Prior AICAR induces elevated glucose uptake concomitant with greater γ3-AMPK activation and reduced membrane cholesterol in skeletal muscle from 26-month-old rats

Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Hanover Park, IL) unless otherwise noted. The reagents and apparatus for SDS-PAGE and nonfat dry milk (no. 170-6404) were obtained from Bio-Rad (Hercules, CA). Pierce MemCode Reversible Protein Stain Kit (#24585), Bicinchoninic acid protein assay (#23225), Tissue Protein Extraction Reagent (TPER; #78510), Protein G magnetic beads (#10004D), and DynaMag^TM-2 magnet (#12321D) were from Thermo Fisher Scientific (Waltham, MA). Anti-phospho Akt Ser⁴⁷³ (pAkt^Ser473; #9271), anti-phospho Akt Thr³⁰⁸ (pAkt^Thr308; #13038), anti-Akt (#4691), anti-phospho AS160 Thr⁶⁴² (pAS160^Thr642; #8881), anti-phospho AS160 Ser⁵⁸⁸ (pAS160^Ser588; #8730), anti-phospho AMPKα Thr¹⁷² (pAMPKα^Thr172; #2531), anti-AMP-activated protein kinase-α (AMPKα; #5831), anti-acetyl CoA carboxylase (ACC; #3676), anti-phospho ACC Ser79 (pACC^Ser79; #3661), anti-TBC1D1 (#5929), anti-hexokinase II (HKII; #2867), anti-insulin receptor (IR; #3025), anti-α-Tubulin (#2144), anti-Na⁺/K⁺-ATPase (#3010) and anti-rabbit IgG horseradish peroxidase conjugate (#7074) were from Cell Signaling Technology (Danvers, MA). Anti-phospho TBC1D1 Thr590 (pTBC1D1^Thr590; #AF2422) was from Sapphire North America (Ann Arbor, MI). Anti-phospho AS160 Ser⁷⁰⁴ (pAS160^Ser704) was provided by Dr. Jonas Thue Treebak (Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark). Anti AMP-activated protein kinase γ3 (γ3-AMPK) was provided by Dr. David Thomson (Brigham Young University, USA) (Hardman et al. 2014). Anti-Akt Substrate of 160 kDa (AS160; #ABS54), Anti-GLUT4 (GLUT4; #CBL243), Anti-phospho-TBC1D1 Ser237 (pTBC1D1^Ser237; #07-2268), P81 Phosphocellulose Squares (#20-134) and enhanced chemiluminescence Luminata Forte Western HRP Substrate (#WBLUF0100) were from MilliporeSigma (Billerica, MA). Anti-HMGCR (HMGCR, #BS-5068 R) and anti-phospho HMGCR Ser872 (pHMGCR^Ser872, # BS-4063 R) were from Bioss Antibodies (Woburn, MA). Anti-ABCA1 (ABCA1, #NB400-105SS) was from Novus Biologicals (Littleton, CO). Anti-phospho ABCA1 Ser2054 (pABCA1^Ser2054; #ab12064) was from Abcam (Cambridge, MA). 3-O-methyl-[³ H] glucose ([³ H]3-MG) was from Sigma-Aldrich, and [¹⁴C] mannitol was from PerkinElmer (Boston, MA). [γ-³³ P]-ATP was from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Liquid scintillation cocktail (#111195-CS) was from Research Products International (Mount Prospect, IL).

Animal care procedures were approved by the University of Michigan Committee on Use and Care of Animals and performed in accordance with the guidelines from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. Male Fischer-344 X Brown Norway rats were obtained from the National Institute of Aging (NIA) rodent colony at approximately 22–23 months old. Rats were individually housed at the University of Michigan (12:12 h light:dark cycle, lights out at 17:00 h), provided with standard rodent chow (Laboratory Diet no. 5L0D; LabDiet, St. Louis, MO) and water ad libitum. The terminal experiment was performed when the rats were approximately 26–27 months old, and the rats were fasted at approximately 17:00 on the night before the experiment.

Rats were deeply anesthetized by an intraperitoneal injection of ketamine–xylazine cocktail (50 mg/kg ketamine and 5 mg/kg xylazine), and both epitrochlearis muscles from each rat were dissected out and longitudinally split into two muscle strips. The muscle strips were placed in vials that were shaken at 45 oscillations per minute and continuously gassed (95% O₂/5% CO₂) in a heated (35 °C) water bath. For muscles analyzed to determine 3-MG uptake and signaling proteins, each muscle strip was incubated with a four-step process (Incubation Protocol 1). During step 1 (60 min), muscle strips were incubated with Krebs-Henseleit Buffer (KHB) supplemented with 8 mM glucose ± 2 mM AICAR (2 mM mannitol for without AICAR). During step 2 (180 min), all muscle strips were incubated in KHB supplemented with 8 mM glucose and 2 mM mannitol in the absence of AICAR. During step 3 (30 min), muscle strips were incubated with KHB supplemented with 0.1% bovine serum albumin (BSA), 2 mM sodium pyruvate, and 6 mM mannitol ± insulin (1.2 nM). During step 4 (20 min), muscle strips were incubated with KHB–BSA, the same concentration of insulin as previous step 3, 8 mM 3-MG (final specific activity of 250 μCi/mmol, [³ H]3-MG), and 2 mM mannitol (final specific activity of 50 μCi/mmol [¹⁴C]-mannitol). After step 4, the final incubation step, muscles were blotted, freeze-clamped, and stored at −80 °C for later processing and analysis.

For muscles analyzed to determine γ3-AMPK activity, two muscle strips from one epitrochlearis muscle were incubated with KHB ± 2 mM AICAR (2 mM mannitol for without AICAR) for 60 min (Incubation Protocol 2), then the muscle strips were blotted, freeze-clamped, and stored at −80 °C for later analysis. Two muscle strips from the contralateral epitrochlearis muscle were incubated with a four-step process (Incubation Protocol 3) in which step 1 and step 2 were the same as Incubation Protocol 1. During steps 3 and 4 (30 min and 20 min), muscle strips were incubated with KHB supplemented with 0.1% BSA, 2 mM sodium pyruvate and 6 mM mannitol with insulin (1.2 nM). For muscles analyzed to determine membrane cholesterol and cholesterol regulatory proteins, each muscle strip from both epitrochlearis muscles was incubated with Incubation Protocol 3. After the final incubation step, muscles were blotted, freeze-clamped, and stored at −80 °C until later processing and analysis.

Frozen muscles were weighed and homogenized with 1 mL ice-cold lysis buffer using a glass pestle attached to motorized homogenizer (Caframo, Georgian Bluffs, ON). For muscle lysates analyzed to determine 3-MG uptake, protein abundance and phosphorylation by immunoblotting, the lysis buffer contained T-PER Tissue Protein Extraction Reagent (#PI-78510; Thermo Scientific, Rockford, IL) supplemented with 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate (NaPPi), 1 mM sodium orthovanadate (Na₃VO₄), 1 mM ß-glycerophosphate, 1 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). For muscle lysates analyzed to determine γ3-AMPK activity, the lysis buffer contained 10% glycerol, 20 mM NaPPi, 1% NP-40, 2 mM PMSF, 150 mM sodium chloride (NaCl), 50 mM HEPES (pH 7.5), 20 mM β-glycerophosphate, 10 mM sodium fluoride (NaF), 1 mM EDTA, 1 mM EGTA, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 2 m MNa₃VO₄. Homogenates were rotated for 1 h at 4 °C prior to centrifugation (15,000 g for 15 min at 4 °C). The supernatants were transferred to microfuge tubes and stored at −80 °C until subsequent analyses. Protein concentration was measured using the bicinchoninic acid procedure.

Aliquots of the supernatants (200 μL) from muscle lysates were pipetted into a vial together with scintillation cocktail. A scintillation counter (PerkinElmer) was used to determine the ³H and ¹⁴C disintegrations per minute. 3-MG uptake was calculated as described by Cartee and Bohn (1995).

The specificity of the γ3-AMPK antibody used for immunoprecipitation (IP) was previously confirmed in Wang et al. (2018). AMPK activity was determined as described by Kjobsted et al. (2015). Briefly, muscle lysates (300 μg protein) were rotated with antibody γ3-AMPK (1:500) and IP buffer [50 mM NaCl, 1% Triton X-100, 50 mM NaF, 5 mM NaPPi, 20 mM Tris-base (pH 7.5), 500 μM PMSF, 2 mM dithiothreitol (DTT), 5 μg/mL leupeptin, 50 μg/mL soybean trypsin inhibitor, 6 mM benzamidine, and 250 mM sucrose] at 4 °C overnight. 50 μL of protein G-magnetic beads were added to each sample, then the samples were rotated for 2 h at 4 °C. DynaMag^TM-2 magnet was used to pellet the protein G-immunocomplex. Each immunopellet was washed once in IP buffer, once in 6 × assay buffer (240 mM HEPES, 480 mM NaCl, pH 7.0), twice in 3 × assay buffer (1:1). The reaction was initiated at 30 °C by the addition of 30 μL of kinase mix buffer (40 mM HEPES, pH 7.5, 80 mM NaCl, 800 μM DTT, 200 μM AMP, 100 μM AMARA peptide, 5 mM magnesium chloride, 200 μM ATP, and 2 μCi of [γ-³³ P]-ATP). After 30 min, the reaction was stopped by the addition of 10 μL of 1% phosphoric acid. Next, 30 μL of supernatant was spotted on P81 phosphocellulose paper. After 3 × 15 min washing with 1% phosphoric acid, followed by 1 × 5 min washing with acetone, the phosphocellulose paper was dried at room temperature and placed in the vials containing 8 mL scintillation cocktail for scintillation counting. Results were expressed relative to the normalized mean of all the samples from each experiment.

Membrane-enriched and cytosol-depleted fractions were obtained by differential centrifugation as described by Grice et al. (2019). Briefly, epitrochlearis muscles were homogenized in ice-cold HES buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, and 250 mM sucrose, 200 μM PMSF, 10 μg/mL pepstatin, and 1 μg/mL leupeptin) with a Polytron PT-3100 homogenizer. The homogenates were centrifuged at 1,380 g for 30 min at 4 °C, and the resulting supernatant was saved. The pellet was resuspended with HES buffer and centrifuged at 1,380 g for 30 min at 4 °C, the resulting supernatant was saved. Then the two supernatants were combined and centrifuged at 17,000 g for 30 min at 4 °C. This combined supernatant (cytosol fraction) was saved. The pellet was washed with HES buffer and centrifuged at 1,380 g for 30 min at 4 °C, the resulting supernatant was saved. Then this supernatant was centrifuged at 17,000 g for 30 min at 4 °C. The resulting pellet (membrane-enriched fraction) was resuspended in HES buffer and stored at −80 °C until analysis.

Membrane enrichment was assessed by immunoblotting with antibodies against membrane marker proteins (insulin receptor, IR; Na⁺/K⁺-ATPase) and a cytosolic marker protein (α-tubulin). Cholesterol content in the membrane-enriched fraction was determined using the Amplex Red Cholesterol Assay Kit (Thermo Scientific; #A12216) as described by Grice et al. (2019).

Immunoblotting procedures were described by Wang et al. (2018). An equal amount of protein from each muscle lysate was mixed with 6 × Laemmli buffer, boiled for 5 min, separated using SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. Equal loading was confirmed using the MemCode protein stain (Antharavally et al. 2004). Membranes were blocked with TBST (Tris-buffered saline, pH 7.5 plus 0.1% Tween-20) that was supplemented with either with 5% BSA or 5% nonfat milk for 1 h at room temperature, incubated with appropriate concentrations of primary and secondary antibodies. Then membranes were subjected to enhanced chemiluminescence and quantified by densitometry (AlphaView; ProteinSimple, San Jose, CA). Results for each sample (densitometric units) were expressed relative to the normalized average of all the samples on the blot. These normalized values were divided by the corresponding MemCode loading control value for each sample (using individual sample MemCode values that were normalized by dividing the mean MemCode values for all samples on each blot). Values for phosphorylated proteins were expressed as the ratio of phosphorylated signaling protein or enzyme to total signaling protein or enzyme (determined for each sample using a separate immunoblot with a primary antibody against the appropriate total signaling protein or enzyme).

Student’s t-test was used for comparisons between two groups. Two-way analysis of variance (ANOVA) was used to identify main effects of insulin (0 or 1.2 nM insulin) and AICAR (0 or 2 mM AICAR). Post-hoc analysis was performed using the Tukey test (SigmaPlot version 14.5; Systat Software, San Jose, CA). Data lacking normal distribution and (or) equal variance were mathematically transformed to achieve normality and equal variance prior to statistical analysis.

Glucose uptake with insulin alone exceeded values without either AICAR or insulin (P < 0.05; Fig. 1), and glucose uptake in the AICAR + insulin group exceeded both the insulin alone group (P < 0.01) and the AICAR alone group (P < 0.05).

To gain insight into the time-course for AICAR effects on γ3-AMPK activity, we determined γ3-AMPK activity immediately post-AICAR treatment (immediate post-AICAR) and 3 h post-AICAR treatment (3 h post-AICAR). γ3-AMPK activity was increased immediate post-AICAR (P < 0.001, Fig. 2A). After 3 h of recovery from AICAR stimulation, muscles with AICAR treatment had greater γ3-AMPK activity compared with unstimulated control muscles (P < 0.01, Fig. 2B). This result indicates a persistent effect of prior AICAR stimulation on γ3-AMPK activity.

For all of the phosphorylated proteins, the data were expressed as a ratio of the phosphorylated to total protein values.

There were no significant effects of insulin or AICAR on total Akt, AMPK, and TBC1D1 abundance. Total AS160 abundance in muscles without insulin was less than in muscles with insulin, either without (∼35%, P < 0.05) or with prior AICAR treatment (∼29%, P < 0.05). ANOVA revealed a significant Insulin × AICAR interaction for total ACC abundance (P < 0.05). Total ACC abundance in the AICAR alone group was less (∼22%, P < 0.01) than in the group without either AICAR or insulin (P < 0.01).

Akt^Thr308 phosphorylation in muscles with insulin was greater than in muscles without either AICAR or insulin (P < 0.001; Fig. 3A), as well as insulin + AICAR exceeded AICAR alone (P < 0.001). Akt^Ser473 phosphorylation was increased by insulin, either in the absence (P < 0.001; Fig. 3B) or presence of AICAR (P < 0.001).

ANOVA revealed a significant Insulin × AICAR interaction for AS160^Ser704 phosphorylation (P < 0.05; Fig. 4A) and AS160^Thr642 phosphorylation (P < 0.05; Fig. 4C). AS160^Ser704 phosphorylation with AICAR alone exceeded without AICAR or insulin (P < 0.05; Fig. 4A), and the AICAR + insulin group was greater than both the insulin alone group (P < 0.001) and the AICAR alone group (P < 0.001). AS160^Ser588 phosphorylation was increased by insulin, either in the absence (P < 0.01; Fig. 4B) or presence of AICAR (P < 0.01). AS160^Thr642 phosphorylation with insulin alone was greater than without either AICAR or insulin, and the AICAR + insulin group exceeded both the insulin alone group (P < 0.01; Fig. 4C) and the AICAR alone group (P < 0.001).

AMPKα^Thr172 (Fig. 5A) or ACC^Ser79 phosphorylation (Fig. 5B) was increased by prior AICAR treatment, regardless of insulin concentration (P < 0.05 without insulin; P < 0.01 with insulin for pAMPKα^Thr172; P < 0.001 without or with insulin for pACC^Ser79).

TBC1D1^Ser237 phosphorylation was increased with prior AICAR treatment, either in the absence (P < 0.05; Fig. 5C) or presence of insulin (P < 0.05). For TBC1D1^Thr590 phosphorylation, there were no significant effects of insulin or AICAR (Fig. 5D).

There were no significant effects of insulin or AICAR on either GLUT4 or HKII abundance. (Fig. 6A and 6B).

The membrane fraction was enriched with membrane marker proteins (IR, Na⁺/K⁺-ATPase) and depleted of the cytosolic marker protein (α-tubulin; Fig. 7A). Skeletal muscle membrane cholesterol content was decreased by prior AICAR treatment (P < 0.05; Fig. 7B).

Phosphorylated HMGCR^Ser872 and phosphorylated ABCA1^Ser2054 were unaltered by prior AICAR treatment (Fig. 7C and 7D).