Maria Berggren-Söderlund

Intralipid decreases apolipoprotein M levels and insulin sensitivity in rats.

Background Apolipoprotein M (ApoM) is a constituent of high-density lipoproteins (HDL). It plays a crucial role in HDL-mediated reverse cholesterol transport. Insulin resistance is associated with decreased ApoM levels. Aims To assess the effects of increased free fatty acids (FFAs) levels after short-term Intralipid infusion on insulin sensitivity and hepatic ApoM gene expression. Methods Adult male Sprague-Dawley (SD) rats infused with 20% Intralipid solution for 6 h. Glucose infusion rates (GIR) were determined by hyperinsulinemic-euglycemic clamp during Intralipid infusion and plasma FFA levels were measured by colorimetry. Rats were sacrificed after Intralipid treatment and livers were sampled. Human embryonic kidney 293T cells were transfected with a lentivirus mediated human apoM overexpression system. Goto-Kakizaki (GK) rats were injected with the lentiviral vector and insulin tolerance was assessed. Gene expression was assessed by real-time RT-PCR and PCR array. Results Intralipid increased FFAs by 17.6 folds and GIR was decreased by 27.1% compared to the control group. ApoM gene expression was decreased by 40.4% after Intralipid infusion. PPARβ/δ expression was not changed by Intralipid. Whereas the mRNA levels of Acaca, Acox1, Akt1, V-raf murine sarcoma 3611 viral oncogene homolog, G6pc, Irs2, Ldlr, Map2k1, pyruvate kinase and RBC were significantly increased in rat liver after Intralipid infusion. The Mitogen-activated protein kinase 8 (MAPK8) was significantly down-regulated in 293T cells overexpressing ApoM. Overexpression of human ApoM in GK rats could enhance the glucose-lowering effect of exogenous insulin. Conclusion These results suggest that Intralipid could decrease hepatic ApoM levels. ApoM overexpression may have a potential role in improving insulin resistance in vivo and modulating apoM expression might be a future therapeutic strategy against insulin resistance in type 2 diabetes.

Palmitic acid suppresses apolipoprotein M gene expression via the pathway of PPARβ/δ in HepG2 cells.

It has been demonstrated that apolipoprotein M (APOM) is a vasculoprotective constituent of high density lipoprotein (HDL), which could be related to the anti-atherosclerotic property of HDL. Investigation of regulation of APOM expression is of important for further exploring its pathophysiological function in vivo. Our previous studies indicated that expression of APOM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF), leptin, hyperglycemia and etc., in vivo and/or in vitro. In the present study, we demonstrated that palmitic acid could significantly inhibit APOM gene expression in HepG2 cells. Further study indicated neither PI-3 kinase (PI3K) inhibitor LY294002 nor protein kinase C (PKC) inhibitor GFX could abolish palmitic acid induced down-regulation of APOM expression. In contrast, the peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) antagonist GSK3787 could totally reverse the palmitic acid-induced down-regulation of APOM expression, which clearly demonstrates that down-regulation of APOM expression induced by palmitic acid is mediated via the PPARβ/δ pathway.

Decreased activities of apolipoprotein m promoter are associated with the susceptibility to coronary artery diseases.

The present study investigated the correlation among genetic polymorphisms of the proximal promoter region of apolipoprotein M (apoM) gene, the polymorphisms in relation to apoM expressions and the susceptibility to coronary artery diseases (CAD) in a Han Chinese population. Four common polymorphic sites, i.e., T-1628G, C-1065A, T-855C and T-778C, were confirmed, and a new deletion mutation C-724del was found, in 206 CAD patients and 209 non-CAD patients using direct DNA sequencing analyses. Occurrences of alleles T-1628G, T-855C and C-724del were significantly higher in CAD patients compared to non-CAD patients. Moreover we examined all these polymorphisms in relation to apoM expression by applying luciferase reporter assay. It demonstrated that constructs -855C and 724del showed obvious decreased luciferase activities, i.e., (0.93±0.15 vs. 2.11±0.15; P=0.012) and (1.13±0.25 vs. 2.11±0.15; P=0.009) respectively, which indicates these two polymorphisms could confer decreased apoM expressions. Meanwhile the occurrences of these two SNP were also significantly higher in the CAD patients than in non-CAD patients. It is therefore reasonable to speculate that down-regulated apoM expressions in relation to these polymorphisms may affect HDL and cholesterol metabolism in vivo and further influence the susceptibility to CAD, although the underlying mechanisms need further investigation.

Rosiglitazone Enhances Apolipoprotein M (Apom) Expression in Rat's Liver

Apolipoprotein M (APOM) has been suggested as a vasculoprotective constituent of high density lipoprotein (HDL), which plays a crucial role behind the mechanism of HDL-mediated anti-atherosclerosis. Previous studies demonstrated that insulin resistance could associate with decreased APOM expressions. In agreement with our previous reports, here, we further confirmed that the insulin sensitivity was also reduced in rats treated with high concentrations of glucose; such effect could be reversed by administration of rosiglitazone, a peroxisome proliferator-activated receptor-γ (PPARγ). The present study shows that Apom expression is significantly affected by either rosiglitazone or hyperglycemia alone without cross interaction with each other, which indicates that the pathway of Apom expression regulating by hyperglycemia might be differed from that by rosiglitazone. Further study indicated that hyperglycemia could significantly inhibit mRNA levels of Lxrb (P=0.0002), small heterodimer partner 1 (Shp1) (P<0.0001), liver receptor homologue-1 (Lrh1) (P=0.0012), ATP-binding cassette transporter 1 (Abca1) (P=0.0012) and Pparb/d (P=0.0043). Two-way ANOVA analysis demonstrated that the interactions between rosiglitazone and infusion of 25% glucose solution on Shp1 (P=0.0054) and Abca1 (4E, P=0.0004) mRNA expression was statistically significant. It is concluded that rosiglitazone could increase Apom expression, of which the detailed mechanism needs to be further investigated. The downregulation of Apom by hyperglycemia might be mainly through decreasing expression of Pparg and followed by inhibiting Lxrb in rats.

TO901317 regulating apolipoprotein M expression mediates via the farnesoid X receptor pathway in Caco-2 cells

BackgroundApolipoprotein M (apoM) may have potential antiatherosclerotic properties. It has been reported that apoM expression could be regulated by many intracellar and extracellar factors. In the present study we further investigated regulation of apoM expression in Caco-2 cells stimulated by a liver X receptor (LXR) agonist, TO901317.Materials and methodsCaco-2 cells were cultured in the presence of either TO901317, farnesoid X receptor (FXR) antagonist guggulsterone or TO901317 together with guggulsterone at different concentrations for 24 hrs. The mRNA levels of ATP-binding cassette transporter A1 (ABCA1), apoA1, apoM, liver receptor homologue-1 (LRH-1) and short heterodimer partner 1 (SHP1) were determined by real-time RT-PCR.ResultsWhen Caco-2 cell cultured with TO901317 alone, the mRNA levels of ABCA1, apoA1, apoM, LRH-1 and SHP1 were significantly increased with dose-dependent manners (p < 0.05), whereas when the cells cultured with guggulsterone alone, the mRNA levels of apoM, SHP1 and LRH-1 (p < 0.05) were strongly inhibited. Moreover, guggulsterone could abolish the TO901317 enhanced mRNA levels of apoA1 apoM, SHP1 and LRH-1.ConclusionThe present study demonstrated that LXR agonist TO901317 induced apoM expression in Caco-2 cells might be mediated via the LXR/FXR pathway.

Effects of simvastatin on apolipoprotein M in vivo and in vitro

ObjectiveTo investigate effects of lipid lowering drug, simvastatin, on apolipoprotein M expression in the hyperlipidemic mice and in hepatic cell line, HepG2 cells.MethodsSwiss male mice were randomly divided into the high fat group and control group, and were intragastrically fed with 0.9% saline (control group) or lipid emulsion (high fat group) at the daily dosage of 15 ml/kg body weight, respectively. After 8 weeks feeding, the hyperlipidemic model was successfully induced and these hyperlipidemic mice were then randomly divided into three experimental groups: vehicle control group, high-dose simvastatin-treated group (100 mg/kg body weight), and low-dose simvastatin-treated group (10 mg/kg body weight). Mice were dosed daily for 6 weeks of simvastatin before mice were sacrificed for determining serum lipid profile and apoM protein levels that was determined by using dot blotting analysis. Effects of simvastatin on apoM mRNA expression in the HepG2 cells were determined by real-time RT-PCR.ResultsComparing to high fat model mice without simvastatin treatment, 100 mg/kg simvastatin could significantly increase serum total cholesterol (P < 0.05). Serum apoM levels, in all mice, were significantly lower in the mice at the age of 26 weeks than the mice at 12 weeks old (P < 0.05), which indicated that serum apoM levels were significantly correlated to the mice age. It demonstrated also that treatment of simvastatin did not influence serum apoM levels in these mouse model, although serum apoM levels were increased by about 13% in the 10 mg/kg simvastatin group than in the vehicle control group without simvastatin. In HepG2 cell cultures, simvastatin could significantly decrease apoM mRNA levels with dose- and time-dependent manners. At 10 μM simvastatin treatment, apoM mRNA decreased by 52% compared to the controls.ConclusionThe present study suggested that simvastatin, in vivo, had no effect on apoM levels in the hyperlipidemic mouse model. ApoM serum levels in mice were significantly correlated to the animals age, whereas in cell cultures simvastatin does inhibit apoM expression in the HepG2 cells. The mechanism behind it is not known yet.

Hyperglycemia-induced downregulation of apolipoprotein M expression is not via the hexosamine pathway.

BackgroundWe previously demonstrated that hyperglycemia could suppress apolipoprotein M (apoM) synthesis both in vivo and in vitro; however, the mechanism of hyperglycemia-induced downregulation of apoM expression is unknown yet.MethodsIn the present study we further examined if hexosamine pathway, one of the most important pathways of glucose turnover, being involved in modulating apoM expression in the hyperglycemia condition. We examined the effect of glucosamine, a prominent component of hexosamine pathway and intracellular mediator of insulin resistance, on apoM expression in HepG2 cells and in rat’s models. In the present study we also determined apolipoprotein A1 (apoA1) as a control gene.ResultsOur results demonstrated that glucosamine could even up-regulate both apoM and apoA1 expressions in HepG2 cell cultures. The glucosamine induced upregulation of apoM expression could be blocked by addition of azaserine, an inhibitor of hexosamine pathway. Moreover, intravenous infusion of glucosamine could enhance hepatic apoM expression in rats, although serum apoM levels were not significantly influences.ConclusionsIt is concluded that both exogenous and endogenous glucosamine were essential for the over-expression of apoM, which may suggest that the increased intracellular content of glucosamine does not be responsible for the depressed apoM expression at hyperglycemia condition.

Decreased Splenic CD4(+) T-Lymphocytes in Apolipoprotein M Gene Deficient Mice.

Spleen T-lymphocytes, especially CD4+ T-cells, have been demonstrated to be involved in broad immunomodulation and host-defense activity in vivo. Apolipoprotein M gene (apoM) may have an important role in the regulation of immunoprocess and inflammation, which could be hypothesized to the apoM containing sphingosine-1-phosphate (S1P). In the present study we demonstrate that the splenic CD4+ T-lymphocytes were obviously decreased in the apoM gene deficient (apoM−/−) mice compared to the wild type (apoM+/+). Moreover, these mice were treated with lipopolysaccharide (LPS) and it was found that even more pronounced decreasing CD4+ T-lymphocytes occurred in the spleen compared to the apoM+/+ mice. The similar phenomena were found in the ratio of CD4+/CD8+ T-lymphocytes. After administration of LPS, the hepatic mRNA levels of tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were markedly increased; however, there were no statistical differences observed between apoM+/+ mice and apoM−/− mice. The present study demonstrated that apoM might facilitate the maintenance of CD4+ T-lymphocytes or could modify the T-lymphocytes subgroups in murine spleen, which may further explore the importance of apoM in the regulation of the host immunomodulation, although the detailed mechanism needs continuing investigation.

Lipid synthesis and secretion in HepG2 cells is not affected by ACTH

Apolipoprotein B (apoB) containing lipoproteins, i.e. VLDL, LDL and Lp(a), are consequently lowered by ACTH treatment in humans. This is also seen as reduced plasma apoB by 20-30% and total cholesterol by 30-40%, mostly accounted for by a decrease in LDL-cholesterol. Studies in hepatic cell line (HepG2) cells showed that apoB mRNA expression is reduced in response to ACTH incubation and is followed by a reduced apoB secretion, which may hypothesize that ACTH lowering apoB containing lipoproteins in humans may be mediated by the inhibition of hepatic apoB synthesis. This was recently confirmed in vivo in a human postprandial study, where ACTH reduced transient apoB48 elevation from the small intestine, however, the exogenic lipid turnover seemed unimpaired. In the present study we investigated if lipid synthesis and/or secretion in HepG2 cells were also affected by pharmacological levels of ACTH to accompany the reduced apoB output. HepG2 cells were incubated with radiolabelled precursors ([14C]acetate and [3H]glycerol) either before or during ACTH stimuli. Cellular and secreted lipids were extracted with chloroform:methanol and separated by the thin layer chromatography (TLC), and [14C]labelled cholesterol and cholesteryl ester and [3H]labelled triglycerides and phospholipids were quantitated by the liquid scintillation counting. It demonstrated that ACTH administration did not result in any significant change in neither synthesis nor secretion of the studied lipids, this regardless of presence or absence of oleic acid, which is known to stabilize apoB and enhance apoB production. The present study suggests that ACTH lowers plasma lipids in humans mainly mediated by the inhibition of apoB synthesis and did not via the reduced lipid synthesis.