Journal: bioRxiv

Article Title: The human AMPKγ3 R225W mutation does neither enhance basal AMPKγ3-associated activity nor glycogen in human or mouse skeletal muscle

doi: 10.1101/2023.08.28.555048

Figure Lengend Snippet: The whole-body and muscle-specific insulin sensitivity of wild-type (WT, black line/white bar) and homozygote AMPKγ3 R225W carrier (KI HOM, blue line/bar) mice was investigated. Intraperitoneal (i.p.) glucose tolerance test (GTT) was performed. Blood glucose response from 0-120 minutes post glucose injection ( A ) and plasma insulin response ( B ) from 0-30 min post glucose injection in WT and KI HOM mice (n = 10-13). I.p. insulin tolerance test (ITT, 0.75 U/kg bodyweight) was performed in WT and KI mice ( C ). Blood glucose response from 0-120 minutes post insulin injection (n=10-13). Submaximal (100 µU/mL) and maximal (10.000 µU/mL) insulin stimulation was performed in isolated m. extensor digitorum longus (EDL; D-F ) and m. soleus (Sol; G-I ) excised from WT and KI HOM mice. Glucose uptake (D, G) and the phosphorylation level of pAkt Thr308 (E, H) and pTBC1D4 Thr649/TBC1D4 (F, I) was measured in EDL and Sol under basal or the two insulin-stimulated conditions ex vivo . All Western-blotting data are normalized to WT basal levels (n=8 both in EDL and Sol). Data are given as mean + SEM including dots to indicate individual values where applicable. Two-way ANOVAs with/without repeated measures and Holm-Sidak/Newman-Keuls post-hoc test (A, C-I) or unpaired t-tests (B) were used (where applicable) to investigate differences between genotypes, glucose/insulin or the interactions. *: (A-C) Indicates differences between genotypes at a given time point (D-I) Indicates differences compared to basal within genotype. #: indicates difference compared to submaximal insulin dose within genotype. *: p<0.05, ***/###: p<0.001. A.U.: Arbitrary Units. ME: Main Effect.

Article Snippet: Primary antibodies against pACC Ser212/222 (3661, mouse/human), pAMPK Thr172 (2531), Erk1/2 (9102), pErk1/2 Thr202/Tyr204 (9101), p38 (9212), p-p38 Thr180/Tyr182 (9211), Akt2 (3063), pAkt Thr308 (9275) and pTBC1D4 Thr649 (8881) were from Cell Signaling Technology (Danvars, MA, USA).

Techniques: Injection, Clinical Proteomics, Isolation, Phospho-proteomics, Ex Vivo, Western Blot

Journal: bioRxiv

Article Title: The human AMPKγ3 R225W mutation does neither enhance basal AMPKγ3-associated activity nor glycogen in human or mouse skeletal muscle

doi: 10.1101/2023.08.28.555048

Figure Lengend Snippet: The whole-body and muscle-specific insulin sensitivity of wild-type (WT, black line/white bar) and homozygote AMPKγ3 R225W carrier (KI HOM, blue line/bar) mice was investigated. Intraperitoneal (i.p.) glucose tolerance test (GTT) was performed. Blood glucose response from 0-120 minutes post glucose injection ( A ) and plasma insulin response ( B ) from 0-30 min post glucose injection in WT and KI HOM mice (n = 10-13). I.p. insulin tolerance test (ITT, 0.75 U/kg bodyweight) was performed in WT and KI mice ( C ). Blood glucose response from 0-120 minutes post insulin injection (n=10-13). Submaximal (100 µU/mL) and maximal (10.000 µU/mL) insulin stimulation was performed in isolated m. extensor digitorum longus (EDL; D-F ) and m. soleus (Sol; G-I ) excised from WT and KI HOM mice. Glucose uptake (D, G) and the phosphorylation level of pAkt Thr308 (E, H) and pTBC1D4 Thr649/TBC1D4 (F, I) was measured in EDL and Sol under basal or the two insulin-stimulated conditions ex vivo . All Western-blotting data are normalized to WT basal levels (n=8 both in EDL and Sol). Data are given as mean + SEM including dots to indicate individual values where applicable. Two-way ANOVAs with/without repeated measures and Holm-Sidak/Newman-Keuls post-hoc test (A, C-I) or unpaired t-tests (B) were used (where applicable) to investigate differences between genotypes, glucose/insulin or the interactions. *: (A-C) Indicates differences between genotypes at a given time point (D-I) Indicates differences compared to basal within genotype. #: indicates difference compared to submaximal insulin dose within genotype. *: p<0.05, ***/###: p<0.001. A.U.: Arbitrary Units. ME: Main Effect.

Article Snippet: Primary antibodies against pACC Ser212/222 (3661, mouse/human), pAMPK Thr172 (2531), Erk1/2 (9102), pErk1/2 Thr202/Tyr204 (9101), p38 (9212), p-p38 Thr180/Tyr182 (9211), Akt2 (3063), pAkt Thr308 (9275) and pTBC1D4 Thr649 (8881) were from Cell Signaling Technology (Danvars, MA, USA).

Techniques: Injection, Clinical Proteomics, Isolation, Phospho-proteomics, Ex Vivo, Western Blot

Journal: Acta physiologica (Oxford, England)

Article Title: The human AMPKγ3 R225W mutation negatively impacts site-1 nucleotide binding and does not enhance basal AMPKγ3-associated activity nor glycogen production in human or mouse skeletal muscle.

doi: 10.1111/apha.14213

Figure Lengend Snippet: FIGURE 4 AMPK activity, glucose uptake, and AMPK signaling in response to AICAR-stimulation in isolated muscle from mouse homozygote AMPKγ3 R225W carriers and wild-type (WT). To understand the function of the AMPKγ3 R225W protein, we isolated m. extensor digitorum longus (EDL) from WT (white bar) and homozygote AMPKγ3 R225W carrier (KI HOM, blue bar) mice and stimulated them with AICAR ex vivo. AMPKα2β2γ3 activity was measured in isolated EDL in the basal or AICAR-stimulated state (2.0 mM) without (A) or with (B) 200 μM AMP added to the activity assay (n = 5–8). Protein expression of AMPKγ3 (C), glucose uptake (D) and phosphorylation levels of pAMPK Thr172 (E), pACC Ser212/ACC (F), and pTBC1D1 Ser231/TBC1D1 (G) were measured in incubated EDL in the basal state or under submaximal (0.25 mM) or maximal (2.0 mM) AICAR stimulation (n = 6–8). The degree of pAMPK Thr172 specifically associated with AMPKα2β2γ3 was measured in AMPKγ3 immunoprecipitates from EDL stimulated with or without AICAR (H, 2.0 mM) and related to the amount of AMPKα2 in the immunoprecipitates to correct for the different AMPKα2 protein levels. Representative blots relating to (C–G) (I). Data are given as means + SEM including dots to indicate individual values. All immunoblotting data is normalized to WT basal levels. Two-way ANOVA with Newman–Keuls post-hoc test was used to evaluate differences between genotype, AICAR, or the interaction. †/‡: indicates effect of AICAR compared to basal within genotype. #: indicates difference between genotypes within AICAR concentration. $: indicates difference between AICAR concentrations within genotype. †/##/$$: p < 0.01, ‡/###/$$$: p < 0.001. AU, arbitrary units; ME, main effect.

Article Snippet: Primary antibodies against pACC Ser212/222 (3661, mouse/human), pAMPK Thr172 (2531), Erk1/2 (9102), pErk1/2 Thr202/Tyr204 (9101), p38 (9212), p- p38 Thr180/Tyr182 (9211), Akt2 (3063), pAkt Thr308 (9275), and pTBC1D4 Thr649 (8881) were from Cell Signaling Technology (Danvars, MA, USA).

Techniques: Activity Assay, Isolation, Ex Vivo, Expressing, Phospho-proteomics, Incubation, Western Blot, Concentration Assay

Journal: Acta physiologica (Oxford, England)

Article Title: The human AMPKγ3 R225W mutation negatively impacts site-1 nucleotide binding and does not enhance basal AMPKγ3-associated activity nor glycogen production in human or mouse skeletal muscle.

doi: 10.1111/apha.14213

Figure Lengend Snippet: FIGURE 5 AMPK activity, glucose uptake, and AMPK signaling in response to muscle contraction in muscle from mouse homozygote AMPKγ3 R225W carriers and wild-type (WT). To understand the function of the AMPKγ3 R225W protein, we contracted one lower hindlimb and isolated m. tibialis anterior (TA) from WT (white bar) and homozygote AMPKγ3 R225W carrier (KI HOM, blue bar) mice and left the contralateral leg as a sham-operated control. AMPKα2β2γ3 activity was measured in the sham-operated or contracted state without (A) or with (B) 200 μM AMP added to the activity assay (n = 8–11). Protein expression of AMPKγ3 (C), glucose uptake (D) and phosphorylation levels of pAMPK Thr172 (E), pACC Ser212/ACC (F), and pTBC1D1 Ser231/TBC1D1 (G) were measured in TA (n = 8–9). The degree of pAMPK Thr172 specifically associated with AMPKα2β2γ3 was measured in AMPKγ3 immunoprecipitates from TA in Sham or Contrx related to the amount of AMPKα2 in the immunoprecipitates to correct for the different AMPKα2 protein levels (H). Representative blots relating to (C–G) (I). Data are given as means + SEM including dots to indicate individual values. All immunoblotting data is normalized to WT Sham levels. A two-way ANOVA with Newman–Keuls post-hoc test was usedto evaluate differences between genotype, contraction, or the interaction. */‡: indicates effect of contraction compared to sham within genotype. #: indicates difference between genotypes within contraction-state. *: p < 0.05, ‡/###: p < 0.001. AU, arbitrary units; ME, main effect.

Article Snippet: Primary antibodies against pACC Ser212/222 (3661, mouse/human), pAMPK Thr172 (2531), Erk1/2 (9102), pErk1/2 Thr202/Tyr204 (9101), p38 (9212), p- p38 Thr180/Tyr182 (9211), Akt2 (3063), pAkt Thr308 (9275), and pTBC1D4 Thr649 (8881) were from Cell Signaling Technology (Danvars, MA, USA).

Techniques: Activity Assay, Isolation, Control, Expressing, Phospho-proteomics, Western Blot