Activating Connexin43 gap junctions primes adipose tissue for therapeutic intervention

Abstract

Adipose tissue is a promising target for treating obesity and metabolic diseases. However, pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization, especially in obese subjects. We have previously shown that during cold exposure, connexin43 (Cx43) gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells. We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue. Using an adipose tissue-specific Cx43 overexpression mouse model, we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of the β ₃-adrenergic receptor agonist Mirabegron and FGF21. Additionally, combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy. In light of these findings, we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it. Thus, Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.

Keywords: Adipose tissue; Connexin43; FGF21; GJA1; Gap junction; Obesity; Type 2 diabetes; β3-Adrenergic receptor agonist.

Graphical abstract

Figure 1

Adipose tissue Cx43 overexpression enhances mirabegron-stimulated mitochondrial respiration and fatty acid utilization. (A) Lucifer yellow (LY) coupling experiments of fresh inguinal white adipose tissue (iWAT) dissected from 12-week-old control (Ctrl) or Cx43 TG mice treated with a chow diet containing 10 mg/kg doxycycline for one week. Quantification of the number of cells coupled to the injected cell is shown on the right (from more than five mice per genotype). (B) Schematic of the experimental design. 12-week-old control (Ctrl) or Cx43 TG mice treated with chow diet containing 10 mg/kg doxycycline at the indicated time. A single dose of mirabegron was administered 90 min before the harvest of the iWAT. (C) Oxygen consumption rates of freshly dissected iWAT measured on a Seahorse instrument (n = 15 pieces fat tissue from five mice). (D) The respiratory exchange ratio (RER) of control (Ctrl) and Cx43 TG mice before and after a single dose oral mirabegron treatment (n = 4–5 mice). Mice were fed a high-fat diet supplemented with 10 mg/kg doxycycline (Dox10 HFD) 24 h before the metabolic cage experiment, and were kept on Dox10 HFD during the metabolic cage recording. The shaded area indicates the dark period during the day. (E) Left: Food intake during the metabolic cage experiment described in Panel D. Right: Accumulative food intake before and after the mirabegron treatment. (n = 4–5 mice). All data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 2

Adipose tissue Cx43 overexpression enhances FGF21's weight loss effects. (A) Weight-loss and food intake of control and Cx43 TG mice treated with PBS or FGF21 (1 mg/kg body weight) on HFD containing 200 mg/kg doxycycline (n = 3–4 mice). 8-week old control or Cx43 TG mice were treated with HFD for 12 weeks to induce obesity, and then both groups were switched to HFD containing 200 mg/kg doxycycline (Dox200 HFD) to induce transgene overexpression and treated with PBS or FGF21 through intraperitoneal injection, daily, for the indicated time. For body weight graph, P value was calculated from two-way ANOVA analysis, indicating the significance of the comparison between indicated groups. (B) Weight-loss and food intake of control and Cx43 TG mice treated with PBS or FGF21 (1 mg/kg body weight) on HFD containing 10 mg/kg doxycycline for 14 days followed by 50 mg/kg doxycycline for additional 10 days (n = 7–9 mice). 8-week old control or Cx43 TG mice were treated with HFD for 12 weeks to induce obesity, and then both groups were switched to HFD containing 10 mg/kg doxycycline (Dox10 HFD) for 14 days, followed by 50 mg/kg doxycycline HFD (Dox50 HFD) treatment. Daily PBS or FGF21 treatment was started at the initiation of Dox10 HFD treatment, through intraperitoneal injection, for the whole duration. For body weight and daily food intake graphs, P values were calculated from two-way ANOVA analysis using data generated on Dox50 HFD treatment, indicating the significance of the comparison between indicated groups. All data are mean ± SEM.

Figure 3

Danegaptide enhances mirabegron's efficacy in improving insulin sensitivity and serum lipid profile. (A) Lucifer yellow coupling experiments of iWAT isolated from 12-week-old normal chow-fed C57BL/6J wild-type mice 1 h after vehicle (Vehi) or danegaptide (Dan, 10 mg/kg body weight) treatment (oral gavage). The number of cells coupled to the injected cell is shown on the right (from at least five mice per treatment). Scale bar = 50 μm. (B) RER of 12-week-old normal chow-fed C57BL/6J wild-type mice treated with danegaptide followed by mirabegron (n = six mice). The shaded area indicates the dark cycle. (C) Insulin sensitivity calculated as HOMA-IR after 9-day daily danegaptide and mirabegron treatment of diet-induced-obese (DIO) C57BL/6J wild-type mice (12 weeks of HFD feeding starting at the age of 8 weeks, n = 5–6 mice). (D) AST and ALT activities after 9-day daily danegaptide and mirabegron treatment of DIO C57BL/6J wild-type mice (12 weeks of HFD feeding starting at the age of 8 weeks, n = 5–6 mice). (E) Serum lipid profiles after 1-week daily danegaptide and mirabegron treatment of DIO C57BL/6J wild-type mice (12 weeks of HFD feeding starting at the age of 8 weeks) (n = 4–5 mice). dHDL: direct high-density lipoprotein; NEFA: nonesterified fatty acids. (F). Representative histology of brown adipose (BAT) and iWAT histology from at least three mice after 1-week daily danegaptide and mirabegron treatment of DIO C57BL/6J wild-type mice (12 weeks of HFD feeding starting at the age of 8 weeks). Scale bar = 100 μm. (G). Adipose tissue beigeing gene expression in the iWAT from 9-day daily danegaptide and mirabegron treatment of DIO C57BL/6J wild-type mice (12 weeks of HFD feeding starting at the age of 8 weeks, n = 10–12 mice). All data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 4

Danegaptide enhances FGF21's metabolic effects. (A) Schematic of the experimental design. 8-week-old C57BL/6J mice were treated with HFD for 12 weeks to induce obesity. Then mice were treated with indicated drugs daily for 16 days; each mouse received vehicle or danegaptide (10 mg/kg body weight) via oral gavage, vehicle, or FGF21 (1 mg/kg body weight) or liraglutide (0.2 mg/kg body weight) via intraperitoneal injection. (B) Enhanced weight loss after adding danegaptide to FGF21 treatment (left). Food intake during the treatment (right) (n = 10 mice). (C) Insulin sensitivity quantified as HOMA-IR after 16-day treatment shown in Panel A (n = 10). (D) Fasting serum lipid profile after 16-day treatment shown in Panel A (n = 10). dHDL: direct high-density lipoprotein; NEFA: nonesterified fatty acids. (E) Representative histology of the liver from at least three mice after 16-day treatment shown in Panel A. Scale bar = 100 μm. All data are mean ± SEM except for Panel B, in which error bars were removed to enhance the view of the data. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

figs1

figs2

figs3

figs4