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

Summary 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.

between mitochondrial CoQ and ceramide levels is intimately linked to the control of cellular insulin sensitivity. For example, increasing mitochondrial ceramide using either chemical or genetic tools, decreased mitochondrial CoQ levels, and induced insulin resistance. Conversely, genetic or pharmacologic manipulations that lowered mitochondrial ceramide levels increased CoQ levels and protected against insulin resistance. Increased mitochondrial ceramides also led to a reduction in several OXPHOS components, hindering mitochondrial respiration and elevating mitochondrial ROS in vitro. This was further supported in human skeletal muscle, where we observed a strong association between insulin sensitivity, abundance of OXPHOS and mitochondrial ceramides. We propose that increased mitochondrial ceramides cause a depletion in various OXPHOS components, leading to mitochondrial malfunction and deficiency in CoQ, resulting in increased ROS and insulin resistance. This provides a significant advance in our understanding of how ceramide causes mitochondrial dysfunction and insulin resistance in mammals.

Palmitate induces insulin resistance by increasing ceramides and lowering CoQ9 levels in L6 -myotubes
Lipotoxicity plays a major role in insulin resistance and Cardiometabolic disease 32 . Excess lipids accumulate in insulin target tissues, such as muscle, impairing insulin-stimulated GLUT4 translocation as well as other metabolic actions of insulin. For this reason, several in vitro models have been employed involving incubation of insulin sensitive cell types with lipids such as palmitate to mimic lipotoxicity in vivo 9 . In this study we have used cell surface GLUT4-HA abundance as the main readout of insulin response. As shown (Fig. 1A), incubation of L6 myotubes with palmitate (150 μM for 16 h) reduced the insulin-stimulated translocation of GLUT4 to the cell surface by~30 %, consistent with impaired insulin action. Despite this marked defect in GLUT4 translocation, we did not observe any defect in proximal insulin signalling as measured by phosphorylation of Akt or TBC1D4 (Fig. 1C & D -100nM insulin; 20mins). Previous studies linking ceramides to defective insulin signalling have utilised the short chain ceramide analogue (C2-ceramide) [24][25][26] . Intriguingly, we were able to replicate that C2-ceramide inhibited both GLUT4 translocation and Akt phosphorylation in L6 myocytes ( Fig. 1B, C & D). One possibility is that palmitate induces insulin resistance in L6 myotubes via a ceramide-independent pathway. However, this is unlikely as palmitate-induced insulin resistance was prevented by the ceramide biosynthesis inhibitor myriocin (Fig. 1A) and we observed a specific increase in C16-ceramide levels in L6 cells following incubation with palmitate, which was also prevented by myriocin ( Fig. 1E & F, Sup. Fig. 1). Based on these data we surmise that C2-ceramide does not faithfully recapitulate physiological insulin resistance, in contrast to that seen with incubation with palmitate.
We previously demonstrated that insulin resistance was associated with CoQ depletion in muscle from high-fat diet fed mice 6 . To test if CoQ supplementation reversed palmitate-induced insulin resistance, L6-myotubes were co-treated with palmitate plus CoQ9. Addition of CoQ9 had no effect on control cells but overcame insulin resistance in palmitate treated cells (Fig. 1A). Notably, the protective effect of CoQ9 appears to be downstream of ceramide accumulation, as it had no impact on palmitate-induced ceramide accumulation (Fig. 1E-F). Strikingly, both myriocin and CoQ9 reversed insulin resistance, suggesting that there might be an interaction between ceramides and CoQ in the induction of insulin resistance with palmitate in these cells. Moreover, we have previously shown that mitochondrial CoQ is a key determinant of insulin resistance 6 suggesting that ceramides and CoQ may interact in mitochondria. To explore this link, we next examined the effect of palmitate on mitochondrial CoQ levels. As shown (Fig.   1G), palmitate lowered mitochondrial CoQ9 abundance by~40 %, and this was prevented with myriocin. To test whether CoQ depletion is downstream of ceramide accumulation, we exposed GLUT4-HA-L6 myotubes to 4-nitrobenzoic acid (4NB) to competitively inhibit 4-hydroxybenzoate:prolyprenyl transferase (Coq2), a limiting step in CoQ9 synthesis 33 . 4NB (2.5 mM for 16 h) decreased mitochondrial CoQ9 to a similar extent as observed in palmitate-treated myocytes (Fig. 1 I) and generated insulin resistance in GLUT4-HA-L6 myotubes ( Fig. 1 H). Notably, 4NB mediated insulin resistance was prevented by provision of CoQ9, as previously described 6 . Interestingly, total ceramide abundance was increased in 4NB treated cells albeit to a lesser extent than observed with palmitate, without affecting other lipid species (   1). One possibility is that CoQ directly controls ceramide turnover 34 . An alternate possibility is that CoQ inside mitochondria is necessary for fatty acid oxidation 12 and CoQ depletion triggers lipid overload in the cytoplasm promoting ceramide production 35 .
In fact, increased fatty acid oxidation is protective against insulin resistance in several model organisms [36][37][38] . Future studies are required to determine how CoQ depletion promotes Cer accumulation. Regardless, these data indicate that ceramide and CoQ have a central role in regulating cellular insulin sensitivity.
Since palmitate treatment can have a number of effects beyond ceramides, we next attempted to increase intracellular ceramides by inhibiting the ceramide degradation pathway. We exposed L6 myotubes to different concentrations of Saclac, an inhibitor of acid ceramidase (Kao et al., 2019), for 24 h. Saclac increases ceramides in L6 cells in a dose-dependent fashion, with the largest effect on C16:0 ceramides (Sup. Fig. 1F).
Consistent with our observation in L6-myotubes, Saclac increased total ceramide levels (~6 fold over basal) (Sup. Fig. 2 A & B) and lowered CoQ levels inside mitochondria (Sup. Fig 2C). Of interest, myriocin prevented Saclac-induced-CoQ depletion demonstrating that there is a similar interaction between ceramide and CoQ levels in this human cell line as observed in L6 cells (Sup. Fig. 2D). Moreover, this was relatively specific to CoQ as we did not observe any change in mitochondrial mass with Saclac Regardless, these data indicate that there is a strong association between ceramide and CoQ and that this has a central role in regulating cellular insulin sensitivity.

Mitochondrial ceramide promotes insulin resistance by lowering CoQ levels.
Although mitochondrial ceramides have been linked with insulin resistance in human skeletal muscle 30 , to date, there is no direct evidence linking mitochondrial ceramides with insulin sensitivity. We wanted to determine if ceramide accumulation specifically in mitochondria is associated with altered CoQ levels and insulin resistance.
To achieve this, we employed doxycycline-Tet-On inducible 40 overexpression of a mitochondrial-targeted Sphingomyelin Phosphodiesterase 5 (mtSMPD5) in GLUT4-HA-L6 cells (GLUT4-HA-L6-mtSMPD5) ( Fig. 2A). SMPD5 is a murine mitochondria-associated enzyme 41 that hydrolyses sphingomyelin to produce ceramides 42 . Thus, overexpressing mtSMPD5 should specifically increase ceramides within mitochondria and avoid potential non-specific effects associated with small molecule inhibitors. As expected, doxycycline induced mitochondrial expression of mtSMPD5, as demonstrated by enrichment of SMPD5 in mitochondria isolated from L6 cells (Fig. 2B) and this was associated with increased total mitochondrial ceramides to the same extent as observed with palmitate treatment (Fig. 2C), with the largest increase in C16-ceramide (Fig. 2D). Importantly, mtSMPD5 overexpression did not affect ceramide abundance in the whole cell lysate nor other lipid species inside mitochondria such as cardiolipin, cholesterol and DAGs (Sup. Fig. 3 A, D-J). Intriguingly, mtSMD5 did not affect sphingomyelin levels in mitochondria (Sup. Fig. 3G), consistent with exchange between mitochondrial and extra-mitochondrial sphingomyelin pools to compensate for the degradation induced by SMPD5 overexpression 43 . Consistent with our hypothesis, mtSMPD5 was sufficient to promote insulin resistance in response to submaximal and maximal insulin doses (Fig. 2E). Furthermore, mtSMPD5 overexpression promoted insulin resistance without affecting Akt phosphorylation ( Fig.   2F -H), and no differences in total GLUT4 levels were observed across the treatments ( Fig. 2F & Sup. Fig. 3B). We next explored if mitochondrial ceramide-induced insulin resistance was mediated by lowering CoQ within mitochondria. In line with our previous results, Mitochondrial CoQ levels were depleted in both palmitate-treated and mtSMPD5-overexpressing cells without any additive effects. This suggests that these strategies to increase ceramides share a common mechanism for inducing CoQ depletion in L6 myotubes (Fig. 2I). Importantly, CoQ9 supplementation prevented both palmitate-and mtSMPD5 induced-insulin resistance (Fig. 2J), suggesting that CoQ depletion is an essential mediator of insulin resistance.

Mitochondrial ceramides are necessary for palmitate-induced CoQ depletion and insulin resistance.
Given increased mitochondrial ceramides are sufficient to induce CoQ depletion and insulin resistance, we next asked whether increased mitochondrial ceramides are necessary to drive these phenotypes. Using the doxycycline-Tet-On inducible system 40 we overexpressed a mitochondrial-targeted Acid Ceramidase 1 (mtASAH1) in GLUT4-HA-L6 cells (GLUT4-HA-L6-mtASAH1) (Fig. 3A). ASAH1 degrades ceramides to fatty acid and sphingosine 44 . Hence, mitochondrial overexpression of ASAH1 was expected to selectively lower ceramides inside mitochondria. Doxycycline increased the abundance of mtASAH1 in the mitochondrial fraction, demonstrating the correct localisation of this construct (Fig. 3B). Furthermore, mtASAH1 induction prevented palmitate-induced mitochondrial ceramide accumulation (total levels and 18:1\16:0 ceramides) (Fig. 3C), indicating the enzyme was functioning as expected. Similar to our observations with mtSMD5 overexpression, mtASAH1 did not alter ceramide abundance in the whole cell lysate or mitochondrial sphingomyelin levels (Sup. Fig. 4 A&F). Notably, mtASAH1 overexpression protected cells from palmitate-induced insulin resistance without affecting basal insulin sensitivity (Fig. 3E). Similar results were observed using insulin-induced glycogen synthesis as an orthologous technique for GLUT4 translocation. These results provide additional evidence highlighting the role of dysfunctional mitochondria in muscle cell glucose metabolism (Sup. Fig. 5K).
Importantly, mtASAH1 overexpression did not rescue insulin sensitivity in cells depleted of CoQ (2.5 mM 4NB for 24 h) supporting the notion that mitochondrial ceramides are upstream of CoQ (Fig. 3E). Neither palmitate nor mtASAH1 overexpression attenuated insulin-dependent Akt phosphorylation (Fig. 3 F-H) nor total GLUT4 abundance (Fig. 3F, Sup. Fig. 4B). Finally, mtASAH1 overexpression increased CoQ levels. In both control and mtASAH1 cells, palmitate induced a depletion of CoQ, however the levels in palmitate treated mtASAH1 cells remained similar to control untreated cells (Fig. 3I).
This suggests that the absolute concentration of CoQ is crucial for insulin sensitivity, rather than the relative depletion compared to basal conditions, thus supporting the causal role of mitochondrial ceramide accumulation in reducing CoQ levels in insulin resistance.
In order to demonstrate the connection between ceramide and CoQ in vivo, we examined whether a reduction of ceramides in mouse skeletal muscle, using a Ceramide Synthase 1 (CerS1) inhibitor, would alter mitochondrial CoQ levels. Treatment of adult mice with the CerS1 inhibitor P053 for 6 wks selectively lowered muscle ceramides without affecting other lipid species (Fig 3J, Sup. Fig 5). Notably, CerS1 11 inhibition increased CoQ in mitochondrial fractions isolated from skeletal muscle (Fig.   3K), similar effect was observed in mice exposed to a high fat diet (HFD) for 5 wks (Supp. Fig. 4H-I). These animals exhibited an improvement in mitochondrial function and reduced muscle triglycerides and adiposity upon HFD (further phenotypic and metabolic characterization of these animals can be found in 45 ) demonstrating the existence of the ceramide/CoQ relationship in muscle in vivo.

Mitochondrial ceramides induce depletion of the electron transport chain.
We have established that both increased mitochondrial ceramides and a loss of mitochondrial CoQ are necessary for the induction of insulin resistance. As such these changes are likely to induce other mitochondrial defects. To gain insight into how increased mitochondrial ceramides drive changes in mitochondrial function we performed MS-based proteomics on L6 cells overexpressing mtSMPD5. mtSMPD5-L6 myotubes were treated with doxycycline for various time points (2, 8, 24, 48 and 72 h) and positive induction was observed after 24 h of treatment ( Fig. Sup. 6A).
Subsequently, control, 24, and 72 h time points were selected for further studies.
Mitochondria were purified via gradient separation and analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-independent acquisition (DIA) mode (Fig. 4A). Across control and mtSMPD5 cells we quantified 2501 proteins where 555 were annotated as mitochondrial proteins (MitoCarta 3.0 and uniprot localisation) 46 .
Analysis of the proteome revealed that 9 and 19 % of mitochondrially annotated proteins were significantly changed at 24 and 72 h respectively (adj. p<0.05, Absolute log2 FC > 0.4) indicative of a temporal progression of changes following induction of mSMPD5 expression (Fig.4 B & C). 60 proteins were decreased at 72 h and of these 47% were functionally annotated as components of oxidative phosphorylation (OXPHOS (rank 1, 28/135 proteins). Within OXPHOS, we observed a significant depletion of the electron transport chain components (ETC). The ETC is composed of several complexes (complex I-V, CI-CV). In this dataset, CI (14/15 decreased), CIII (5/6 decreased) and CIV (13/13 decreased) but not CII (0/3 decreased) or CV (3/15 decreased) were depleted after mtSMPD5 overexpression (Fig. 4D). Despite the bulk downregulation of CI, III and IV, the assembly machinery associated with each complex was either upregulated or unchanged after mtSMPD5 overexpression (Fig. 4J) suggesting that mitochondrial ceramides somehow alter ETC stability. Intriguingly, neither CII nor CV were affected by mtSMPD5 suggesting that ceramides preferentially affect those ETC complexes that are part of structures known as supercomplexes (SCs) 47 . Importantly, as part of CoQ is found in SCs binding CI (Fig. 4F) we mapped the levels of individual subunits of CI onto the recently solved structure of bovine CI 48 . This revealed the loss of subunits around the N-module (Ndufs1, Ndufs4, Ndufs6, Ndufv2 & Ndufv3) and Q-module (Ndufa5 & Ndufs2) in CI (Fig. 4H). Importantly, CI downregulation was not associated with reduction in gene expression as shown in Sup.

Mitochondrial ceramides impair mitochondrial function
Based on the ceramide-dependent depletion of ETC members, we hypothesised that mitochondrial ceramides would impair mitochondrial function. To test this, we evaluated several aspects of mitochondrial function upon mtSMPD5 overexpression. Mitochondrial respiration is broadly considered to be the best measure for describing mitochondrial activity 22,49 . Respiration was assessed in intact mtSMPD5-L6 myotubes treated with Notably, we did not observe evidence of energy deficiency in our model (data not shown). Interestingly, CoQ9 supplementation partially recovered basal and ATP-linked mitochondrial respiration, suggesting that part of the mitochondrial defects are induced by CoQ9 depletion. The attenuation in mitochondrial respiration is consistent with a depletion of the ETC subunits observed in our proteomic dataset (Fig. 4). Since mitochondrial respiratory activity is limited by several factors including nutrient supply, bioenergetic demands, among others 50 we tested whether the ETC generally or a specific respiratory complex was affected by mtSMPD5 overexpression. We measured the activity of the respiratory chain by providing substrates for each respiratory chain complex to permeabilized cells and analysed oxygen consumption. mtSMPD5 overexpressing cells exhibited attenuated mitochondrial respiration irrespective of the substrate provided (  5N). Collectively, these data suggest that increased mitochondrial ceramides cause a loss of mitochondrial respiratory capacity and an increase in ROS production as a result of ETC depletion in L6-myotubes.

Association of mitochondrial proteome with insulin sensitivity and mitochondrial ceramides in human muscle
To further characterise the effect of mitochondrial ceramides on ETC abundance in a more physiological context we performed a cross-sectional study assessing the mitochondrial lipid profile and protein abundance in muscle biopsies obtained from four groups of people (athletes, lean, obese and type 2 diabetics (T2D). The demographic information and detailed lipidomic analysis of these individuals was previously (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint (Fig. 6A). A total of 2,058 unique protein groups were quantified in at least one sample, where 571 were annotated as mitochondrial associated proteins (Human MitoCarta 3.0) 46 . After filtering (proteins in >50% of samples within each group), 492 mitochondrial proteins were reliably quantified across 67 samples (Fig. 6B). We noted that the mitochondrial fraction from athletes were enriched for mitochondrial proteins, and this could be corrected by global median normalisation (Fig. Sup. 7D). Pairwise comparison of the mitochondrial proteome between all groups revealed differences between groups, although relatively small in effect size ( Fig. 6C & D). For instance, 16% of all mitochondrial proteins were significantly different between T2D and athletes ( Fig. 6C), however 56% (45/80) of these proteins were changed by less than 1.5-fold. This trend was even stronger when comparing the obese group to the athletes, where 18% of mitochondrial proteins were changed, but~80% were changed less than 1.5-fold. Gene set enrichment revealed a highly significant general trend following the difference in insulin sensitivity (measure by the rate of glucose disappearance -Rd-using a stable isotope -[6,6-2 H 2 ]glucose -during a hyperinsulinemic-euglycemic clamp 30 , where TCA cycle and respiratory electron transport and Complex I biogenesis were enriched as follows: Athletes > Lean > Obese > T2D (Sup. Table 2). Of note, the T2D group had an enrichment of mitochondrial translation compared to the obese group.
To further explore the relationship between mitochondria and insulin sensitivity, the mitochondrial proteome was correlated to the muscle insulin sensitivity measured using 2 H 2 glucose Rd. As a group, all detected proteins within CI of the ETC were highly correlated with muscle insulin sensitivity (p = 4e-15) (Fig. 6E), and to a lesser extent proteins within CIV (p=0.08) and CV (p= 0.09; Fig. Sup. 7E). The abundance of CII and 16 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint CIII, together with the small and large mitochondrial ribosome subunits, were not associated with insulin sensitivity across all the samples (Fig. Sup. 7F). Next, we determined the association between the mitochondrial proteome and the levels of C18:0 ceramide in the mitochondria/ER fraction. In line with our previous observations, as a group, CI proteins were inversely correlated with mitochondrial ceramides (p = 8e- 14) and no association was observed between CII and C18:0 ceramides across samples ( Fig. 6F). Furthermore, components of CIV were also negatively correlated with mitochondrial ceramides although to a lesser extent (Sup. Fig. 7F, p = 0.026) and CV was not associated with mitochondrial ceramides (Sup. Fig. 7F, p = 0.737). According to these results, ETC subunits exhibit differential sensitivity to mitochondrial ceramides, with CI subunits being the most sensitive in human muscle. To uncover structural changes in CI that could correlate with increased ceramide we mapped those proteins significatively associated with mitochondrial ceramides to the bovine CI structure 48 .
Consistent with L6-mtSMPD5 myotubes, the N and Q modules were the regions with the most negative associated subunits with mitochondria ceramides in human muscle ( Fig. 6G). To determine the conservation in the changes in the mitochondrial proteome induced by increased ceramides, we compared the proteomes of mtSMPD5-L6-myotubes (72 h after induction) and human muscle biopsies. We observed that across the two datasets, CI and CIV subunits were downregulated after mtSMPD5 overexpression and were negatively associated with C18 ceramides in human samples (Fig. 6H). In turn, CI and CIV were positively associated with muscle insulin sensitivity (Fig. 6I), suggesting that these ETC subunits exhibited a conserved sensitivity to ceramide accumulation with a potential role in insulin sensitivity.
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Discussion
Insulin resistance is characterised by attenuated insulin-dependent glucose uptake in relevant target tissues, such as muscle and fat, and it plays a central role in cardiometabolic diseases 3 . In skeletal muscle, mitochondrial ceramides have been linked to insulin resistance 30 , however, to date no direct link connecting mitochondrial ceramides with insulin resistance has been established. Furthermore, CoQ depletion and defective mitochondria have also been independently associated with insulin resistance 6,7 . In the current study we present evidence suggesting that these factors are mechanistically linked inside mitochondria. Our data demonstrate that increased mitochondrial ceramides are both necessary and sufficient to induce insulin resistance in skeletal muscle. This is likely a function of increased ROS production that results from the specific depletion of the OXPHOS subunits and the concomitant loss of CoQ.
Analysis of the human muscle mitochondrial proteome strongly supports mitochondrial ceramide linked changes in the OXPHOS machinery as major drivers of insulin sensitivity. In this study, we mainly utilised L6-myotubes, which share many important characteristics with primary muscle fibres. Both types of cells exhibit high sensitivity to insulin and respond similarly to maximal doses of insulin, with GLUT4 translocation stimulated between 2 to 4 times over basal levels in response to 100 nM insulin (as shown in Fig. 1-4, 51,52 ). Additionally, mitochondrial respiration in L6-myotubes have a similar sensitivity to mitochondrial poisons, as observed in primary muscle fibres (as shown in Fig. 5, 53 ). Finally, inhibiting ceramide production increases CoQ levels in both L6-myotubes and adult muscle tissue (as shown in Fig. 2-3). Therefore, L6-myotubes 18 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint possess the necessary metabolic features to investigate the role of mitochondria in insulin resistance, and this relationship is likely applicable to primary muscle fibres.
Many stressors, including chronic inflammation and anticancer drugs, stimulate endogenous ceramide generation 54 and CoQ depletion in mitochondria 55,56 .
Nevertheless, experimental evidence testing the link between these molecules has been lacking. We observed that increased mitochondrial ceramides drive a depletion of mitochondrial CoQ leading to insulin resistance ( Fig. 1 & 2), and that reducing mitochondrial ceramide protects against the loss of CoQ and IR (Fig. 3). These findings align with our earlier observations demonstrating that mice exposed to HFHSD exhibit mitochondrial CoQ depletion in skeletal muscle 6 . Given that CoQ supplementation is sufficient to overcome ceramide induced-IR ( Fig. 1 & 2), but a reduction of mitochondrial ceramide does not overcome a loss of CoQ (Fig. 3), our data support a pathway whereby an increase in mitochondrial ceramides precedes loss of CoQ. Interestingly, inhibition of CoQ synthesis also increased ceramides, suggesting a bidirectionality to the ceramide-CoQ nexus. That said, this effect was modest ( Fig. 1) and we cannot exclude off target effects of the inhibitor. It is possible that CoQ directly controls ceramide turnover 34 or alternatively that CoQ inside mitochondria is necessary for fatty acid oxidation 12 and CoQ depletion triggers lipid overload in the cytoplasm promoting ceramide production 35 . Further studies will be needed to determine how CoQ depletion promotes ceramide accumulation.
Our proteomics analysis revealed that the loss of CoQ parallels a loss of mitochondrial ETC complexes CI, CIII and CIV. These are known to form supercomplexes or respirasomes where~25 -35 % of CoQ is localised in mammals 57,16 . This bulk 19 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint downregulation of the respirasome induced by ceramides may lead to CoQ depletion.
The observation that both palmitate and SMPD5 overexpression trigger CoQ depletion without additive effects support the notion that ceramides may trigger the depletion of a specific CoQ9 pool localised within the inner mitochondrial membrane. Despite the significant impact of ceramide on mitochondrial respiration, we did not observe any indications of cell damage in any of the treatments, suggesting that our models are not explained by toxicity and increased cell death (Sup. Fig. 2H & J). Whilst the physiological role of respirasomes is still a subject of discussion, it has been suggested that they may enhance energy generation by optimising electron flow while reducing production of ROS 58,59 and therefore their loss can be predicted to increase ROS generation. The 2 major mechanisms that might account for the loss of the respirasomes are decreased synthesis or increased degradation. Proteomics data suggests no deficiency in the OXPHOS biosynthetic machinery or assembly proteins and an increase in the machinery for protein import (Fig. 4). In addition, the absence of mRNA downregulation in mtSMPD5 overexpressing cells strongly suggests that at least a portion of the observed protein depletion within CI is attributed to diminished protein stability. It therefore seems reasonable to speculate that the loss of these mitochondrial complexes is driven by increased degradation. Interestingly, pharmacological CIII inhibition leads to respirasome degradation via oxidative stress produced by reverse electron transfer (RET) 60 . Since ceramides can directly inhibit CIII 61 it is possible that a similar mechanism mediates the effect of ceramides on the respirasome (Fig. 5). This suggests that defective respirasome activity (e.g. induced by ceramides) triggers ROS, which over time depletes respirasome subunits and a stoichiometric CoQ depletion, 20 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint leading to further ROS production as a consequence. Another possibility is that, because of its highly hydrophobic nature, ceramides impact membrane fluidity promoting a gel/fluid phase transition 62 . These alterations in membrane fluidity could decrease respirasome stability. It is likely that bound lipids stabilise the interactions between the complexes in the respirasome and that this is impaired by ceramides. In fact, bound lipid molecules are observed in the structure of the porcine respirasome 63 and the isolated bovine CI 63 but none of the lipids identified thus far directly bridge different complexes. In order to understand the role of lipids in stabilising respirasomes and the role of ceramides in such stabilisation, higher-resolution structures will be required 64 .
The current studies pose a number of key unanswered questions. First, how does ceramide accumulate in mitochondria in insulin resistance? This could involve transfer from a different subcellular compartment 65,66 or in situ mitochondrial ceramide synthesis.
Consistent with the latter, previous studies have suggested that various enzymes involved in ceramide metabolism are specifically found in mitochondria 41,[67][68][69][70][71] . Notably, CerS1-derived ceramide induces insulin-resistance in skeletal muscle 72 . Although this enzyme has not been reported as a mitochondrial protein, it can be transferred from the endoplasmic reticulum surface to the mitochondria under cellular stress in metabolically active tissues such as muscle and brain 73 . This provides a potential mechanism where cellular stress, like nutrient overload, may induce transfer of CerS1 to mitochondria, increasing mitochondrial ceramide to trigger insulin resistance.
A further question is how ceramide regulates insulin sensitivity. We present evidence that mitochondrial dysfunction precedes insulin resistance. However, previous studies 21 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint have failed to observe changes in mitochondrial morphology, respiration or ETC components during early stages of insulin resistance 22,74 . However, in many cases such studies fail to document changes in insulin-dependent glucose metabolism in the same tissue as was used for assessment of mitochondrial function. This is crucial because we and others do not observe impaired insulin action in all muscles from high fat fed mice for example 6,9 . In addition, surrogate measures such as insulin-stimulated Akt phosphorylation may not accurately reflect tissue specific insulin action as demonstrated in figure 1C. Thus, further work is required to clarify some of these inconsistencies. We observed that mitochondrial ceramides were associated with the loss of CoQ, increased production of mitochondrial ROS and impaired mitochondrial respiration 6,9,10 . As we discussed above, this is likely a direct result of respirasome depletion. The molecular linkage between ROS production and IR remains unknown.
Early studies suggested that ceramides and ROS impaired canonical insulin signalling [24][25][26] , however, our current data do not support this, with the caveat that these were static signalling measures. One possibility is the release of a signalling molecule from the mitochondria that impairs insulin action 75 . The mitochondrial permeability transition pore (mPTP) is an attractive candidate for this release since its activity is increased by mitochondrial ROS 76 and ceramides 77 . It has been shown that mPTP inhibition protects against insulin resistance in either palmitate-or ceramide-induced L6-myotubes and mice on a high-fat diet 78 . Furthermore, mPTP deletion in the liver protects against liver steatosis and insulin resistance in mice 79 . Strikingly, CoQ is an antioxidant and also an inhibitor of mPTP suggesting that part of the protective mechanism of CoQ may involve the mPTP 80 . Because CoQ can accumulate in various 22 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint intracellular compartments, it's important to consider that its impact on insulin resistance might be due to its overall antioxidant properties rather than being limited to a mitochondrial effect. Excitingly, mtSMPD5 increased the abundance of mPTP associated proteins suggesting a role of this pore in ceramide induced insulin resistance (Sup. Fig. 6E). It is also possible that ceramides generated within mitochondria in SMPD5 cells leak out from the mitochondria into other membranes (e.g. PM and GLUT4vesicles) affecting other aspects of GLUT4 trafficking and insulin action.
However, the observation that ASAH1 overexpression reversed IR without affecting whole cell ceramides argues against this possibility.
Ultimately, the significant challenge for the field is the discovery of the unknown factor(s) released from mitochondria that cause insulin resistance, their molecular target(s), and the transduction mechanism(s).
The observations described above led us to speculate on whether there is a teleological reason for why these mitochondrial perturbations occur and why they drive insulin resistance? Under conditions of stress, nutrient incorporation into the cell needs to be adjusted to keep the balance between energy supply and utilisation. In situations where the mitochondrial respirasome is depleted, the mitochondria's ability to oxidise nutrients can be easily overwhelmed without a corresponding reduction in nutrient uptake. In this scenario, insulin resistance may be a protective mechanism to prevent mitochondrial nutrient oversupply 9 . Beyond nutrient uptake, the respirasome depletion could also affect the ability of the mitochondria to switch between different energy substrates depending on fuel availability, named "metabolic Inflexibility" 81 this mechanism may 23 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint potentially play a role in the ectopic lipid accumulation seen in individuals with obesity, a condition linked with cardio-metabolic disease.
In summary, our results provide evidence for the existence of a mechanism inside mitochondria connecting ceramides, mitochondrial respiratory complexes, CoQ and mitochondrial dysfunction as part of a core pathway leading to insulin resistance. We identified that CoQ depletion links ceramides with insulin resistance and define the respirasome as a critical connection between ceramides and mitochondrial dysfunction.     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 29 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023.      (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint diacylglycerol (I -J) (as indicated) were determined by lipidomics and normalised against mitochondrial protein concentration. Cells were exposed to doxycycline for 3 d for SMPD5 induction. N= 3, Mean ± S.E.M. ****p< 0.0001.

Figure S4
A) Total ceramide levels extracted from whole lysate of L6-myotubes exposed to different treatments (as indicated). B-C) Densitometric analysis of Figure 3F  Insulin-induced glycogen synthesis. L6-myotubes were exposed to different treatments (as indicated) and glycogen storage was measured by 14-C glucose (as indicated in material and method section). Data is expressed as a percentage of increased 14-glycogen after insulin stimulation relative to control cells. N = 4, mean ± S.E.M. *p<0.05. H-I) Total ceramides (H) and mitochondrial CoQ levels (I) in skeletal muscle from mice exposed to a high-fat diet and exposed to either vehicle (DMSO) or the inhibitor of CerS1 P053 (5 mg/Kg) in drinking water for 6 wks as described in 45

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, David James (David.james@sydney.edu.au) or James Burchfield (James.burchfield@sydney.edu.au)

Materials availability
This study generated two new molecular tools to overexpress mitochondria targeted SMPD5 and ASAH1. The plasmids are available upon request.

36
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint All Lipidomic analyses are available as supplementary information.

Administration of P053 to mice
Mice of the C57BL6/J strain were obtained from the Animal Resources Centre of Perth (WA, Australia). Mice were housed in a controlled 12:12 h light-dark cycle, and they had ad libitum access to water and food. The oral gavage administration of P053 (5 mg/kg) was performed daily, while the control animals received vehicle (2% DMSO). The experiments were approved by the UNSW animal care and ethics committee (ACEC

15/48B), and followed guidelines issued by the National Health and Medical Research
Council of Australia.

Cell lines
Mycoplasma-free L6 myotubes overexpressing HA-GLUT4 and HeLa cell lines were used for all in vitro experiments (detailed below each legend). HA-GLUT4 overexpression is essential for studying insulin sensitivity in vitro as we previously

Lipid quantification
Lipids were quantified by selected reaction monitoring on a TSQ Altis triple quadrupole mass spectrometer coupled to a Vanquish HPLC system (ThermoFisher Scientific).
Lipids were separated on a 2.1 100 mm Waters Acquity UPLC C18 column (1.7 µM pore size) using a flow rate of 0.28 mL/min. Mobile phase A was 0.1% formic acid, 10 mM ammonium formate in 60% acetonitrile/40% water. Mobile phase B was 0.1% formic acid and 10mM ammonium formate in 90% isopropanol/10% acetonitrile. Total 39 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Mass spectrometry sample preparation
Isolated mitochondria were defrosted and centrifuged at 4°C at 4,000 x g for 15 min, and supernatant was removed. The mitochondrial pellet was resuspended in 100 uL 2 % SDC in Tris-HCl buffer (100 mM; pH 8.0) and the protein concentration determined by BCA assay. 10 ug of each sample was aliquoted and volume adjusted to 50 uL with milli-Q water, and samples were reduced and alkylated by addition of TCEP and CAA (10 and 40 mM respectively) at 60°C for 20 minutes. Once cooled to room temperature, 0.4 ug MS grade trypsin and Lys-C were added to each sample, and proteins were digested overnight (16 h) at 37°C. Peptides were prepared for MS analysis by 40 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ;

SDB-RPS stage tips. 2 layers of SDB-RPS material was packed into 200 µL tips and
washed by centrifugation of StageTips at 1,000 x g for 2 min in a 96-well adaptor with 50 µL acetonitrile followed by 0.2% TFA in 30% methanol and then 0.2% TFA in water. 50 µL of samples were loaded to StageTips by centrifugation at 1,000 g for 3 min. Stage tips were washed with subsequent spins at 1,000 g for 3 min with 50 uL 1% TFA in ethyl acetate, then 1% TFA in isopropanol, and 0.2% TFA in 5% ACN. Samples were eluted by addition of 60 µL 60% ACN with 5% NH4OH4. Samples were dried by vacuum centrifugation and reconstituted in 30 µL 0.1% TFA in 2% ACN.

Mass spectrometry acquisition and analysis
Samples were analysed using a Dionex UltiMate™ 3000 RSLCnano LC coupled to a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. with an HCD collision energy at 30% and MS2 spectra collected between m/z 300-2000 at resolution of 30,000, with an AGC target of 3e5 and the maximum injection time set to automatic. Raw data files were searched using DIA-NN using library generated from a 16-fraction high pH reverse phase library 83

Matrigel-coated plates
Matrigel diluted 1:100 v/v in ice-cold PBS was dispensed into 96-well plates (Eppendorf Cell Imaging plate, UNSPSC 41122107; and Perkin Elmer Cell Carrier Ultra, Cat# 6055300) and incubated for 2 h at room temperature. Before use, plates were washed twice in PBS at room temperature.

HA-GLUT4 assay
HA-GLUT4 levels on the plasma membrane were determined as previously described 9,91 . L6-myotubes stably overexpressing HA-Glut4 were washed twice with warm PBS and serum-starved for two hours (in DMEM/0.2% BSA/GlutaMAX/with 220 mM bicarbonate (pH 7.4) at 37°C, 10 % CO2). Cells were then stimulated with insulin for twenty minutes, after which the cells were placed on ice and washed three times with ice cold PBS. Cells were blocked with ice cold 10 % horse serum in PBS for 20 min, fixed with 4 % paraformaldehyde (PFA) for 5 min on ice and 20 min at room temperature. PFA was quenched with 50 mM glycine in PBS for 5 min at room temperature. We measured the accessibility of the HA epitope to an anti-HA antibody 44 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint (Covance, 16B12) for 1 h at room temperature. Cells were then incubated with 20 mg/mL goat anti-mouse Alexa-488-conjugated secondary antibody (Thermo Fisher Scientific) for 45 min at room temperature. The determination of total HA-GLUT4 was performed in a separate set of cells following permeabilization with 0.01% saponin (w/v) and anti-HA staining (as above). Each experimental treatment group had its own total HA-GLUT4. A FLuostar Galaxy microplate reader (BMG LABTECH) was used to measure fluorescence (excitation 485 nm/emission 520 nm). Surface HA-GLUT4 was expressed as a fold over control insulin condition.

Induction of insulin resistance
To promote insulin resistance, cells were stimulated for 16 h with 150 μM palmitate-BSA or EtOH-BSA as control. The palmitate was complexed with BSA as previously described 9

Glycogen synthesis assay
L6 myotubes overexpressing ASAH were grown and differentiated in 12-well plates, as described in the Cell lines section, and stimulated for 16 h with palmitate-BSA or EtOH-BSA, as detailed in the Induction of insulin resistance section.
On day seven of differentiation, myotubes were serum starved in plain DMEM for 3 and a half hours. After incubation for 1 hour at 37°C with 2 µCi/ml D-[U-14C]-glucose in the presence or absence of 100 nM insulin, glycogen synthesis assay was performed, as previously described 92 45 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint

Coenzyme Q determination
CoQ9 and CoQ10 content in cell lysates and mitochondrial fractions were determined as described previously (Burger et al., 2020). Aliquots of 15 µg mitochondrial protein as prepared below were adjusted to a volume of 100 µL with water and subsequently mixed with 250 μL ice-cold methanol containing 0.1% HCl, 20 µL internal standard CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Mitochondrial isolation
Mitochondrial isolation from cultured L6-myotubes and was performed as described elsewhere 93,94 . Briefly, cells were homogenised in an ice-cold mitochondrial isolation buffer (5 mM HEPES, 0.5 mM EGTA, 200 mM mannitol and 0.1 % BSA, pH 7.4 containing protease inhibitors) using a Cell Homogenizer with 18-micron ball. Cells were passed through the Cell Homogenizer 10 times using 1 mL syringe. Cell Homogenizer was equilibrated with 1 mL of ice-cold isolation buffer prior to the experiment.
Homogenates were centrifuged at 700 g for 10 min and the supernatant centrifuged at 10,300 g for 10 min to generate the crude mitochondrial pellet. The 10,300 g pellet was resuspended in 1 mL of isolation buffer and transferred into a polycarbonate tube containing 7.9 mL of 18% Percoll in the homogenization buffer and centrifuged at 95,000 g at 4°C for 30 min. The mitochondrial pellet was collected and diluted in a homogenization buffer (1 mL) and centrifuged at 10,000 g for 10 min at 4°C. The supernatant was discarded, and the pellet was washed with a homogenization buffer without BSA followed by protein quantification with BCA protein assay.

47
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ; https://doi.org/10.1101/2023.03.10.532020 doi: bioRxiv preprint Mitochondria from adult skeletal muscle (from mixed hindlimb muscle) were isolated by differential centrifugation as described previously 95 . Briefly, muscle was diced in CP-1 medium (100 mM KCl, 50 mM Tris/HCl, pH 7.4, and 2 mM EGTA), digested on ice for 3 min in CP-2 medium [CP-1, to which was added 0.5% (w/v) BSA, 5 mM MgCl2, 1 mM ATP and 2.45 units ml-1 Protease Type VIII (Sigma P 5380)] and homogenised using an ultra-turrax homogenizer. The homogenate was spun for 5 min at 500 g and 4°C.
The resulting supernatant was subjected to a high-speed spin (10,600 g, 10 min, 4°C) and the mitochondrial pellet was resuspended in CP-1. The 10,600 g spin cycle was repeated, the supernatant removed and the mitochondrial pellet snapped frozen.

Western Blotting
After insulin stimulation or mitochondrial isolation, samples were tip sonicated in 2% SDS-RIPA. Insoluble material was removed by centrifugation at 21,000 g x 10 min.
Protein concentration was determined by bicinchoninic acid method (Thermo Scientific).
10 μg of protein was resolved by SDS-PAGE and transferred to PDVF membranes.
Membranes were blocked in Tris-buffered saline (TBS) 4 % skim milk for 30 min at room temperature, followed by primary antibody incubation (detailed antibody is provided in "Key Resource Table"). Membranes were washed in TBS 0.1% tween (TBS-T) and incubated with appropriate secondary antibodies (IRDye700-or IRDye800-conjugated) in TBS-T 2% skim milk for 45 min at room temperature. Images were obtained by using 700-or 800-nm channels using Odyssey IR imager. Densitometry analysis of immunoblots was performed using Image Studio Lite (version 5. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Complex I  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 19, 2023. ;