Lack of Adipocyte AMPK Exacerbates Insulin Resistance and Hepatic Steatosis through Brown and Beige Adipose Tissue Function - PubMed (original) (raw)

. 2016 Jul 12;24(1):118-29.

doi: 10.1016/j.cmet.2016.06.006.

Eric M Desjardins 1, Justin D Crane 1, Brennan K Smith 1, Alex E Green 1, Serge Ducommun 2, Tora I Henriksen 3, Irena A Rebalka 4, Aida Razi 5, Kei Sakamoto 2, Camilla Scheele 6, Bruce E Kemp 7, Thomas J Hawke 4, Joaquin Ortega 5, James G Granneman 8, Gregory R Steinberg 9

Affiliations

Lack of Adipocyte AMPK Exacerbates Insulin Resistance and Hepatic Steatosis through Brown and Beige Adipose Tissue Function

Emilio P Mottillo et al. Cell Metab. 2016.

Abstract

Brown (BAT) and white (WAT) adipose tissues play distinct roles in maintaining whole-body energy homeostasis, and their dysfunction can contribute to non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes. The AMP-activated protein kinase (AMPK) is a cellular energy sensor, but its role in regulating BAT and WAT metabolism is unclear. We generated an inducible model for deletion of the two AMPK β subunits in adipocytes (iβ1β2AKO) and found that iβ1β2AKO mice were cold intolerant and resistant to β-adrenergic activation of BAT and beiging of WAT. BAT from iβ1β2AKO mice had impairments in mitochondrial structure, function, and markers of mitophagy. In response to a high-fat diet, iβ1β2AKO mice more rapidly developed liver steatosis as well as glucose and insulin intolerance. Thus, AMPK in adipocytes is vital for maintaining mitochondrial integrity, responding to pharmacological agents and thermal stress, and protecting against nutrient-overload-induced NAFLD and insulin resistance.

Copyright © 2016 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Norepinephrine Activates AMPK in Human Brown Adipocytes but AMPK Does Not Regulate Lipolysis In Vivo

(A) Primary human brown adipocytes were treated with 1 μM norepinephrine (NE) or vehicle for 45 min before measuring pAMPKa T172, total AMPKa, pACC S79, total ACC, and UCP1. *p < 0.05, **p < 0.01 denotes a NE effect as determined by Student’s t test. (B) AMPKβ1 and β2, pAMPKα T172, total AMPKα, pACC S79, and total ACC levels in Control (CreERT2 −) or iβ1β2AKO mice (CreERT2 +) in BAT, iWAT, gWAT, liver, quadriceps muscle, and heart. (C–J) Isolated adipocytes from Control (−) and iβ1β2AKO (+) mice were untreated (Basal) or treated with 10 μM isoproterenol (Iso) for 45 min and the amount of FFA (D) or glycerol (E) released into the media was quantified. Adipocytes were immunoblotted for total and phosphorylated ACC, HSL, ATGL, AMPKα, and total levels of AMPK β1 and β2 and β-tubulin (C). The ratios of phospho-AMPKαT172/AMPKα (F), phospho-ACCS79/ACC (G), phospho-HSLS565/HSL (H), phospho-HSLS660/HSL (I), and phospho-ATGLS406/ATGL (J) were quantified (n = 3 mice per genotype, from three independent experiments). Data are means ± SEM. ***p < 0.001 denotes a genotype effect within groups, and †p < 0.05, ††p < 0.01, and †††p < 0.001 denote an Iso effect as determined by Student’s t test (D) or two-way ANOVA and Bonferroni post hoc test. N.D., not determined. See also Figure S1.

Figure 2

Figure 2. Adipocyte AMPK Is Required for Acute BAT-Mediated Thermogenesis

(A) Timeline, in weeks of age, for tamoxifen (TMX) treatment and cold exposure in female Control and iβ1β2AKO mice. (B–D) Core body temperature (B), thermal images (C), and interscapular BAT temperature (D) in Control and iβ1β2AKO mice at 30°C, and 4°C for 1 and 2 hr (n = 6–9 per group). (E) Triglyceride levels in BAT of mice kept at thermoneutrality (30°C) or exposed to cold (4°C) (n = 4–7 per group). (F–I) Phosphorylation of AMPK ([F] and [G], pAMPKαT172/AMPKα) and ACC ([F] and [H], pACCS79/ACC), and total UCP1 protein levels ([F] and [I]) in response to cold (4°C) in BAT of Control and iβ1β2AKO mice (n = 4–6 per group). *p < 0.05 and ***p < 0.001 denotes a cold (4°C) effect within genotypes, and ††p < 0.01 and †††p < 0.001 denote a genotype effect as determined by two-way ANOVA and Bonferroni post hoc test. (J) Timeline, in weeks of age, for tamoxifen (TMX) and acute CL-316,243 (CL, 0.033 nmol/kg) treatment in female Control and iβ1β2AKO mice (n = 8 mice per group). (K–N) Oxygen consumption (VO2; [K]), representative thermal images of mice in given groups (L), interscapular BAT surface temperature (M), and plasma FFA (N) in response to a single injection of saline or CL (0.033 nmol/kg, 20 min; n = 8 per group). Data are means ± SEM. *p < 0.05 denotes a genotype effect within groups, and †††p < 0.001 denotes a CL effect as determined by two-way ANOVA and Bonferroni post hoc test. See also Figure S2.

Figure 3

Figure 3. Adipocyte AMPK Is Important for Maintaining Mitochondrial Homeostasis in BAT

(A–C) Representative electron micrographs of mitochondria at two different magnifications (A), quantification of percentage mitochondria with disrupted cristae (B) and total number of mitochondria per micrograph (C) from BAT of chow-fed Control and iβ1β2AKO mice (n = 3 to 4 mice per genotype). (D) Respiration in isolated BAT mitochondria with malate and L-carnitine (M + L-Carn; 2 mM and 2.5 mM) and after the addition of palmitoyl-CoA (PCoA; 30 μM [n = 7 BAT mitochondrial isolations per genotype]). (E) Phospho-ULK1S555 protein levels normalized to total ULK1 levels from BAT of Control and iβ1β2AKO mice (n = 11 to 12 mice per genotype). (F and G) immunoblot analysis of the LC3BII/LCBI ratio (F) and p62 levels normalized to β-tubulin (G) in whole BAT tissue from chow-fed Control and iβ1β2AKO mice (n = 6 to 7 per group). Data are means ± SEM. *p < 0.05, **p < 0.01, and **p < 0.001 denote a genotype effect within groups as determined using a Student’s t test. See also Figure S3.

Figure 4

Figure 4. Adipocyte AMPK Is Required for the Adaptive Response to Thermogenesis and the Browning Program

(A) Timeline, in weeks of age, for tamoxifen (TMX) and 5-day CL 316,243 (CL) treatment in female Control and iβ1β2AKO mice. Mice were treated with 0.5 mg/kg CL for four consecutive days and 1 mg/kg on the final day. (B) Whole-body oxygen consumption (VO2) under basal conditions or 6 hr post-CL injection on indicated days (n = 6–9 per group). (C and D) COX activity (C) and representative OXPHOS subunit immunoblotting with quantification (D) (n = 8–10 per group) in BAT of Control and iβ1β2AKO mice treated with saline or CL for 5 days (5D CL). (E) Representative iWAT histological images (10× magnification) of Control and iβ1β2AKO mice treated with CL for 5 days. (F) mRNA expression of browning markers (Cidea, Ppara, Pdk4) in iWAT of Control and iβ1β2AKO mice treated with saline or CL for 5 days (n = 6–9 per group). (G and H) iWAT Ucp1 mRNA (G) and protein levels (H) in Control and iβ1β2AKO treated with saline or CL for 5 days (5D CL) (n = 6–9 per group). Data are means ± SEM. *p < 0.05 and **p < 0.01 denote a genotype effect within groups, and †p < 0.05, ††p < 0.01, and †††p < 0.001 denote a CL effect within genotype as determined by two-way ANOVA and Bonferroni post hoc test. See also Figure S4.

Figure 5

Figure 5. Adipocyte AMPK Protects against High-Fat-Diet-Induced Insulin Resistance

(A) Timeline, in weeks of age, for tamoxifen (TMX) and chow or HFD treatments in male Control and iβ1β2AKO mice (n = 8 to 9 for chow fed, n = 11 to 12 for HFD). (B) Total and phosphorylated (T172) levels of AMPKα in BAT of HFD-fed Control (−) and iβ1β2AKO (+) mice (n = 4 to 5). (C and D) Body weight curves (C) and adiposity (D) in Control and iβ1β2AKO mice on indicated diet for 12 weeks. (E and F) Fasting blood glucose (E) and plasma insulin concentrations (F) in Control and iβ1β2AKO mice on indicated diet for 10 weeks. (G and H) Glucose tolerance test (GTT; [G]) and insulin tolerance test (ITT; [H]) performed after 10 to 11 weeks of HFD in Control and iβ1β2AKO mice. AUC, area under the curve. Data are means ± SEM. *p < 0.05 and **p < 0.01 denote a genotype effect within groups as determined by a Student’s t test ([B], AUC of [G] and [H]), two-way ANOVA (E, F), or two-way repeated-measures ANOVA ([C], glucose curves of [G] and [H]) and Bonferroni post hoc test. See also Figure S5.

Figure 6

Figure 6. Deletion of Adipocyte AMPK Promotes Hepatic Lipid Accumulation

(A) Timeline, in weeks of age, for tamoxifen (TMX) treatment and experimental procedures in male Control and iβ1β2AKO mice on HFD. (B) 3H-2-deoxy-D-glucose (2-DG) uptake in BAT of Control and iβ1β2AKO mice on HFD, fasted for 6 hr and injected with 0.7 U/kg insulin (n = 3 to 4). (C) Change in oxygen consumption in response to CL-316,243 (0.033 nmol/kg, β3-AR test) in Control and iβ1β2AKO mice on HFD (n = 7 to 8). (D) Plasma triglyceride (TGs) levels in HFD-treated mice in fed and fasted (10 hr) states (n = 8 to 9 for fed, n = 11 to 12 for fasted). (E) Levels of total AKT and phospho-AKTS473 (pAKTS473) under basal conditions (Basal) and in response to an IP injection of insulin (1.0 U/kg for 15 min) in the liver, BAT, gWAT, and quadriceps muscle (Quad) of 12 week HFD-treated Control (−) and iβ1β2AKO (+) mice (n = 5–7). (F–H) Liver weights (F), representative H&E stains (20×; [G]) and liver triglyceride (TGs; [H]) levels in 12 weeks HFD-treated Control and iβ1β2AKO mice (n = 18–20 per group). (I) Plasma ALT levels in HFD fed Control and iβ1β2AKO mice (n = 14 to 15). Data are means ± SEM. *p < 0.05 and **p < 0.01 denote a genotype effect within groups, and †p < 0.05 and ††p < 0.01 denote an insulin effect within genotypes as determined using a Student’s t test ([B], [C], [F], [H]) or, where appropriate, by two-way ANOVA and Bonferroni post hoc test. See also Figure S6.

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