Distinct mechanisms involving diacylglycerol, ceramides, and inflammation underlie insulin resistance in oxidative and glycolytic muscles from high fat-fed rats

Abstract

This study investigated whether oxidative and glycolytic skeletal rat muscles respond differently to a high-fat (HF) sucrose-enriched diet with respect to diacylglycerol (DAG) and ceramides accumulation, protein kinase C (PKC) activation, glucose metabolism, and the expression of inflammatory genes. HF diet (8 weeks) suppressed insulin-stimulated glycogen synthesis and glucose oxidation in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. However, DAG and ceramides levels increased in Sol and EDL, but not in Epit muscles of HF-fed rats. Additionally, membrane-bound PKC-delta and PKC-theta increased in Sol and EDL, whereas in Epit muscles both PKC isoforms were reduced by HF diet. In Epit muscles, HF diet also increased the expression of tumor necrosis factor-α (TNF-α) receptors (CD40 and FAS), toll-like receptor 4 (TLR4), and nuclear factor kappa light polypeptide gene enhancer in B cells (NF-kB), whereas in Sol and EDL muscles the expression of these inflammatory genes remained unchanged upon HF feeding. In conclusion, HF diet caused DAG and ceramides accumulation, PKC activation, and the induction of inflammatory pathways in a fiber type-specific manner. These findings help explain why oxidative and glycolytic muscles similarly develop insulin resistance, despite major differences in their metabolic characteristics and responsiveness to dietary lipid abundance.

The ketogenic diet prevents steatosis and insulin resistance by reducing lipogenesis, diacylglycerol accumulation and protein kinase C activity in male rat liver

“…After the 1 h incubation period, liver slices were quickly removed and the medium was acidified with 0.2 ml of H 2 SO 4 (5 M), and the flasks were maintained sealed at 37°C for an additional 1 h for collection of the 14 CO 2 released. Subsequently, the filter papers were carefully removed and transferred to scintillation vials for radioactivity counting (Jani et al., 2021).…”

“…An ultra‐high‐pressure liquid chromatography system (UHPLC‐UV, Nexera X2, Shimadzu, Kyoto, Japan) was used to measure total amounts of DAG, TAG and ceramides (Jani et al., 2021) in lipid samples extracted using Folch's method (Folch et al., 1987). Briefly, 50 mg of liver tissue was homogenized in 200 μl of chloroform:methanol (MeOH) (2:1 v/v), dried overnight under nitrogen gas, and resuspended in 100 μl of 2‐propanol‐hexane (ProHex, 5:4 v/v) prior to chromatographic analysis.…”

“…PKCε protein levels were determined in the cytosolic and membrane fractions by immunoblotting (Jani et al, 2021). Liver homogenates (50 mg) were used to obtain the cytoplasmic and membrane protein fractions using a subcellular protein fractionation kit from Thermo Fisher J Physiol 600.18…”