PCSK9 plays a significant role in cholesterol homeostasis and lipid transport in intestinal epithelial cells.

OBJECTIVES: The proprotein convertase subtillisin/kexin type 9 (PCSK9) regulates cholesterol metabolism via degradation of low-density lipoprotein receptor (LDLr). Although PCSK9 is abundantly expressed in the intestine, limited data are available on its functions. The present study aims at determining whether PCSK9 plays important roles in cholesterol homeostasis and lipid transport in the gut. METHODS AND RESULTS: Caco-2/15 cells were used allowing the exploration of the PCSK9 secretory route through the apical and basolateral compartments corresponding to intestinal lumen and serosal circulation, respectively. The output of PCSK9 occurred through the basolateral membrane, a site characterized by the location of LDLr. Co-immunoprecipitation studies indicated an association between PCSK9 and LDLr. Addition of purified recombinant wild type and D374Y gain-of function PCSK9 proteins to the basolateral medium was followed by a decrease in LDLr concomitantly with the accumulation of both forms of PCSK9. Furthermore, the latter caused a significant enhancement in cholesterol uptake also evidenced by a raised protein expression of cholesterol transporters NPC1L1 and CD36 without changes in SR-BI, ABCA1, and ABCG5/G8. Moreover, exogenous PCSK9 altered the activity of HMG-CoA reductase and acylcoenzyme A: cholesterol acyltransferase, and was able to enhance chylomicron secretion by positively modulating lipids and apolipoprotein B-48 biogenesis. Importantly, PCSK9 silencing led to opposite findings, which validate our data on the role of PCSK9 in lipid transport and metabolism. Moreover, PCSK9-mediated changes persisted despite LDLr knockdown. CONCLUSIONS: These findings indicate that, in addition to its effect on LDLr, PCSK9 modulates cholesterol transport and metabolism, as well as production of apo B-containing lipoproteins in intestinal cells.

Expression of Sar1b enhances chylomicron assembly and key components of the coat protein complex II system driving vesicle budding.

OBJECTIVE: SAR1b plays a significant role in the assembly, organization, and function of the coat protein complex II, a critical complex for the transport of proteins from the endoplasmic reticulum to the Golgi. Recently, mutations in SARA2 have been associated with lipid absorption disorders. However, functional studies on Sar1b-mediated lipid synthesis pathways and lipoprotein packaging have not been performed. METHODS AND RESULTS: Sar1b was overexpressed in Caco-2/15 cells and resulted in significantly augmented triacylglycerol, cholesteryl ester, and phospholipid esterification and secretion and markedly enhanced chylomicron production. It also stimulated monoacylglycerol acyltransferase/diacylglycerol acyltransferase activity and enhanced apolipoprotein B-48 protein synthesis, as well as elevated microsomal triglyceride transfer protein activity. Along with the enhanced chylomicrons, microsomes were characterized by abundant Sec12, the guanine exchange factor that promotes the localization of Sar1b in the endoplasmic reticulum. Furthermore, coimmunoprecipitation experiments revealed high levels of the complex components Sec23/Sec24 and p125, the Sec23-interacting protein. Finally, a pronounced interaction of Sec23/Sec24 with sterol regulatory element binding protein (SREBP) cleavage-activating protein and SREBP-1c was noted, thereby permitting the transfer of the transcription factor SREBP-1c to the nucleus for the activation of genes involved in lipid metabolism. CONCLUSION: Our data suggest that Sar1b expression may promote intestinal lipid transport with the involvement of the coat protein complex II network and the processing of SREBP-1c.

Renal and metabolic disorders depend on the renin-angiotensin system in Lyon hypertensive rats associated with diabetes.

BACKGROUND: In Lyon genetically hypertensive (LH) rats with diabetes, the effects of angiotensin converting enzyme (ACE) inhibition with perindopril on the prevention of dyslipidemia and proteinuria were evaluated by comparison with a nonspecific antihypertensive treatment. METHODS: Diabetes was induced in 2-day-old male LH rats by intraperitoneal injection of streptozotocin (75 mg/kg). Glucose tolerance (glucose 2 g/kg by gavage), blood pressure (BP), plasma lipids, and urinary protein excretion were studied in: (i) untreated diabetic LH rats, (ii) diabetic LH rats treated from 8 to 16 weeks of age with oral perindopril at a low dose (0.01 mg/kg/day), (iii) similar rats treated with oral perindopril for the same duration at a high dose (1 mg/kg/day), and (iv) similar rats treated for the same duration with a triple therapy regimen consisting of hydralazine, hydrochlorothiazide, and reserpine (75, 15, and 0.75 mg/kg/day, respectively). RESULTS: The neonatal administration of streptozotocin in LH rats increased nonfasting glycemia and induced a marked glucose intolerance which was accompanied by further increases in BP, plasma cholesterol, and urinary protein excretion. None of the treatments was able to modify glucose tolerance in diabetic LH rats. The low dose of perindopril was ineffective in the prevention of hypertension, dyslipidemia, and proteinuria in diabetic LH rats, while the high dose of perindopril normalized the BP, reduced the plasma lipids, and lowered the proteinuria. However, in spite of significant reduction in BP, the triple therapy failed to improve dyslipidemia and proteinuria; on the contrary, the therapy worsened these two conditions. CONCLUSIONS: In diabetic LH rats, only ACE inhibition is of benefit to the kidney and lipidemia, thereby demonstrating that antihypertensive regimens may differ in their capacity to protect the target organs and lipid metabolism in a diabetic setting.

Neonatal streptozotocin-induced glucose intolerance: different consequences in Lyon normotensive and hypertensive rats.

BACKGROUND: Lyon hypertensive (LH) rats exhibit a mild hypertension associated with excessive body weight, spontaneous hyperlipidemia, elevated insulin/glucose ratio and exaggerated urinary protein excretion. AIMS: We aimed to develop, in LH rats and their normotensive control (LL) rats, a moderate non-insulin-dependent diabetic model to study the different consequences on metabolic and renal functions. METHODS: Non-insulin-dependent diabetes was induced by intraperitoneal injection of streptozotocin (STZ) at 2 days of age (50, 75 or 100 mg/kg for LH and 75, 100 or 125 mg/kg for LL rats). The evolution, with age, of glycemia, glucose tolerance (glucose 2 g/kg by gavage), blood pressure, plasma lipids and urinary protein and albumin excretions were studied in control and STZ-treated LH and LL rats. RESULTS: Although fasting glycemia was not significantly changed, the neonatal administration of STZ increased non-fasting glycemia and induced a marked glucose intolerance that were comparable between LH rats receiving 75 mg/kg and LL rats receiving 100 mg/kg of STZ. Interestingly, in treated LH rats only, the impaired glucose tolerance was accompanied by further metabolic and renal dysfunctions characterized by additional increases in plasma cholesterol (+28%) and triglycerides (+105%) and accelerated progression of proteinuria (+36%) and albuminuria (+48%). CONCLUSIONS: These observations indicate that susceptibility to diabetic metabolic disorders and renal diseases may be linked to the genetic predisposition to hypertension. This new model offers a reasonable reflection of the human situation, where hypertension and non-insulin-dependent diabetes often coincide, suitable for molecular, biochemical and pharmacological investigations.

Role of purinergic cotransmission in the sympathetic control of arterial pressure variability in conscious rats.

Previous studies have shown that the sympathetically mediated oscillations of arterial pressure (AP), the so-called Mayer waves, are shifted from 0.4 to 0.6 Hz after acute alpha-adrenoceptor blockade in conscious rats. This raises the possibility that, under physiological conditions, Mayer waves are mediated by the conjoint action of norepinephrine and other sympathetic cotransmitters. To evaluate the possible role of the cotransmitter ATP in determining the frequency of Mayer waves, AP and renal sympathetic nerve activity (RSNA) were simultaneously recorded in 10 conscious rats with cardiac autonomic blockade before and after acute blockade of P2 receptors with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. P2 receptor blockade did not alter the mean level and overall variability of AP and RSNA but shifted peak coherence between AP and RSNA from 0.43 +/- 0.02 to 0.22 +/- 0.01 Hz. A model of the sympathetic limb of the arterial baroreceptor reflex was designed to simulate Mayer waves at 0.2 and 0.6 Hz, with norepinephrine and ATP, respectively, acting as the sole sympathetic cotransmitter. When both cotransmitters acted in concert, a single oscillation was observed at 0.4 Hz when the gain ratio of the adrenergic to the purinergic components was set at 15. The model thus accounted for an important role for ATP in determining Mayer wave frequency, but not in sustaining the mean level of AP or controlling its overall variability.