Intramyocellular Ceramide Research Articles

Preliminary evidence for reduced adipose tissue inflammation in vegetarians compared with omnivores

BackgroundThere are links between obesity and inflammation that may relate activation of pro-inflammatory pathways by dietary factors. Because dietary fat intake of vegetarians is thought to be more beneficial than that of omnivores, we hypothesized that obese vegetarians would have less adipose tissue inflammation and lower intramyocellular ceramide concentrations than equally obese omnivores.MethodsEight obese vegetarian (1 male) and 8 obese omnivore volunteers (1 male) completed a Food Frequency Questionnaire, underwent body composition measures, subcutaneous adipose tissue and muscle biopsies. We used immunohistochemistry to measure adipose macrophage (ATM) and senescent cells. Plasma free fatty acid (FFA), adipose FA and muscle ceramide profiles were measured using liquid chromatography/mass spectrometry. Student t tests were used for the comparison of primary outcomes; univariate regression analysis was used to test for associations between dietary patterns and ATMs (secondary analysis).ResultsThere were no differences in age (38 ± 8 vs. 39 ± 8 years), BMI (32.2 ± 2.6 vs. 33.3 ± 1.9 kg/m2) or percent body fat (44 ± 8 vs. 45 ± 4) between the vegetarians and omnivores. Vegetarians consumed 42% (P = 0.02) less saturated fat and 50% (P = 0.04) less cholesterol than the omnivores. Plasma FFA of vegetarians had lesser proportions of palmitic acid (24 ± 3 vs. 29 ± 4%, P = 0.02) and vegetarians had fewer femoral pro-inflammatory ATMs than omnivores (3.6 ± 2.8 vs. 7.9 ± 4.4 per 100 adipocytes, respectively; P = 0.02). Omnivores had 50% greater (P = 0.01) expression of TNF mRNA in abdominal fat. We found no significant between group differences in muscle ceramide concentrations.ConclusionsAlthough the sample size is small, these results may indicate that dietary patterns play a role in adipose tissue inflammation, as reflected by reduced number of femoral ATMs in obese vegetarians than obese omnivores.

Corrigendum.

CorrigendumCORRIGENDUMPublished Online:08 May 2018https://doi.org/10.1152/ajpendo.zh1-7894-corr.2018Original articleMoreSectionsPDF (27 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Chung JO, Koutsari C, Blachnio-Zablieska AU, Hames KC, Jensen MD. Effects of meal ingestion on intramyocellular ceramide concentration and fractional de novo synthesis in humans. Am J Physiol Endocrinol Metab 314: E105–E114, 2018. First published September 26, 2017; doi: 10.1152/ajpendo.00153.2017.—In the published article, the third coauthor’s name was misspelled in the author line and in the abstract. The correct spelling is Agnieszka Urszula Blachnio-Zabielska and Blachnio-Zabielska AU, respectively.This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformation Related ArticlesEffects of meal ingestion on intramyocellular ceramide concentrations and fractional de novo synthesis in humans 16 Mar 2018American Journal of Physiology-Endocrinology and Metabolism More from this issue > Volume 314Issue 5May 2018Pages E530-E530 Copyright & PermissionsCopyright © 2018 the American Physiological Societyhttps://doi.org/10.1152/ajpendo.zh1-7894-corr.2018PubMed29737204History Published online 8 May 2018 Published in print 1 May 2018 Metrics

Effects of meal ingestion on intramyocellular ceramide concentrations and fractional de novo synthesis in humans.

We investigated the effects of meal ingestion on intramyofibrillar (IMF) and subsarcolemmal (SS) ceramide metabolism in volunteers ranging from lean to obese. Thirty-eight women and men underwent a steady-state meal ingestion protocol that included a 6.5-h infusion of [U-13C]palmitate and muscle biopsies 1.5 and 6.5 h after starting the tracer infusion. We measured IMF and SS sphingolipid concentrations and the contribution of plasma palmitate to intramyocellular C16:0 ceramide by use of LC-MS-MS. In response to meal ingestion SS C24 ceramide concentrations, but not C14-C20 concentrations, increased significantly. IMF ceramide concentrations did not change. The increases in SS C24 ceramides were negatively related to parameters of insulin resistance. The fractional contribution of plasma palmitate to intramyocellular C16:0 ceramides in both IMF and SS fractions was inversely related to overweight status (β = -0.432, P = 0.0095 and β = -0.443, P = 0.0058, respectively). These data indicate that meal ingestion has differing effects on SS ceramide subspecies and suggest that the fractional de novo synthesis of intramyocellular ceramide from plasma palmitate in the postprandial condition is reduced in those who are overweight.

Intramyocellular Ceramides: Subcellular Concentrations and Fractional De Novo Synthesis in Postabsorptive Humans.

We investigated the relationship between insulin resistance markers and subsarcolemmal (SS) and intramyofibrillar (IMF) ceramide concentrations, as well as the contribution of plasma palmitate (6.5-h infusion of [U-13C]palmitate) to intramyocellular ceramides. Seventy-six postabsorptive men and women had muscle biopsies 1.5, 6.5, and 24 h after starting the tracer infusion. Concentrations and enrichment of muscle ceramides were measured by liquid chromatography-tandem mass spectrometry. We found that HOMA of insulin resistance, plasma insulin, and triglyceride concentrations were positively correlated with SS C16:0 and C18:1 ceramide, but not SS C14:0-Cer, C20:0-Cer, C24:0-Cer, and C24:1-Cer concentrations; IMF ceramide concentrations were not correlated with any metabolic parameters. The fractional contribution of plasma palmitate to 16:0 ceramide was greater in SS than IMF (SS, 18.2% vs. IMF, 8.7%; P = 0.0006). Plasma insulin concentrations correlated positively with the fractional contribution of plasma palmitate to SS 16:0 ceramide. The fractional contribution of plasma palmitate to intramyocellular SS 16:0 ceramide was positively correlated with SS C16:0 ceramide concentrations (γ = 0.435; P = 0.002). We conclude that skeletal muscle SS ceramides, especially C16 to C18 chain lengths and the de novo synthesis of intramyocellular ceramide from plasma palmitate are associated with markers of insulin resistance.

Intramyocellular ceramides and skeletal muscle mitochondrial respiration are partially regulated by Toll-like receptor 4 during hindlimb unloading.

Physical inactivity and disuse result in skeletal muscle metabolic disruption, including insulin resistance and mitochondrial dysfunction. The role of the Toll-like receptor 4 (TLR4) signaling pathway in contributing to metabolic decline with muscle disuse is unknown. Therefore, our goal was to determine whether TLR4 is an underlying mechanism of insulin resistance, mitochondrial dysfunction, and skeletal muscle ceramide accumulation following muscle disuse in mice. To address this hypothesis, we subjected (n = 6-8/group) male WT and TLR4-/- mice to 2 wk of hindlimb unloading (HU), while a second group of mice served as ambulatory wild-type controls (WT CON, TLR4-/- CON). Mice were assessed for insulin resistance [homeostatic model assessment-insulin resistance (HOMA-IR), glucose tolerance], and hindlimb muscles (soleus and gastrocnemius) were used to assess muscle sphingolipid abundance, mitochondrial respiration [respiratory control ratio (RCR)], and NF-κB signaling. The primary finding was that HU resulted in insulin resistance, increased total ceramides, specifically Cer18:0 and Cer20:0, and decreased skeletal muscle mitochondrial respiration. Importantly, TLR4-/- HU mice were protected from insulin resistance and altered NF-κB signaling and were partly resistant to muscle atrophy, ceramide accumulation, and decreased RCR. Skeletal muscle ceramides and RCR were correlated with insulin resistance. We conclude that TLR4 is an upstream regulator of insulin sensitivity, while partly upregulating muscle ceramides and worsening mitochondrial respiration during 2 wk of HU.

CrossTalk proposal: Intramyocellular ceramide accumulation does modulate insulin resistance.

CrossTalk proposal: Intramyocellular ceramide accumulation does modulate insulin resistance.

CrossTalk opposing view: Intramyocellular ceramide accumulation does not modulate insulin resistance.

CrossTalk opposing view: Intramyocellular ceramide accumulation does not modulate insulin resistance.

In vitro palmitate treatment of myotubes from postmenopausal women leads to ceramide accumulation, inflammation and affected insulin signaling.

Menopause is associated with an increased incidence of insulin resistance and metabolic diseases. In a chronic palmitate treatment model, we investigated the role of skeletal muscle fatty acid exposure in relation to the metabolic deterioration observed with menopause. Human skeletal muscle satellite cells were isolated from premenopausal (n = 6) and postmenopausal (n = 5) women. In an in vitro model, the myotubes were treated with palmitate (300 µM) for one-, two- or three days during differentiation. Effects on lipid accumulation, inflammation and insulin signaling were studied. Palmitate treatment led to a 108% (CI 95%: 50%; 267%) increase in intramyocellular ceramide in the myotubes from the postmenopausal women (post-myotubes) compared with a 26% (CI 95%: −57%; 96%) increase in myotubes from the premenopausal women (pre-myotubes), (p<0.05). Furthermore, post-myotubes had a 22% (CI 95%: 4%; 34%) increase in pJNK (p = 0.04) and a 114% (CI 95%: 50%; 177%) increase in Hsp70 protein expression (p = 0.03) after three days of palmitate treatment, compared with pre-myotubes, in which no increase in either pJNK (−12% (CI 95: −26%; 2%)) or Hsp70 (7% (CI 95: −78%; 91%)) was detected. Furthermore, post-myotubes showed a blunted insulin stimulated phosphorylation of AS160 in response to chronic palmitate treatment compared with pre-myotubes (p = 0.02). The increased intramyocellular ceramide content in the post-myotubes was associated with a significantly higher mRNA expression of Serine Palmitoyltransferase1 (SPT1) after one day of palmitate treatment (p = 0.03) in post-myotubes compared with pre-myotubes. Our findings indicate that post-myotubes are more prone to develop lipid accumulation and defective insulin signaling following chronic saturated fatty acid exposure as compared to pre-myotubes.

A liquid chromatography/tandem mass spectrometry method for measuring the in vivo incorporation of plasma free fatty acids into intramyocellular ceramides in humans

Sphingolipids are important components of cell membranes that serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. De novo ceramide biosynthesis depends on fatty acid availability, but whether muscle uses circulating free fatty acids or pre-existing intracellular stores is unknown. Our goal was to develop a method to detect the incorporation of intravenously infused [U-(13)C]palmitate into intramyocellular ceramides. We used liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipid species and (13)C-isotopic enrichment of 16:0-ceramide. Chromatographic separation was performed using ultra-performance liquid chromatography. The analysis was performed on a triple quadrupole mass spectrometer using a positive ion electrospray ionization source with selected reaction monitoring (SRM). The sphingolipids ions, except enriched ceramide, were monitored as [M+2+H](+). The [(13)C(16)]16:0-ceramide was monitored as [M+16+H](+). By monitoring two different transitions of the [(13)C(16)]16:0-ceramide (554/536 and 554/264) we could indirectly measure enrichment of the palmitate that is not a part of the sphingoid base. Concentration and enrichment could be measured using 20 mg of muscle obtained from volunteers receiving a low dose [U-(13)C]palmitate infusion. LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo.

Inhibition of De Novo Ceramide Synthesis Reverses Diet-Induced Insulin Resistance and Enhances Whole-Body Oxygen Consumption

OBJECTIVEIt has been proposed that skeletal muscle insulin resistance arises from the accumulation of intramyocellular lipid metabolites that impede insulin signaling, including diacylglycerol and ceramide. We determined the role of de novo ceramide synthesis in mediating muscle insulin resistance.RESEARCH DESIGN AND METHODSMice were subjected to 12 weeks of diet-induced obesity (DIO), and then treated for 4 weeks with myriocin, an inhibitor of serine palmitoyl transferase-1 (SPT1), the rate-limiting enzyme of de novo ceramide synthesis.RESULTSAfter 12 weeks of DIO, C57BL/6 mice demonstrated a doubling in gastrocnemius ceramide content, which was completely reversed (141.5 ± 15.8 vs. 94.6 ± 10.2 nmol/g dry wt) via treatment with myriocin, whereas hepatic ceramide content was unaffected by DIO. Interestingly, myriocin treatment did not alter the DIO-associated increase in gastrocnemius diacyglycerol content, and the only correlation observed between lipid metabolite accumulation and glucose intolerance occurred with ceramide (R = 0.61). DIO mice treated with myriocin showed a complete reversal of glucose intolerance and insulin resistance which was associated with enhanced insulin-stimulated Akt and glycogen synthase kinase 3β phosphorylation. Furthermore, myriocin treatment also decreased intramyocellular ceramide content and prevented insulin resistance development in db/db mice. Finally, myriocin-treated DIO mice displayed enhanced oxygen consumption rates (3,041 ± 124 vs. 2,407 ± 124 ml/kg/h) versus their control counterparts.CONCLUSIONSOur results demonstrate that the intramyocellular accumulation of ceramide correlates strongly with the development of insulin resistance, and suggests that inhibition of SPT1 is a potentially promising target for the treatment of insulin resistance.

Modulating serine palmitoyl transferase (SPT) expression and activity unveils a crucial role in lipid-induced insulin resistance in rat skeletal muscle cells

Saturated fatty acids, such as palmitate, promote accumulation of ceramide, which impairs activation and signalling of PKB (protein kinase B; also known as Akt) to important end points such as glucose transport. SPT (serine palmitoyl transferase) is a key enzyme regulating ceramide synthesis from palmitate and represents a potential molecular target in curbing lipid-induced insulin resistance. In the present study we explore the effects of palmitate upon insulin action in L6 muscle cells in which SPT expression/activity has been decreased by shRNA (small-hairpin RNA) or sustained incubation with myriocin, an SPT inhibitor. Incubation of L6 myotubes with palmitate (for 16 h) increases intramyocellular ceramide and reduces insulin-stimulated PKB activation and glucose uptake. PKB inhibition was not associated with impaired IRS (insulin receptor substrate) signalling and was ameliorated by short-term treatment with myriocin. Silencing SPT expression (approximately 90%) by shRNA or chronic cell incubation with myriocin (for 7 days) markedly suppressed SPT activity and palmitate-driven ceramide synthesis; however, challenging these muscle cells with palmitate still inhibited the hormonal activation of PKB. This inhibition was associated with reduced IRS1/p85-PI3K (phosphoinositide 3-kinase) coupling that arises from diverting palmitate towards greater DAG (diacylglycerol) synthesis, which elevates IRS1 serine phosphorylation via activation of DAG-sensitive PKCs (protein kinase Cs). Treatment of SPT-shRNA cells or those treated chronically with myriocin with PKC inhibitors antagonized palmitate-induced loss in insulin signalling. The findings of the present study indicate that SPT plays a crucial role in desensitizing muscle cells to insulin in response to incubation with palmitate. While short-term inhibition of SPT ameliorates palmitate/ceramide-induced insulin resistance, sustained loss/reduction in SPT expression/activity promotes greater partitioning of palmitate towards DAG synthesis, which impacts negatively upon IRS1-directed insulin signalling.

Gender-Related Differences in the Metabolic Response to Fasting

Free fatty acids (FFA) may induce insulin resistance via synthesis of intramyocellular ceramide. During fasting, women have lower plasma glucose levels than men despite higher plasma FFA, suggesting protection from FFA-induced insulin resistance. We studied whether the relative protection from FFA-induced insulin resistance during fasting in women is associated with lower muscle ceramide concentrations compared with men. MAIN OUTCOME MEASURES AND DESIGN: After a 38-h fast, measurements of glucose and lipid fluxes and muscle ceramide and fatty acid translocase/CD36 were performed before and after a hyperinsulinemic euglycemic clamp. Plasma glucose levels were significantly lower in women than men with a trend for a lower endogenous glucose production in women, whereas FFA and lipolysis were significantly higher. Insulin-mediated peripheral glucose uptake was not different between sexes. There was no gender difference in muscle ceramide in the basal state, and ceramide did not correlate with peripheral glucose uptake. Muscle fatty acid translocase/CD36 was not different between sexes in the basal state and during the clamp. After 38 h of fasting, plasma FFA were higher and plasma glucose was lower in women compared with men. The higher plasma FFA did not result in differences in peripheral insulin sensitivity, possibly because of similar muscle ceramide and fatty acid translocase/CD36 levels in men and women. We suggest that during fasting, women are relatively protected from FFA-induced insulin resistance by preventing myocellular accumulation of ceramide.

Chronic Treatment with Pioglitazone Does Not Protect Obese Patients with Diabetes Mellitus Type II from Free Fatty Acid-Induced Insulin Resistance

Thiazolidinediones increase peripheral insulin sensitivity and decrease plasma free fatty acids (FFA). However, their exact mechanism of action has not been fully elucidated. We studied the protective effect of pioglitazone on FFA-induced insulin resistance and the effects on intramyocellular glycosphingolipids. We studied glucose metabolism in the basal state and during a hyperinsulinemic euglycemic clamp by using stable isotopes. Studies were performed at baseline and after 4 months of treatment with pioglitazone. Patients were then studied on a third occasion during infusion of a lipid emulsion to increase plasma FFA to pretreatment levels. All studies were combined with muscle biopsies to measure intramyocellular ceramide and glycosphingolipids. Patients were obese with poorly controlled type 2 diabetes mellitus. Patients were treated with 30 mg pioglitazone once daily. The change in peripheral insulin sensitivity after treatment with pioglitazone and during the infusion of the lipid emulsion was the main outcome measure. Peripheral glucose uptake (Rd) increased significantly, but returned to baseline levels after increasing plasma FFA to pretreatment levels. Insulin-mediated suppression of FFA was increased significantly. Intramyocellular ceramide concentrations were higher during the hyperinsulinemic clamp after treatment with pioglitazone, but not in the basal state. The intramyocellular content of glycosphingolipids and plasma concentrations of ceramide and glycosphingolipids did not change. Pioglitazone increases Rd and insulin-mediated suppression of plasma FFA, but does not protect patients with type 2 diabetes mellitus from FFA-induced insulin resistance. This effect of pioglitazone is not attained via a decrease in intramyocellular concentrations of ceramide or glycosphingolipids.

Effects of chronic activation of peroxisome proliferator-activated receptor-α or high-fat feeding in a rat infarct model of heart failure

Intracardiac accumulation of lipid and related intermediates (e.g., ceramide) is associated with cardiac dysfunction and may contribute to the progression of heart failure (HF). Overexpression of nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) increases intramyocellular ceramide and left ventricular (LV) dysfunction. We tested the hypothesis that activation of fatty acid metabolism with fat feeding or a PPARalpha agonist increases myocardial triglyceride and/or ceramide and exacerbates LV dysfunction in HF. Rats with infarct-induced HF (n = 38) or sham-operated rats (n = 10) were either untreated (INF, n = 10), fed a high-fat diet (45% kcal fat, INF + Fat, n = 15), or fed the PPARalpha agonist fenofibrate (150 mg.kg(-1).day(-1), INF + Feno, n = 13) for 12 wk. LV ejection fraction was significantly reduced with HF (49 +/- 6%) compared with sham operated (86 +/- 2%) with no significant differences in ejection fraction (or other functional or hemodynamic measures) among the three infarcted groups. Treatment with the PPARalpha agonist resulted in LV hypertrophy (24% increase in LV/body mass ratio) and induced mRNAs encoding for PPARalpha-regulated genes, as well as protein expression and activity of medium chain acyl-CoA dehydrogenase (compared with INF and INF + Fat groups). Myocardial ceramide content was elevated in the INF group compared with sham-operated rats, with no further change in the INF + Fat or INF + Feno groups. Myocardial triglyceride was unaffected by infarction but increased in the INF + Fat group. In conclusion, LV dysfunction and dilation are not worsened despite upregulation of the fatty acid metabolic pathway and LV hypertrophy or accumulation of myocardial triglyceride in the rat infarct model of HF.