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. 2014 Apr 14;9(4):e94689.
doi: 10.1371/journal.pone.0094689. eCollection 2014.

Activation of AMPKα2 is not crucial for mitochondrial uncoupling-induced metabolic effects but required to maintain skeletal muscle integrity

Mario Ost  1 , Franziska Werner  1 , Janine Dokas  1 , Susanne Klaus  1 , Anja Voigt  1
Affiliations

Activation of AMPKα2 is not crucial for mitochondrial uncoupling-induced metabolic effects but required to maintain skeletal muscle integrity

Mario Ost et al. PLoS One. .

Abstract

Transgenic (UCP1-TG) mice with ectopic expression of UCP1 in skeletal muscle (SM) show a phenotype of increased energy expenditure, improved glucose tolerance and increase substrate metabolism in SM. To investigate the potential role of skeletal muscle AMPKα2 activation in the metabolic phenotype of UCP1-TG mice we generated double transgenic (DTG) mice, by crossing of UCP1-TG mice with DN-AMPKα2 mice overexpressing a dominant negative α2 subunit of AMPK in SM which resulted in an impaired AMPKα2 activity by 90±9% in SM of DTG mice. Biometric analysis of young male mice showed decreased body weight, lean and fat mass for both UCP1-TG and DTG compared to WT and DN-AMPKα2 mice. Energy intake and weight-specific total energy expenditure were increased, both in UCP1-TG and DTG mice. Moreover, glucose tolerance, insulin sensitivity and fatty acid oxidation were not altered in DTG compared to UCP1-TG. Also uncoupling induced induction and secretion of fibroblast growth factor 21 (FGF21) from SM was preserved in DTG mice. However, voluntary physical cage activity as well as ad libitum running wheel access during night uncovered a severe activity intolerance of DTG mice. Histological analysis showed a progressive degenerative morphology in SM of DTG mice which was not observed in SM of UCP1-TG mice. Moreover, ATP-depletion related cellular stress response via heat shock protein 70 was highly induced, whereas capillarization regulator VEGF was suppressed in DTG muscle. In addition, AMPKα2-mediated induction of mitophagy regulator ULK1 was suppressed in DTG mice, as well as mitochondrial respiratory capacity and content. In conclusion, we demonstrate that AMPKα2 is dispensable for SM mitochondrial uncoupling induced metabolic effects on whole body energy balance, glucose homeostasis and insulin sensitivity. But strikingly, activation of AMPKα2 seems crucial for maintaining SM function, integrity and the ability to compensate chronic metabolic stress induced by SM mitochondrial uncoupling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phenotypic characterization of DTG mice.
(A) Representative western blots of Quadriceps muscle from 12-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice, Mitofusin-2 (MFN2) was used as a loading control (n = 2 out of 6–8 analyzed per group). (B) Activity assay of catalytic AMPKα1 and AMPKα2 subunits in Gastrocnemius muscle from 12-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice (n = 5 per group). (C–E) Development of body weight and body composition from week 4 up to week 12 of age (n = 9–10 per goup). Data are the mean ± SEM. Means with different letters are significantly different (1way ANOVA and Bonferroni's multiple comparisons test, p<0.05).
Figure 2
Figure 2. Improved systemic glucose homeostasis is independent of AMPKα2 activation.
(A) Blood glucose levels, area under curve (AUC) of blood glucose and (B) plasma insulin levels during an oral glucose tolerance test (oGTT) in 10-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice (n = 9–10 per group). (C) Ex vivo basal and insulin-stimulated glucose uptake in intact Extensor digitorum longus (EDL) muscle from 12- to 17-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice (n = 10–15 per group). Data are the mean ± SEM. Means with different letters are significantly different (1way ANOVA and Bonferroni's multiple comparisons test, p<0.05).
Figure 3
Figure 3. Fatty acid utilization is not disturbed in DTG mice.
(A) Ex vivo basal and AICAR-stimulated fatty acid (FAO, [3H]-palmitate) oxidation in isolated intact Soleus muscle from 12- to 17-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice (n = 10-15 per group). (B) Representative western blots of Quadriceps muscle from 12-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice, Mitofusin-2 (MFN2) was used as a loading control (n = 2 out of 6–8 analyzed per group). Means with different letters are significantly different (1way ANOVA and Bonferroni's multiple comparisons test, p<0.05).
Figure 4
Figure 4. Impaired activity tolerance and skeletal muscle morphology in DTG mice.
(A) Cage activity and (B) voluntary running wheel activity over 24 hrs of 11-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice (n = 6–10 per group). (C) Representative H&E staining of M. tibialis anterior (TA) muscle from 20-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice showing progressive muscle degeneration, including central nuclei (black arrows) in DTG mice, (scale bars 50 µm). (D) Quantification of cross-sectional area (CSA) of myofibers (n = 3 per group). (E) Gene expression analysis of muscle fiber-type markers in Quadriceps muscle of 12-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice by quantitative RT-PCR (n = 8 per group). Means with different letters are significantly different (1way ANOVA and Bonferroni's multiple comparisons test, p<0.05).
Figure 5
Figure 5. Increased cellular stress response in muscle of DTG mice.
(A) Gene expression analysis of FGF21 in Quadriceps muscle by quantitative RT-PCR (n = 8 per group) and (B) FGF21 plasma concentration of 12-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice (n = 9–10 per group). (B) Representative western blots of proteins involved in integrated stress response and FGF21 induction and (C) of cellular stress response markers HSP25 and HSP70 in Quadriceps muscle from 12-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice, Mitofusin-2 (MFN2) was used as a loading control (n = 2 out of 6–8 analyzed per group). Means with different letters are significantly different (1way ANOVA and Bonferroni's multiple comparisons test, p<0.05).
Figure 6
Figure 6. Diminished skeletal muscle mitochondrial respiratory capacity in DTG mice.
(A–C) Ex vivo mitochondrial function assessed by measuring oxygen consumption of permeabilized muscle fibers of Soleus muscle from 17- to 23-wk-old WT, DN-AMPKα2, UCP1-TG and DTG mice; (A) upon substrates only (MO, malate + octanoylcarnitine, state 2 respiration), (B) ADP-stimulated respiration (state 3 respiration) fuelled by different substrates for complex I (MOPG, MO + pyruvate + glutamate) and complex II (MOPGS, MOPG + succinate), and (C) maximal uncoupled respiration (state U) after addition of the chemical uncoupler FCCP (n = 6–8 per group). (D) Citrate synthase (CS) activity in Quadriceps muscle of same mice as indicator of total mitochondrial capacity (n = 5–6 per group). Means with different letters are significantly different (1way ANOVA and Bonferroni's multiple comparisons test, p<0.05).
Figure 7
Figure 7. Suppressed mitochondrial OXPHOS induction in DTG mice.
(A) Representative western blots and relative quantification of OXPHOS proteins and (B) of key regulators of mitochondrial biogenesis in Quadriceps muscle from WT, DN-AMPKα2, UCP1-TG and DTG mice. Mitofusin-2 (MFN2) was used as a loading control (n = 2 out of 6–8 analyzed per group). Means with different letters are significantly different (1way ANOVA and Bonferroni's multiple comparisons test, p<0.05).
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Grants and funding

This research received funding from the European Union's Seventh Framework Program FP7 2007-2013 under grant agreement n° 244995 (BIOCLAIMS Project) and from the Leibniz Society (SAW-2013-FBN-3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.