Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Journal of Lipid Research · 5 authors, 4 centres

AI SUMMARY

FIDELITY 100%

POPULATION10-week-old male and female C57Bl/6 mice

INTERVENTIONHigh-fat diet (60% energy from fat) for 2 weeks

COMPARISONControl diet (10% energy from fat) for 2 weeks

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TL;DR

This study developed a novel method using oral 13C3,15N L-serine to track ceramide 16:0 synthesis in mouse liver and mitochondria. After 2 weeks of high-fat diet, mitochondrial ceramide content and synthesis increased significantly, while total liver ceramide content did not change. This method enables in vivo measurement of ceramide turnover, which may help understand ceramide's role in metabolic diseases.

Full summary

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**Background:** Ceramides (CERs) are central to sphingolipid metabolism and are implicated in metabolic dysfunction and cardiovascular risk. Mitochondrial CERs are associated with decreased respiration and increased apoptosis. While CER concentrations are well-studied, methods to measure CER kinetics in vivo are limited. This study aimed to develop a stable isotope method using oral 13C3,15N L-serine to quantify CER 16:0 production in whole liver and isolated hepatic mitochondria in mice fed a control diet (CD) or high-fat diet (HFD) for 2 weeks.

**Methods:** 10-week-old male (n=24) and female (n=24) C57Bl/6 mice were fed CD (10% fat) or HFD (60% fat) for 2 weeks. Mice received an oral bolus of 13C3,15N L-serine (20 mg/kg BW) by intraperitoneal injection on day 0, then in drinking water (0.90 mg/ml) until euthanasia. Four mice per group (2 males, 2 females) were euthanized on days 0, 1, 2, 4, 7, and 12. CERs and dihydroceramides (dhCERs) were extracted from whole liver and isolated hepatic mitochondria and analyzed by HPLC-tandem MS with multiple reaction monitoring. Percent enrichment of labeled CER 16:0 was calculated. Fractional synthetic rates (FSRs, pools/day) were determined by fitting single exponential curves to enrichment data. Absolute synthetic rates (ASRs, pmol/mg tissue/day) were calculated as FSR × pool size. Protein expression of CER synthetic enzymes and mitochondrial markers were measured by Western blot and enzymatic assays.

**Key Results:** After 2 weeks, HFD-fed animals had significantly greater body weight (27.1±4.7 vs 23.4±3.4 g, p<0.001), body fat (23±4% vs 13±4%, p<0.001), liver triglyceride content (20.9±5.5 vs 17.7±5.7 mg/g, p<0.001), serum FFA (0.31±0.05 vs 0.26±0.04 mmol/l, p=0.002), and serum glucose (13.7±2.4 vs 11.7±3.0 mmol/l, p=0.007) compared to CD. Total liver CER concentrations did not differ between diets, but saturated species 16:0, 18:0, and 20:0 were significantly higher in HFD (30%, 100%, and 115% respectively). Total mitochondrial CER concentrations were significantly greater with HFD, with increases in 16:0 (46%), 18:0 (128%), 20:0 (255%), 22:0 (68%), and 24:0 (116%). A positive correlation between total liver and mitochondrial CER was found only in HFD animals (r=0.360, p=0.012). HFD animals had a greater proportion of total liver CER in the mitochondrial pool (19±5% vs 12±4%, p<0.0001). Fractional synthesis of liver CER 16:0 was similar between diets (CD: ~0.5 pools/day, HFD: ~0.4 pools/day), but mitochondrial fractional synthesis was significantly greater in HFD (p<0.05). In CD, mitochondrial FSR was 31% lower than liver FSR; in HFD, mitochondrial FSR was 34% higher. ASR for liver 16:0 was similar between diets (15% difference), while mitochondrial ASR was 59% greater in HFD (p<0.05). Half-life of mitochondrial 16:0 CER was significantly longer with CD. Protein expression of SPT3 was higher in HFD, while CERS2 was lower (−22%) and DEGS1 was lower (−20%). Mitochondrial citrate synthase activity and electron transport chain complexes I–III and V were lower in HFD.

**Clinical Implications:** This study provides a novel, non-invasive method to measure in vivo CER synthesis in liver and mitochondria using oral serine isotope. The finding that short-term HFD increases mitochondrial CER content and turnover without altering total liver CER suggests that mitochondrial CER accumulation may be an early event in diet-induced metabolic dysfunction. This method can be used to investigate therapies targeting CER synthesis or degradation to reduce lipotoxicity in metabolic diseases such as type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular disease.

PICO

PPOPULATION

10-week-old male and female C57Bl/6 mice

IINTERVENTION

High-fat diet (60% energy from fat) for 2 weeks

OOUTCOME

Ceramide 16:0 content and synthesis rates in whole liver and isolated hepatic mitochondria

STUDY TYPE

other

SPECIALTY

endocrinology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

100% · A

ORIGINAL PAPER

PMC10193228

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Journal of Lipid Research

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ABSTRACT

Abstract Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered 13 C 3 , 15 N l -serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations ( P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions. Supplementary key words: ceramides, kinetics, lipidomics, liver, mitochondria, stable isotope tracers

CITATION EXPORT

Justine M Mucinski, Jonas M McCaffrey, R Scott Rector et al.. Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis. Journal of Lipid Research. (2023). https://doi.org/10.1016/j.jlr.2023.100366

AUTHORS

5 · 4 CENTRES

Justine M Mucinski, Jonas M McCaffrey, R Scott Rector, Takhar Kasumov, Elizabeth J Parks

1Department of Nutrition and Exercise Physiology, University of Missouri, Columbia, Missouri, USA
2Division of Gastroenterology and Hepatology, Department of Medicine, School of Medicine, University of Missouri, Columbia, Missouri, USA
3Research Service, Harry S Truman Memorial Veterans Medical Center, Columbia, Missouri, USA
4Department of Pharmaceutical Sciences, College of Pharmacy, Northeast Ohio Medical University, Rootstown, Ohio, USA