ucp1 and Serpina3k mRNA expression in BAT from 3- to 4-mo-old male control and Ad-GsKO mice (n = 3 per group). (C) Rectal temperature in 3- to 4-mo-old male control and Ad-GsKO mice maintained on standard diet at room temperature (time 0) and hourly after being placed at 4 °C (n = 7 per group). (D) Ucp1 mRNA levels in BAT of male control and Ad-GsKO mice at baseline (24 °C) and after 3 h at 4 °C (Cold) (n = 3 per group). (E) O2 consumption in 4-mo-old female control and Ad-GsKO mice on a standard diet measured at 30 °C before and after administration of the β3-adrenergic receptor agonist CL316243 (0.01 mg/kg i.p.; n = 5–6 per group). (F) Respiratory exchange ratios in control and Ad-GsKO mice before and after CL316243 administration. Differences before and after treatment were significantly different between groups. Data are mean ± SEM *P < 0.05 or ***P < 0.001 vs. controls; #P < 0.05 vs. room temperature or baseline.
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Table 1. Serum chemistries in control and Ad-GsKO mice Metabolite/factor Random Glucose, mg/dL Insulin, ng/mL FFAs, mM TGs, mg/dL Cholesterol, mg/dL Leptin, ng/mL FABP4, ng/mL FGF21, pg/mL RBP4, μg/mL AdipoQ, μg/mL T4, μg/dL T3, ng/dL Starved Glucose, mg/dL Insulin, ng/mL
Control 90 0.58 0.39 139 91 11 223 100 29 16.8 3.46 90.0
± ± ± ± ± ± ± ± ± ± ± ±
6 0.10 0.05 6 8 3 41 11 1 1.8 0.18 2.7
48 ± 2 0.49 ± 0.06
Ad-GsKO 89 0.37 0.23 104 95 10 121 92 27 11.0 3.73 92.1
± ± ± ± ± ± ± ± ± ± ± ±
4 0.05 0.04* 13* 11 2 19* 8 1 1.4* 0.24 4.2
48 ± 2 0.34 ± 0.01*
Data are mean ± SEM n = 6–8 per group. Blood was collected from 12- to 16-wk-old female mice. *P < 0.05 vs. controls. AdipoQ, adiponectin.
We also observed no differences in body weight, body composition, food intake, or energy expenditure when mice were maintained at thermoneutral temperature (30 °C) for 8 wk (Fig. S3). This is in contrast to Ucp1 knockout mice, which were shown to have increased weight gain and metabolic efficiency when maintained at thermoneutrality (2). Gsα Is Required for BAT Thermogenesis and Browning of WAT. Consistent with their increased weight (Fig. 1G), interscapular BAT pads from Ad-GsKO mice were grossly enlarged and pale in comparison with BAT from controls. Histology revealed that BAT from Ad-GsKO mice contained enlarged adipocytes with unilocular lipid droplets (Fig. 2A), which were morphologically similar to WAT adipocytes. Consistent with this, Ucp1 mRNA expression was decreased by ∼92% in BAT from Ad-GsKO, whereas expression of Serpina3k, a relatively WAT-specific marker (16), was increased >10-fold (Fig. 2B). Next, we investigated the impact of Gsα deficiency on the process of browning of WAT, a feature normally most prominent in s.c. WAT. Histological examination of s.c. Ing WAT from control mice raised at 22 °C showed areas of browning (smaller adipocytes with multiple lipid droplets), whereas browning was absent in Ing WAT from Ad-GsKO mice (Fig. 2A). Consistent with this, Ucp1 mRNA could be detected in control Ing WAT by RT-PCR (Cт: 30.0 ± 1.8) but was undetectable in Ad-GsKO Ing WAT (Cт > 40). BAT and beige adipose tissue are known to be required for mice to maintain normal body temperature (Tb) in a cold environment. In contrast to controls, Ad-GsKO mice were unable to maintain their Tb when placed at 4 °C (Fig. 2C), consistent with the presence of inactive BAT and absence of beige fat. Induction of BAT Ucp1 mRNA in the cold was almost completely absent (Fig. 2D), which would be expected because the absence of Gsα would make BAT resistant to SNS stimulation. The ability of a β3-adrenergic receptor agonist (CL31624), which is relatively specific for adipose tissue, to stimulate whole-animal energy expenditure (O2 consumption rate) was also markedly impaired in Ad-GsKO mice, although not completely lost (Fig. 2E), a result very similar to that observed in Ucp1 knockout mice (17). Compared with controls, mutants also showed a lower reduction in the RER (vCO2/vO2) in response to CL31624 (Fig. 2F), indicating a lower stimulation of fatty acid metabolism in response to the drug. Interestingly, Ad-GsKO mice had a slightly higher baseline Tb at 22 °C that was statistically significant during the light phase 448 | www.pnas.org/cgi/doi/10.1073/pnas.1517142113
(Table S1). There were no associated differences in activity levels during either the light or dark phase (Table S1), and quadratic fit of activity vs. Tb showed that Tb was higher in the dark phase independent of the activity level (Fig. S4A). When mice were placed in a temperature gradient during the light phase we observed no difference in temperature preference between Ad-GsKO mice and controls (Fig. S4 B and C), suggesting that the mutants prefer their slightly higher Tb and did not perceive this as hyperthermia (18). Although the mechanism responsible for this small shift in Tb is unclear, our results suggest that mice can chronically defend against hypothermia at a temperature moderately below thermoneutrality by BAT/beige fat-independent mechanisms. Our results are consistent with the small increase in Tb (0.1–0.3 °C) that has also been observed in Ucp1 knockout mice maintained at 20 °C (19). In summary, Ad-GsKO mice raised at 22 °C showed many metabolic and thermogenic features in common with Ucp1 knockout mice (normal energy balance on standard diet and HFD, similar loss of metabolic response to a β3 adrenergic agonist, small increase in baseline Tb, and inability to defend Tb acutely at 4 °C) (17, 19). These changes in temperature in Ad-GsKO mice were not associated with any changes in circulating levels of thyroid hormones [triiodothyronine (T3) and thyroxine (T4); Table 1]. Both Adipose Tissue Lipolysis and Lipogenesis Are Reduced in Ad-GsKO Mice. Ad-GsKO mice had significant down-regulation of multiple
genes in BAT required for lipolysis, including those encoding perilipin 1 (plin1), hormone-sensitive lipase (lipe), and adipose triglyceride lipase (ATGL, pnpla2) and its coactivator CGI-58 (abhd5) (20) (Fig. 3A). Similar reductions of plin1, lipe, and pnpla2 mRNA, but not abhd5, mRNA were also observed in Ad-GsKO Ing WAT (Fig. 3B). Consistent with markedly reduced expression of genes related to lipolysis, Ad-GsKO mice had reduced levels of serum free fatty acids (FFA; Fig. 3E and Table 1) and glycerol (Fig. 3F) at baseline, which probably reflect reduced lipolysis but could also be due to changes in FFA and triglyceride (TG) turnover. In contrast to controls, Ad-GsKO mice showed minimal induction of either plasma FFA or glycerol in response to CL31624 (Fig. 3 E and F). This latter observation is likely secondary to both loss of β-adrenergic responsiveness in adipose tissue due to Gsα deficiency and marked reduction in expression of lipolysis-related genes and is consistent with the impaired induction of energy expenditure and reduced lowering of RER in response to CL31624 that we also observed in these mice (Fig. 2 E and F), although direct measures of lipolytic protein and phosphorylation levels were not examined. We also observed markedly reduced expression of genes required for glucose uptake and fatty-acid synthesis, including genes encoding glucose transporter 4 (Glut4), pyruvate carboxylase (Pcx), malic enzyme 1 (Me1), acyl-CoA synthetase longchain family member 1 (Ascl1), fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1), and carbohydrate response element binding protein (Chrebp) in BAT and Ing WAT of AdGsKO mice (Fig. 3 C and D). There was no change in expression of malic enzyme 2 (Me2) in BAT or Ing WAT of Ad-GsKO mice (Fig. 3 C and D). Consistent with these gene expression results, 2-deoxyglucose (DG) uptake in BAT after i.p. administration of insulin in vivo was significantly reduced in BAT (Fig. 4E) and tended to be lower in Ing and Epi WAT (Fig. 4 F and G) of AdGsKO mice. In addition, Ad-GsKO mice had reduced rates of incorporation of glucose into lipid in BAT and epididymal WAT in vitro (Fig. 3F). Ad-GsKO also had reduced expression of genes involved in fatty acid uptake [e.g., lipoprotein lipase (Lpl), fatty acid translocase (Cd36)] and esterification [e.g., phosphoenolpyruvate carboxykinase (Pepck), diacylglycerol acyltransferase 1 (Dgat1), and mitochondrial glycerol-3-phosphate acyltransferase (Gpam)] in both BAT (Fig. S5A) and Ing WAT (Fig. S5B). Pepck, a gene involved in glycerol-3-phosphate production, was also significantly Li et al.
Fig. 3. Reduced lipolysis and lipogenesis in adipose tissues of Ad-GsKO mice. (A and B) mRNA expression of lipolysis-related genes in BAT (A) and Ing WAT (B) from 3- to 4-mo-old male control and Ad-GsKO mice maintained on standard diet (n = 3–5 per group). Abhd5, abhydrolase domain containing 5 (CGI-58); Lipe, hormone-sensitive lipase; Plin1, perilipin 1; Pnpla2, adipose triglyceride lipase. (C and D) mRNA expression of glucose uptake and de novo lipogenesis related genes in interscapular BAT (C) and Ing WAT (D) from 3- to 4-mo-old male control and Ad-GsKO mice maintained on standard diet (n = 3–5 per group). Ascl1, acyl-CoA synthetase long-chain family member 1; Chrebp, carbohydrate responsive element binding protein; Fasn, fatty acid synthase; Glut4, glucose transporter 4; Me1 and Me2, malic enzymes 1 and 2; Pcx, pyruvate carboxylase; Scd1, stearoyl-CoA desaturase 1. (E and F) Plasma FFA (E) and glycerol (F) levels in 8-mo-old male control and Ad-GsKO mice after i.p. injection of saline or CL316243 (CL; n = 5–7 per group). (G) D-[14C(U)]-glucose incorporation into lipid in BAT and Epi WAT isolated from 4-mo-old male control and Ad-GsKO mice (n = 3–5 per group). Results are normalized to tissue weight and expressed as percent of control. Data are mean ± SEM *P < 0.05 vs. controls; #P < 0.05 vs. baseline.
Despite a reduction in adipose tissue lipid metabolism, AdGsKO mice were able to maintain a normal rate of whole-body fatty acid oxidation in vivo (Fig. S6A). To what extent increased metabolism in skeletal muscle prevented the development of obesity in Ad-GsKO mice is not fully clear. It is interesting to note that in these animals gastrocnemius muscle had increased insulin-stimulated glucose uptake (Fig. 4 F and G) and soleus muscle had increased expression of the peroxisome proliferatoractivated receptor-γ coactivator-1α (PGC-1α) and cytochrome c genes, although no increase in expression of the mitochondrial genes Tfam or Nrf1 was observed (Fig. S6B). We also did not observe an increase in expression of the gene encoding sarcolipin, a protein implicated in nonshivering thermogenesis in skeletal muscle (21), in soleus muscle of Ad-GsKO mice. Rather, its expression was decreased (Fig. S6B).
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down-regulated in Epi WAT in both fed and fasted Ad-GsKO mice (Fig. S5C). Consistent with these changes in gene expression, we showed that glucose incorporation into TGs and fatty acids (both free and associated with TG) were markedly reduced in explanted BAT and Ing and Epi WAT (Fig. S5D). The effect of insulin on glucose incorporation was not examined. Incorporation of extracellular oleic acid to TG in explanted BAT and Epi WAT of Ad-GsKO mice were also significantly reduced (Fig. S5E), which could be due to either reduced lipid uptake, esterification, or both. Overall our results suggest that glucose and lipid uptake, fatty acid synthesis, and esterification may all be reduced in adipose tissue of Ad-GsKO mice. At least for WAT, normal adipocyte cell size and lipid content may be the result of similar reductions in lipolysis and lipid synthesis, which counterbalance their opposing effects on lipid storage.
Fig. 4. Improved glucose metabolism and insulin sensitivity in Ad-GsKO mice. (A and B) Results of glucose tolerance tests on 4- to 5-mo-old male control and Ad-GsKO mice maintained on standard diet (A) or after 8 wk of HFD (B). (C and D) Results of insulin tolerance tests on 4- to 5-mo-old control and Ad-GsKO mice maintained on standard diet (C) or 8 wk of HFD (D) (n = 5–8 per group). (E–G) In vivo 2-DG uptake in interscapular BAT (E), Epi WAT (F), Ing WAT (G), gastrocnemius muscle (H), and cardiac muscle (I) of 2-mo-old male control and Ad-GsKO mice after administration of insulin (Humulin, 0.75 mU/kg i.p.; n = 3–5 per group). (J) Blood glucose levels in 4-mo-old male control and Ad-GsKO mice before and after administration of pyruvate (2 g/kg i.p.; n = 5–8 per group). (K and L) Liver (K) and soleus muscle (L) TG content in 4-mo-old male control and Ad-GsKO mice (n = 6 per group). (M) mRNA expression of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in Epi WAT from 3-mo-old male control and Ad-GsKO mice (n = 6 per group). Area under curves for glucose, insulin, and pyruvate tolerance tests were all significantly different between control and Ad-GsKO mice. Data are mean ± SEM *P < 0.05 vs. controls.
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Gsα Deficiency in Fat Tissues Improved Glucose Metabolism and Insulin Sensitivity. Ad-GsKO mice had normal fed and starved
serum glucose levels, whereas serum insulin levels were significantly lower in the starved state and tended to be lower in the fed state (Table 1). Glucose and insulin tolerance tests showed AdGsKO mice to have significantly improved glucose tolerance and insulin sensitivity on both standard diet and HFD (Fig. 4 A–D and Fig. S2C). Consistent with this finding, insulin-stimulated 2-DG uptake was significantly increased in vivo in both skeletal (gastrocnemius) and cardiac muscle in Ad-GsKO mice (Fig. 4 H and I). In addition, Ad-GsKO mice showed a smaller rise in serum glucose after administration of pyruvate, consistent with reduced hepatic gluconeogenesis, although this difference could reflect increased pyruvate oxidation in liver to compensate for reduced energy substrate availability (Fig. 4J). Glycogen levels (measured at 1300 hours) were reduced by 15% in livers of AdGsKO mice whereas they were unchanged in soleus muscle (Fig. S7 A and B). AMP kinase phosphorylation, a downstream marker of insulin signaling, was unaffected in liver but increased by 41% in soleus muscle of Ad-GsKO mice (Fig. S7 C–F). Improved insulin sensitivity in Ad-GsKO mice may be partially explained by improved lipid status, because Ad-GsKO mice had lower serum FFA and TG levels, as well as liver and muscle TG levels (Fig. 4 K and L). Expression of genes involved in lipid synthesis were either unchanged or reduced (e.g., Pcx, Chrebp, Cd36, and Gpam) in livers from Ad-GsKO mice (Fig. S8A) and liver TG secretion rates were significantly reduced in Ad-GsKO mice (Fig. S8B). We also examined several circulating factors that have been shown to affect glucose metabolism and insulin sensitivity. Serum retinol binding protein 4 (RBP4) and fibroblast growth factor 21 (FGF21) levels were unaffected (Table 1). Serum adiponectin levels were reduced in Ad-GsKO mice, consistent with our prior results showing that adiponectin expression in adipose tissue is induced by stimulation with a β3 agonist (22). However, reduced serum adiponectin levels are associated with impaired, rather than improved, peripheral glucose metabolism. Serum levels of FABP4, an adipokine that is markedly elevated in obese/diabetic patients and that stimulates hepatic gluconeogenesis (23–25), were markedly reduced by almost 50% in Ad-GsKO mice (Table 1), making this a possible mechanism contributing to the improved glucose metabolism present in Ad-GsKO mice. There were no differences in expression of the inflammatory genes interleukin-6 and tumor necrosis factor-α in Epi fat from Ad-GsKO and control mice (Fig. 4M), making it unlikely that differences in adipose tissue inflammation are contributing to the observed differences in glucose metabolism. Discussion In this paper we generated mice with loss of Gsα signaling (and therefore resistance to activation by SNS and other hormones) in mature BAT and WAT adipocytes and examined the effects on temperature maintenance, adipocyte metabolism, energy balance, and glucose homeostasis. Our results provide important insights into on how these physiological parameters are regulated by SNS activation of adipose tissue. As expected, Ad-GsKO mice showed marked reductions in BAT activation, cold tolerance, cold-induced Ucp1 up-regulation, and browning of WAT. This result is expected, given the known role of SNS activation of BAT and WAT in these processes, and is consistent with the cold intolerance observed in both our prior adiposespecific Gsα knockout model (10) and Ucp1 knockout mice (17). Similar to Ucp1 knockout mice (17), we observed a small increase in Tb at room temperature in Ad-GsKO mice. Presumably this reflects increased thermogenesis in other tissue(s), although the mechanisms responsible for these observations are unclear. Although it has been shown that loss of BAT function can be compensated for by increased browning of WAT (26), this 450 | www.pnas.org/cgi/doi/10.1073/pnas.1517142113
is not an explanation in our model because WAT browning was absent. The observed absence of a detectable change in energy expenditure despite a small increase in Tb may reflect compensation by the small, though not significant, reduction in activity levels or may reflect the limitations of indirect calorimetry to detect small differences in energy expenditure. Despite loss of BAT and WAT activation by SNS, Ad-GsKO mice had normal body weight and adiposity on both standard diet and HFD. One potential explanation for this observation is that loss of Gsα in adipocytes led not only to reduced lipolysis, a process known to be activated by Gsα and cAMP, but also to a similar reduction in expression of genes critical for glucose and lipid uptake, fatty acid synthesis, and esterification in both BAT and WAT, associated with reduced incorporation of glucose and fatty acids into TG. This finding is consistent with a recent study showing that β3-adrenergic stimulation increased both lipolysis and de novo lipogenesis in BAT and WAT and that induction of de novo lipogenesis may be coupled to lipolysis (27). Another study showed that although norepinephrine acutely inhibited lipogenesis in cultured BAT cells, longer treatments led to stimulation of lipogenesis (28). It is interesting to note that mutations of lipolytic genes encoding ATGL and hormone-sensitive lipase are associated with only small changes in adiposity and reduced lipogenesis in adipose tissue, suggesting that lipid anabolic and catabolic pathways may be coupled (29, 30). It is also possible that the reduced lipogenesis observed in Ad-GsKO mice may reflect fundamental changes in the adipogenic program, because a profound effect of Gsα on adipogenesis was observed in our prior FGsKO mouse model in which Gsα deletion occurred at an earlier stage of adipogenesis (12). Further studies will be required to understand the mechanisms by which Gsα seems to affect both lipolysis and lipogenesis in the same direction. The role of SNS-induced BAT activation and browning of WAT in diet-induced thermogenesis has great appeal and stimulation of these responses has been proposed as a potential treatment for obesity (1, 2, 8, 9). However, whether BAT function is necessary for the maintenance of normal body weight is still not definitively answered. Ablation of sympathetic nerves (4–6) or their neurotransmitters (norepinephrine and epinephrine) (7) does not result in obesity, even when BAT is functionally inactive. Similarly, Ucp1 knockout mice fed on either a standard chow or HFD do not develop obesity relative to littermate controls at room temperature (17). Although Ucp1 knockout mice showed increased gain in fat mass and diet-induced thermogenesis when maintained at thermoneutrality and this was ascribed to loss of diet-induced thermogenesis (2), we observed no changes in fat mass or energy expenditure in Ad-GsKO maintained at thermoneutrality. The absence of greater adiposity of Ad-GsKO mice on either standard diet or HFD provides further direct evidence that activation of BAT or browning of WAT is not required for maintenance of normal energy balance. Another striking finding in Ad-GsKO mice is significant improvement in insulin sensitivity and peripheral glucose metabolism despite the fact that BAT is inactive and the absence of improvement in whole-body adiposity. This effect of glucose metabolism is probably independent of changes in Ucp1 expression, because Ucp1 knockout mice on standard diet were shown to have no differences in glucose tolerance or insulin levels (31). We observed no differences in circulating FGF21 or RBP4 levels to help explain this effect, and circulating levels of adiponectin, a factor associated with improved glucose metabolism, were actually decreased in Ad-GsKO mice. In addition, we found no evidence for differences in WAT inflammation that might contribute to alterations in glucose metabolism. One potential mechanism contributing to improved insulin sensitivity and glucose metabolism in Ad-GsKO mice is reduced lipolysis in adipose tissue resulting in lower circulating FFA levels. It is well established that FFAs, the major product of lipolysis, cause deleterious effects on insulin Li et al.
sensitive organs (32, 33) and that genetic or pharmacologic inhibition of adipose tissue lipolysis in mice improves glucose metabolism and insulin sensitivity (29, 34). Moreover, high lipolysis rate is associated with low insulin sensitivity in humans (34). The lower TG content in liver present in Ad-GsKO may also contribute to improved glucose metabolism in these mice. FABP4 is a recently identified adipokine released from adipocyte that leads to impaired glucose metabolism via increased hepatic glucose production and perhaps other mechanisms as well, although alternative mechanisms have not been directly examined (23, 25). FABP4 release from adipocytes is stimulated by β-adrenergic receptor agonists that activate Gsα, and circulating FABP4 levels have been shown to be markedly elevated in obese/diabetic patients (23, 24). Conversely, reduced plasma FABP4 levels are associated with improved glucose metabolism and insulin sensitivity (23, 35). Therefore, inhibiting Gsα signaling pathway in adipocytes may improve insulin sensitivity and glucose metabolism via reduced lipolysis, release of FABP4, and perhaps other additional mechanisms. In conclusion, although the level of functional BAT is inversely correlated with adiposity in humans (36, 37) and positively
correlated with improved insulin sensitivity and glucose metabolism in mice and humans (38–40), our results suggest that reduced BAT function alone may be insufficient for the development of obesity or insulin resistance. Moreover, specific inhibition of adipose tissue Gsα signaling seems to dramatically affect whole-body glucose metabolism in the absence of an effect on body weight.
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Materials and Methods All studies were approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee. Gsα exon 1-floxed mice (E1fl/fl) (41) were mated with adiponectin-cre mice (15) to generate Ad-GsKO mice (adiponectin-cre+, E1fl/fl). Animals were maintained on a 12-h light/dark cycle (6:00 AM/6:00 PM) and standard pellet diet (NIH-07, 5% fat by weight) unless indicated. HFD (Bio-Serv) consisted of 59.4 kcal% fat, 16.2 kcal% protein, and 24.5 kcal% carbohydrate. All data are expressed as mean ± SEM and analyzed by Student’s t test with differences considered significant at P < 0.05. Detailed methods are described in SI Materials and Methods.
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ACKNOWLEDGMENTS. We thank M. Reitman and A. Cypess for suggestions and technical assistance. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.
