Human adipose beiging in response to cold and mirabegron

Abstract

Background: The induction of beige adipocytes in s.c. white adipose tissue (WAT) depots of humans is postulated to improve glucose and lipid metabolism in obesity. The ability of obese, insulin-resistant humans to induce beige adipose tissue is unknown.

Methods: We exposed lean and obese research participants to cold (30-minute ice pack application each day for 10 days of the upper thigh) or treated them with the β3 agonist mirabegron. We determined beige adipose marker expression by IHC and quantitative PCR, and we analyzed mitochondrial bioenergetics and UCP activity with an Oxytherm system.

Results: Cold significantly induced UCP1 and TMEM26 protein in both lean and obese subjects, and this response was not associated with age. Interestingly, these proteins increased to the same extent in s.c. WAT of the noniced contralateral leg, indicating a crossover effect. We further analyzed the bioenergetics of purified mitochondria from the abdominal s.c. WAT of cold-treated subjects and determined that repeat ice application significantly increased uncoupled respiration, consistent with the UCP1 protein induction and subsequent activation. Cold also increased State 3 and maximal respiration, and this effect on mitochondrial bioenergetics was stronger in summer than winter. Chronic treatment (10 weeks; 50 mg/day) with the β3 receptor agonist mirabegron induces UCP1, TMEM26, CIDEA, and phosphorylation of HSL on serine660 in obese subjects.

Conclusion: Cold or β3 agonists cause the induction of beige adipose tissue in human s.c. WAT; this phenomenon may be exploited to increase beige adipose in older, insulin-resistant, obese individuals.

Trial registration: Clinicaltrials.gov NCT02596776, NCT02919176.

Funding: NIH (DK107646, DK112282, P20GM103527, and by CTSA grant UL1TR001998).

Keywords: Adipose tissue; Clinical Trials; Metabolism; Obesity.

Conflict of interest statement

Conflict of interest: The authors declare that no conflict of interest exists.

Figures

Figure 1. Repeated cold exposure induces uncoupling protein 1 (UCP1) in human thigh s.c. white adipose tissue (WAT).

An ice pack was applied to the thigh for 30 minutes each day for 10 consecutive days. S.c. WAT was isolated and subjected to mRNA analysis and UCP1 IHC as described in Methods. (A) UCP1 mRNA expression was determined in lean (n = 16) and obese (n = 8) biopsies. (B and C) adipose tissue sections (10 μm) were stained with rabbit anti-UCP1 antibody; a representative image of UCP1 staining at baseline and after 10 days of icing in the iced and contralateral legs of a lean (B) and obese (C) subjects is shown. Scale bars: 50 μm. (D) UCP1 staining was quantified in the lean (n = 17) and obese (n = 8) subjects. The data are expressed as area of UCP1 staining (μm2) per adipocyte number. The data were analyzed by a repeated-measures one-way MANOVA as described in Methods. Data represent mean ± SEM; *P < 0.05; ****P < 0.0001; #P < 0.1.

Figure 2. Repeated cold exposure induces transmembrane protein 26 (TMEM26) in human thigh s.c. white adipose tissue (WAT).

S.c. WAT described in Figure 1 was isolated and subjected to mRNA analysis and TMEM26 IHC. (A) TMEM26 mRNA expression was determined in lean (n = 16) and obese (n = 8) biopsies. (B and C) Adipose tissue sections (10 μm) were stained with rabbit anti-TMEM26 antibody; a representative image of TMEM26 staining at baseline and after 10 days of icing in the iced and contralateral legs of a lean (B) and obese (C) subject is shown. Scale bars: 50 μm. (D) TMEM26 staining was quantified in the lean (n = 17) and obese (n = 8) subjects. The data are expressed as area of TMEM26 staining (μm2) per adipocyte number. The data were analyzed by a repeated-measures one-way MANOVA as described in Methods. Data represent mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. Localization of uncoupling protein 1 (UCP1) in s.c. white adipose tissue (WAT).

S.c. WAT from a cold-treated leg described in Figure 1 was stained with rabbit anti-UCP1 and cluster of differentiation 31 (CD31) (A) or lectin (B). (C) Green autofluorescence demonstrates the outline of adipocytes. Arrows point to UCP1 localized to adipocytes. Scale bars: 50 μm. This is a representative image of an experiment done on obese and lean subjects (n = 3).

Figure 4. Regression analysis of the beiging response to cold with age, BMI, and insulin sensitivity (SI).

The change in uncoupling protein 1 (UCP1) and transmembrane protein 26 (TMEM26) staining in s.c. white adipose tissue of the cold-treated leg was calculated (cold-baseline). Regression analysis of the change in UCP1 (A–C) and TMEM26 (D–F) staining versus age (n = 25), BMI (n = 25), and SI (Matsuda Index; n = 20) is shown. Spearman correlation coefficients and significant P values from unpaired, two tailed student’s t-tests are indicated.

Figure 5. Cold stimulates s.c. white adipose tissue (WAT) mitochondrial bioenergetics.

(A) Abdominal s.c. WAT was isolated from subjects before and after cold exposure, mitochondria were purified, and the bioenergetics were analyzed using an Oxytherm system as described in Methods. An example trace shows the O2 level in the chamber during the course of the experiment for 1 subject before and after cold exposure. The substrates pyruvate (Pyr) and malate (Mal), adenosine diphosphate (ADP), oligomycin (Oligo), free fatty acid (FFA; 60 uM linoleic acid), fatty acid free BSA, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP; 10 μm) were sequentially added at the indicated times. The oxygen consumption rate (OCR; nmoles/min) was determined during each step. (B and C) Analysis of mitochondrial bioenergetics before and after 10 days of repeated cold exposure. (D) Uncoupled respiration was determined by calculating the difference between the Oligo and FFA OCRs. (E) Maximal respiration was calculated by determining the difference between Oligo and FCCP OCRs. Data are represented as mean ± SEM (n = 11). The data were analyzed by a paired, 2-tailed student’s t test; *P < 0.05; #P < 0.1.

Figure 6. Cold changes mitochondrial bioenergetics more in the summer than winter.

The oxygen consumption rate (OCR) data from Figure 5 were analyzed by season. (A and B) Mitochondrial bioenergetics at baseline and after cold in the summer (n = 5). (C and D) Mitochondrial bioenergetics at baseline and after cold in the winter (n = 6). (E–G) The baseline and cold OCRs for State 3 respiration, uncoupling protein–mediated uncoupled respiration, and maximal uncoupled respiration at baseline and in response to cold in the summer; these values were calculated as described in Figure 5. (H and I) The change (cold-baseline) in State 3 and maximal OCRs stimulated by cold in summer and winter. Data are represented as mean ± SEM. The data were analyzed by a paired, 2-tailed student’s t test; *P < 0.05; #P < 0.1.

Figure 7. Mirabegron treatment induces beige adipocyte markers in obese subjects.

S.c. white adipose tissue (WAT) was isolated from obese subjects before and after treatment with 50 mg mirabegron per day for 10 weeks. (A and B) Uncoupling protein 1 (UCP1), (C and D) transmembrane protein 26 (TMEM26), and (E and F) cell death–inducing DNA fragmentation factor-α–like effector A (CIDEA) were analyzed by IHC as described in Methods and quantified. Scale bars: 50 μm. Data represent mean ± SEM (n = 6) and were analyzed by a paired, 2-tailed student’s t test; ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 8. Mirabegron treatment increases hormone sensitive lipase (HSL) serine660 (P-Ser660) phosphorylation but does not induce peroxisome proliferator–activated receptor γ coactivator 1-α (PGC1α) expression.

(A and B) HSL phosphorylation on residue serine660 was characterized in abdominal s.c. white adipose tissue (WAT)before and after mirabegron treatment by IHC and quantified as described in Methods. Scale bars: 10 μM. (C) HSL phosphorylation on serine565 was determined by IHC. (D) PGC1A mRNA expression was determined by quantitative PCR as described in Methods. (E) PGC1α protein expression was determined by immunoblotting as described in Methods. Inset, PGC1α and actin immunoblots (uncropped blots are shown in Supplemental Figure 3). (F) The mitochondrial DNA/nuclear DNA ratio was determined as described in Methods. The data represent mean ± SEM. The data in B–F were analyzed by a paired student’s, 2-tailed t test (n = 6, except E [n = 3]; *P < 0.05).