Novel roles of hepatic lipase and phospholipid transfer protein in VLDL as well as HDL metabolism

Objective

Elevated plasma phospholipid transfer protein (PLTP) expression may increase atherosclerosis in mice by reducing plasma HDL and increasing hepatic VLDL secretion. Hepatic lipase (HL) is a lipolytic enzyme involved in several aspects of the same pathways of lipoprotein metabolism. We investigated whether the effects of elevated PLTP activity are compromised by HL deficiency.

Methods and results

HL deficient mice were crossbred with PLTP transgenic (PLTPtg) mice and studied in the fasted state. Plasma triglycerides were decreased in HL deficiency, explained by reduced hepatic triglyceride secretion. In PLTPtg mice, a redistribution of HL activity between plasma and tissue was evident and plasma triglycerides were also decreased. HL deficiency mitigated or even abolished the stimulatory effect of elevated PLTP activity on hepatic triglyceride secretion. HL deficiency had a modest incremental effect on plasma HDL, which remained present in PLTP transgenic/HL^−/− mice, thereby partially compensating the decrease in HDL caused by elevation of PLTP activity. HDL decay experiments showed that the fractional turnover rate of HDL cholesteryl esters was delayed in HL deficient mice, increased in PLTPtg mice and intermediate in PLTPtg mice in an HL^−/− background.

Conclusions

HL affects hepatic VLDL. Elevated PLTP activity lowers plasma HDL-cholesterol by stimulating the plasma turnover and hepatic uptake of HDL cholesteryl esters. HL is not required for the increase in hepatic triglyceride secretion or for the lowering of HDL-cholesterol induced by PLTP overexpression.     

Reduction of HDL levels lowers plasma PLTP and affects its distribution among lipoproteins in mice

Phospholipid transfer protein (PLTP) is associated with HDL particles in plasma, where it transfers phospholipids between lipoproteins and remodels HDL particles. Tangier disease patients, with a mutated ABCA1 transporter, have extremely low plasma HDL concentration and reduced PLTP activity levels, a phenotype that is also observed in mice lacking ABCA1. We investigated whether low HDL levels and low PLTP activity are mechanistically related. Firstly, we studied PLTP expression and distribution among lipoproteins in mice lacking ABCA1 (ABCA1^−/−). Parallel to the strong reduction in PLTP activity in plasma of ABCA1^−/− mice, decreased PLTP protein levels were observed. Neither PLTP synthesis in liver or macrophages nor the ability of the macrophages to secrete PLTP were impaired in ABCA1^−/− mice. However, the PLTP activity level in the medium of cultured macrophages was determined by HDL levels in the medium. PLTP was associated with HDL particles in wild type mice, whereas in ABCA1^−/− mice, PLTP was associated with VLDL and LDL particles. Secondly, we treated different mouse models with varying plasma HDL and PLTP levels (wild type, ABCA1^−/−, apoE^−/− and PLTPtg mice, overexpressing human PLTP) with a synthetic LXR ligand, and investigated the relationship between LXR-mediated PLTP induction and HDL levels in plasma. Plasma PLTP activity in wild type mice was induced 5.6-fold after LXR activation, whereas in ABCA1^−/−, apoE^−/− and PLTPtg mice, all having reduced HDL levels, induction of PLTP activity was 2.4- , 3.2- and 2.0-fold, respectively. The less pronounced PLTP induction in these mice compared to wild type mice was not caused by a decreased PLTP gene expression in the liver or macrophages. Our findings indicate that the extent of LXR-mediated PLTP induction depends on plasma HDL levels. In conclusion, we demonstrate that ABCA1 deficiency in mice affects plasma PLTP level and distribution through an indirect effect on HDL metabolism. In addition, we show that the extent of LXR-mediated PLTP induction is HDL-dependent. These findings indicate that plasma HDL level is an important regulator of plasma PLTP and might play a role in the stabilization of PLTP in plasma.     

Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.     

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset.

Objective

To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism.

Methods

25-OH vitamin D and 1,25-(OH)₂ vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia.

Results

In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)₂ vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001).

Summary

Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.     

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion bunding capacity of the chelators.     

In-vivo phosphorylation of the cardiac L-type calcium channel beta-subunit in response to catecholamines

In canine myocardium, the -subunit of the L-type Ca²⁺ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217–222, 1993). We have assessed the identity of the -subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca²⁺ channel -subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the -subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dt_max. The results imply the involvement of a PKA-mediated phosphorylation of the Ca²⁺ channel -subunit in the adrenergic stimulation of intact canine myocardium.