Mitochondrial electron transport chain, ceramide and Coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle

Abstract

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (under chow and high fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

Figure 1.. Palmitate increases ceramides, decreases CoQ and induces insulin resistance in L6-myotubes.

A) Insulin-induced GLUT4 translocation in L6-HA-GLUT4 myotubes exposed to palmitate (150 μM for 16 h, Palm) or BSA (Control) in presence of DMSO (control), myriocin (10 μM for 16 h) or CoQ9 (10 μM for 16 h). Plasma membrane GLUT4 (PM-GLUT4) abundance was normalised to insulin-treated control cells. N = 4, mean ± S.E.M. *p< 0.05 vs Control ins, # p< 0.5 vs Palm ins B) Insulin-induced GLUT4 translocation in L6-HA-GLUT4 myotubes exposed to C2-ceramide, Dihydroceramide (100 μM, DHC) or DMSO (Control) for 2 h. Plasma membrane GLUT4 (PM-GLUT4) abundance was normalised to insulin-treated control cells. N = 5, mean ± S.E.M. **p< 0.01vs control ins, # p< 0.5 vs 100 μM C2 Ceramide ins. (C and D) L6-HA-GLUT4 myotubes were serum-starved after BSA (Control for 16 h), Palmitate (Palm for 16 h), C2-ceramide (100 μM for 2 h, C2) or dihydroceramide (100 μM for 2 h, DHC) treatment and acute insulin (Ins) was added where indicated. Phosphorylation status of indicated sites was assessed by immunoblot (C). Immunoblots were quantified by densitometry and normalised to insulin-treated control cells (indicated by dotted line). N = 3, mean ± S.E.M. **p< 0.01, ***p< 0.001 (E and F) Endogenous ceramides levels in L6-HA-GLUT4 myotubes treated for 16 h with BSA (control), palmitate (150 μM, Palm), myriocin (10 μM for 16 h) or CoQ (10 μM for 16 h) as indicated in the graph. Total (E) and specific (F) ceramide species were quantified. N = 4, mean ± S.E.M. **p< 0.01, ****p< 0.001vs Control, ### p < 0.001 vs Palm, ρ p<0.01 vs Palm. G) CoQ9 level in mitochondrial fraction obtained from L6-HA-GLUT4 myotubes. N = 4, mean ± S.E.M. ***p< 0.001 vs Control, ## p<0.01 vs Palm H) Insulin-induced GLUT4 translocation in L6-HA-GLUT4 myotubes exposed to 4-NB (2.5 mM for 16 h) or DMSO (Control) in presence of CoQ9 (10 μM for 16 h). Plasma membrane GLUT4 (PM-GLUT4) abundance was normalised to insulin-treated control cells. N = 4, mean ± S.E.M. ***p< 0.001 vs Control ins, ## p<0.01, ### p<0.001 vs 4NB I) CoQ9 level in mitochondrial fraction obtained from L6-HA-GLUT4 myotubes exposed to DMSO (Control) or 4NB for 16 h. N = 4, mean ± S.E.M. *p< 0.05 (J and K) Total (J) and specific (K) ceramide species quantified in L6-HA-GLUT4 myotubes treated for 16 h with DMSO (control) or 4NB (2.5 mM for 16 h).. N = 4, mean ± S.E.M. **p< 0.01, ****p< 0.0001 L) Insulin-induced GLUT4 translocation in L6-HA-GLUT4 myotubes exposed to Saclac (10 μM for 24 h) or EtOH (Control) in presence of DMSO (control), myriocin (10 μM for 16 h) or CoQ9 (10 μM for 16 h). Plasma membrane GLUT4 (PM-GLUT4) abundance was normalised to insulin-treated control cells. N = 5, mean ± S.E.M. ***p< 0.001 vs Control Ins, ### p < 0.001 vs Saclac Ins.

Figure 2.. Mitochondrial overexpression of SMPD5 induces insulin resistance by lowering CoQ9 in L6-myotubes.

A) Schematic representation of doxycycline-inducible overexpression of mitochondrial targeted sphingomyelinase 5 (SMPD5). L6-HA-GLUT4 myotubes were exposed to 1 μg/mL of Doxycycline from day 3 to day 6 of differentiation. Experiments were performed on day 7 of differentiation. B) Determination of SMPD5 expression in mitochondrial fraction obtained from L6-HA-GLUT4. Doxycycline was added where indicated. (C and D) Levels of endogenous ceramides in mitochondrial fraction from L6-HA-GLUT4 myotubes treated with BSA (Control) palmitate (150 μM for 16 h, palm) or doxycycline. Total (C) and specific (D) ceramide species were quantified. N = 3, mean ± S.E.M. *p< 0.05, ** p<0.01 and ****p< 0.0001 vs Control. E) Insulin-induced GLUT4 translocation in L6-HA-GLUT4 myotubes exposed to Doxycycline (1 μg/mL for 3 d). Plasma membrane GLUT4 (PM-GLUT4) abundance was normalised to 100 nM insulin-treated control cells. N = 6, mean ± S.E.M. *p< 0.05, **p<0.01, ***p<0.001 vs control ins. (F, G and H) L6-HA-GLUT4 myotubes were serum-starved after BSA (Control), Palmitate (150 μM for 16 h, palm) or Doxycycline (1 μg/mL for 3 d) treatment and acute insulin (Ins) was added where indicated. Phosphorylation status of indicated sites was assessed by immunoblot. Immunoblots were quantified by densitometry and normalised to insulin-treated control cells (indicated by dotted line). N = 3, mean ± S.E.M. * p<0.05, *** p<0.001 vs Basal I) CoQ9 level in mitochondrial fraction obtained from L6-HA-GLUT4 myotubes exposed to doxycycline (1 μg/mL for 3 d) N = 4, mean ± S.E.M. **p<0.001. J) Insulin-induced GLUT4 translocation in L6-HA-GLUT4 myotubes exposed to Doxycycline (1 μg/mL for 3 d). Control or doxycycline treated cells were exposed to BSA (control), Palmitate (150 μM for 16 h, palm) or CoQ9 (10 μM for 16 h). Plasma membrane GLUT4 (PM-GLUT4) abundance was normalised to insulin-treated control cells. N = 5, mean ± S.E.M. **p< 0.01 vs Control ins, ## p<0.01 vs palm ins

Figure 3.. Mitochondrial overexpression of ASAH1 protects against insulin resistance and increases CoQ levels in L6-myotubes.

A) Schematic representation of doxycycline-inducible overexpression of mitochondrial targeted Acid Ceramidase 1 (ASAH1). L6-HA-GLUT4 myotubes were exposed to 1 μg/mL of Doxycycline from day 3 to day 6 of differentiation. Experiments were performed on day 7 of differentiation. B) Determination of ASAH1 expression in mitochondrial fraction obtained from L6-HA-GLUT4. Doxycycline was added where indicated. (C and D) Endogenous ceramides levels in mitochondrial fraction from L6-HA-GLUT4 myotubes treated with BSA (Control) palmitate (150 μM for 16 h, palm) or doxycycline. Total (C) and specific (D) ceramide species were quantified. N = 3, mean ± S.E.M. *p< 0.05 vs Control, ## p<0.01 vs Palm E) Insulin-induced GLUT4 translocation in L6-HA-GLUT4 myotubes exposed to Doxycycline (1 μg/mL for 3 d). Control or doxycycline treated cells were exposed to BSA (control), Palmitate (150 μM for 16 h, palm) or 4NB (2.5 mM for 16 h). Plasma membrane GLUT4 (PM-GLUT4) abundance was normalised to insulin-treated control cells. N = 6, mean ± S.E.M. **p<0.01 vs Control ins, ### p<0.001 vs Palm ins (F, G and H) L6-HA-GLUT4 myotubes were serum-starved after BSA (Control), Palmitate (150 μM for 16 h, palm) or Doxycycline (1 μg/mL for 3 d) treatment and acute insulin (Ins) was added where indicated. Phosphorylation status of indicated sites was assessed by immunoblot. Immunoblots were quantified by densitometry and normalised to insulin-treated control cells (indicated by dotted line). N = 3, mean ± S.E.M. ***p<0.001 vs Basal, # p<0.05 vs Control ins. I) CoQ9 level in mitochondrial fraction obtained from L6-HA-GLUT4 myotubes exposed to doxycycline (1 μg/mL for 3 d). Control or doxycycline treated cells were exposed to BSA (control) or palmitate (150 μM for 16 h, palm) N = 4, mean ± S.E.M. *p<0.05 vs control, ## p<0.01 vs Palm. J) Levels of total ceramides in skeletal muscle of mice fed chow with vehicle or 5 mg/kg P053 for 6 wks. N = 7, Mean ± S.E.M. ***p< 0.001. K) Levels of CoQ in mitochondrial fraction isolated from skeletal muscle of mice fed chow with vehicle or 5 mg/kg P053 for 6 wks. N = 7, Mean ± S.E.M. *p<0.05.

Figure 4.. Mitochondrial ceramides induce a selective depletion of supercomplexes associated proteins in L6-myotubes.

A) Workflow schematics. B & C) Pairwise comparisons of mitochondrial proteome between all four groups. Cut-off for −log10 adjusted p-value (-log10(p-value)) was set at 2 and Log2(FC) at 0.5 (blue). D) Quantification of the OXPHOS protein complexes generated by the summed abundance of all subunits within a given complex. E) Quantification of the Complex Q protein complexes generated by the summed abundance of all subunits within the complex. F) Schematics of CoQ distribution between CI-binding and free CoQ G) Schematics of OXPHOS subunits (top) and assembly factors (bottom) significatively up-regulated (light red), down-regulated (blue) and no change (grey) after 72 h of mtSMPD5 overexpression. H) Subunit levels for proteins after mtSMPD5 overexpression mapped to the complex I structure. The colours were calculated with an in-house python script and the resultant model was rendered using ChimeraX. Grey, no detected; Purple, below cut off (Log2FC = 0.4).

Figure 5.. Mitochondrial ceramides impair mitochondrial function.

A) mtSMPD5 overexpression decreases oxygen consumption rate (JO₂) (means ± SEM; n = 6 – 8 biological replicates) measured by mitochondrial stress test. After 1 h no CO₂ environment, cells were stimulated with oligomycin (Oligo), Carbonyl cyanide-p-trifluoromethoxyphenylhydrazon (FCCP) and antimycin A (AA) and Rotenote (Rot) at indicated time points. (means ± SEM; n = 6 – 8 biological replicates). B - F) Quantification of JO₂ measured by mitochondrial stress test from Fig 6A as described in material and method. (means ± SEM; n = 6 – 8 biological replicates). * p<0.05, ** p<0.01, *** p<0.001 vs control G-L) mtSMPD5 overexpression diminishes respiratory CI (G & J), CII (H & K) and CIV (I & L). JO₂ was performed in permeabilized cells supplemented with adenosine diphosphate (ADP) and CI to IV substrates (means ± SEM; n = 3 biological replicates). Mal, Malate; Glut, Glutamate; Rot, Rotenone; Succ, Succinate; TMPD, tetramethyl-phenylenediamine; Asc, Ascorbic acid; Oligo, Oligomycin. J, K and L are quantifications from graphs G, H and I respectively. * p<0.05, ** p<0.01 vs control. M) mtSMPD5 overexpression increased mitochondrial oxidative stress. Cells were loaded with the redox sensitive dye MitoSOX and the mitochondrial marker mitoTracker deep Red for 30 min before imaging in a confocal microscope (see method). (means ± SEM; n = 10 biological replicates). AA, Antimycin A. ** p<0.01, *** p<0.001 vs control N) mtSMPD5 overexpression does not alter mitochondrial membrane potential. Cells were loaded with the potentiometric dye Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRM⁺) in non quenching mode and the mitochondrial marker mitoTracker deep Red for 30 min before imaging in a confocal microscope (see method). (means ± SEM; n > 10 biological replicates).

Figure 6.. Mitochondrial proteome profiling associates complex I with muscle insulin sensitivity.

A) Quantification of proteins across samples. B) Number of significant deferential regulated (DR) proteins by pairwise comparison.Groups not shown have no significantly regulated proteins after correcting for multiple testing. C) Gene set enrichment between all comparisons. D) Relative protein abundance in isolated mitochondria from human skeletal muscle muscle cells. Comparisons are shown on top of each graph. Light blue, significatively regulated proteins (-log10(p-val) = 2). E) Proteins rank against rate glucose disappearance during clamp (Rd) correlation. Proteins within complex I of the electron transport chain are highly significant with Rd. F) Proteins rank against mitochondrial ceramide (Cer) 18:0 abundance. Proteins within complex I of the electron transport chain are highly significant with Cer18:0. G) Subunit levels associated with mitochondrial ceramides mapped to the complex I structure. The colours were calculated with an in-house python script and the resultant model was rendered using ChimeraX. Grey, no detected; Purple, below cut off (Corr. Coef. = - 0.25 to 0.25). H) Quantification of the mitochondrial proteins generated by the summed abundance of all subunits associated with a specific function from mtSMPD5-L6 myotubes after 72 h vs summed mitochondrial proteins (function) associated with either C18:0 ceramide (left panel) or insulin sensitivity (right panel) from human samples.