Two weeks of metformin treatment enhances mitochondrial respiration in skeletal muscle of AMPK kinase dead but not wild type mice

Abstract

Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5'AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α(2) (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.

Figure 1

Figure 1A+B: Mitochondrial respiratory capacity in TA muscles from WT and KD mice treated with metformin or saline for two weeks. A: Mitochondrial respiration (O₂ flux) related to milligram muscle. B: Mitochondrial respiration (O₂ flux) related to milligram muscle and expressed relative to mitochondrial content (CS activity). Dark grey bars shows results in TA muscles from saline treated WT mice. Light grey bars shows results in TA muscles from saline treated KD mice. Dark grey bars with slashes shows results in TA muscles from metformin treated WT mice. Light grey bars with slashes shows results in TA muscles from metformin treated KD mice. ‡: Indicate significant difference between genotype within intervention (P<0.05). #: Indicate significant difference between interventions in AMPK KD mice (P<0.05). †: Indicate significant interaction between genotype and intervention (P<0.05). *: Indicate significant main effect of genotype (P<0.05). n = 11–14. Values are means ± S.E.M. Figure 1C: Substrate control ratio (SCR) is calculated as state 3 respiration with complex I and II linked substrates divided by state 3 respiration with complex I linked substrates. White bars shows results in TA muscles from saline treated mice. Black bars shows results in TA muscles from metformin treated mice. n = 11–14. Values are means ± S.E.M.

Figure 2. Protein expressions of Complex I–V and Cytochrome C of the mitochondria electron transport chain in TA muscles from WT and KD mice treated with metformin or saline for two weeks.

White and black bars shows results from saline treated and metformin treated mice respectively. A: Complex I protein expression. B: Comp lex II protein expression. C: Complex III protein expression. D: Cytochrome C protein expression. E: Complex IV protein expression. F: Complex V protein expression. *: Indicates significant difference between genotype (P<0.05). (*): P = 0.059. N = 12–17. Values are S.E.M.

Figure 3. Representative Western blots of Complex I–V, Cytochrome C, Pyruvate Dehydrogenase (PDH) subunit E1α protein, PDH site1 and PDH site2 phosphorylation and ACC^Ser211 phosphorylation.

Figure 4. PDH site 1 and 2 phosphorylation and protein expression in TA muscles from WT and KD mice treated with metformin or saline for two weeks.

White and black bars shows results from saline treated and metformin treated mice respectively. A: PDH protein expession. B: PDH site 1 phosphorylation. C: PDH site 1 phosphorylation related to PDH protein expression. D: PDH site 2 phosphorylation. E: PDH site 1 phosphorylation related to PDH protein expression. *: Indicates significant difference between genotype (P<0.05). N = 12–17. Values are S.E.M.

Figure 5. HAD and CS activity in TA muscles from WT and KD mice treated with metformin or saline for two weeks.

White and black bars shows results from saline treated and metformin treated mice respectively. A: HAD activity. B: CS activity. *: Indicates significant difference between genotype (P<0.05), N = 12–17. Values are S.E.M.

Figure 6. Protein expression of AMPKα subunits and ACC Ser²²¹ phosphorylation.

A: Representative Western blot of AMPK α₂ in AMPK WT and KD muscles. The absence of a detectable band at the expected molecular weight of ∼63 kDa in KD mice muscles indicates a strongly reduced endogenous α₂ protein expression. Instead the slightly heavier band in KD muscles is detection of the myc-tag transgenic α₂ protein. B: α₁ protein expression in WT and AMPK KD muscles. *: Indicates significant difference between genotype, P<0.001, n = 10. Values are means ± S.E.M. C: ACC Ser²²¹ phosporyaltion in AMPK WT and KD muscle lysates (TA) from chronic saline (white bars) and metformin (black bars) treated mice. *: Indicates significant difference between genotype, P<0.001, n = 11–14. Values are means ± S.E.M.