Insulin-resistant female rat skeletal muscles display diacylglycerol-mediated protein kinase C activation and inflammation without ceramide accumulation

This study investigated the role of diacylglycerol (DAG)-mediated protein kinase C (PKC) activation, ceramide accumulation and inflammation in insulin-resistant female oxidative and glycolytic skeletal muscles induced by an obesogenic high-fat sucrose-enriched (HFS) diet. The HFS diet impaired insulin-stimulated AKT Thr308 phosphorylation and glycogen synthesis, whereas rates of fatty acid oxidation and basal lactate production were significantly elevated in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. Insulin resistance was


Introduction
Skeletal muscles account for ∼30% and 40% of body mass in women and men, respectively (Elia, 1992). Given the large mass and its capacity to take up relatively high amounts of glucose from the circulation, the skeletal muscle compartment plays a significant role in the maintenance of whole-body glucose homeostasis (Rasool et al., 2018). Interestingly, even though women have approximately two-thirds the skeletal muscle mass and twice the adipose mass of their male counterparts, impaired fasting glucose has been reported to be higher in men (17%) than in women (13%) (Lundsgaard & Kiens, 2014). Furthermore, the rate of blood glucose clearance during an intravenous glucose tolerance test has been reported to be 15% higher in women than men (Clausen et al., 1996). Even in rodent models of glucose intolerance, insulin resistance and diabetes, males show a stronger phenotype than females (Frias et al., 2001;Macotela et al., 2009). These observations indicate that in addition to major body composition differences between males and females, sex has a profound impact on glucose and fat metabolism, conferring on women a favourable effect for insulin sensitivity. At the whole-body level, insulin sensitivity is regulated by the ability of several organs and tissues, such as liver, adipose tissue and skeletal muscles, to metabolize glucose. In this context, as the capacity of the adipose tissue to store fat reaches its maximum (e.g. obesity), lipotoxicity resulting from ectopic lipid deposition causes insulin resistance in peripheral organs (Abdul-Ghani & DeFronzo, 2010;Defronzo & Tripathy, 2009), including liver and skeletal muscle. It could be that because men have lower fat mass than women, the susceptibility to dysfunctional metabolic alterations caused by lipotoxicity could be higher in men than in women. Indeed, it has been reported that women are less prone to fatty acid-induced peripheral insulin resistance than men (Frias et al., 2001). This was based on the observation that acutely increased levels of circulating non-esterified fatty acids (NEFA) inhibited peripheral tissue insulin sensitivity in men, but not in women (Frias et al., 2001). However, no distinction has been reported regarding the contribution of specific peripheral organs (e.g. liver and skeletal muscle) to NEFA-induced insulin resistance in men versus women.
One of the proposed mechanisms by which excess intracellular fat accumulation causes insulin resistance involves the accumulation of lipid intermediates such as diacylglycerol (DAG) and ceramides in myocytes (Goldberg et al., 2009). According to this model, elevated levels of the bioactive signalling lipids DAG or ceramides, formed during the process of triacylglycerol (TAG) synthesis and storage in skeletal muscles, activate protein kinase C (PKC, mainly the θ and δ isoforms in skeletal muscles). In its activated state, PKC has been demonstrated to inhibit the kinase activity of the insulin receptor (Morino et al., 2006), which then impairs all subsequent steps of the intracellular insulin signalling cascade and glucose metabolism in skeletal muscles. Similarly, it has been proposed that ceramides promote the activation of atypical PKC isoforms (PKCζ /λ), which also impairs insulin-stimulated AKT phosphorylation and its downstream signalling steps (Sokolowska & Blachnio-Zabielska, 2019). Recent work from our lab (Jani et al., 2021) confirmed that male Wistar rats fed for 8 weeks an obesogenic high-fat sucrose-enriched (HFS) diet increased DAG and ceramide contents in Sol (highly oxidative muscle rich in type I fibres) (Ariano et al., 1973) and EDL (mixed muscle, rich in type I and IIa fibres) (Ariano et al., 1973), whereas in Epit muscles (highly glycolytic, rich in type IIa and IIb fibres) (Nesher et al., 1980) neither DAG nor ceramides were significantly elevated by the HFS diet. Moreover, whereas membrane-bound PKCδ and PKCθ was increased in Sol and EDL, both PKC isoforms were reduced in Epit muscle from obese rats (Jani et al., 2021). Hence, data from our previous studies provide evidence that glycerolipid and ceramide accumulation, as well as DAG-induced PKC activation, follows a fibre type-dependent pattern under conditions of diet-induced obesity, and also that distinct mechanisms drive insulin resistance in oxidative and glycolytic muscles from male rats fed an obesogenic diet (Jani et al., 2021). Whether this is also the case in female skeletal muscles remains to be determined. Furthermore, it has been shown that despite having 47% more TAG content, lean moderately trained women displayed 29% higher skeletal muscle insulin sensitivity than matched men (Høeg et al., 2009). It could be that a higher capacity to store intramuscular TAG prevented DAG and ceramide accumulation in female skeletal muscles and consequently led to a lower induction of PKC activation. This could be one of the potential mechanisms underlying higher peripheral insulin sensitivity in females than males. To address this hypothesis, in this study we provide a detailed analysis of glycerolipid and ceramide content, PKC activation, as well as rates of basal and insulin-stimulated AKT phosphorylation, glycogen synthesis and glucose oxidation in oxidative and glycolytic muscles from female rats fed an obesogenic diet for 8 weeks.

Reagents
Fatty acid (FA)-free bovine serum albumin (BSA), glycogen and palmitic acid were obtained from Sigma-Aldrich (

Animals
Wistar strain female albino rats ordered from Envigo (Indianapolis, IN, USA) weighing 150−200 g (initial weight) were maintained in a constant temperature (23°C), with a fixed (12 h-12 h) light-dark cycles and fed for 8 weeks ad libitum. The diet was either a standard rat chow (SC) providing 27.0%, 13.0% and 60.0% of calories from protein, fat and carbohydrates, respectively (energy density = 3.43 kcal/g) or a high-fat sucrose-enriched (HFS) diet purchased from Research Diets (New Brunswick, NJ, USA; cat. no. D12492) providing 20.0%, 60.0% and 20.0% of calories from protein, fat and carbohydrates, respectively (energy density = 5.24 kcal/g).

Ethics approval
The protocol containing all animal procedures described in this study was specifically approved by the Committee on the Ethics of Animal Experiments of York University J Physiol 601.10 (York University Animal Care Committee, YUACC, permit number: 2021-03) and performed strictly in accordance with the YUACC guidelines. All tissue extraction procedures were performed under ketamine/xylazine anaesthesia (90 mg and 10 mg/100 g body weight (BW)), and all efforts were made to minimize suffering. All experiments in this study were carried out in compliance with the ARRIVE guidelines. The investigators understand the ethical principles under which the journal operates and that their work complies with its animal ethics checklist.

Glucose monitoring and glucose tolerance test
Rats were bled from the saphenous vein to assess glycaemia and insulinaemia in the fed state after 8 weeks of dietary intervention. Plasma glucose was determined by using the OneTouch UltraMini blood glucose monitoring system from LifeScan Canada Ltd (Burnaby, BC, Canada). Insulin measurements were conducted using an ELISA kit purchased from Millipore-Sigma (Burlington, MA, USA). For the glucose tolerance test (GTT), the animals were fasted overnight and had their basal glucose measured. Subsequently, each animal received an intraperitoneal (i.p.) injection of 1.75 g of glucose/kg BW (30% glucose solution in saline). Blood was then collected after 15, 30, 60, 90 and 120 min for the determination of serum glucose and insulin concentrations (Sepa-Kishi et al., 2018).

Muscle isolation and incubation for measurement of glucose oxidation, glycogen synthesis and lactate production
After anesthetizing with ketamine/xylazine (90 mg and 10 mg/100g BW) tissue samples were immediately extracted. Muscles chosen were based on their wide range of reported fibre-type distributions with distinct mitochondrial contents and oxidative capacities. The percentages of type I, type IIa and type IIb in Sol, EDL and Epit muscles are 84/16/0, 3/57/40 (Ariano et al., 1973) and 15/20/65 (Nesher et al., 1980), respectively. Upon extraction, muscle strips (18-22 mg) were mounted onto thin stainless steel wire clips to maintain optimal resting length and then quickly added to 2 ml of pre-gassed (30 min with O 2 :CO 2 95:5% (v/v)) Krebs-Ringer bicarbonate (KRB) buffer containing 4% fat-free BSA and 6 mM glucose in scintillation vials. Subsequently, the vials were sealed and continuously gasified for the entire 1 h-pre-incubation period. Muscle strips were then transferred to vials containing 2 ml of the same KRB buffer plus d-[U-14 C]glucose (0.2 μCi/ml) and incubated for one additional hour under continuous gasification in either the absence (basal) or the presence of insulin (100 nM) for the determination of glucose oxidation, glycogen synthesis and lactate production (Fediuc et al., 2006). To assess glucose oxidation, a small Eppendorf tube containing a loosely folded piece of filter paper moistened with 0.2 ml of 2-phenylethylamine/methanol (1:1, v/v) was inserted into the scintillation vial with the muscles. After the 1 h-incubation period, the muscles were removed to measure glycogen synthesis and samples (100 μl) of the incubation medium were collected for lactate production. The remaining medium were acidified with 0.2 ml of H 2 SO 4 (5 N) at 37°C for an additional 1 h to collect 14 CO 2 released. Subsequently, the filter papers were carefully removed and processed for radioactivity counting to assess glucose oxidation. Immediately after incubation, muscle strips were quickly washed in ice-cold phosphate-buffered saline, blotted on filter paper, snap frozen with liquid nitrogen (N 2 ) and digested in 0.5 ml of 1 mol/l KOH at 70°C for 45 min. To the digested muscle, glycogen carrier was added and allowed to precipitate overnight in 100% ethanol at −20°C. Next morning, samples were centrifuged (2300 g), the supernatant was discarded and 0.5 ml of water was added to each precipitate to count radioactivity and determine rates of glycogen synthesis. For lactate production, the medium aliquots were first deproteinated using 10 kDa filters (centrifuged at 15,700 g for 15 min at 4°C) and then assayed using a colorimetric assay kit following manufacturer's instructions.

Measurement of palmitate oxidation in isolated muscles
After extraction, muscle strips were mounted onto thin stainless steel wire clips and then quickly added to 2 ml of pre-gassed (30 min with O 2 :CO 2 95:5% (v/v)) KRB buffer containing 4% fat-free BSA and 6 mM glucose in scintillation vials. Subsequently, the pre-incubated muscle strips were gasified and incubated in 2 ml of KRB buffer plus 0.2 mM of cold palmitic acid previously complexed with fatty acid-free BSA and [1-14 C]palmitic acid (0.2 μCi/ml) for one more hour. A tube with a loosely folded filter paper was moistened with 0.2 ml of 2-phenylethylamine/methanol (1:1, v/v) was added inside the scintillation vial. After the 1 h-incubation period, the muscles were removed and the media was sealed and acidified with 0.2 ml of H 2 SO 4 (5 N), at 37°C for an additional 1 h for collection of the 14 CO 2 released. Finally, the filter papers extracted and processed for radioactivity counting (Vitzel et al., 2013).
For determination of AKT phosphorylation, total PKCδ and PKCθ contents, as well as their cytoplasmic and membrane fractions in Sol, EDL and Epit muscles, samples of Sol, EDL and Epit muscles were homogenized in a buffer containing 25 mmol/l Tris-HCl and 25 mmol/l NaCl (pH 7.4), 1 mmol/l MgCl 2 , 2.7 mmol/l KCl, 1% Triton X-100 and protease and phosphatase inhibitors (0.5 mmol/l Na 3 VO 4 , 1 mmol/l NaF, 1 μmol/l leupeptin, 1 μmol/l pepstatin and 20 mmol/l phenylmethylsulfonyl fluoride). Subsequently, muscle homogenates were centrifuged, and the supernatants were collected and used for the determination of total protein content. For fractionation of cytoplasmic and membrane fractions, a protein fractionation kit from Thermo Fisher Scientific was then used for each muscle. The Bradford assay was used to measure protein in samples of fractionated muscle tissue. An aliquot of each subcellular fraction was diluted 1:1 (v/v) with 2 × Laemmli sample buffer, heated to 95°C for 5 min and subjected to SDS-PAGE. Primary antibodies for PKCδ and PKCθ were used in a dilution of 1:2000. β-Actin were used as loading control for total PKCδ and PKCθ, while Na,K-ATPase was used as a loading control for the membrane fractions of PKCδ and PKCθ and glyceraldehyde 3-phosphate dehydrogenase was used as loading control for cytoplasmic fractions of PKCδ and PKCθ.

Determination of TAG, DAG and ceramide contents in Sol, EDL and Epit muscles
DAG, TAG and ceramide content was quantified using the ultra-high-pressure liquid chromatography system (UHPLC-UV, Nexera X2, Shimadzu, Kyoto, Japan) as described earlier (Jani et al., 2021). Briefly, lipid samples were extracted from Sol, EDL and Epit muscles using Folch's method (Folch et al., 1957). About 100 mg of muscle tissue were homogenized in 200 μl of chloroform:methanol (MeOH) (2:1 v/v), dried overnight under nitrogen gas (N 2 ) and resuspended in 100 μl of elution medium of 2-propanol-hexane (ProHex, 5:4 v/v); 50 μl of the sample was injected automatically into a reverse phase column (C18 5 μm 250 × 4.6 mm). The chromatography conditions were set to 40°C for 20 min using a gradient of MeOH and ProHex:100% MeOH from 0 to 10 min, followed by 50% MeOH and 50% ProHex for 10 min, maintained with isocratic elution for 10 min. Diolein and Triolein (0.25 μg/μl) were used to obtain a standard curve (Carvalho et al., 2012). Quantification was performed using the UHPLC-UV detection machine. In order to analyse total ceramide content, a small volume of the lipid extract obtained after Folch's extraction was transferred into new pre-weighed Eppendorf tubes as previously described (Błachnio-Zabielska et al., 2008). The organic phase was hydrolysed in 1 M KOH at 90°C for 60 min. The sphingosine liberated from ceramides was analysed by means of UHPLC by mixing it with 15 μl o-phthalaldehyde reagent and allowing it to derivatize for 20 min at room temperature. Subsequently, the samples were reconstituted in 100 μl of chloroform-methanol-acetic acid-water (50:37.5:3.5:2 v/v/v/v) and run through a porous silica column (ARC-18 1.8 μm 100 × 2.1 mm). Elution was conducted with heptane-isopropyl ether-acetic acid (60:40:3 v/v/v) at a gradient from 0% to 100% in 30 min at a flow rate of 0.8 ml/min followed by isocratic elution with acetonitrile: deionized distilled water (90:10, v/v) and a flow rate of 1 ml/min (Dobrzyń & Górski, 2002). The calibration curve was prepared using N-acetyl-d-sphingosine as a standard. The column was then equilibrated with chloroform-methanol-acetic acid-water (50:37.5:3.5:2 v/v/v/v) for 10 min at the same flow rate. Due to limited amount of tissue obtained from Epit muscles, two muscles from animals from the same treatment group were combined to be able to run all assays in duplicate. Therefore, for some experiments the graphed data show n = 4 for Epit. For Sol and EDL, this was not the case because these muscles are much larger and provide enough material to run all assays without combining material.

Quantitative analysis of mRNA expression changes in skeletal muscle with HFS diet
Primers were designed using the software PrimerQuest (IDT) and the Affymetrix database (NetAffx Analysis Center, Thermo Fisher Scientific) for each given gene. RNA was isolated from Sol, EDL and Epit using the RNeasy kit (Germantown, Maryland, MD, USA), followed by DNase treatment to remove genomic DNA carry-over. RT-PCR reactions were carried out at amplification conditions as follows: 95°C (10 min); 40 cycles of 95°C (15 s), 60°C (60 s). Quantitative PCR was performed using the CFX96 Real-Time System from Bio-Rad Laboratories (Mississauga, ON, Canada). All genes were normalized to the control gene TBP, and values are expressed as fold changes relative to SC. Primers sequences used are shown in Table 1.

Statistical analyses
Statistical analyses were performed by using two-way ANOVA with Holm-Bonferroni comparison post-hoc test or Student's t test as indicated in the figure legends. Normality was evaluated using the Kolmogorov-Smirnov normality test. Data are presented as means ± SD. The level of significance was set to P < 0.05.

Energy intake, body weight, adiposity and muscle weight
Despite having similar total energy intake during the feeding period amongst the groups, at the end of the J Physiol 601.10

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Cd40 8-week dietary intervention period, the HFS-fed group gained significantly more body weight (BW) than SC-fed rats (Table 2). HFS-fed rats also had significantly higher weight of Sol, EDL and Epit muscles in comparison to control SC rats. In addition, HFS-fed rats displayed significantly elevated blood glucose and insulin levels in comparison to SC-fed rats (Table 2).

Insulin-induced phosphorylation of AKT Thr308 and rates of glycogen synthesis in Sol, EDL and Epit muscles
As expected, AKT Thr308 phosphorylation increased robustly in Sol, EDL and Epit muscles of SC-fed rats upon insulin stimulation ( Fig. 1A-C). However, in all muscles from HFS rats insulin-stimulated AKT Thr308 phosphorylation was significantly impaired (Fig. 1A-C). Under basal conditions, glycogen synthesis rates were similar in all three muscles when comparing SC-and HFS-fed rats. Also, rates of insulin-stimulated glycogen synthesis increased by 1.5-fold in Sol (Fig. 1D), 1.57-fold in EDL (Fig. 1E) and 1.42-fold in Epit (Fig. 1F) in SC-fed rats. In contrast, all muscles from HFS-fed rats displayed a stunted glycogen synthesis response when stimulated with insulin (Fig. 1F). These findings clearly show that insulin signalling is defective in skeletal muscles of rats fed a HFS diet, which is consistent with the reduction in insulin-stimulated glycogen synthesis in oxidative and glycolytic muscles of these animals.

Insulin-stimulated lactate production and glucose oxidation in Sol, EDL and Epit muscles
All three muscles from rats fed a HFS diet had a higher basal rate of lactate production than SC-fed muscles ( Fig. 2A-C). In fact, HFS diet significantly increased basal lactate production by 2.88-, 2.56-and 3.8-fold in Sol, EDL and Epit muscles, respectively, in comparison to the SC diet. However, the production of lactate in response to insulin by muscles from rats fed a HFS diet was impaired. Similarly, in the presence of insulin, glucose oxidation was significantly increased by 1.43-fold in Sol (Fig. 2D), 1.28-fold in EDL (Fig. 2E) and 1.58-fold in Epit (Fig. 2F) muscles of SC rats, whereas in HFS-fed rats insulin-stimulated glucose oxidation was potently suppressed in all three muscles (Fig. 2D-F).

Palmitate oxidation and TAG accumulation in Sol, EDL and Epit muscles
Rates of palmitate oxidation in Sol, EDL and Epit muscles ( Fig. 3A-C) were significantly elevated with a HFS diet by ∼1.4-fold in all three muscles. Similarly, TAG accumulation was increased by 1.78-, 1.71-and 2.56-fold in Sol, EDL and Epit muscles, respectively, by the HFS diet ( Fig. 3D-F).

DAG and ceramide contents and mRNA expression of Dgat1 and Dgat2 in Sol, EDL, and Epit muscles
In SC-fed rats, DAG content was highest in EDL (5.63 ± 1.08 μg/mg of tissue) followed by Sol (2.4 ± 0.2 μg/mg of tissue) and Epit (1.3 ± 0.016 μg/mg of tissue) muscles ( Fig. 4A-C). HFS feeding significantly increased DAG content by 2.33-, 2.01-and 1.76-fold in Sol, EDL and Epit, respectively. In SC-fed rats, ceramide content was also highest in EDL (0.36 ± 0.18 μg/mg of tissue), followed by Epit (0.12 ± 0.046 μg/mg of tissue) and the least in Sol (0.058 ± 0.0093 μg/mg of tissue) muscles ( Fig. 4D-F). Upon HFS feeding, ceramide content in Sol, EDL and Epit muscles remained unaltered. These findings indicate that despite fibre-type differences, DAG accumulation increased in all three muscles, whereas the ceramide content was not affected by the obesogenic HFS diet. Quantitative PCR analysis revealed that the mRNA levels of Dgat1 in Sol, EDL and Epit muscles did not differ between SC-and HFS-fed rats (Fig. 4G-I). However, Dgat2 mRNA levels were 4.37-fold higher in Sol, 2.61-fold higher in EDL and 4.41-fold higher in Epit muscles of HFS than SC-fed rats (Fig. 4G-I).

PKCθ and PKCδ content and localization in Sol, EDL and Epit muscles
PKCθ and PKCδ are the most abundant isoforms of these kinases in skeletal muscle (Kolczynska et al., 2020;Osada et al., 1992). In this context, we found that total PKCδ levels remained unaltered in Sol, EDL and Epit muscles,

HFS diet causes impairment of insulin-induced AKT Thr308 phosphorylation and glycogen synthesis in oxidative and glycolytic muscles under basal (B) and insulin-stimulated (I) conditions
AKT phosphorylation (A-C) and glycogen synthesis (D-F) in Sol, EDL and Epit, respectively. Data are means ± SD. * P < 0.05 versus SC basal, two-way ANOVA; n = 6-11 for AKT phosphorylation and n = 8-10 for glycogen synthesis.
J Physiol 601.10 whereas total PKCθ levels were significantly reduced in Sol and EDL, but not in Epit muscle from rats fed a HFS diet (Fig. 5A-C). We then determined the activation of PKCθ and PKCδ by measuring its content in the membrane and cytoplasmic fractions in Sol, EDL and Epit (Itani et al., 2000;Jani et al., 2022;Li et al., 2015). With chronic HFS feeding Sol membrane/cytoplasm ratios for PKCδ and PKCθ were significantly increased by 1.26and 1.51-fold ( Fig. 6A and D). In EDL muscles, HFS diet increased by 1.8-fold the PKCθ membrane/cytoplasm ratio, whereas PKCδ remained unchanged ( Fig. 6B and

. The HFS diet increases palmitate oxidation and TAG accumulation in oxidative and glycolytic muscles
Palmitate oxidation (A-C) and TAG accumulation (D-F) in Sol, EDL and Epit muscles, respectively. Data are means ± SD. * P < 0.05 versus SC, t test; n = 5−9 for palmitate oxidation and n = 5−11 for TAG content.
fractions from Epit PKCθ and PKCδ were unaltered by the HFS diet ( Fig. 6C and F). These findings indicate that HFS diet increased PKCθ membrane/cytoplasm ratio in Sol and EDL, but not in Epit muscles, which did not display a significant increase in the activity of either PKC isoform.

Discussion
The main findings of this study were that HFS diet-induced obesity led to a significant elevation in intramuscular DAG and TAG contents without any alteration in ceramide levels in insulin-resistant female oxidative and glycolytic muscles. Moreover, a pro-inflammatory response was observed in skeletal muscles of rats fed a HFS diet, although this occurred in a fibre type-specific pattern. In fact, muscles rich in type IIa and type IIb fibres (EDL and Epit) (Ariano et al., 1973;Nesher et al., 1980) displayed a marked increase in the mRNA expression of Tlr4, Il6, Nfkb and Fas, whereas in Sol muscles (rich in type I fibres) these markers of inflammation did not differ from SC-fed rats. Despite these fibre type-specific alterations in the expression of inflammatory mediators, all muscles from female rats fed the obesogenic HFS diet significantly enhanced their rates of fatty acid oxidation and displayed marked impairments in insulin-stimulated AKT Thr308 phosphorylation, glycogen synthesis and glucose oxidation. These effects, also previously reported in male rats (Jani et al., 2021), were accompanied by elevated glycaemia and insulinaemia, which are consistent with whole-body insulin resistance in female rats fed a HFS diet. We have also observed that despite similar energy intake between SC-and HFS-fed rats, adiposity was significantly higher in the latter than the former group of animals. The apparent disproportionate increase in adipose tissue growth in HFS-fed rats in comparison to SC-fed rats could be at least partially attributed to reduced energy expenditure in HFS-fed rats. This is supported by our previous observations that rats fed a HFS diet for 8 weeks displayed ∼30% lower ambulatory activity (Araujo et al., 2010;So et al., 2011;Wu et al., 2014). Moreover, we have recently provided evidence that the HFS diet causes whitening of the brown adipose tissue in rats (Da Eira et al., 2023), an effect that likely attenuated diet-induced thermogenesis and favoured adipose tissue growth in these animals. This is also consistent with enhanced energy efficiency we have reported in rats fed a HFS diet (So et al., 2011). One of the mechanisms associated with skeletal muscle insulin resistance in diet-induced obesity involves the activation of PKC by elevated DAG levels in myocytes (Jornayvaz et al., 2010). Here, we investigated the activation of the two most prominent PKC isoforms (PKCδ and PKCθ) in skeletal muscles (Yu et al., 2002). We observed that the total levels of PKCδ in all muscles studied were not affected by feeding the HFS diet; however, total PKCθ content was significantly reduced in Sol and EDL, whereas in Epit muscles it remained unaltered. Importantly, analysis of the cytosolic versus membrane fractions revealed that in Sol and EDL female muscles the translocation of PKCθ was significantly elevated by the HFS diet. This latter finding was similar to what we had previously reported for male Sol and EDL muscles in which insulin resistance was accompanied by elevated PKCδ and PKCθ translocation upon HFS feeding (Jani et al., 2021). However, in female Epit both PKC isoforms were reduced by HFS feeding. Thus, in both males (Jani et al., 2021) and females the mechanism underlying HFS diet-induced insulin resistance in Epit muscles appears to be independent of DAG-induced PKC activation. This is consistent with observations that subcellular glycerolipid distribution in skeletal muscle does not explain sex differences that exist in obese humans with respect to insulin sensitivity (Broussard et al., 2021). Thus, even though females have been reported to accumulate higher amounts of TAG than males in skeletal muscles (Høeg et al., 2009), it did not protect against DAG-induced PKC activation and the development of insulin resistance in both oxidative and glycolytic female muscles under conditions of HFS diet-induced obesity. In this context, both male (Jani et al., 2021) and female Epit muscles elicited a significant pro-inflammatory response under obesogenic conditions, which likely played a preponderant role in driving insulin resistance in this glycolytic muscle chronically exposed to the HFS diet. Because TLR4 has been reported to selectively increase sphingolipid levels within the cell (Dobrzyń & Górski, 2002;Holland et al., 2011) and the HFS diet caused a marked increase in Tlr4 expression in female EDL and Epit muscles, one would expect that this would be followed by an increase in ceramides in both muscles. Surprisingly, despite increased Tlr4 mRNA expression, we found that ceramides remained unaltered by the HFS diet in all female muscles studied. This differs from our previous observations that in male rats Sol and EDL muscles displayed an increase in ceramide content, whereas in Epit it remained unaltered by feeding a HFS diet (Jani et al., 2021). In this scenario, our findings support that DAG-induced PKC activation was a key factor in promoting insulin resistance in EDL and Epit muscles of female rats fed a HFS diet. Furthermore, our qPCR analysis revealed that the mRNA levels of Dgat2 were also elevated in Sol, EDL and Epit muscles, whereas Dgat1 remained unaltered in muscles of female rats fed the HFS diet in comparison to SC-fed animals. Diacylglycerol acyltransferase (DGAT) catalyses the conversion of DAG into TAG and both Dgat1 and Dgat2 mRNA expression can be regulated transcriptionally, although evidence exists that the activity of these enzymes can also be regulated post-translationally (Chen et al., 2022), but the exact mechanisms remain poorly understood. Thus, our finding that Dgat2 mRNA expression was elevated in all three female muscles could signify that the majority of the intramyocellular acyl-CoAs were directed towards TAG formation rather than being used for ceramide synthesis in these muscles. Besides DAG-induced PKC activation, it is also possible that alterations in other parameters of lipid metabolism not measured in this study (e.g. accumulation of muscle acylcarnitines) (Broussard et al., 2021) could explain obesity-induced muscle insulin resistance in female muscles. In this context, here we show that muscles from HFS-fed female rats displayed enhancement (∼40%) in rates of fatty acid oxidation, which is lower than what we had previously reported (60-80%) for male rats of the same strain (Wistars) and age after 8 weeks of HFS feeding (Jani et al., 2021). Thus, lower capacity to enhance fatty acid oxidation compared to males under conditions of dietary lipid abundance likely led to a metabolic shift that contributed to DAG-induced PKC activation and insulin resistance. This is compatible with our findings that insulin-stimulated glucose oxidation was impaired, whereas basal lactate production was markedly increased in all three muscles from female rats fed the HFS diet. These findings are consistent with previous observations that PKC-induced impairment of insulin signalling also caused suppression of glycogen synthesis (Abdul-Ghani & DeFronzo, 2010;Dresner et al., 1999), the pathway essentially responsible for the entire non-oxidative glucose metabolism in skeletal muscles (Defronzo & Tripathy, 2009). Under such conditions, even though insulin resistance limited glucose uptake, the impairment of glycogen synthesis likely led to a diversion of intramyocyte glucose toward lactate synthesis in rats fed a HFS diet. Interestingly, lactate has been shown to suppress glycolysis in rats (Choi et al., 2002), whereas lactate-induced lactylation promotes IRS-1 Ser636 phosphorylation, which has been associated with impairment of insulin signalling in human skeletal muscle (Maschari et al., 2022). Thus, increased lactate production itself could be contributing to aggravating insulin resistance in skeletal muscles of HFS-fed rats. It is possible that substrate competition also contributed to enhance lactate formation, given that fatty acid oxidation was significantly increased in all muscles from female rats fed the HFS diet. This would be consistent with the tenets of the metabolic inflexibility hypothesis (Kelley et al., 1999) and the Randle cycle (Randle et al., 1963). However, more recent studies in muscles of insulin-resistant HF-fed male rats and obese insulin-resistant humans (Song et al., 2020) provided evidence that the physiopathology of muscle insulin resistance is dissociated from alterations in mitochondrial substrate preference. This was supported by findings that the ratio of mitochondrial pyruvate oxidation (V PDH ) to rates of mitochondrial citrate synthase (V CS ) flux did not differ between insulin-sensitive and insulin-resistant Sol and quadriceps rat muscles (Song et al., 2020). Importantly, these V PDH /V CS data were from male Sprague-Dawley rats fed a HF diet for 3 weeks, whereas in our study we used female Wistar rats fed a HFS diet for 8 weeks. Thus, potential metabolic differences regarding rat strain, length of feeding and sex should be considered and further explored with respect to the role of diet-induced obesity and insulin resistance in mitochondrial function in future studies. In summary, by looking at muscles with different fibre type compositions we were able to assess distinct metabolic pathways to integrate insulin signalling responses with the accumulation of lipid intermediates (e.g. DAG and ceramides), isoform-specific PKC activation, lactate production and inflammatory markers. Thus, through a more integrative approach, our female rat studies allowed us to identify meaningful pathways that lead to impairment in skeletal muscle metabolism and insulin sensitivity. In fact, our data from female rats provide evidence that DAG-induced PKC activation along with inflammation are the main drivers of insulin resistance in oxidative and glycolytic skeletal muscles under conditions of HFS diet-induced obesity. This was supported by impairments in insulin-stimulated AKT phosphorylation and glycogen synthesis, whereas rates of fatty acid oxidation and basal lactate production were significantly elevated. We also found that, despite developing insulin resistance and increasing Tlr4 expression in response to HFS feeding, none of the muscles studied displayed alterations in ceramide content. This could be explained by a significant increase in Dgat2 mRNA expression in all three female muscles studied. In this context, upregulation of DGAT2 likely diverted the majority of the intramyocellular acyl-CoAs to TAG synthesis instead of ceramides. These findings differ from what we had previously reported for male rats in which insulin resistance was accompanied by increases in both DAG and ceramides in Sol and EDL muscles. However, for Epit muscles, DAG accumulation and inflammation were associated with insulin resistance, since in both males (Jani et al., 2021) and females ceramides remained unaltered in Epit from HFS-fed rats. Overall, this study helps elucidate the molecular mechanisms underlying insulin resistance in female skeletal muscles under conditions of diet-induced obesity.