A: 2-DG uptake by epitrochlearis muscle with (■) or without (□) insulin stimulation. Paired epitrochlearis muscles from the same rat underwent either the standard incubation protocol ([³H]-2-DG and [¹⁴C]mannitol) or the revised incubation protocol ([³H]-2-DG only, with three washes). Data are means ± SEM (n = 7–9 muscles per protocol at each insulin concentration). B: Correlations between the standard and revised protocols from muscles used in A (n = 7–9 muscles per protocol and insulin concentration). Each symbol represents data from paired muscles after incubation in the respective protocol, with (■) or without (○) insulin. Without insulin: R = 0.88, P < 0.05; with insulin: R = 0.91, P < 0.05. C: 2-DG uptake by epitrochlearis muscles with (■) or without (□) insulin stimulation, followed by a 60-min incubation in media with (+) or without (−) collagenase. Data are means ± SEM (n = 6 per bar). D: Correlations between the 2-DG uptake by fiber bundles (expressed as micromoles per microgram protein) vs. mean 2-DG uptake by single fibers isolated from their corresponding fiber bundle (~10–20 fibers, expressed as nanomoles per microliter), represented by each symbol, with (■) or without (○) insulin. Without insulin: R = 0.68, P < 0.05; with insulin: R = 0.63, P < 0.05.

Representative SDS–polyacrylamide gel of single fibers isolated from rat epitrochlearis muscle visualized using Coomassie Brilliant Blue staining. Lane 1: E+S, pooled sample from rat extensor digitorum longus and soleus muscles expressing MHC types I, IIA, IIB, and IIX. Lane 2: Ladder, molecular mass standard. Lane 3: Epitrochlearis single fiber expressing type IIB/X (hybrid fiber). Lane 4: Epitrochlearis single fiber expressing type IIB/X (hybrid fiber). Lane 5: Epitrochlearis single fiber expressing type IIB. Lane 6: Epitrochlearis single fiber expressing type IIA. Lane 7: Epitrochlearis single fiber expressing type IIX.

A: Mean 2-DG uptake by fibers expressing the same MHC isoform, with (■) or without (□) insulin stimulation. For basal (no insulin) fibers, the number of fibers for each MHC type is in parentheses: IIB (105), IIA (16), IIX (7), and IIB/X (30). For insulin-stimulated fibers, the number of fibers for each MHC type is in parentheses: IIB (117), IIA (15), IIX (13), and IIB/X (41). Data are means ± SEM. ANOVA revealed a significant difference related to MHC expression for insulin-stimulated fibers (P < 0.05). Post hoc analysis of insulin-stimulated fibers revealed significant differences (P < 0.05) for 1) *IIA vs. all other fiber types; and 2) †IIB/X vs. IIB fibers. B: Mean 2-DG uptake by fibers isolated from epitrochlearis muscle that were incubated without (□) or with (■) CB. For basal (no insulin) fibers, without CB, the number of fibers for each MHC type is in parentheses: IIB (56), IIA (11), IIX (9), and IIB/X (23). For basal fibers, with CB, the number of fibers for each MHC type is in parentheses: IIB (58), IIA (6), IIX (11), and IIB/X (25). For insulin-stimulated fibers, without CB, the number of fibers for each MHC type is in parentheses: IIB (37), IIA (5), IIX (6), and IIB/X (19). For insulin-stimulated fibers, with CB, the number of fibers for each MHC type is in parentheses: IIB (40), IIA (6), IIX (5), and IIB/X (16). Data are means ± SEM. For fibers without insulin, there were significant main effects of CB status (no CB greater than with CB; P < 0.001) and fiber type (P < 0.001). Post hoc analysis indicated for fibers without insulin, no CB is greater than with CB for IIB/X fibers (§P < 0.05). For fibers with insulin, there were significant main effects of CB status (no CB greater than CB; P < 0.001) and fiber type (P < 0.001) and a significant CB × fiber-type interaction (P < 0.001). Post hoc analysis revealed that for insulin-stimulated fibers 1) #P < 0.05 for CB vs. no CB for all fiber types; and 2) for fibers with no CB, *P < 0.05 for IIA vs. IIB, IIX, and IIB/X fibers and †P < 0.05 for IIB/X vs. IIB fibers.

Mean 2-DG uptake by fibers expressing the same MHC isoform, with (■) or without (□) AICAR stimulation. For basal (no AICAR) fibers, the number of fibers for each MHC type is within parentheses: IIB (27), IIA (6), IIX (6), and IIB/X (1). For AICAR-stimulated fibers, the number of fibers for each MHC type is within parentheses: IIB (51), IIA (21), IIX (13), and IIB/X (15). Data are means ± SEM. ANOVA revealed a significant difference related to MHC expression for AICAR-stimulated fibers (P < 0.05). Post hoc analysis of AICAR-stimulated fibers revealed a significant difference. *P < 0.05 for IIB vs. IIA fibers.

Mean 2-DG uptake by fibers isolated from epitrochlearis muscles from lean Zucker (LZ) (□) or obese Zucker (OZ) (■) rats. For basal (no insulin) fibers from LZ rat muscles, the number of fibers for each MHC type is in parentheses: IIB (51), IIA (10), IIX (8), and IIB/X (11). For basal fibers from OZ rat muscles, the number of fibers for each MHC type is in parentheses: IIB (71), IIA (10), IIX (5), and IIB/X (14). For insulin-stimulated fibers, from LZ rat muscles, the number of fibers for each MHC type is in parentheses: IIB (73), IIA (13), IIX (15), and IIB/X (19). For insulin-stimulated fibers from OZ rat muscles, the number of fibers for each MHC type is in parentheses: IIB (99), IIA (9), IIX (7), and IIB/X (25). Data are means ± SEM. For fibers with insulin, there were significant main effects of genotype (LZ greater than OZ; P < 0.001) and fiber type (P < 0.001) and a significant genotype × fiber-type interaction (P < 0.01). Post hoc analysis of insulin-stimulated fibers revealed 1) #P < 0.05 for insulin-stimulated OZ vs. LZ for all fiber types; and 2) for insulin-stimulated fibers within only the LZ group, *P < 0.05 for insulin-stimulated IIA vs. IIB, IIX, and IIB/X fibers.

IκB-β (A) and APPL1 (B) protein abundance in single fibers isolated from epitrochlearis muscles from LZ (□) or OZ (■) rats. Representative blots of protein abundance (IκB-β or APPL1) and corresponding Coomassie Brilliant Blue-stained gels of posttransfer MHC (loading control) are provided. The density value for each protein (IκB-β or APPL1) was normalized to the density value for the loading control. A: There was a significant main effect of genotype (LZ greater than OZ; *P < 0.005) but no significant fiber-type difference for IκB-β. Data are means ± SEM. n = 8 fibers per bar. B: There was no significant genotype or fiber-type difference for APPL1. Data are means ± SEM. n = 8 fibers per bar.