AMP-Activated Protein Kinase (AMPK) Regulates Energy Metabolism through Modulating Thermogenesis in Adipose Tissue

Abstract

Obesity occurs when excess energy accumulates in white adipose tissue (WAT), whereas brown adipose tissue (BAT), which is specialized in dissipating energy through thermogenesis, potently counteracts obesity. White adipocytes can be converted to thermogenic "brown-like" cells (beige cells; WAT browning) under various stimuli, such as cold exposure. AMP-activated protein kinase (AMPK) is a crucial energy sensor that regulates energy metabolism in multiple tissues. However, the role of AMPK in adipose tissue function, especially in the WAT browning process, is not fully understood. To illuminate the effect of adipocyte AMPK on energy metabolism, we generated Adiponectin-Cre-driven adipose tissue-specific AMPK α1/α2 KO mice (AKO). These AKO mice were cold intolerant and their inguinal WAT displayed impaired mitochondrial integrity and biogenesis, and reduced expression of thermogenic markers upon cold exposure. High-fat-diet (HFD)-fed AKO mice exhibited increased adiposity and exacerbated hepatic steatosis and fibrosis and impaired glucose tolerance and insulin sensitivity. Meanwhile, energy expenditure and oxygen consumption were markedly decreased in the AKO mice both in basal conditions and after stimulation with a β3-adrenergic receptor agonist, CL 316,243. In contrast, we found that in HFD-fed obese mouse model, chronic AMPK activation by A-769662 protected against obesity and related metabolic dysfunction. A-769662 alleviated HFD-induced glucose intolerance and reduced body weight gain and WAT expansion. Notably, A-769662 increased energy expenditure and cold tolerance in HFD-fed mice. A-769662 treatment also induced the browning process in the inguinal fat depot of HFD-fed mice. Likewise, A-769662 enhanced thermogenesis in differentiated inguinal stromal vascular fraction (SVF) cells via AMPK signaling pathway. In summary, a lack of adipocyte AMPKα induced thermogenic impairment and obesity in response to cold and nutrient-overload, respectively, whereas chronic AMPK activation by A-769662 promoted WAT browning in inguinal WAT and protected against HFD-induced obesity and related metabolic dysfunction. These findings reveal a vital role for adipocyte AMPK in regulating the browning process in inguinal WAT and in maintaining energy homeostasis, which suggests that the targeted activation of adipocyte AMPK may be a promising strategy for anti-obesity therapy.

Keywords: A-769662; AMP-activated protein kinase; PGC-1α; energy metabolism; thermogenesis; white adipose browning.

Figure 1

Adipose AMPK deficiency impaired cold tolerance and suppressed cold-induced thermogenesis specifically in inguinal white adipose tissue. (A) Western blot analysis of UCP1, p-AMPKα (T172) and AMPKα protein levels in the interscapular brown adipose tissue (BAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) of 9-week-old male wild-type (WT) mice housed at RT or 4°C for 24 h. β-actin was used as a loading control. n = 4. (B) Western blot analysis of AMPKα1, α2, β1, β2, γ1, γ2 protein expression levels in the BAT, iWAT, eWAT and liver of 10-week-old male AKO mice and age-matched floxed littermates. β-actin was used as a loading control. n = 3. (C) Rectal temperature of 8-week-old chow-fed AKO and floxed mice at 4°C for 8 h. n = 9. (D–M) 8-week-old male chow-fed AKO mice and floxed mice were housed at 4°C for 48 h. Representative transmission electronic microscopy images of iWAT (D) and BAT (G), total number of mitochondria per micrograph in iWAT (E) and BAT (H) and the percentage of mitochondria with disrupted cristae over total mitochondria in iWAT (F) and BAT (I) from AKO mice and floxed mice were shown. n = 3. Scale bar: 2 μm in low magnification (×5,000, upper) and 0.5 μm in high magnification (×20,000, bottom). The relative mRNA levels of thermogenic genes and fatty acid oxidation (FAO)-related genes in the iWAT (J) and BAT (K) of AKO mice and floxed mice were analyzed by quantitative RT-PCR (normalized to 36b4). n = 8–9. The expression levels of AMPKα, p-AMPKα (T172), ACC, p-ACC (S79), UCP1 and PGC-1α in the iWAT (L) and BAT (M) of AKO mice and floxed mice were determined by western blot analysis. Data are presented as the means ± SEM. Student's t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the indicated control group.

Figure 2

Adipose-specific AMPKα deletion exacerbated high-fat diet (HFD)-induced adiposity and glucose intolerance. (A) Change in body weight of AKO mice and age-matched floxed littermates during the indicated period. (B) Representative images of 41-week-old male AKO mice and age-matched flox/flox mice. (C) Fat mass, fluid mass and lean mass relative to the total body weight of 40-week-old mice. (D) Relative weight of the liver, iWAT, eWAT, pWAT, and BAT to the total body weight of mice. (E) Blood glucose levels from the intraperitoneal glucose tolerance test (ipGTT) of 20-week-old mice following a single injection of glucose (2 g/kg) (left). The area under the curve (AUC) of the ipGTT during the indicated times was calculated as the blood glucose multiplied by the time (mM^*min) (right). (F) Blood glucose levels of 30-week-old mice following a single injection of insulin (0.75 U/kg) in the intraperitoneal insulin tolerance test (ITT) (left). The AUC of the ITT is shown (right). Chow-Flox: AMPK α1/α2-floxed mice fed a chow diet, Chow-AKO: adipose tissue-specific AMPK α1/α2 KO mice fed a chow diet, HFD-Flox: AMPK α1/α2-floxed mice fed a HFD, HFD-AKO: adipose tissue-specific AMPK α1/α2 KO mice fed a HFD. Data are presented as the means ± SEM. n = 11–15. Student's t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the corresponding Flox group.

Figure 3

Lack of adipocyte AMPKα increased adipocyte size in inguinal white adipose tissue. (A) Representative H&E stained images of iWAT, eWAT, and BAT in AKO mice and age-matched floxed littermates fed a chow diet (left) or a HFD (right). Scale bar = 100 μm. (B–D) Average adipocyte area in iWAT (B) and eWAT (C) and average lipid droplet area in the BAT (D) in AKO mice and floxed littermates fed a chow diet or a HFD. Data are presented as the means ± SEM. n = 5-7. Student's t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the corresponding Flox group.

Figure 4

Deletion of adipocyte AMPKα reduced basal and β3-adrenergic-activated energy expenditure in chow- and HFD-fed mice. (A–D) Metabolic cage analyses of 40-week-old AKO and floxed mice fed a chow or a HFD were performed. (A) The O₂ consumption of chow-fed mice during a 12-h light-dark cycle and after an injection of CL 312,643 (1 mg/kg) (left). Average basal and CL 312,643-stimulated O₂ consumption of chow-fed mice (right). (B) The energy expenditure (EE) of chow-fed mice during a 12-h light-dark cycle and after an injection of CL 312,643 (1 mg/kg) (left). Average basal and CL 312,643-stimulated EE of chow-fed mice (right). (C) The O₂ consumption of HFD-fed mice during a 12-h light-dark cycle and after an injection of CL 312,643 (1 mg/kg) (left). Average basal and CL 312,643-stimulated VO₂ consumption of HFD-fed mice (right). (D)The EE of HFD-fed mice during a 12-h light-dark cycle and after an injection of CL 312,643 (1 mg/kg) (left). Average basal and CL 312,643-stimulated EE of HFD-fed mice (right). Data are presented as the means ± SEM. n = 8. Student's t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the corresponding Flox group.

Figure 5

Adipocyte AMPK deficiency promoted the development of liver steatosis and fibrosis. (A) Representative H&E stained (top) and Sirius red stained (bottom) images of liver in AKO mice and age-matched floxed littermates fed with a chow diet (left) or a HFD (right). Scale bar = 100 μm. (B) Average lipid droplet area per cell in liver in AKO mice and floxed littermates fed with a chow diet or a HFD. (C) Fibrosis area in liver were evaluated in chow- or HFD-fed AKO mice and floxed littermates. (D,E) Liver TG (D) and TC (E) levels in chow- or HFD-fed AKO mice and floxed littermates. (F) Liver hydroxyproline level in chow- or HFD-fed AKO mice and floxed littermates. (G,H) The relative mRNA levels of lipogenesis genes (G) and fibrosis-related genes (H) in the liver of in chow- or HFD-fed AKO mice and floxed littermates were analyzed by quantitative RT-PCR (normalized to Actb). n = 8–9. (I) Absolute liver weight in chow- or HFD-fed AKO mice and floxed littermates. (J) The expression levels of AKT, p-AKT (S473), IR, p-IR (Y1162) in the liver of HFD-fed AKO mice and floxed littermates were determined by western blot analysis. (K,L) Relative phosphorylation levels of AKT and IR were determined by densitometric quantification of the immunoblots shown in (J). n = 3. (M,N) Plasma ALT and AST levels in chow- or HFD-fed AKO mice and floxed littermates. Data are presented as the means ± SEM. Student's t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the corresponding Flox group.

Figure 6

Chronic AMPK activation by A-769662 protected against HFD-induced obesity and dysregulated glucose metabolism (A) Body weight (left) and average body weight gain (right) of chow- and HFD-fed mice during the 6-week treatment period. (B) Average food intake of chow- and HFD-fed mice during the 6-week treatment period. (C) Absolute weights of the liver, iWAT, eWAT, pWAT and BAT of chow- and HFD-fed mice after 6-week treatment. (D,E) Liver TG (D) and TC (E) levels of chow- and HFD-fed mice after 6-week treatment. (F) Fasting blood glucose was measured during the 4th week of treatment. (G,H) Intraperitoneal glucose tolerance tests of chow- and HFD-fed mice were conducted at week 4 of treatment (G). The AUC from 0 to 120 min was calculated (H). (I,J) Insulin tolerance test of chow- and HFD-fed mice were conducted at week 5 of treatment (I). The AUC from 0 to 120 min was calculated (J). Chow-Veh: mice fed a chow diet and treated with vehicle, Chow-A-769662: mice fed a chow diet and treated with A-769662, HFD-Veh: mice fed a HFD and treated with vehicle, HFD-A-769662: mice fed a HFD and treated with A-769662. Data are presented as the means ± SEM. n = 5–9. Student's t-test. ^*P < 0.05, ^**P < 0.01 compared with the indicated control group.

Figure 7

A-769662 promoted energy expenditure and cold tolerance in HFD-fed mice. (A,B) During the 4th week of chronic administration, indirect calorimetry was used to investigate the energy metabolism of HFD-fed mice. Mice were dosed with vehicle or A-769662 (30 mg/kg) and then acclimated to the chamber for 12 h. Change of O₂ consumption (A left), EE (B left) during the indicated periods and average O₂ consumption (A right), EE (B right) during the whole period of measurement were assessed. n = 8. (C) Change in body temperature of HFD-fed mice after cold exposure at 4°C. n = 11–12. Data are presented as the means ± SEM. Student's t-test. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared with the indicated control group.

Figure 8

A-769662 induced browning in the inguinal WAT of HFD-fed mice. (A) Representative H&E-stained images of the iWAT, eWAT and BAT of HFD-fed mice. Scale bar = 100 μm. (B–D) Average adipocyte area in the iWAT (B) and eWAT (C) and average lipid droplet area in the BAT (D) of HFD-fed mice. n = 4–6. (E) Mitochondrial DNA copy number of iWAT, eWAT, and BAT in HFD-fed mice. (F) Relative mRNA levels of thermogenic genes and brown-selective genes in the iWAT of HFD-fed mice after 6 weeks of treatment. n = 6–8. (G) Western blot analysis of UCP1, AMPKα, p-AMPKα (T172), ACC, and p-ACC (S79) expression levels in the iWAT of HFD-fed mice after 6 weeks of treatment. β-actin was used as a loading control. (H) Relative protein expression level of UCP1 and the relative phosphorylation levels of AMPKα and ACC were determined by densitometric quantification of the immunoblots shown in (G). n = 4. Data are presented as the means ± SEM. Student's t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the indicated control group.

Figure 9

A-769662 facilitated thermogenesis in differentiated iWAT-SVF cells through AMPK signaling pathway. (A–D) iWAT-SVF cells were induced to differentiation toward brown-like adipocytes and were treated with the indicated compounds on day 7. (A) Relative mRNA levels of thermogenic genes in differentiated iWAT-SVF cells treated with the indicated compounds for 6 h were analyzed by quantitative RT-PCR. n = 4. (B) Western blot analysis of UCP1, PGC-1α, p-AMPKα (T172), AMPKα, p-ACC (S79), and ACC expression levels in differentiated iWAT-SVF cells treated with indicated compounds on day 7 for 24 h. (C) Relative protein expression levels of UCP1 and PGC-1α and the relative phosphorylation levels of AMPKα and ACC were determined by densitometric quantification of the immunoblots shown in (B). n = 3. (D) Basal and uncoupled oxygen consumption rate (OCR) of differentiated iWAT-SVF cells treated with NE (10 μM) for 3 h or with DMSO or A-769662 at different concentrations on day 7 for 12 h. n = 4. (E–G) iWAT-SVF cells were isolated from iWAT of 5-week-old AMPK α1/α2-floxed mice and induced to differentiate toward beige adipocytes. Cells were infected with NC and Cre lentivirus on day 6 to knockdown AMPKα expression and were treated with the indicated compounds on day 8. (E) Relative mRNA levels of the indicated genes in differentiated iWAT-SVF cells treated with the indicated compounds for 6 h on day 8 were analyzed by quantitative RT-PCR. n = 4. (F) Western blot analysis of UCP1, PGC-1α, p-AMPKα (T172), AMPKα, p-ACC (S79), and ACC expression levels in differentiated iWAT-SVF cells treated with indicated compounds on day 8 for 24 h. (G) Relative protein expression levels of UCP1 and PGC-1α and the relative phosphorylation levels of AMPKα and ACC were determined by densitometric quantification of the immunoblots shown in (F). n = 3. Data are presented as the means ± SEM. One-way ANOVA. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with the indicated DMSO group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with the indicated NC group.