Ceramide In Tissues Research Articles

Ceramides-Emerging Biomarkers of Lipotoxicity in Obesity, Diabetes, Cardiovascular Diseases, and Inflammation.

Abnormalities in lipid homeostasis have been associated with many human diseases, and the interrelation between lipotoxicity and cellular dysfunction has received significant attention in the past two decades. Ceramides (Cers) are bioactive lipid molecules that serve as precursors of all complex sphingolipids. Besides their function as structural components in cell and mitochondrial membranes, Cers play a significant role as key mediators in cell metabolism and are involved in numerous cellular processes, such as proliferation, differentiation, inflammation, and induction of apoptosis. The accumulation of various ceramides in tissues causes metabolic and cellular disturbances. Recent studies suggest that Cer lipotoxicity has an important role in obesity, metabolic syndrome, type 2 diabetes, atherosclerosis, and cardiovascular diseases (CVDs). In humans, elevated plasma ceramide levels are associated with insulin resistance and impaired cardiovascular and metabolic health. In this review, we summarize the role of ceramides as key mediators of lipotoxicity in obesity, diabetes, cardiovascular diseases, and inflammation and their potential as a promising diagnostic tool.

Metformin Blunts Bed Rest-induced Skeletal Muscle Ceramide Accumulation in Older Adults

Skeletal muscle insulin resistance is an undesirable outcome during periods of physical inactivity in older adults, which can lead to a variety of metabolic diseases. Insulin sensitive tissue (e.g., liver, and skeletal muscle) derived ceramides are well known to promote insulin resistance. Evidence suggests that metformin can regulate tissue ceramide accumulation. Therefore, we hypothesized that metformin treatment during a short-term period of bed rest in male and female older adults would reduce skeletal muscle ceramide accumulation and prevent insulin resistance. Our study design consisted of older adults (66 ± 4.5 y/o placebo, n=10; 71.7 ± 5.1 y/o metformin, n=10) who were subjected to 5 days of bed rest. Participants were randomly assigned to placebo or metformin treatment groups. Drug or placebo treatments started two weeks prior to bed rest and continued throughout the 5 days of bed rest. Prior to and after bed rest, participants underwent a euglycemic-hyperinsulinemic clamp. Muscle biopsies (from the vastus lateralis) were taken prior to and after each clamp both before and after bed rest. We found that insulin-stimulated muscle ceramides were increased after bed rest but were blunted in older adults who took metformin. In addition, muscle insulin resistance correlated with the increase in ceramides. We conclude that metformin was effective to reduce ceramide accumulation in muscle during a period of bed rest in older adults. R21AG064576 awarded to M.J.D. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Podocyte-specific silencing of acid sphingomyelinase gene to abrogate hyperhomocysteinemia-induced NLRP3 inflammasome activation and glomerular inflammation.

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.

Hypoxia induced deregulation of sphingolipids in colon cancer is a prognostic marker for patient outcome

Sphingolipids are important for the physicochemical properties of cellular membranes and deregulated in tumors. In human colon cancer tissue ceramide synthase (CerS) 4 and CerS5 are reduced which correlates with a reduced survival probability of late-stage colon cancer patients. Both enzymes are reduced after hypoxia in advanced colorectal cancer (CRC) cells (HCT-116, SW620) but not in non-metastatic CRC cells (SW480, Caco-2). Downregulation of CerS4 or CerS5 in advanced CRC cells enhanced tumor formation in nude mice and organoid growth in vitro. This was accompanied by an enhanced proliferation rate and metabolic changes leading to a shift towards the Warburg effect. In contrast, CerS4 or CerS5 depletion in Caco-2 cells reduced tumor growth in vivo. Lipidomic and proteomic analysis of membrane fractions revealed significant changes in tumor-promoting cellular pathways and cellular transporters. This study identifies CerS4 and CerS5 as prognostic markers for advanced colon cancer patients and provides a comprehensive overview about the associated cellular metabolic changes. We propose that the expression level of CerS4 and CerS5 in colon tumors could serve as a basis for decision-making for personalized treatment of advanced colon cancer patients.Trial registration: The study was accredited by the study board of the Deutsche Krebsgesellschaft (Registration No: St-D203, 2017/06/30, retrospectively registered).

Dietary delivery of glycomacropeptide within the whey protein matrix is not effective in mitigating tissue ceramide deposition and obesity in mice fed a high-fat diet

Obesity is often accompanied by heightened circulating and tissue inflammation along with an increase in sphingolipids (e.g., ceramides) in metabolically active and insulin-sensitive organs. Whey protein isolate (WPI) has been shown to decrease inflammation and increase insulin sensitivity when given during a high-fat diet (HFD) intervention in rodents. The whey protein bioactive peptide glycomacropeptide (GMP) has also been linked to having anti-inflammatory properties and regulating lipogenesis. Therefore, the purpose of the study was to determine the effect of dietary GMP within the whey protein matrix on tissue inflammation, adiposity, and tissue ceramide accumulation in an obesogenic rodent model. Young adult male mice (10 wk old) underwent a 10-wk 60% HFD intervention. Glycomacropeptide was absent in the control low-fat diet and HFD WPI (-GMP) groups. The HFD WPI (1×GMP) treatment contained a standard amount of GMP, and HFD WPI (2×GMP) had double the amount. We observed no differences in weight gain or reductions in adiposity when comparing the GMP groups to HFD WPI (-GMP). Similarly, insulin resistance and glucose intolerance were not offset with GMP, and skeletal muscle and liver tissue ceramide content was unaltered with the GMP intervention. In contrast, the additional amount of GMP (2×GMP) might adversely affect tissue obesity-related pathologies. Together, dietary GMP given in a whey protein matrix during an HFD intervention does not alter weight gain, insulin resistance, glucose intolerance, and sphingolipid accumulation in the liver and skeletal muscle.

Inhibitory Effect of Podocyte‐Specific Silencing of Acid Sphingomyelinase Gene on NLRP3 Inflammasome Activation and Glomerular Injury During Hyperhomocysteinemia

Acid Sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)‐induced NLRP3 inflammasome activation in podocytes and glomerular sclerosis. The present study was designed to test whether somatic podocyte‐specific silencing of ASM gene (its mouse code is Smpd1) attenuates hHcy‐induced NLRP3 inflammasome activation and associated exosome release in podocytes and thereby suppresses glomerular inflammatory response and injury. We first constructed a floxed Smpd1 shRNA construct (Vigene Bioscience, Cat# IS100001) that has both RFP and GFP as reporter genes, where RFP is expressed if there is no cre excision. If cre excision occurs, GFP is expressed. This floxed Smpd1 shRNA was transfected into podocytes isolated from WT/Podocre mice, which decreased ASM mRNA and protein levels by 90% and 60%, respectively. This suggests efficient gene silencing in podocytes with cre gene. When this floxed Smpd1 shRNA was transfected into mouse kidney by intravenous infusion and ultrasound‐microbubble directed introduction, ASM expression decreased by 65% in WT/Podocre mice and 51% in Smpd1trg/Podocre mice after 8 weeks of shRNA transfection. It was also found that transfection of floxed Smpd1 shRNA substantially attenuated hHcy‐induced increase in NLRP3 inflammasome product, IL‐1β. By nanoparticle tracking analysis, floxed Smpd1 shRNA transfection was shown to significantly block hHcy‐induced elevation of urinary exosome excretion, an indicative of NLRP3 inflammasome product release from the cells. In Smpd1trg/Podocre mice, hHcy was found to induce exaggerated NLRP3 inflammasome activation, exosome excretion and glomerular inflammatory response such as immune cell infiltration compared to WT/WT mice. This exaggerated glomerular pathological response to hHcy was also significantly suppressed by podocyte‐specific Smpd1 gene silencing. Besides, podocyte‐specific Smpd1 gene deletion substantially attenuated proteinuria and albuminuria. Taken together, our results suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and associated exosome release and that podocyte‐specific silencing of ASM gene may be an important therapeutic strategy for treatment of glomerular injury and ultimate sclerosis during hHcy.

Effects of serine palmitoyltransferase inhibition by myriocin in ad libitum-fed and nutrient-restricted ewes.

The fungal isolate myriocin inhibits serine palmitoyltransferase and de novo ceramide synthesis in rodents; however, the effects of myriocin on ceramide concentrations and metabolism have not been previously investigated in ruminants. In our study, 12 non-lactating crossbred ewes received an intravenous bolus of myriocin (0, 0.1, 0.3, or 1.0 mg/kg/body weight [BW]; CON, LOW, MOD, or HIGH) every 48 h for 17 d. Ewes consumed a high-energy diet from day 1 to 14 and were nutrient-restricted (straw only) from day 15 to 17. Blood was collected preprandial and at 1, 6, and 12 h relative to bolus and nutrient restriction. Tissues were collected following euthanasia on day 17. Plasma was analyzed for free fatty acids (FFAs), glucose, and insulin. Plasma and tissue ceramides were quantified using mass spectrometry. HIGH selectively decreased metabolizable energy intake, BW, and plasma insulin, and increased plasma FFA (Dose, P < 0.05). Myriocin linearly decreased plasma very-long-chain (VLC) ceramide and dihydroceramide (DHCer) by day 13 (Linear, P < 0.05). During nutrient restriction, fold-change in FFA was lower with increasing dose (P < 0.05). Nutrient restriction increased plasma C16:0-Cer, an effect suppressed by MOD and HIGH (Dose × Time, P < 0.05). Myriocin linearly decreased most ceramide and DHCer species in the liver and omental and mesenteric adipose, VLC ceramide and DHCer in the pancreas, and C18:0-Cer in skeletal muscle and subcutaneous adipose tissue (Linear, P ≤ 0.05). We conclude that the intravenous delivery of 0.3 mg of myriocin/kg of BW/48 h decreases circulating and tissue ceramide without modifying energy intake in ruminants.

CrossTalk proposal: Intramuscular lipid accumulation causes insulin resistance.

CrossTalk proposal: Intramuscular lipid accumulation causes insulin resistance.

The Role of Ceramides in Insulin Resistance.

Resistance to insulin is a pathophysiological state related to the decreased response of peripheral tissues to the insulin action, hyperinsulinemia and raised blood glucose levels caused by increased hepatic glucose outflow. All the above precede the onset of full-blown type 2 diabetes. According to the World Health Organization (WHO), in 2016 more than 1.9 billion people over 18 years of age were overweight and about 600 million were obese. Currently, the primary hypothesis explaining the probability of occurrence of insulin resistance assigns a fundamental role of lipids accumulation in adipocytes or nonadipose tissue (muscle, liver) and the locally developing chronic inflammation caused by adipocytes hypertrophy. However, the major molecular pathways are unknown. The sphingolipid ceramide is the main culprit that combines a plethora of nutrients (e.g., saturated fatty acids) and inflammatory cytokines (e.g., TNFα) to the progression of insulin resistance. The accumulation of sphingolipid ceramide in tissues of obese humans, rodents and Western-diet non-human primates is in line with diabetes, hypertension, cardiac failure or atherosclerosis. In hypertrophied adipose tissue, after adipocytes excel their storage capacity, neutral lipids begin to accumulate in nonadipose tissues, inducing organ dysfunction. Furthermore, obesity is closely related to the development of chronic inflammation and the release of cytokines directly from adipocytes or from macrophages that infiltrate adipose tissue. Enzymes taking part in ceramide metabolism are potential therapeutic targets to manipulate sphingolipids content in tissues, either by inhibition of their synthesis or through stimulation of ceramides degradation. In this review, we will evaluate the mechanisms responsible for the development of insulin resistance and possible therapeutic perspectives.

Gastric Bypass Surgery Improves the Skeletal Muscle Ceramide/S1P Ratio and Upregulates the AMPK/ SIRT1/ PGC-1α Pathway in Zucker Diabetic Fatty Rats.

Roux-en-Y gastric bypass (RYGB) is associated with remission of type 2 diabetes. However, the cellular and molecular mechanisms remain unknown. We hypothesized that RYGB would increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (SIRT1), AMPK/pAMPK, and citrate synthase (CS) protein expression and decrease insulin resistance and these changes would bemediated by sphingolipids, including ceramides and the sphingolipid metabolite sphingosine-1 phosphate (S1P). Male ZDF rats were randomized to RYGB (n = 7) or sham surgery (n = 7) and harvested after 28days. Total tissue ceramide, ceramide subspecies (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1), and S1P were quantified in the white gastrocnemius muscle using LC-ESI-MS/MS after separation with HPLC. Total SIRT1, AMPK, PGC-1α, and CS protein expression were measured by Western blot. Body weight, fasting glucose, insulin, and HOMA-IR decreased significantly after RYGB compared with sham control. These changes were paralleled by lower total ceramide (483.7 ± 32.3 vs. 280.1 ± 38.8nmol/g wwt), C18:0 ceramide subspecies (P < 0.05), higher S1P (0.83 ± 0.05 vs. 1.54 ± 0.21nmol/g wwt, P < 0.05), and a lower ceramide/S1P ratio (P < 0.05) in the RYGB versus sham group. AMPK, pAMPK, SIRT1, PGC-1α, and CS protein expression was also higher after RYGB (P < 0.05). The ceramide/S1P ratio correlated with weight loss (r = 0.48, P = 0.08), insulin resistance (r = 0.61, P = 0.02), PGC-1α (r = - 0.51, P < 0.06), CS (r = - 0.63, P = 0.01), and SIRT1 (r = - 0.54, P < 0.04). Our data demonstrate that sphingolipid balance, and increased AMPK, SIRT1, PGC-1α, and CS protein expression are part of the mechanism that contributes to the remission of diabetes after RYGB surgery.

Sphingosine-1-Phosphate Signaling and Metabolism Gene Signature in Pediatric Inflammatory Bowel Disease: A Matched-case Control Pilot Study.

The aim of this study was to investigate gene expression levels of proteins involved in sphingosine-1-phosphate (S1P) metabolism and signaling in a pediatric inflammatory bowel disease (IBD) patient population. IBD is a debilitating disease affecting 0.4% of the US population. The incidence of IBD in childhood is rising. Identifying effective targeted therapies that can be used safely in young patients and developing tools for selecting specific candidates for targeted therapies are important goals. Clinical IBD trials now underway target S1PR1, a receptor for the pro-inflammatory sphingolipid S1P. However, circulating and tissue sphingolipid levels and S1P-related gene expression have not been characterized in pediatric IBD. Pediatric IBD patients and controls were recruited in a four-site study. Patients received a clinical score using PUCAI or PCDAI evaluation. Colon biopsies were collected during endoscopy. Gene expression was measured by qRT-PCR. Plasma and gut tissue sphingolipids were measured by LC-MS/MS. Genes of S1P synthesis (SPHK1, SPHK2), degradation (SGPL1), and signaling (S1PR1, S1PR2, and S1PR4) were significantly upregulated in colon biopsies of IBD patients with moderate/severe symptoms compared with controls or patients in remission. Tissue ceramide, dihydroceramide, and ceramide-1-phosphate (C1P) levels were significantly elevated in IBD patients compared with controls. A signature of elevated S1P-related gene expression in colon tissues of pediatric IBD patients correlates with active disease and normalizes in remission. Biopsied gut tissue from symptomatic IBD patients contains high levels of pro-apoptotic and pro-inflammatory sphingolipids. A combined analysis of gut tissue sphingolipid profiles with this S1P-related gene signature may be useful for monitoring response to conventional therapy.

Enzyme replacement therapy for Farber disease: Proof-of-concept studies in cells and mice

A series of studies were carried out in Farber disease (OMIM #228000) cells and mice to evaluate the feasibility of enzyme replacement therapy (ERT) for this disorder. Media from Chinese hamster ovary (CHO) cells overexpressing human recombinant acid ceramidase (rhAC) was used to treat fibroblasts from a Farber disease patient, leading to significantly reduced ceramide. We also found that chondrocytes from Farber disease mice had a markedly abnormal chondrogenic phenotype, and this was corrected by rhAC as well. Acute dosing of rhAC in Farber mice confirmed the enzyme's bioactivity in vivo, and showed that it could be safely administered at doses up to 50mg/kg. These studies also revealed little or no re-accumulation of ceramide in tissues for at least 7days after enzyme administration. Once weekly administration of rhAC moderately improved survival of the mice, which could be enhanced by starting enzyme administration at an earlier age (3days vs. 3weeks). Repeat administration of the enzyme also led to normalization of spleen size, significantly reduced plasma levels of monocyte chemoattractant protein 1 (MCP-1), reduced infiltration of macrophages into liver and spleen, and significantly reduced ceramide and sphingosine in tissues. Overall, we conclude that ERT should be further developed for this debilitating and life-threatening disorder.

Targeting ceramide metabolism in obesity.

Obesity is a major health concern that increases the risk for insulin resistance, type 2 diabetes (T2D), and cardiovascular disease. Thus, an enormous research effort has been invested into understanding how obesity-associated dyslipidemia and obesity-induced alterations in lipid metabolism increase the risk for these diseases. Accordingly, it has been proposed that the accumulation of lipid metabolites in organs such as the liver, skeletal muscle, and heart is critical to these obesity-induced pathologies. Ceramide is one such lipid metabolite that accumulates in tissues in response to obesity, and both pharmacological and genetic strategies that reduce tissue ceramide levels yield salutary actions on overall metabolic health. We will review herein why ceramide accumulates in tissues during obesity and how an increase in intracellular ceramide impacts cellular signaling and function as well as potential mechanisms by which reducing intracellular ceramide levels improves insulin resistance, T2D, atherosclerosis, and heart failure. Because a reduction in skeletal muscle ceramide levels is frequently associated with improvements in insulin sensitivity in humans, the beneficial findings reported for reducing ceramides in preclinical studies may have clinical application in humans. Therefore, modulating ceramide metabolism may be a novel, exciting target for preventing and/or treating obesity-related diseases.

Ischemia/Reperfusion Injury Alters Sphingolipid Metabolism in the Gut

Background: Intestinal ischemia/reperfusion injury (I/R) is a significant cause of morbidity and mortality in surgical patients. Ceramide is a mediator of apoptosis and has been implicated as increasing bacterial infection susceptibility. The metabolite of ceramide, sphingosine, was recently shown to play an important role in the cell-autonomous, innate immune response of the upper respiratory tract by killing bacterial pathogens. The role of ceramide and/or sphingosine after mesenteric I/R is unknown. We investigated the specific effects of intestinal I/R on tissue ceramide and sphingosine concentration and resulting susceptibility to bacterial invasion. Methods: To simulate intestinal I/R, C57BL/6 mice underwent 30 minutes of vascular clamp-induced occlusion of the superior mesenteric artery followed by variable reperfusion times. Jejunum segments and intraluminal contents were analyzed for ceramide, sphingosine and bacteria using immunohistochemistry. Jejunum samples were also homogenized and cultured to quantify bacterial presence in the proximal intestine. Results: We hypothesized that I/R induces an increase of ceramide in the intestine resulting in increased permeability, while a concomitant decrease of sphingosine may permit bacterial overgrowth. Control mice had no measurable bacteria in their proximal jejunum as measured by tissue culture and immunohistochemistry. After I/R, bacterial counts in the jejunum increased in a time-dependent manner, reaching a peak at 12 hours after reperfusion. Immunohistochemical analysis revealed a marked increase in ceramide in the vasculature of jejunal villi. In contrast, while ceramide concentrations in the epithelial cells decreased after I/R, sphingosine levels appeared to remain unchanged. Surprisingly, bacteria present in the jejunal lumen following I/R contained a ceramide coat. Conclusion: These data indicate that intestinal I/R leads to small intestine bacterial overgrowth as well as ceramide formation in the jejunal vasculature, which may contribute to the gut permeability associated with this injury. Moreover, our novel finding of ceramide in bacterial membranes represents a new opportunity to investigate the dynamic pathogenicity of the gut microbiome. The hypothesis that a decrease of sphingosine after I/R permits bacterial overgrowth in the intestine was not confirmed.

Positive effects of voluntary running on metabolic syndrome-related disorders in non-obese hereditary hypertriacylglycerolemic rats.

While metabolic syndrome is often associated with obesity, 25% of humans suffering from it are not obese and the effect of physical activity remains unclear in such cases. Therefore, we used hereditary hypertriaclyglycerolemic (HHTg) rats as a unique model for studying the effect of spontaneous physical activity [voluntary running (VR)] on metabolic syndrome-related disorders, such as dyslipidemia, in non-obese subjects. Adult HHTg males were fed standard (CD) or high-sucrose (HSD) diets ad libitum for four weeks. Within both dietary groups, some of the rats had free access to a running wheel (CD+VR, HSD+VR), whereas the controls (CD, HSD) had no possibility of extra physical activity. At the end of the four weeks, we measured the effects of VR on various metabolic syndrome-associated parameters: (i) biochemical parameters, (ii) the content and composition of triacylglycerols (TAG), diacylglycerols (DAG), ceramides and membrane phospholipids, and (iii) substrate utilization in brown adipose tissue. In both dietary groups, VR led to various positive effects: reduced epididymal and perirenal fat depots; increased epididymal adipose tissue lipolysis; decreased amounts of serum TAG, non-esterified fatty acids and insulin; a higher insulin sensitivity index. While tissue ceramide content was not affected, decreased TAG accumulation resulted in reduced and modified liver, heart and skeletal muscle DAG. VR also had a beneficial effect on muscle membrane phospholipid composition. In addition, compared with the CD group, the CD+VR rats exhibited increased fatty acid oxidation and protein content in brown adipose tissue. Our results confirm that physical activity in a non-obese model of severe dyslipidemia has many beneficial effects and can even counteract the negative effects of sucrose consumption. Furthermore, they suggest that the mechanism by which these effects are modulated involves a combination of several positive changes in lipid metabolism.

Liver and skeletal muscle ceramides in alloxan-induced diabetes

Aim. To study of liver and sceletal muscle ceramides in alloxan-induced diabetes at different periods after alloxan exposure. Methods. Repeated experiments were performed on white male rats. Diabetes was modeled by alloxan hydrochloride solution intraperitoneal injection. The disease development was monitored using clinical and laboratory parameters. Ceramides in tissues were determined in intact animals (control), at the 10, 20, 30, 45 and 60 day after the alloxan injection. Ceramides level in the plates was determined by thin layer chromatography using the external standard. Results. In intact animals, ceramides level in liver was twice as high as in muscle. At all terms after alloxan injection, ceramide level was significantly higher compared to controls, and was in muscle compared to liver, with similar change pattern. Up to the 20th day, a significant increase of ceramide level was observed compared to control (eightfold in liver and sevenfold in muscle). By 30th day, a partial recovery was registered, followed by subsequent increase of ceramide level. This indicates the influence of compensation mechanisms in the early stages of the experiment with further decompensation on late stages. Conclusion. In alloxan-induced type 1 diabetes, ceramide level in insulin-dependent tissues depends on the term after the alloxan injection; the findings are of great importance for the research of secondary insulin resistance, as well as other diabetes-associated pathological conditions, exact mechanisms.

Treating fatty liver and insulin resistance

Treating fatty liver and insulin resistance

Heart failure increases neutral sphingomyelinase activity and ceramide content in rat diaphragm

Patients with chronic heart failure (CHF) have skeletal muscle weakness and elevated secretory sphingomyelinase activity. Sphingomyelinase (SMase) acts through ceramide to depress skeletal muscle force in vitro. We hypothesized that diaphragm weakness in CHF is accompanied by increases in SMase activity and tissue ceramide content. We studied rats 14–16 weeks after myocardial infarction (n = 5–10) and sham surgery (n = 10–13). CHF was confirmed by a decrease in left‐ventricular fractional shortening measured by echocardiography (Sham 38 ± 2%, CHF 28 ± 1%; P &lt; 0.05). Skeletal muscle dysfunction in CHF was confirmed by decreased maximal tetanic force of diaphragm strips (in N/cm2: Sham 30 ± 0.6, CHF 26 ± 0.5; P &lt; 0.05). CHF elicited a 27% increase (P &lt; 0.05) in neutral SMase (N‐SMase) activity. Consistent with this finding, total ceramide content was elevated in CHF diaphragm (in pmol/mg protein: Sham, 280 ± 5 and CHF, 343 ± 16; P &lt; 0.05)—subspecies heightened (P &lt; 0.05) in CHF were C18, C18:1, C20, C20:1, C20:4, C22:1. Acid SMase activity was unchanged in CHF. Similarly, there were no changes in the content of either sphingosine or sphingosine‐1‐phosphate (P &gt; 0.05). Our data suggests that increased diaphragm ceramide content is a consequence of N‐SMase activation in CHF. Activation of N‐SMase and heightening of ceramide content may contribute to skeletal muscle weakness.Funding support: NIH 1K99HL098453‐01, 4R00HL098453‐02

Increase in plasma ceramide levels via secretory sphingomyelinase activity in streptozotocin-induced diabetic rats

Ceramide mediates apoptosis and is upregulated by oxidative stress. To reveal the causative agent of diabetes-induced complications, we examined the changes in ceramide metabolism during diabetes. Two and 8 weeks after intraperitoneal injection of streptozotocin (STZ: 40 mg kg−1 body weight) to rats, tissue ceramide levels were analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). Blood glucose was significantly increased 2 weeks after STZ administration. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) levels were also increased in the diabetic rats, suggesting that hepatic and renal damage was induced by STZ administration. Vitamin C, an indicator of oxidative stress, was significantly decreased in the plasma, liver, and kidney of rats 2 weeks after STZ administration. Although no differences in hepatic ceramide levels were observed between the control and diabetic rats, plasma and renal ceramide levels were significantly increased 8 weeks after STZ administration. In the liver and kidney, acid and neutral sphingomyelinase (SMase) activities were not increased, while secretory sphingomyelinase (sSMase) activity was increased in the plasma of diabetic rats after STZ administration. These data indicated that STZ administration induced the increase in plasma ceramide levels via the increase in sSMase activity. It was suggested that increased plasma ceramide levels were involved in the renal damage induced by STZ in diabetic rats accompanied with the enhancement of oxidative stress.

A Natural Tetrahydropyrimidine Protects Small Bowel from Cold Ischemia and Subsequent Warm in vitro Reperfusion Injury

Objective: Ischemia/reperfusion (I/R) injury is a major difficulty in small bowel transplantation. Many postoperative complications are caused by damage to the intestinal mucosa following organ retrieval and during cold storage. Here we investigate whether 1-, 4-, 5-, 6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid (THP) can protect ileal mucosa and muscularis against cold ischemia and subsequent warm in vitro reperfusion injury. Methods: Small rat bowels were stored in the absence or presence of THP or its synthetic derivative lauryl-THP for 18 h at 4°C. Several biochemical parameters were evaluated following isolated reperfusion in tissue grafts and/or effluent solution. Graft morphology was analyzed by electron microscopy. Ceramide was quantified as a potential target of THP action. Results: THP considerably inhibited apoptosis and improved all parameters illustrating membrane and subcellular integrity and functionality. Parallel studies with lauryl-THP failed to show a similar protective effect. Lauryl-THP, in contrast to THP, also failed to decrease tissue ceramide content, indicating the involvement of this well-known mediator of cellular stress response. Conclusions: THP effectively protects ileal mucosa and muscularis against ischemia and reperfusion injury, indicating for the first time the clinical relevance of this new and promising molecule that might become a central component in tissue and organ protection in the near future.