Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice.

BACKGROUND: The prevalence of dyslipidemia and obesity resulting from excess energy intake and physical inactivity is increasing. The liver plays a pivotal role in systemic lipid homeostasis. Effective, natural dietary interventions that lower plasma lipids and promote liver health are needed. OBJECTIVE: Our goal was to determine the effect of dietary sphingolipids on plasma lipids and liver steatosis. DESIGN: APOE*3Leiden mice were fed a Western-type diet supplemented with different sphingolipids. Body cholesterol and triacylglycerol metabolism as well as hepatic lipid concentrations and lipid-related gene expression were determined. RESULTS: Dietary sphingolipids dose-dependently lowered both plasma cholesterol and triacylglycerol in APOE*3Leiden mice; 1% phytosphingosine (PS) reduced plasma cholesterol and triacylglycerol by 57% and 58%, respectively. PS decreased the absorption of dietary cholesterol and free fatty acids by 50% and 40%, respectively, whereas intestinal triacylglycerol lipolysis was not affected. PS increased hepatic VLDL-triacylglycerol production by 20%, whereas plasma lipolysis was not affected. PS increased the hepatic uptake of VLDL remnants by 60%. Hepatic messenger RNA concentrations indicated enhanced hepatic lipid synthesis and VLDL and LDL uptake. The net result of these changes was a strong decrease in plasma cholesterol and triacylglycerol. The livers of 1% PS-fed mice were less pale, 22% lighter, and contained 61% less cholesteryl ester and 56% less triacylglycerol than livers of control mice. Furthermore, markers of liver inflammation (serum amyloid A) and liver damage (alanine aminotransferase) decreased by 74% and 79%, respectively, in PS-fed mice. CONCLUSION: Sphingolipids lower plasma cholesterol and triacylglycerol and protect the liver from fat- and cholesterol-induced steatosis.

Response of apolipoprotein E*3-Leiden transgenic mice to dietary fatty acids: combining liver proteomics with physiological data.

Dietary fatty acids have a profound impact on atherosclerosis, but mechanisms are not fully understood. We studied the effects of a saturated fat diet supplemented with fish oil, trans10,cis12 conjugated linoleic acid (CLA), or elaidic acid on lipid and glucose metabolism and liver protein levels of APOE*3 Leiden transgenic mice, a model for lipid metabolism and atherosclerosis. Fish oil lowered plasma and liver cholesterol and triglycerides, plasma free fatty acids, and glucose but increased plasma insulin. CLA lowered plasma cholesterol but increased plasma and liver triglycerides, plasma beta-hydroxybutyrate, and insulin. Elaidic acid lowered plasma and liver cholesterol. Proteomics identified significant regulation of 65 cytosolic and 8-membrane proteins. Many of these proteins were related to lipid and glucose metabolism, and to oxidative stress. Principal component analysis revealed that fish oil had a major impact on cytosolic proteins, and elaidic acid on membrane proteins. Correlation analysis between physiological and protein data revealed novel clusters of correlated variables, among which a metabolic syndrome cluster. The combination of proteomics and physiology gave new insights in mechanisms by which these dietary fatty acids regulate lipid metabolism and related pathways, for example, by altering protein levels of long-chain acyl-CoA thioester hydrolase and adipophilin in the liver.

Apolipoprotein C3 deficiency results in diet-induced obesity and aggravated insulin resistance in mice.

Our aim was to study whether the absence of apolipoprotein (apo) C3, a strong inhibitor of lipoprotein lipase (LPL), accelerates the development of obesity and consequently insulin resistance. Apoc3(-/-) mice and wild-type littermates were fed a high-fat (46 energy %) diet for 20 weeks. After 20 weeks of high-fat feeding, apoc3(-/-) mice showed decreased plasma triglyceride levels (0.11 +/- 0.02 vs. 0.29 +/- 0.04 mmol, P < 0.05) and were more obese (42.8 +/- 3.2 vs. 35.2 +/- 3.3 g; P < 0.05) compared with wild-type littermates. This increase in body weight was entirely explained by increased body lipid mass (16.2 +/- 5.9 vs. 10.0 +/- 1.8 g; P < 0.05). LPL-dependent uptake of triglyceride-derived fatty acids by adipose tissue was significantly higher in apoc3(-/-) mice. LPL-independent uptake of albumin-bound fatty acids did not differ. It is interesting that whole-body insulin sensitivity using hyperinsulinemic-euglycemic clamps was decreased by 43% and that suppression of endogenous glucose production was decreased by 25% in apoc3(-/-) mice compared with control mice. Absence of apoC3, the natural LPL inhibitor, enhances fatty acid uptake from plasma triglycerides in adipose tissue, which leads to higher susceptibility to diet-induced obesity followed by more severe development of insulin resistance. Therefore, apoC3 is a potential target for treatment of obesity and insulin resistance.

Acute inhibition of hepatic beta-oxidation in APOE*3Leiden mice does not affect hepatic VLDL secretion or insulin sensitivity.

Hepatic VLDL and glucose production is enhanced in type 2 diabetes and associated with hepatic steatosis. Whether the derangements in hepatic metabolism are attributable to steatosis or to the increased availability of FA metabolites is not known. We used methyl palmoxirate (MP), an inhibitor of carnitine palmitoyl transferase I, to acutely inhibit hepatic FA oxidation and investigated whether the FAs were rerouted into VLDL secretion and whether this would affect hepatic glucose production. After an overnight fast, male APOE3*Leiden transgenic mice received an oral dose of 10 mg/kg MP. Administration of MP led to an 83% reduction in plasma beta-hydroxybutyrate (ketone body) levels compared with vehicle-treated mice (0.47 +/- 0.07 vs. 2.81 +/- 0.16 mmol/l, respectively; P < 0.01), indicative of impaired ketogenesis. Plasma FFA levels were increased by 32% and cholesterol and insulin levels were decreased by 17% and 50%, respectively, in MP-treated mice compared with controls. MP treatment led to a 30% increase in liver triglyceride (TG) content. Surprisingly, no effect on hepatic VLDL-TG production was observed between the groups at 8 h after MP administration. In addition, the capacity of insulin to suppress endogenous glucose production was unaffected in MP-treated mice compared with controls. In conclusion, acute inhibition of FA oxidation increases hepatic lipid content but does not stimulate hepatic VLDL secretion or reduce insulin sensitivity.

Cell cycle perturbations and radiosensitization effects in a human prostate cancer cell line.

PURPOSE: To test the hypothesis that radiation-induced, transient G2/M arrest could potentially sensitize tumor cells to a subsequent, well-timed radiation dose. METHODS: PC-3 human prostate cancer cells were treated using either radiotherapy or (186)Re-labeled hydroxyethylidene diphosphonate ((186)Re-HEDP) treatment in different combinations. The resulting cell cycle shift and clonogenic cell death were analyzed by DNA flow cytometry and colony forming cell assay, respectively. RESULTS: Radiation doses of 4 Gy and 8 Gy induced a transient G2/M arrest, with a maximum after approximately 16 h. The presence of 2 mM pentoxifylline effectively abrogated this radiation-induced G2 M arrest, confirming a cell-cycle checkpoint-mediated effect. A second dose of 4 Gy, timed at the height of the G2/M arrest, significantly increased clonogenic cell-kill compared to delivery after a suboptimal interval (10 h, 20 h or 25 h after the first radiation fraction). Moreover, timed second doses of 2 Gy, 3 Gy or 4 Gy yielded improved normalized treatment effects compared to non-pretreated control. Radionuclide treatment of PC-3 cells, using (186)Re-HEDP (0.74 MBq/ml and 1.48 MBq/ml; total dose: 4.1 and 8.2 Gy, respectively) also induced a dose-dependent G2/M accumulation, which sensitized the cells to a subsequent external radiation dose of 2 Gy or 4 Gy. The observed pattern of cell-cycle shift towards a predominance of the G2/M phase is in line with the lack of functional p53 in this cell line. CONCLUSIONS: Radiation-induced cell-cycle shift was shown to effectively confer increased radiosensitivity to prostate tumor cells. Optimally timed combination of radiotherapy and radionuclide therapy could thus significantly increase treatment efficacy.