A Novel Role for DOC2B in Ameliorating Palmitate-Induced Glucose Uptake Dysfunction in Skeletal Muscle Cells via a Mechanism Involving β-AR Agonism and Cofilin

Abstract

Diet-related lipotoxic stress is a significant driver of skeletal muscle insulin resistance (IR) and type 2 diabetes (T2D) onset. β₂-adrenergic receptor (β-AR) agonism promotes insulin sensitivity in vivo under lipotoxic stress conditions. Here, we established an in vitro paradigm of lipotoxic stress using palmitate (Palm) in rat skeletal muscle cells to determine if β-AR agonism could cooperate with double C-2-like domain beta (DOC2B) enrichment to promote skeletal muscle insulin sensitivity under Palm-stress conditions. Previously, human T2D skeletal muscles were shown to be deficient for DOC2B, and DOC2B enrichment resisted IR in vivo. Our Palm-stress paradigm induced IR and β-AR resistance, reduced DOC2B protein levels, triggered cytoskeletal cofilin phosphorylation, and reduced GLUT4 translocation to the plasma membrane (PM). By enhancing DOC2B levels in rat skeletal muscle, we showed that the deleterious effects of palmitate exposure upon cofilin, insulin, and β-AR-stimulated GLUT4 trafficking to the PM and glucose uptake were preventable. In conclusion, we revealed a useful in vitro paradigm of Palm-induced stress to test for factors that can prevent/reverse skeletal muscle dysfunctions related to obesity/pre-T2D. Discerning strategies to enrich DOC2B and promote β-AR agonism can resist skeletal muscle IR and halt progression to T2D.

Keywords: DOC2B; GLUT4; cofilin; glucose uptake; insulin resistance; lipotoxic stress; palmitate; skeletal muscle; type 2 diabetes; β-adrenergic receptor.

Figure 1

β-AR agonism enhances insulin-stimulated GLUT4 translocation to the PM. (A) Analysis of ADRB mRNA family member expression (genes encoding the β-AR family members) relative to Hypoxanthine Phosphoribosyltransferase (HPRT) housekeeping gene in male C57BL/6J mouse quadricep skeletal muscle (n = 7 mice). (B) ADRB mRNA expression (relative to HPRT housekeeping gene) in L6-GLUT4myc myotubes (n = 7 independent passages). (C) ADRB mRNA expression (relative to HPRT housekeeping gene) in L6-GLUT4myc myoblasts (n = 5 independent passages). (D,E) The effect of β-AR agonists (BRL or ISO) on PM GLUT4myc accumulation ± insulin (INS) in L6-GLUT4myc myoblasts (n = 4 independent passages/condition). In (E), cells were pretreated with β-blocker (Pro) prior to β-AR agonist application. The immunofluorescence intensity of PM GLUT4myc was normalized to the fluorescence intensity of the nuclear DNA stain SYTO 60. (A–E) Data are expressed as mean ± standard error of the mean (SEM). ** p < 0.01, *** p < 0.001, **** p < 0.0001. Green indicates mRNA expression; red indicates the β-AR blocker propranolol treatment; light blue indicates the β-AR agonist BRL treatment; dark blue indicates the β-AR agonist ISO treatment.

Figure 2

Lipotoxic stress impairs insulin-stimulated- and β-AR agonism-induced-GLUT4 translocation to the PM. (A–C) Analysis of 2-DG uptake (A) and PM GLUT4 levels (B) in L6-GLUT4myc myotubes treated with Palm or vehicle (Veh) ± insulin (INS) stimulation. n = 6 independent experiments. (C) Myoblasts subjected to Palm stress were also incubated with β-AR agonists (BRL or ISO) for 2 h prior to insulin stimulation. n = 5–6 independent experiments. The immunofluorescence intensity of PM GLUT4myc was normalized to the fluorescence intensity of the nuclear DNA stain SYTO 60. (D) Assessment of cell viability in L6-GLUT4myc myoblasts grown under stated conditions. n = 5 independent experiments. (E,F) Quantification of GLUT4 protein abundance in L6-GLUT4myc myotubes (E) and L6-GLUT4myc myoblasts (F) grown under stated conditions. n = 5 independent experiments. Representative immunoblots are shown. Ponceau S (Ponc. S) staining of the full-length PVDF membrane was used as a loading control. (A–F) Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Palm stress impairs DOC2B expression. (A) Immunoblot analysis and quantification of DOC2B protein abundance in male C57BL/6J mouse skeletal muscle. Mice were fed an HFD (n = 15) or chow diet (n = 13). The protein level was normalized to HPRT. (B,C) Immunoblot analysis and quantification of DOC2B protein abundance in L6-GLUT4myc myotubes (B) and myoblasts (C) under stated conditions (n = 5 independent experiments). Representative immunoblots are shown. Ponceau S (Ponc. S) staining of the full-length PVDF membrane was used as loading control. (D) Analysis of DOC2B mRNA expression relative to HPRT housekeeping gene in male C57BL/6J mouse skeletal muscle. As in (A), mice were fed an HFD (n = 3) or chow diet (n = 4). (E,F) Analysis of DOC2B mRNA expression relative to HPRT housekeeping gene in L6-GLUT4myc myotubes (E) and L6-GLUT4myc myoblasts (F) under stated conditions (n = 5 independent experiments). (G) Immunoblot analysis and quantification of DOC2B protein abundance in L6-GLUT4myc myoblasts under stated conditions (n = 3 independent experiments). A representative immunoblot is shown. Ponceau S staining of the full-length PVDF membrane was used as a loading control. (A–G) Data are expressed as mean ± SEM. N.S.; not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

DOC2B enrichment in combination with β-AR agonism promotes insulin-stimulated glucose uptake and GLUT4 translocation under Palm-stress conditions. (A,B) Analysis of 2-DG uptake levels (A) and surface GLUT4myc levels (B) in control GFP or DOC2B-enriched L6-GLUT4myc muscle cells subjected to stated conditions (n = 6–8 independent experiments). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

DOC2B protects against Palm-induced cofilin phosphorylation in L6-GLUT4myc myotubes. (A) Effect of adenoviral transduced DOC2B enrichment (or GFP alone) ± insulin (INS) stimulation on p-Cofilin^Ser3 under Palm-stress (or Veh.) conditions. A representative immunoblot is shown (n = 4 independent experiments). (B) Effect of adenoviral transduced DOC2B enrichment (or GFP alone) and ± insulin (INS) stimulation on downstream insulin signaling effector p-AKT^Ser473 under Palm-stress (or Veh.) conditions. Under some conditions, the added effect of β-AR agonism (BRL or ISO) was analyzed. A representative immunoblot is shown (n = 3 independent experiments). Bar graphs show densitometric analysis of phosphorylated AKT^Ser473 normalized to total AKT. Black vertical dashed lines indicate the splicing of lanes from within the same gel exposure. (A,B) Ponceau S staining of the full-length PVDF membrane was used as a loading control. Data are expressed as mean ± SEM. N.S.; not significant, * p < 0.05, ** p < 0.01.

Figure 6

Schematic diagram of DOC2B-mediated GLUT4 translocation in response to β-AR agonists and insulin under palmitate-induced lipotoxic stress. Palmitate-induced lipotoxic stress results in a decrease in DOC2B expression, β-AR, and insulin signaling and an increase in cofilin phosphorylation, leading to reduced glucose uptake. Overexpressing DOC2B reverses this effect, triggering cofilin dephosphorylation. This process facilitates actin remodeling, ultimately restoring glucose uptake in myotubes. Red arrows indicate the negative effects of palmitate-induced lipotoxic stress. Green arrows indicate the positive changes associated with DOC2B overexpression.