Chronic β3-AR stimulation activates distinct thermogenic mechanisms in brown and white adipose tissue and improves systemic metabolism in aged mice

Abstract

Adipose thermogenesis has been actively investigated as a therapeutic target for improving metabolic dysfunction in obesity. However, its applicability to middle-aged and older populations, which bear the highest obesity prevalence in the United States (approximately 40%), remains uncertain due to age-related decline in thermogenic responses. In this study, we investigated the effects of chronic thermogenic stimulation using the β3-adrenergic (AR) agonist CL316,243 (CL) on systemic metabolism and adipose function in aged (18-month-old) C57BL/6JN mice. Sustained β3-AR treatment resulted in reduced fat mass, increased energy expenditure, increased fatty acid oxidation and mitochondrial activity in adipose depots, improved glucose homeostasis, and a favorable adipokine profile. At the cellular level, CL treatment increased uncoupling protein 1 (UCP1)-dependent thermogenesis in brown adipose tissue (BAT). However, in white adipose tissue (WAT) depots, CL treatment increased glycerol and lipid de novo lipogenesis (DNL) and turnover suggesting the activation of the futile substrate cycle of lipolysis and reesterification in a UCP1-independent manner. Increased lipid turnover was also associated with the simultaneous upregulation of proteins involved in glycerol metabolism, fatty acid oxidation, and reesterification in WAT. Further, a dose-dependent impact of CL treatment on inflammation was observed, particularly in subcutaneous WAT, suggesting a potential mismatch between fatty acid supply and oxidation. These findings indicate that chronic β3-AR stimulation activates distinct cellular mechanisms that increase energy expenditure in BAT and WAT to improve systemic metabolism in aged mice. Considering that people lose BAT with aging, activation of futile lipid cycling in WAT presents a novel strategy for improving age-related metabolic dysfunction.

Keywords: adipose metabolism; aging; and fatty acid oxidation; beta‐3 adrenergic agonists; energy expenditure; futile substrate cycling; glucose metabolism; lipolysis; thermogenesis; uncoupling protein 1.

FIGURE 1

Metabolic effects of chronic β3‐AR treatment in aged mice. Aged C57BL/6J mice were treated with CL 316,243 (1 mg/kg BW; osmotic pump infusion for 4 weeks). Relative changes in (a–c) body weight (BW), and fat and lean masses (n = 8/group); (d–f) fasting glucose, insulin, and HOMA‐IR (n = 5–8/group, both sexes); (g, h) glucose values during intraperitoneal glucose tolerance test and AUC analysis (n = 6/group, both sexes); (i) adiponectin and leptin levels (n = 7–12, both sexes) and (j) Representative blots of pAKT/AKT and their densitometry analysis expressed as fold change over controls in the liver and muscle (n = 3, males). Data are shown as average ± SEM, with significance determined using Student's t‐test (*p < 0.05 between the groups).

FIGURE 2

Effects of chronic β3‐AR treatment on energy expenditure and mitochondrial activity in the adipose tissue depots of aged mice. Chronic CL 316,243 treatment‐induced changes in (a, b) energy expenditure; (c, d) oxygen consumption; (e) activity levels assessed by distance (m/mouse); and (f) RER in light and dark cycles (n = 3–4/group; females). (g) Mitochondrial respiration assessed by measuring the reduction of the electron acceptor dye TTC in BAT, iWAT, and eWAT depots (n = 7–8/group; both sexes). Data are shown as average ± SEM, with significance determined using Student's t‐test (*p < 0.05 between the groups).

FIGURE 3

Effects of chronic β3‐AR treatment on adipose fatty acid oxidation and adipocyte size distribution in aged mice. Chronic CL 316,243 treatment‐induced changes in (a) fatty acid β‐oxidation in iWAT and BAT depots (n = 6–9/group, males) and (b) Representative MCAD western blots and their normalized values are expressed as fold change over controls (n = 4–5/group, males). (c) Representative images of H&E stained adipose sections. Black arrows indicate multilocular adipocytes. (d) Adipocyte size distribution in iWAT and eWAT. Data are shown as average ± SEM; *p < 0.05 between the groups based on the Student's t‐test analysis.

FIGURE 4

Effects of chronic β3‐AR treatment on UCP1 expression in fat depots. (a) Western blot images and densitometry analysis for UCP1 in adipose depots (n = 4–5/group, males). (b) Representative images of UCP1 immunohistochemistry in adipose tissue sections of aged control and CL‐treated mice. Please note that UCP1 expression (brown staining) was increased in the BAT but not in the eWAT and iWAT of CL‐treated aged animals. Data are shown as average ± SEM; *p < 0.05 between the groups based on the Student's t‐test analysis.

FIGURE 5

Effects of chronic β3‐AR activation on lipid turnover and DNL in adipose tissues. (a) Western blot images and densitometry analysis for quantitation of PEPCK, GYK, ACSL1, and DGAT1 protein levels in adipose depots (n = 4/group, males). Normalized protein values are expressed as fold change over controls. (b) De novo synthesis of palmitate and (c) glycerol in TG determined using the D₂O technique (n = 4–6/group, males). In both b and c, the top panels shows the data expressed as fractional de novo synthesis (%) and the bottom panel shows the enrichment de novo synthesis rates normalized to tissue weights. Data are shown as average ± SEM; *p < 0.05 between the groups based on the Student's t‐test analysis.

FIGURE 6

Changes in inflammatory mediators in serum and adipose tissue following CL treatment in aged mice. (a–d) Protein levels of inflammatory mediators were assessed in tissue lysates of eWAT, iWAT, and BAT, and serum samples of aged controls and CL‐treated mice (n = 6–8/group, males). Data are shown as average ± SEM; *p < 0.05 between the groups based on the Student's t‐test analysis.

FIGURE 7

Effects of a reduced dose of CL on metabolic parameters and IL‐6 levels in aged mice. Aged mice (18 months old; males) were treated with a reduced dose of CL 316,243 (0.5 mg/kg BW via osmotic pump infusion) for 4 weeks. (a) Final BW; (b) fat mass; (c) In situ ETC activity in adipose depots, and (d) Glucose values during intraperitoneal GTT and its AUC analysis. IL6 protein levels in (e) iWAT and (f) serum (n = 6–7/group). Data are shown as average ± SEM; *p < 0.05 between the groups based on the Student's t‐test analysis.