Exquisite regulation of energy homeostasis protects from nutrient deprivation but causes metabolic dysfunction upon nutrient excess. In human and murine adipose tissue, the accumulation of ligands of the receptor for advanced glycation end products (RAGE) accompanies obesity, implicating this receptor in energy metabolism. Here, we demonstrate that mice bearing global- or adipocyte-specific deletion of Ager, the gene encoding RAGE, display superior metabolic recovery after fasting, a cold challenge, or high-fat feeding. The RAGE-dependent mechanisms were traced to suppression of protein kinase A (PKA)-mediated phosphorylation of its key targets, hormone-sensitive lipase and p38 mitogen-activated protein kinase, upon β-adrenergic receptor stimulation—processes that dampen the expression and activity of uncoupling protein 1 (UCP1) and thermogenic programs. This work identifies the innate role of RAGE as a key node in the immunometabolic networks that control responses to nutrient supply and cold challenges, and it unveils opportunities to harness energy expenditure in environmental and metabolic stress.

Here, we show that mice bearing adipocyte-specific deletion of Ager display significant protection from HFD-induced obesity and insulin resistance and exhibit a superior ability to thermoregulate during a cold challenge, compared to mice in which adipocytes express Ager. WT mice that underwent a surgical transplantation of either interscapular BAT (iBAT) or subcutaneous inguinal WAT (iWAT) from mice with adipocyte-specific deletion of Ager demonstrate significant protection from HFD-induced obesity and insulin resistance, compared to WT mice transplanted with iBAT or iWAT in which adipocytes expressed Ager. We traced these adipocyte-intrinsic, RAGE-dependent mechanisms to RAGE ligand-mediated suppression of PKA-dependent phosphorylation of HSL and p38 MAPK—processes that, collectively, dampen UCP1 and thermogenic programs.

The immunoglobulin superfamily molecule, the receptor for advanced glycation end (RAGE) products, binds to a distinct repertoire of molecules, such as the carboxymethyllysine (CML)-advanced glycation end products (AGEs), high-mobility group box 1 (HMGB1), and S100/calgranulins, which accumulate in metabolic stress (). Although these ligands are classically linked to diabetes and inflammation, recent evidence places these molecules and RAGE itself in human and murine obese adipose tissue (). RAGE localization in human obese adipose tissue aligns with our recent finding that mice globally devoid of Ager (the gene encoding RAGE) are protected from obesity and insulin resistance when fed a high-fat diet (HFD) compared to wild-type (WT) mice (). Yet, the mediating mechanisms have remained elusive.

The diverse ligand repertoire of the receptor for advanced glycation endproducts and pathways to the complications of diabetes.

White adipose tissue (WAT) and brown adipose tissue (BAT) in mammals store energy in the form of triglycerides and release fatty acids and glycerol in response to catecholaminergic stimulation by sympathetic nerves (). Whereas WAT liberates fatty acids into the circulation, BAT preferentially oxidizes fatty acids and dissipates energy through uncoupled respiration and the production of heat (). The recent identification of BAT in adult humans has rekindled an interest in harnessing brown fat to spur cellular energy expenditure (). The physiologic process in which energy is dissipated in the form of heat in response to stressors such as cold challenges or over-feeding is termed “adaptive thermogenesis” and is dependent on the expression and activity of uncoupling protein 1 (UCP1) in brown adipocytes (). Catecholamines stimulate β-adrenergic receptors (ARBs), culminating in increased activity of protein kinase A (PKA) and leading to phosphorylation of adipocyte lipases, such as hormone-sensitive lipase (HSL) and lipolysis, and of p38 mitogen-activated protein kinase (MAPK) and regulation of genes linked to lipid oxidation in brown adipocytes, including Ucp1 and Ppargc1a (peroxisome proliferator-activated receptor γ [PPARγ]-coactivator 1α) (). Though a process classically ascribed to BAT, during prolonged starvation or a cold challenge, white adipocytes undergo “beiging” or “browning,” in which WAT assumes increased UCP1-expressing adipocytes bearing thermogenic capacity (). Despite the profound importance of such metabolic plasticity, the natural “brakes” in adipose tissues—the best described of which is insulin ()—remain incompletely understood.

Studies on the metabolism of adipose tissue. XVII. In vitro effects of insulin upon the metabolism of the carbohydrate and triglyceride stores of adipose tissue from fasted-refed rats.

Studies on the metabolism of adipose tissue. XII. The effects of insulin and epinephrine on free fatty acid and glycerol production in the presence and absence of glucose.

The epidemic of obesity and its metabolic sequelae profoundly impact human health and longevity (). Maintaining a precise balance between energy intake and expenditure is essential for an organism’s ability to store or utilize nutrients. Yet, hoarding energy is a double-edged sword, albeit, salutary in nutrient deprivation; in over-feeding, excess energy storage confers susceptibility to obesity and type 2 diabetes.

There were no Ager-dependent differences in the protein levels of two other lipolytic molecules, adipose triglyceride lipase or monoacylglycerol lipase, upon treatment with CL316,243 ( Figures S7 G and S7H), and there were no genotype-dependent differences in the levels of cyclic AMP (cAMP) between WT and Ager null CL-316,243-treated primary adipocytes from iBAT, iWAT, or eWAT ( Figure S7 I).

On account of the demonstrated roles for a PKA-independent, β3-adrenergic-mediated stimulation of extracellular signal-related kinase (ERK) MAPK on lipolysis in, we tested the effects of the RAGE ligand, CML-AGE. In C3H10T1/2 cells, treatment with CL316,243 resulted in significantly increased phospho/total ERK MAPK, which was significantly suppressed by CML-AGE; the suppressive effects of CML-AGE were prevented by treatment with the RI ( Figure S7 E). However, in primary WT or Ager null adipocytes retrieved from iBAT or iWAT, no significant effects of CML-AGE on phospho/total ERK MAPK in CL316,243-treated cells were observed ( Figure S7 F).

We next tested primary adipocytes from iBAT and iWAT. In WT, but not in Ager null primary adipocytes derived from iBAT ( Figure 7 E) and iWAT ( Figure 7 F), treatment with CML-AGE in CL316,243-treated cells resulted in significant suppression of phosphorylation of pHSL Serine563/total HSL and p38/total p38 MAPK. In primary iBAT- and iWAT-derived adipocytes, treatment with CL316,243 resulted in significantly increased phosphorylated HSL Serine563/total HSL and increased phosphorylated p38/total p38 MAPK, which was significantly attenuated by H89 ( Figure S7 C). Treatment of Ager null adipocytes from iBAT or iWAT with CL316,243, CML-AGE, and H89 resulted in significant decreases in phosphorylated HSL Serine563/total HSL and phosphorylated p38/total p38 MAP kinase, compared to cells treated with CL316,243 and CML-AGE alone ( Figures 7 G and 7H). Thus, direct catalytic inhibition of PKA significantly reduced the effects of deletion of Ager on rescuing the suppression of CML-AGE on phosphorylation of these two PKA targets. In vivo, expression of Lipe (gene encoding HSL), a target of activated PKA (), was significantly higher in the floating adipocyte fraction retrieved from the iWAT of Ager null than from the WT mice ( Figure S7 D).

We assessed the effects of RAGE on phosphorylation of HSL Serine563 and p38 MAPK. Treatment of C3HT101/2 cells with CL316,243 resulted in significantly higher phospho/total pHSL Serine563 and phospho/total p38 MAPK compared to vehicle, which were both significantly reduced by treatment with CML-AGE. However, in cells treated with CL316,243 and CML-AGE, treatment with the RI resulted in significantly higher phospho-p38/total p38 MAPK and trended to higher phospho-HSL Serine563/total HSL, compared to treatment with CL316,243 and CML-AGE alone ( Figure 7 C). We hypothesized that if these effects of RAGE were through a blockade of phosphorylation of PKA targets, then direct catalytic inhibition of PKA should blunt the effects of the RI. First, we tested the efficacy of H89, a commonly used compound to inhibit PKA activity, on the suppression of the effects of CL316,243 (). In C3HT101/2 cells, treatment with H89 blocked the increased phosphorylated HSL Serine563/total HSL ratio and the increased phosphorylated p38/total p38 MAPK induced by CL316,243 ( Figure S7 B). We then tested if H89 blocked the effects of the RI. Compared to cells treated with CL316,243, CML-AGE, and the RI, the addition of H89 resulted in a significant reduction in phosphorylation of both HSL Serine563 and p38 MAPK ( Figure 7 D). Thus, direct catalytic inhibition of PKA blunted the effects of the RI on rescuing the suppressive effects of CML-AGE on the phosphorylation of pHSL Serine563 and p38 MAPK.

To trace the fate of lipolysis and signaling pathways downstream of PKA that regulate adaptive thermogenesis, we focused on two PKA targets: HSL and p38 MAP kinase (MAPK) (). Based on the discovery that the cytoplasmic domain of RAGE is essential for RAGE signaling, in part through its binding to DIAPH1 (), we recently described the small molecule antagonist, 2- [4- (acetylamino) phenyl] −4- Quinolinecarboxylic acid, methyl ester (“RAGE inhibitor”), which binds to the cytoplasmic domain of RAGE, blocks its interaction with DIAPH1, and suppresses RAGE ligand-stimulated signal transduction in cellular and murine models (). Significantly higher levels of glycerol release were observed in C3HT101/2 cells treated with CL316,243 and the RAGE inhibitor (RI) versus CL316,243 alone ( Figure 7 A). The RI had no independent effect on the numbers of lipid droplets, as assessed by staining with BODIPY ( Figure 7 B), nor did it affect the relative mitochondrial DNA content ( Figure S7 A).

(E–H) Primary adipocytes from (E and G) iBAT or (F and H) iWAT of WT and Ager null mice were treated with the RAGE ligand CML-AGE (300 μg/ml) for 1 h, CL316,243 (10 μM) for 15 min, and with or without H89 (20 μM) for 30 min prior to CL. Cells were lysed and western blotting performed for the detection of phosphorylated HSL Serine563, total HSL, phosphorylated p38 MAPK, total p38 MAPK, and GAPDH. Band intensities were normalized to the respective total HSL or p38 MAPK, and the relative fold change is presented as mean ± SEM from N = 4 pooled mice/group in three independent experiments with at least 2–3 technical replicates per experiment.

(D) Differentiated adipocytes from C3H10T1/2 cells were treated as in (C) with or without the addition of H89 (20 μM) for 30 min prior to CL316,243 (10 μM). Cells were lysed and western blotting performed for phosphorylated HSL Serine563, total HSL, phosphorylated p38 MAPK, total p38 MAPK, and GAPDH. Band intensities were normalized to the respective total HSL or total p38 MAPK, and the relative fold change is presented as mean ± SEM. Each experiment consisted of at least three independent studies with at least three technical replicates.

(C and D) C3H10T1/2 cells were differentiated and treated with a vehicle or CML-AGE (300 μg/ml) for 75 min alone or after pre-treatment with the RAGE inhibitor (1 μM) for 30 min followed by CL316,243 (10 μM) treatment for 15 min.

(B) C3H10T1/2 cells were differentiated to adipocytes and pre-incubated with a vehicle or the RAGE inhibitor (1μM) for 90 min prior to CL316,243 (10 μM). Lipid droplets were stained using BODIPY and nuclei stained with DAPI. A representative fluorescence micrograph and mean ± SEM is shown. Scale bar: 100 μm.

(A) Differentiated adipocytes from C3H10T1/2 cells were serum starved for 3 h and pretreated with the RAGE inhibitor (2- [4- (acetylamino) phenyl] −4- Quinolinecarboxylic acid, methyl ester) (1 μM) for 1 h prior to CL316,243 (10 μM) for 15 min. The mean glycerol content normalized to total protein per well ± SEM is reported from three independent experiments.

Interaction of the RAGE cytoplasmic domain with diaphanous-1 is required for ligand-stimulated cellular migration through activation of Rac1 and Cdc42.

To determine if the effects of RAGE ligands were via direct action on Ucp1 transcription, we employed Ucp1 promoter luciferase constructs ( Figure 6 I) transfected into C3H10T1/2 cells. Compared to the vehicle, treatment with NE resulted in a significant increase in Ucp1 promoter luciferase activity, which was suppressed by CML-AGE ( Figure 6 I). We prepared mutants of the Ucp1 promoter in which only the proximal promoter, which possesses functional CRE-binding elements, or the enhancer elements, were expressed ( Figure 6 J). Treatment with CML-AGE exerted only modest effects on the suppression of basal but not NE-mediated promoter luciferase activities, indicating that the entire Ucp1 promoter was required to detect the effects of CML on transcriptional activity. On account of central roles for PKA in the β-adrenergic stimulation of thermogenic gene programs in adipocytes, we tested the effects of CML-AGE on PKA activity. In primary iBAT-derived adipocytes treated with CL316,243 and CML-AGE, a significant attenuation in PKA activity was observed ( Figure 6 K).

RAGE and its ligand, carboxymethyllysine (CML)-AGE, are highly expressed in obese human adipose tissue (). We previously showed that CML-AGEs were specific ligands of RAGE (). WT primary adipocytes were treated with vehicle or NE, alone or with CML-AGE. In the basal state and upon treatment with NE, incubation with CML-AGE resulted in a significant suppression of Ucp1 and Ppargc1a mRNA expression in primary adipocytes from iBAT and iWAT ( Figures 6 F and 6G). In eWAT-derived adipocytes, CML-AGE treatment decreased Ucp1 and Ppargc1a at baseline and significantly suppressed the NE-stimulated induction of Ucp1 with a similar trend noted for Ppargc1a ( Figure S6 F). In iBAT-derived adipocytes, treatment with CML-AGE significantly decreased the expression of UCP1 ( Figure 6 H).

We differentiated iBAT- and iWAT-derived adipocytes from WT and Ager null mice and stimulated them with CL316,243 (CL) (ARB3-specific agonist) to test the effects on lipolysis. In iBAT adipocytes, significantly higher CL316,243-stimulated levels of glycerol, and both basal and CL316,243-stimulated levels of non-esterified fatty acids (NEFAs), were observed in Ager null than in WT mice ( Figure 6 A). In iWAT adipocytes, basal and CL316,243-stimulated glycerol levels were significantly higher in adipocytes derived from Ager null mice, as was the CL316,243-mediated stimulation of the NEFA release ( Figure 6 B). Significantly higher oxygen consumption rates (OCRs) were observed in primary iBAT adipocytes from Ager null than from WT mice after treatment with isoproterenol (non-specific ARB agonist), which was minimally suppressed by oligomycin in the Ager null adipocytes, suggestive of maximum mitochondria uncoupling ( Figure 6 C). When primary iBAT adipocytes were treated with vehicle or norepinephrine (NE) + T3 (thyroid hormone), significantly higher Ucp1 mRNA transcripts were observed in adipocytes devoid of Ager versus WT at baseline and after NE/T3 ( Figure 6 D). Primary iBAT-derived adipocytes from Ager null versus WT mice displayed significantly higher levels of UCP1 ( Figure 6 E). These data point to adipocyte-intrinsic roles for RAGE in β-adrenergic stimulation of lipolysis and upregulation of Ucp1 expression and activity.

In (A)–(E), WT (black) and Ager null (green). Data analysis: (A–D, repeated measures) two-way ANOVA followed by post hoc Bonferroni test; (E and H) two-tailed Student’s t test; (F, G, and I–K) one-way ANOVA followed by a post hoc Tukey’s HSD test; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

(K) PKA activity was determined in primary iBAT adipocytes from WT mice treated with CL316,243 (10 μM) for 15 min alone or after pre-incubation with the RAGE ligand CML-AGE (300 μg/ml) for 16 h. The mean ± SEM of N = 8 mice/group is presented.

(J) Ucp1 promoter luciferase constructs were generated and transfected into undifferentiated C3H10T1/2 cells and the cells cultured in medium containing NE (5 μM) for the last 6 h or CML-AGE (300 μg/ml) for 16 h. Normalized luciferase activities are shown relative to vehicle control. The mean ± SEM of three independent experiments is presented.

(I) Schematic of Ucp1 promoter with enhancer elements. Undifferentiated C3H10T1/2 cells were transfected with mouse 3.1 kB Ucp1 promoter luciferase construct (Ucp1-luc) and stimulated for the last 6 h with NE (5 μM) and/or CML-AGE (300 μg/ml) for 16 h. Normalized luciferase activities are shown as fold-change compared to vehicle control.

(H) Cell lysates from primary adipocytes from iBAT of WT mice treated as in (F) and (G) were used for the detection of UCP1/TUBULIN. The mean ± SEM is reported in cell lysates from N = 3 mice/group.

(F and G) Primary adipocytes from (F) iBAT and (G) iWAT of WT mice were incubated with vehicle or the RAGE ligand CML-AGE (300 μg/ml) for 16 h alone or with NE (5 μM) for the final 6 h. qRT-PCR for detection of relative Ucp1 or Ppargc1a mRNA expression was performed. The mean ± SEM is reported from N = 5 mice/group.

(E) Western blotting was performed for detection of UCP1/TUBULIN from primary iBAT adipocytes of 8-week-old WT and Ager null mice. The mean ± SEM is reported in N = 6 independent cell lysates per group along with representative blots.

(D) Primary iBAT-derived adipocytes from iBAT of WT or Ager null mice were stimulated with norepinephrine (NE; 5 μM) and T3 (thyroid hormone, 2 nM) for 6 h, and relative Ucp1 mRNA expression was determined by qRT-PCR. The mean ± SEM is reported in N = 4 mice/group.

(C) A representative OCR curve for iBAT-derived adipocytes during a mitochondrial stress test is shown under basal condition and following isoproterenol (10 μM). This experiment was performed three times using cells derived from N = 3 mice/group per experiment.

(A and B) iBAT- (A) and iWAT- (B) derived cells were incubated for 3 h in Krebs-Ringer bicarbonate buffer (KRB) and subjected to vehicle or CL316,243 (CL) (10 μM) for 3 h; glycerol and non-esterified fatty acids (NEFAs) in the media were measured. The means normalized to total protein per well ± SEM are reported from triplicate, independent experiments from cells derived from N = 5 mice/group.

Compared to thermoneutrality, exposure to 4°C results in significant increases in sympathetic nerve innervation to the eWAT and iWAT (). As β-adrenergic stimulation of adipocytes is a primary mechanism for lipolysis and the generation of fatty acid products that regulate Ucp1 expression and activity (), we tested whether RAGE plays a role in this pathway in primary and cultured adipocytes.

To assess the effects of RAGE ligand sequestration, we previously treated WT mice fed a HFD for 18 weeks with soluble RAGE (sRAGE), the extracellular ligand-binding domains of RAGE, which act as a decoy receptor and thus suppress RAGE signaling. When treatment was given concurrently with a HFD, compared to vehicle-treated mice, mice treated with sRAGE displayed less body weight gain (). Compared to vehicle-treated mice, in the iWAT of sRAGE-treated mice, we noted the following: expression of Ucp1 mRNA was significantly higher; expression of Cidea was significantly higher in the eWAT, iWAT, and iBAT; expression of Ppargc1a mRNA was significantly higher in the eWAT and iBAT; and expression of Dio2 mRNA was significantly higher in eWAT ( Figures S6 A–S6D). Immunohistochemistry revealed more pronounced UCP1 immunoreactive epitopes in the eWAT, iWAT, and iBAT of sRAGE-treated mice ( Figure S6 E). Thus, both pharmacological and genetic approaches indicate that RAGE suppresses adipose tissue thermogenic programing.

After 20 weeks of the HFD, in native iBAT, WT mice receiving AgerCre (+) versus Cre (–) iWAT displayed a significantly higher expression of Ppargc1a and Pnpla2 mRNA transcripts, with no statistically significant differences in levels of Ucp1, Dio2, or Cpt2a mRNA transcripts ( Figure 5 I). Immunohistochemistry revealed that the native iBAT of mice that were transplanted with AgerCre (+) versus Cre (–) iWAT displayed a significantly higher UCP1-positive area ( Figure 5 J). In native iWAT, WT mice receiving AgerCre (+) versus Cre (–) iWAT displayed significantly higher levels of Ucp1, Dio2, and Pnpla2 mRNA transcripts; significantly lower levels of Ppargc1a mRNA; and no differences in Cpt2a mRNA transcripts ( Figure 5 . Immunohistochemistry revealed that there were no significant differences in the UCP1-positive area in the iWAT of mice that had been transplanted with AgerCre (+) or Cre (–) iWAT ( Figure 5 L). In the tiWAT, the WT mice recipients of AgerCre (+) versus Cre (–) iWAT displayed significantly higher levels of Ucp1 and Pnpla2 mRNA transcripts, a trend to higher levels of Dio2 expression, lower levels of Ppargc1a, and no significant differences in Cpt2a mRNA transcripts ( Figure 5 M). In native eWAT, significantly higher levels of Ucp1, Ppargc1a, and Pnpla2 mRNA transcripts were observed in the recipients of AgerCre (+) than of Cre (–) iWAT, with no statistically significant differences in Dio2 or Cpt2a mRNA transcripts ( Figure S5 C). Thus, the deletion of Ager in iBAT or iWAT adipocytes transmits protection from diet-induced obesity and insulin and glucose intolerance and enhances expression of thermogenic genes.

We next transplanted iWAT expressing or devoid of adipocyte Ager into young, lean, WT mice and established the presence of vascularized non-necrotic grafts (representative image; Figure S5 A). Four days after the transplantation, mice were switched to a HFD for an additional 20 weeks. Compared to recipients of AgerCre (–) iWAT, WT mice receiving AgerCre (+) iWAT displayed significantly less weight gain after 12 weeks of the HFD ( Figures 5 A and 5B ). The WT mice recipients of AgerCre (+) iWAT displayed significantly less fat mass than recipients of Cre (–) iWAT ( Figure 5 C). No differences in food intake or physical activity were observed by iWAT donor genotype ( Figures 5 D and 5E). As with iBAT transplants, significantly higher energy expenditure was observed in WT recipients of AgerCre (+) than Cre (–) iWAT after 16 weeks of the HFD ( Figure 5 F). The WT mice receiving AgerCre (+) versus Cre (–) iWAT displayed superior glucose tolerance ( Figure 5 G) and insulin sensitivity while on the HFD ( Figures 5 H and S5 B).

(A–M) Source of donor iWAT: Ager flox/flox Cre (–) shown as red and Ager flox/flox Cre (+) shown as green. Data analysis: (B, G, and H, repeated measures, and C, independent samples) two-way ANOVA followed by a post hoc Bonferroni test; (D–H, area under the curve, and I–M) two-tailed Student’s t test. Where group mean variances were statistically different (p < 0.05), data were analyzed post hoc using the non-parametric Mann-Whitney U test; ∗ p < 0.05; ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

(J and L) In (J, native iBAT) and (L, native iWAT), immunohistochemistry for detection of UCP1 protein was performed and image analysis employed to quantify UCP1-positive area; representative images at 20× magnification are shown. The mean ± SEM is reported in N = 5 mice/group. Scale bar: 100 μm.

(I, K, and M) The relative mRNA expression by qRT-PCR for Ucp1, Dio2, Ppargc1a, Cpt2a, and Pnpla2 mRNA transcripts in (I) native iBAT, (K) native iWAT, and (M) transplanted iWAT (tiWAT) was examined, and the mean ± SEM is reported in N = 3–4 mice/group.

(G and H) Glucose (G) and insulin tolerance (H) tests were performed after 12 and 13 weeks of a HFD, respectively. The mean ± SEM is reported in N = 5–6 mice/group.

(D–F) Average daily food intake (D), physical activity (E), and energy expenditure (F) were assessed after 16 weeks of a HFD. In (B)–(F), the mean ± SEM is reported in N = 7–9 mice/group.

(A) Representative photographs from mice receiving surgical transplantation of either Ager flox/flox Cre (–) (red, left) or Ager flox/flox Cre (+) iWAT (green, right) are shown after 20 weeks on a HFD.

We retrieved native iBAT and iWAT, transplanted iBAT (tiBAT) and eWAT, and assessed the expression of thermogenic genes after 20 weeks of a HFD. In native iBAT, the levels of Dio2, Cpt2a, and Pnpla2 mRNA levels were significantly higher in WT mice receiving AgerCre (+) than those receiving Cre (–) iBAT, while levels of Ucp1 and Ppargc1a mRNA were generally, but not significantly, higher ( Figure 4 I). Immunohistochemistry revealed that the native iBAT of mice receiving AgerCre (+) versus Cre (–) iBAT transplants displayed a significantly higher UCP1-positive area ( Figure 4 J). In native iWAT, there were no significant differences in levels of Ucp1, Dio2, Ppargc1a, Cpt2a, or Pnpla2 mRNA in WT mice receiving AgerCre (+) versus Cre (–) iBAT transplants ( Figure 4 K). However, immunostaining revealed that the native iWAT of mice that were transplanted with AgerCre (+) iBAT displayed significantly higher UCP1-positive areas than mice that had been transplanted with Cre (–) iBAT ( Figure 4 L). In the transplanted iBAT, levels of Ucp1, Dio2, Ppargc1a, Cpt2a, and Pnpla2 mRNA were significantly higher in transplanted iBAT originating from AgerCre (+) than from Cre (–) mice ( Figure 4 M). In native eWAT, levels of Ucp1, Ppargc1a, Cpt2a, and Pnpla2 mRNA transcripts were significantly higher in the mice receiving AgerCre (+) than those receiving Cre (–) iBAT, while levels of Dio2 mRNA transcripts were significantly lower ( Figure S4 C). Figure S4 D is a representative image of the secondary antibody-alone control for the UCP1 immunostaining.

As the Adipoq Cre-recombinase strategy employed above affected levels of Ager in both brown and white adipocytes, the relative contribution of each depot could not be discerned. To address this point, we performed an adipose tissue surgical transplantation of iBAT bearing adipocyte-specific deleted Ager or adipocyte-expressing Ager into lean, young, WT mice. Figure S4 A is a representative image demonstrating a well-vascularized, non-necrotic iBAT graft. After 4–5 days’ recovery from surgery, mice were switched to a HFD and were monitored over 20 weeks. Beginning at 4 weeks post-HFD, body weights in WT mice receiving AgerCre (+) iBAT were significantly lower than those of mice receiving AgerCre (–) iBAT, an observation that continued and was magnified throughout the HFD feeding period ( Figures 4 A and 4B ). Fat mass was significantly lower in the WT mice receiving AgerCre (+) versus Cre (–) iBAT after 14 weeks of the HFD ( Figure 4 C). Although there were no differences in food intake or physical activity between the AgerCre (+) and Cre (–) mice iBAT donors ( Figures 4 D and 4E), energy expenditure was significantly higher in the mice receiving AgerCre (+) than those receiving Cre (–) iBAT after 16 weeks of the HFD ( Figure 4 F). After 12 weeks of the HFD, WT mice receiving AgerCre (+) iBAT were more glucose tolerant than mice receiving Cre (–) iBAT ( Figure 4 G). Insulin sensitivity was improved in mice receiving the AgerCre (+) than the Cre (–) iBAT ( Figures 4 H and S4 B).

(A–M) Source of donor iBAT: Ager flox/flox Cre (–) shown as red and Ager flox/flox Cre (+) shown as green. Data analysis: (B, G, and H, repeated measures, and C, independent samples) two-way ANOVA followed by a post hoc Bonferroni test; (D and F–H, area under the curve, and I–M) two-tailed Student’s t test. Where group mean variances were statistically different (p < 0.05), data were analyzed post hoc using the non-parametric Mann-Whitney U test; ∗ p < 0.05; ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

(J and L) In (J, native iBAT) and (L, native iWAT), immunohistochemistry for the detection of UCP1 was performed and image analysis employed to quantify UCP1-positive area; representative images are shown at 20× magnification. The mean ± SEM is reported in N = 5 mice/group. Scale bar: 100 μm.

(I, K, and M) The relative mRNA expression by qRT-PCR for Ucp1, Dio2, Ppargc1a, Cpt2a, and Pnpla2 for (I) native iBAT, (K) native iWAT, and (M) transplanted iBAT (tiBAT) retrieved from the WT recipient mice after 20 weeks on a HFD is reported in N = 4–6 mice/group.

(G and H) Glucose (G) and insulin tolerance (H) tests were performed after 12 or 13 weeks on a HFD, respectively. The mean ± SEM is reported in N = 10–11 mice/group.

(D–F) Average daily food intake (D), physical activity (E), and energy expenditure (F) were assessed after 16 weeks on a HFD. For (B)–(F), the mean ± SEM is reported in N = 15 mice/group.

(A) Representative photograph of mice surgically transplanted with iBAT from either Ager flox/flox Cre (–) (red, left) or Ager flox/flox Cre (+) iBAT (green, right) after 20 weeks on a HFD.

We next exposed littermate AgerCre (+) and Cre (–) mice to a cold challenge. Compared to AgerCre (–) mice, Cre (+) mice exhibited significant protection from a loss of body temperature after 24 h of exposure to 4°C ( Figure 3 H), with no genotype-dependent differences observed in body weight changes ( Figure 3 I). We examined the expression of genes associated with thermogenic responses in iBAT and iWAT after cold exposure. In iBAT, there were no genotype-dependent differences in Ucp1, Dio2, Ppargc1a, or Cidea during the cold challenge, and only significantly lower Ucp1 was observed in AgerCre (+) at baseline ( Figure 3 J). In iWAT, after 24 h of 4°C, levels of Ucp1, Dio2, Ppargc1, and Cidea mRNA were significantly higher in AgerCre (+) than in Cre (–) mice ( Figure 3 K). In eWAT, levels of Ucp1 were significantly higher in AgerCre (+) than in Cre (–) mice at 4°C; no genotype-dependent differences were observed in Ppargc1a mRNA at 4°C but the levels of Ucp1 and Ppargc1a were significantly higher at room temperature in the AgerCre (+) than the Cre (–) mice ( Figure S3 F). These data support adipocyte-intrinsic roles for RAGE in adaptive thermogenic gene program responses after the HFD and 4°C cold challenge.

We determined if a modulation of adipose inflammation accompanied the metabolic protection observed in AgerCre (+) mice after the HFD. In eWAT, but not iWAT, mRNA levels of Emr1 (F4/80) and Ccl2 were significantly lower in AgerCre (+) than in Cre (–) mice ( Figure S3 E). No differences in Tnfa or Il10 expression were observed in eWAT or iWAT between the genotypes ( Figure S3 E). Transcript levels for Irf4, which increases adipocyte lipolysis (), were significantly higher in the eWAT, iWAT, and iBAT of AgerCre (+) mice than Cre (–) mice ( Figure S3 E). Tlr4, which has been linked to endotoxin-mediated stimulation of lipolysis in adipocytes (), was significantly higher in eWAT, iWAT, and iBAT of AgerCre (+) than Cre (–) mice ( Figure S3 E), and levels of Tlr2 mRNA were significantly higher in eWAT and iWAT, not iBAT, of AgerCre (+) than of Cre (–) mice ( Figure S3 E). Thus, decreased adipose inflammation accompanied the observed protection in AgerCre (+) mice from diet-induced obesity and insulin intolerance.

We phenotyped male littermate AgerCre (+) and AgerCre (–) mice at baseline while they were fed a standard chow diet and then after 6 weeks of HFD feeding (60% kcal/fat). At baseline, body weight and composition, average food intake, physical activity, and energy expenditure rates were indistinguishable between AgerCre (+) (green lines) and AgerCre (–) (red lines) mice ( Figures 3 A–3E). However, when fed a HFD, AgerCre (+) mice weighed significantly less than Cre (–) mice ( Figure 3 A). Whole body fat mass, measured usingH-MRS, was significantly lower in the AgerCre (+) than in Cre (–) mice after 3 and 6 weeks of the HFD ( Figure 3 B). Average food consumption did not differ by genotype, except after 6 weeks of the HFD, in which lower food consumption was noted in AgerCre (+) than in Cre (–) mice ( Figure 3 C). No genotype-dependent differences in physical activity were noted at baseline or after 3 and 6 weeks of the HFD ( Figure 3 D). AgerCre (+) mice displayed significantly higher energy expenditure rates than Cre (–) mice after 3 weeks of the HFD, with trends in the same direction at 6 weeks of the HFD ( Figure 3 E). No differences in glucose-tolerance testing (GTT) were observed after 12 weeks of the HFD ( Figure 3 F). AgerCre (+) mice exhibited significantly greater insulin sensitivity than Cre (–) mice during an insulin-tolerance test (ITT) after 12 weeks of the HFD ( Figures 3 G and S3 D).

In (F)–(K), the mean ± SEM is reported in N = 4–5 mice/group. In (A)–(K), Ager flox/flox Cre (–) shown as red and Ager flox/flox Cre (+) shown as green. Data analysis: (A–G, repeated measures, and J and K, independent samples) two-way ANOVA followed by a post hoc Bonferroni test; (H and I) two-tailed Student’s t test; ∗ p < 0.05; ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

(J and K) Relative mRNA expression of Ucp1, Dio2, Ppargc1a, and Cidea mRNA was assessed in (J) iBAT and (K) iWAT retrieved from mice housed at room temperature or after termination of a 24-h cold challenge (4°C).

(F and G) Glucose (F) and insulin tolerance (G) tests were performed after 12 weeks of a HFD. The mean ± SEM is reported in N = 4–5 mice/group.

(B–E) Body composition (B), average food intake (C), physical activity (D), and energy expenditure (E) were assessed in mice fed standard chow (baseline) or after 3 or 6 weeks of a HFD. In (A)–(E), the mean ± SEM is reported in N = 8 mice/group.

In addition to adipocytes, immune and vascular cells and sympathetic fibers populate adipose tissue and might contribute to the impact of RAGE expression. To definitively probe for roles for RAGE in adipocyte-intrinsic responses to the HFD and cold challenge, we generated Ager floxed mice ( Figures S3 A and S3B). Agermice were bred with adiponectin (Adipoq) Cre-recombinase mice to yield AgerAdipoq Cre (+) (adipocyte-specific Ager-deleted mice) and littermate AgerAdipoq Cre (–) (Ager WT) mice. This resulted in the deletion of Ager from iWAT, eWAT, and iBAT, but not from the pancreas, muscle, and liver ( Figure S3 C). Hereafter, these animals will be referred to as AgerCre (+) and AgerCre (–) mice.

As an alternative paradigm to induce lipolysis and thermogenic programs, we performed a cold stress (). Compared to room temperature, the cold challenge resulted in a significantly increased expression of Ager in iBAT after 6 or 12 h at 4°C. ( Figure 2 E). In iWAT and eWAT, Ager expression was significantly higher after 12 h of exposure to 4°C, compared to room temperature ( Figures 2 F and S2 C). To assess whether upregulation of Ager in iBAT and iWAT in mice subjected to the 4°C cold challenge affected thermoregulation in vivo, we assessed their core body temperature after 48 h of exposure to 4°C. Mice devoid of Ager exhibited significantly less loss of body temperature than did WT mice over 48 h ( Figure 2 G). In iBAT, at 6 or 12 h of exposure to 4°C, levels of Ucp1 mRNA did not differ between Ager null and WT mice; at room temperature, levels of Ucp1 mRNA transcripts were significantly higher in Ager null than in WT iBAT ( Figure 2 H). In iWAT, levels of Ucp1 mRNA were significantly higher in Ager null mice at room temperature and after 6 or 12 h in 4°C ( Figure 2 I). In eWAT, levels of Ucp1 were significantly lower in Ager null than in WT mice at room temperature and after 6 or 12 h at the 4°C cold challenge ( Figure S2 D). Thus, deletion of Ager protects against the loss of body temperature in mice subjected to the 4°C environment, which was accompanied by RAGE-dependent regulation of Ucp1 in iWAT.

First, we used a fasting-refeeding paradigm. After 24 h of fasting, WT mice displayed a significantly higher expression of Ager in iBAT, but not in iWAT. In iBAT, after 24 h of refeeding, the expression of Ager was significantly lower after fasting and returned to baseline (fed) levels ( Figure 2 A). Similar expression patterns were observed in eWAT under these conditions ( Figure S2 A). After 24 h of fasting, mice globally devoid of Ager displayed significantly less loss of body temperature than the WT mice did ( Figure 2 B). We examined the expression of Acox1 and Cpt2a, genes that are associated with fatty acid beta oxidation. In iBAT, levels of Acox1 and Cpt2a mRNA were significantly higher in the fasted Ager null than in the WT mice ( Figure 2 C), and in iWAT, significantly higher levels of Acox1 mRNA, but not Cpt2a mRNA, were observed in fasted mice devoid of Ager than in the WT mice ( Figure 2 D). In eWAT, there were no genotype-dependent differences in the fed or the fasted states ( Figure S2 B).

In (B)–(D) and (G)–(I): WT (black bars) and Ager null (green bars) mice. Data are presented as mean ± SEM in N = 4–6 mice/group. Data analysis: (A, E, and F) one-way ANOVA with post hoc Tukey’s HSD test; (B and G) two-tailed Student’s t test; (C, D, H, and I) two-way ANOVA followed by a post hoc Sidak test or Tukey’s test, as appropriate; ∗ p < 0.05; ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

(H and I) WT or Ager null mice were housed at room temperature (∼23°C) or exposed to a 4°C cold challenge for 6 or 12 h. iBAT (H) and iWAT (I) were dissected after and subjected to qRT-PCR for detection of relative Ucp1 mRNA expression.

(C and D) Relative mRNA expression of Acox1 and Cpt2a was measured by qRT-PCR in (C) iBAT and (D) iWAT from fed and fasted (24 h) WT and Ager null mice.

(B) Change in core body temperature, [(Δ) temperature], between the fasted and the refed states was measured in WT or Ager null male mice housed at room temperature (RT).

Our findings thus far indicate that the deletion of Ager upregulates adipocyte thermogenic gene programs in iBAT and iWAT. In adipocytes, β-adrenergic signals upregulate Ucp1 transcription, and, in part through lipolysis, the released fatty acids are proposed to enhance UCP1 activity in the mitochondria (). We tested these concepts in settings in which adaptive thermogenic programs are engaged.

Although eWAT is not primarily a thermogenic adipose tissue depot, we nevertheless examined the above markers. In eWAT, the levels of Creb, Dio2, Fasn, Cebpa, and Tfam were significantly higher in Ager null than in WT; there were no differences in Cidea, Ucp1, Pnpla, and Pparg; and the levels of Prdm16, Ppargc1a, and Adrb3 were significantly lower in the eWAT of Ager null than of WT mice ( Figure S1 J).

To address this point, we retrieved adipose tissues from WT mice and mice globally devoid of Ager fed standard chow at room temperature. Ager deletion resulted in a significantly higher expression of thermogenic genes (Prdm16, Ppargc1a, Creb, Dio2, Cidea, and Ucp1) in iBAT and iWAT. Transcript levels of Adrb3 were significantly higher in iBAT, but not iWAT, of Ager null than of WT mice ( Figure 1 F). Regarding lipolysis (Pnpla2) and lipogenesis (Fasn, Cebpa, and Pparg), mRNA levels of these genes were significantly higher in Ager null than in WT iBAT and iWAT. In relation to mitochondrial properties, levels of Tfam in iBAT (), but not iWAT, were significantly higher in Ager null than in WT mice ( Figure 1 F), thus suggesting that mitochondrial biogenesis was higher in iBAT of Ager null than of WT mice.

As the highest expression of Ager in the adipose depots was in iBAT, we probed the effect of Ager deletion on mitochondrial function in iBAT-derived primary adipocytes from mice fed standard chow. Basal respiration rates and ATP production were significantly higher in adipocytes derived from Ager null than from WT mice ( Figure 1 E). The adipocytes derived from Ager null iBAT exhibited pronounced mitochondrial activity, as assessed by MitoTracker Red CMXRos () ( Figure S1 I). These findings suggest that RAGE contributes to the regulation of thermogenic programs in iBAT and to browning or beiging in iWAT.

Although core body temperature at thermoneutrality (30°C) did not differ between the WT and Ager null mice ( Figure 1 C), at room temperature, mice devoid of Ager displayed a significantly higher core body temperature than the WT mice ( Figure 1 D). Body weight, adiposity, and plasma norepinephrine levels did not differ between the Ager null and the WT mice fed standard chow ( Figures S1 D–S1H).

We determined the expression patterns of RAGE in WAT and BAT in WT mice fed standard chow and found that Ager is expressed in BAT and WAT. Compared to epididymal adipose tissue (eWAT) or iWAT, a significantly higher expression of Ager mRNA was observed in iBAT ( Figure 1 A). In the iWAT and eWAT depots, a significantly higher expression of Ager mRNA transcripts was observed in the floating adipocytes than in the stromal vascular fraction (SVF) ( Figure S1 A). When preadipocytes from the SVF of iBAT, iWAT, and eWAT were differentiated into adipocytes, in each depot, a significantly higher expression of Ager mRNA was noted on days 3 or 8 of differentiation versus day 0, which paralleled time-dependent increases in Fasn, a marker for adipocyte differentiation ( Figure 1 . There were no discernible differences in the morphology of iBAT-, iWAT-, or eWAT-derived primary adipocytes or in the neutral lipid content on day 7 of differentiation from eWAT-derived adipocytes ( Figures S1 B and S1C). Thus, increasing Ager expression accompanies, but is not required for, the differentiation of primary adipocytes from iBAT, iWAT, and eWAT.

Unless otherwise stated, the data are presented as mean ± SEM in N = 4–5 mice/group. Data analysis: (A and B) independent samples one-way ANOVA followed by a post hoc Tukey’s HSD test; (C and D) two-tailed Student’s t test; and (E, repeated measures, and F, independent samples) two-way ANOVA followed by post hoc Sidak test; ∗ p < 0.05; ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

(F) iBAT and iWAT retrieved from 8-week-old WT or Ager null mice housed at room temperature were analyzed via qRT-PCR for detection of relative mRNA expression of Prdm16, Ppargc1a, Creb, Adrb3, Dio2, Cidea, Ucp1, Pnpla2, Fasn, Cebpa, Pparg, and Tfam.

(E) Oxygen consumption rates (OCRs) normalized to total protein were determined in primary adipocytes differentiated from iBAT from WT and Ager null mice. The means ± SEM are reported from five technical replicates with three biological replicates per group.

(C and D) Core body temperature of 8-week-old male WT or Ager null mice determined in mice housed at (C) thermoneutrality (30°C) for 24 h and (D) at room temperature (∼23°C).

(B) Primary adipocytes from iBAT, iWAT, and eWAT of WT mice fed standard chow were subjected to qRT-PCR for detection of relative Ager and Fasn mRNA expression on day 0, 3, and 8 of differentiation.

Discussion

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Our data indicate that in states of RAGE ligand enrichment, such as in high-fat feeding, the activity of the RAGE axis is heightened. Although iBAT from adipocyte Ager-deleted mice displays lower levels of Ucp1 and Dio2 at room temperature and in low-fat feeding (i.e., LOW RAGE LIGAND environment) when compared to iBAT from adipocyte Ager-expressing mice ( Figure 3 J), when these iBAT tissues are transplanted into environmentally-stressed WT mice fed a HFD (i.e, HIGH RAGE LIGAND environment), the absence of RAGE in adipocytes unrestrains thermogenic programming, as evidenced by the increased expression of Ucp1, Dio2, Ppargc1a, Cpt2a, and Pnpla2 mRNA in the transplanted iBAT of the adipocyte Ager-deleted versus Ager-expressing iBAT ( Figure 4 M). These considerations are supported by the work presented in Figures 6 and 7 , in which the RAGE ligand CML-AGE suppresses lipolysis, phosphorylation of p38 MAPK, and thermogenic gene programs.

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Lai X. Lethally hot temperatures during the Early Triassic greenhouse. In the broader context of evolution and adaptive selection, then, what forces drive this metabolic braking role for Ager? Selective pressures in evolution favor phenotypes that safeguard survival in the face of paucities and extreme stresses (). Hence, when nutrient supplies are unlimited and selective survival pressures are lost, the urge to hoard energy, if unchecked, may become a vulnerability. The gene AGER, located on chromosome 6 in the major histocompatibility complex (MHC) III humans (), first appeared in Laurasiatheria (), a superorder of placental mammals that is part of the larger group of mammals classified as Eutheria. Eutheria are mammalian clades, and the oldest Eutherian species is believed to be Juramaia sinensis, which dates to 160 million years ago (mya) (). A key property of the Eutherians is the expression of UCP1 in BAT, which imbues the capacity for non-shivering thermogenesis (). Mammals first appeared during the Mesozoic era, about 250 mya, which followed the period known as the “Great Dying,” in which a massive extinction of plants and land species ensued consequent to a period of intense global warming (). Perhaps AGER evolved as a defense against starvation or swings in ambient temperatures through its ability to suppress adaptive thermogenesis.

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In summary, this work demonstrates that RAGE suppresses adaptive thermogenesis in both BAT and WAT during a physiological response to fasting, a cold challenge, or high-fat feeding; adds Ager to the cadre of genes that tether inflammation to the regulation of energy homeostasis; defines the endogenous function of RAGE in energy metabolism; and pinpoints tractable therapeutic targets to harness energy expenditure in metabolic disorders through the blockade of RAGE signal transduction.