Protein expression of AMPKα subunits and ACC Ser²²¹ phosphorylation.

<p>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. <b>*</b>: 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. <b>*</b>: Indicates significant difference between genotype, P<0.001, n = 11–14. Values are means ± S.E.M.</p

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.

<p>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.</p

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.

<p>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.</p

Figure 1

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053533#pone-0053533-g001" target="_blank">Figure 1A+B</a>: 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053533#pone-0053533-g001" target="_blank">Figure 1C</a>: 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.</p

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

<p>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.</p

DataSheet1_Metabolic effects of alternate-day fasting in males with obesity with or without type 2 diabetes.DOCX

Alternate-day fasting induces oscillations in energy stores. We hypothesized that repeated oscillations increases insulin secretion and sensitivity, and improve metabolic health in patients with obesity with or without type 2 diabetes (T2DM). Twenty-three male patients fasted every other day for 30 h for 6 weeks. Experiments included resting energy expenditure, continuous glucose monitoring, intravenous glucose tolerance test, euglycemic hyperinsulinemic clamp, body composition, hepatic triglyceride content, muscle biopsies which were performed at baseline, during 3 weeks without allowed weight loss, and after additional 3 weeks with weight loss. Bodyweight decreased ∼1% and further ∼3% during weeks one to three and four to six, respectively (p Clinical Trial registration: (ClinicalTrials.gov), (ID NCT02420054).</p