David Santos

Hepatic lipase- and endothelial lipase-deficiency in mice promotes macrophage-to-feces RCT and HDL antioxidant properties

Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol (HDLc) levels and presumably could affect two main HDL atheroprotective functions, macrophage-to-feces reverse cholesterol transport (RCT) and HDL antioxidant properties. In this study, we assessed the effects of both HL and EL deficiency on macrophage-specific RCT process and HDL ability to protect against LDL oxidation. HL- and EL-deficient and wild-type mice were injected intraperitoneally with [(3)H]cholesterol-labeled mouse macrophages, after which the appearance of [(3)H]cholesterol in plasma, liver, and feces was determined. The degree of HDL oxidation and the protection of oxidative modification of LDL co-incubated with HDL were evaluated by measuring conjugated diene kinetics. Plasma levels of HDLc, HDL phospholipids, apoA-I, and platelet-activated factor acetyl-hydrolase were increased in both HL- and EL-deficient mice. These genetically modified mice displayed increased levels of radiolabeled, HDL-bound [(3)H]cholesterol 48h after the label injection. The magnitude of macrophage-derived [(3)H]cholesterol in feces was also increased in both the HL- and EL-deficient mice. HDL from the HL- and EL-deficient mice was less prone to oxidation and had a higher ability to protect LDL from oxidation, compared with the HDL derived from the wild-type mice. These changes were correlated with plasma apoA-I and apoA-I/HDL total protein levels. In conclusion, targeted inactivation of both HL and EL in mice promoted macrophage-to-feces RCT and enhanced HDL antioxidant properties.

PPAR-β/δ activation promotes phospholipid transfer protein expression

The peroxisome proliferator-activated receptor (PPAR)-β/δ has emerged as a promising therapeutic target for treating dyslipidemia, including beneficial effects on HDL cholesterol (HDL-C). In the current study, we determined the effects of the PPAR-β/δ agonist GW0742 on HDL composition and the expression of liver HDL-related genes in mice and cultured human cells. The experiments were carried out in C57BL/6 wild-type, LDL receptor (LDLR)-deficient mice and PPAR-β/δ-deficient mice treated with GW0742 (10mg/kg/day) or a vehicle solution for 14 days. GW0742 upregulated liver phospholipid transfer protein (Pltp) gene expression and increased serum PLTP activity in mice. When given to wild-type mice, GW0742 significantly increased serum HDL-C and HDL phospholipids; GW0742 also raised serum potential to generate preβ-HDL formation. The GW0742-mediated effects on liver Pltp expression and serum enzyme activity were completely abolished in PPAR-β/δ-deficient mice. GW0742 also stimulated PLTP mRNA expression in mouse J774 macrophages, differentiated human THP-1 macrophages and human hepatoma Huh7. Collectively, our findings demonstrate a common transcriptional upregulation by GW0742-activated PPAR-β/δ of Pltp expression in cultured cells and in mouse liver resulting in enhanced serum PLTP activity. Our results also indicate that PPAR-β/δ activation may modulate PLTP-mediated preβ-HDL formation and macrophage cholesterol efflux.

Quantification of In Vitro Macrophage Cholesterol Efflux and In Vivo Macrophage-Specific Reverse Cholesterol Transport

Promotion of reverse cholesterol transport (RCT) is thought to be a major HDL-mediated mechanism for protecting against atherosclerosis. Preclinical studies support the concept that increasing cholesterol efflux from macrophages may confer atheroprotective benefits independently of the plasma HDL-cholesterol concentration. The application of the macrophage-to-feces RCT method in genetically engineered mice has provided evidence that this major HDL property correlates closely with changes in atherosclerosis susceptibility. This chapter provides details on the methodologies currently used to measure in vitro cholesterol efflux from macrophages or in vivo macrophage-specific RCT. The general principles and techniques described herein may be applied to measure the in vitro cholesterol efflux capacity of human serum in macrophage cultures and to evaluate the effect of different experimental pathophysiological conditions or the efficacy of different therapeutic strategies on the modulation of in vivo macrophage-RCT in mice.

Consumption of polyunsaturated fat improves the saturated fatty acid-mediated impairment of HDL antioxidant potential.

SCOPE The present study aimed to compare the effects of diets containing high-fat, high-cholesterol and saturated fatty acids (HFHC-SFA) and HFHC-polyunsaturated fatty acids-containing (HFHC-PUFA) diets on two major antiatherogenic functions of HDL, the HDL antioxidant function and the macrophage-to-feces reverse cholesterol transport. METHODS AND RESULTS Experiments were carried out in mice fed a low-fat, low-cholesterol (LFLC) diet, an HFHC-SFA diet or an HFHC-PUFA diet in which SFAs were partly replaced with an alternative high-linoleic and α-linolenic fat source. The HFHC-SFA caused a significant increase in serum HDL cholesterol and phospholipids as well as elevated levels of oxidized HDL and oxidized LDL. Replacing SFA with PUFA significantly reduced the levels of these oxidized lipoproteins and enhanced the ability of HDL to protect LDL from oxidation. The SFA-mediated impairment of HDL antioxidant potential was not associated with the cholesterol content of the diet, obesity or insulin resistance. In contrast, the effect of the HFHC diets on fecal macrophage-derived cholesterol excretion was independent of the fatty acid source. CONCLUSION SFA intake impairs the antioxidant potential of HDL and increases serum levels of oxidized lipoprotein species whereas the antioxidant potential of HDL is enhanced after PUFA consumption.

LXR-dependent regulation of macrophage-specific reverse cholesterol transport is impaired in a model of genetic diabesity

&NA; Diabesity and fatty liver have been associated with low levels of high‐density lipoprotein cholesterol, and thus could impair macrophage‐specific reverse cholesterol transport (m‐RCT). Liver X receptor (LXR) plays a critical role in m‐RCT. Abcg5/g8 sterol transporters, which are involved in cholesterol trafficking into bile, as well as other LXR targets, could be compromised in the livers of obese individuals. We aimed to determine m‐RCT dynamics in a mouse model of diabesity, the db/db mice. These obese mice displayed a significant retention of macrophage‐derived cholesterol in the liver and reduced fecal cholesterol elimination compared with nonobese mice. This was associated with a significant downregulation of the hepatic LXR targets, including Abcg5/g8. Pharmacologic induction of LXR promoted the delivery of total tracer output into feces in db/db mice, partly due to increased liver and small intestine Abcg5/Abcg8 gene expression. Notably, a favorable upregulation of the hepatic levels of ABCG5/G8 and NR1H3 was also observed postoperatively in morbidly obese patients, suggesting a similar LXR impairment in these patients. In conclusion, our data show that downregulation of the LXR axis impairs cholesterol transfer from macrophages to feces in db/db mice, whereas the induction of the LXR axis partly restores impaired m‐RCT by elevating the liver and small intestine expressions of Abcg5/g8.

Human hepatic lipase overexpression in mice induces hepatic steatosis and obesity through promoting hepatic lipogenesis and white adipose tissue lipolysis and fatty acid uptake

Human hepatic lipase (hHL) is mainly localized on the hepatocyte cell surface where it hydrolyzes lipids from remnant lipoproteins and high density lipoproteins and promotes their hepatic selective uptake. Furthermore, hepatic lipase (HL) is closely associated with obesity in multiple studies. Therefore, HL may play a key role on lipid homeostasis in liver and white adipose tissue (WAT). In the present study, we aimed to evaluate the effects of hHL expression on hepatic and white adipose triglyceride metabolism in vivo. Experiments were carried out in hHL transgenic and wild-type mice fed a Western-type diet. Triglyceride metabolism studies included β-oxidation and de novo lipogenesis in liver and WAT, hepatic triglyceride secretion, and adipose lipoprotein lipase (LPL)-mediated free fatty acid (FFA) lipolysis and influx. The expression of hHL promoted hepatic triglyceride accumulation and de novo lipogenesis without affecting triglyceride secretion, and this was associated with an upregulation of Srebf1 as well as the main genes controlling the synthesis of fatty acids. Transgenic mice also exhibited more adiposity and an increased LPL-mediated FFA influx into the WAT without affecting glucose tolerance. Our results demonstrate that hHL promoted hepatic steatosis in mice mainly by upregulating de novo lipogenesis. HL also upregulated WAT LPL and promoted triglyceride-rich lipoprotein hydrolysis and adipose FFA uptake. These data support the important role of hHL in regulating hepatic lipid homeostasis and confirm the broad cardiometabolic role of HL.

Phytosterol‐mediated inhibition of intestinal cholesterol absorption in mice is independent of liver X receptor

SCOPE Previous studies have proposed that phytosterols activate liver X receptors (LXR) in the intestine, thereby reducing intestinal cholesterol absorption and promoting fecal cholesterol excretion. METHODS AND RESULTS In the present study, we examined the effects of dietary phytosterol supplementation on intestinal cholesterol absorption and fecal neutral sterol excretion in LXRαβ-deficient mice, and wild-type mice treated with synthetic high-affinity LXRαβ agonists. LXRαβ deficiency led to an induction of intestinal cholesterol absorption and liver cholesterol accumulation. Phytosterol feeding resulted in an approximately 40% reduction of intestinal cholesterol absorption both in wild-type and LXRαβ-deficient mice, reduced dietary cholesterol accumulation in liver and promoted the excretion of fecal cholesterol-derived compounds. Furthermore, phytosterols produced additive inhibitory effects on cholesterol absorption in mice treated with LXRαβ agonists. CONCLUSIONS Our data confirm the effect of LXR in regulating intestinal cholesterol absorption and demonstrate that the cholesterol-lowering effects of phytosterols occur in an LXR-independent manner.

Alteraciones en el contenido proteico y disfunción de lipoproteínas de alta densidad en ratones hiperhomocisteinémicos

AIM The aim of this study was to evaluate the proteic changes in high-density lipoproteins (HDL) induced by methionine-induced hyperhomocysteinemia in mice and its relationship with two of their main antiatherogenic properties. METHODS AND RESULTS The oral administration of methionine resulted in an elevation (~8 times) in the plasma concentration of homocysteine. Hyperhomocysteinemia was inversely correlated with the plasma concentration of HDL cholesterol and its main protein component of HDL, apolipoprotein (apo) A-I, respectively. The cholesterol efflux in vivo from macrophages to HDL was decreased in hyperhomocysteinemic mice compared with the control mice. However, the reverse cholesterol transport from macrophages to feces remained unchanged. On the other hand, the ability of HDL from hyperhomocysteinemic mice to prevent the oxidative modification of low-density lipoproteins (LDL) was found decreased and associated with a concomitant reduction in the plasma activity of paraoxonase-1 (PON1) and the plasma concentration of apoA-I, and with a relative reduction in the apoA-IV content (~1.5 times) in the hyperhomocysteinemic HDL, respectively. CONCLUSION The decrease in the ability of HDL from hyperhomocysteinemic mice to prevent LDL from oxidation was associated with a decrease in the apoA-I, PON1 and apoA-IV.

ApoA-I mimetic administration, but not increased apoA-I-containing HDL, inhibits tumour growth in a mouse model of inherited breast cancer

Low levels of high-density lipoprotein cholesterol (HDLc) have been associated with breast cancer risk, but several epidemiologic studies have reported contradictory results with regard to the relationship between apolipoprotein (apo) A-I and breast cancer. We aimed to determine the effects of human apoA-I overexpression and administration of specific apoA-I mimetic peptide (D-4F) on tumour progression by using mammary tumour virus-polyoma middle T-antigen transgenic (PyMT) mice as a model of inherited breast cancer. Expression of human apoA-I in the mice did not affect tumour onset and growth in PyMT transgenic mice, despite an increase in the HDLc level. In contrast, D-4F treatment significantly increased tumour latency and inhibited the development of tumours. The effects of D-4F on tumour development were independent of 27-hydroxycholesterol. However, D-4F treatment reduced the plasma oxidized low-density lipoprotein (oxLDL) levels in mice and prevented oxLDL-mediated proliferative response in human breast adenocarcinoma MCF-7 cells. In conclusion, our study shows that D-4F, but not apoA-I-containing HDL, hinders tumour growth in mice with inherited breast cancer in association with a higher protection against LDL oxidative modification.

Administration of CORM-2 inhibits diabetic neuropathy but does not reduce dyslipidemia in diabetic mice

The antinociceptive effects of the carbon monoxide-releasing molecule tricarbonyldichlororuthenium (II) dimer (CORM-2) during chronic pain are well documented, but most of its possible side-effects remain poorly understood. In this work, we examine the impact of CORM-2 treatment on the lipoprotein profile and two main atheroprotective functions attributed to high-density lipoprotein (HDL) in a mouse model of type 1 diabetes while analyzing the effect of this drug on diabetic neuropathy. Streptozotocin (Stz)-induced diabetic mice treated with CORM-2 (Stz-CORM-2) or vehicle (Stz-vehicle) were used to evaluate the effect of this drug on the modulation of painful diabetic neuropathy using nociceptive behavioral tests. Plasma and tissue samples were used for chemical and functional analyses, as appropriate. Two main antiatherogenic properties of HDL, i.e., the ability of HDL to protect low-density lipoprotein (LDL) from oxidation and to promote reverse cholesterol transport from macrophages to the liver and feces in vivo (m-RCT), were also assessed. Stz-induced diabetic mice displayed hyperglycemia, dyslipidemia and pain hypersensitivity. The administration of 10 mg/kg CORM-2 during five consecutive days inhibited allodynia and hyperalgesia and significantly ameliorated spinal cord markers (Cybb and Bdkrb1expression) of neuropathic pain in Stz mice, but it did not reduce the combined dyslipidemia shown in Stz-treated mice. Its administration to Stz-treated mice led to a significant increase in the plasma levels of cholesterol (∼ 1.4-fold vs. Ctrl, ∼ 1.3- fold vs. Stz-vehicle; p < 0.05) and was attributed to significant elevations in both non-HDL (∼ 1.8-fold vs. Ctrl; ∼ 1.6-fold vs. Stz-vehicle; p < 0.05) and HDL cholesterol (∼ 1.3-fold vs. Ctrl, ∼ 1.2-fold vs. Stz-vehicle; p < 0.05). The increased HDL in plasma was not accompanied by a commensurate elevation in m-RCT in Stz-CORM-2 compared to Stz-vehicle mice; instead, it was worsened as revealed by decreased [3H]-tracer trafficking into the feces in vivo. Furthermore, the HDL-mediated protection against LDL oxidation ex vivo shown by the HDL isolated from Stz-CORM-2 mice did not differ from that obtained in Stz-vehicle mice. In conclusion, the antinociceptive effects produced by a high dose of CORM-2 were accompanied by antioxidative effects but were without favorable effects on the dyslipidemia manifested in diabetic mice.