Variable effects of gender and Western diet on lipid and glucose homeostasis in aged PCSK9-deficient C57BL/6 mice CSK9PC57BL/6.

BACKGROUND: Proprotein convertase subtilisin/kexin-type 9 (PCSK9) downregulates clearance of plasma cholesterol by liver. Its inactivation increases this clearance, reducing cardiovascular risk. However, a lack of PCSK9 could also lead to cholesterol accumulation in pancreatic islet beta cells, impairing insulin secretion. We reported earlier that 4-month-old male PCSK9-deficient (KO) C57BL/6 mice were hyperglycemic and insulin-insufficient relative to their wild-type (WT) counterparts. Here, we examined how gender and diet affect lipid and glucose homeostasis in these mice at 8 months of age. METHODS: After being fed a normal diet or a Western diet for over 6 months, KO mice were compared with same-gender WT mice for fasting plasma levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose, and insulin; for glucose disposal and glucose-stimulated insulin secretion (GSIS); and for pancreatic islet morphology. RESULTS: A. Females: On normal diet, KO mice showed lower plasma TC, HDL-C, and LDL-C, higher plasma glucose, and normal glucose disposal despite impaired GSIS. On Western diet, they showed comparable plasma TC and HDL-C, but lower LDL-C, higher plasma glucose, and normal glucose disposal despite impaired GSIS. B. Males: On normal and Western diets, KO mice showed lower plasma TC, HDL-C, and LDL-C, similarly elevated plasma glucose, glucose intolerance, and impaired GSIS. C. Both: KO mice on either diet showed pancreatic islet dysmorphism, with larger, possibly immature secretory granules. CONCLUSIONS: Lower LDL-C and impaired GSIS are two major phenotypes in aged PCSK9-deficient C57BL/6 mice. These phenotypes are modulated by gender and diet.

The precursor to the germ cell-specific PCSK4 proteinase is inefficiently activated in transfected somatic cells: evidence of interaction with the BiP chaperone.

Proprotein convertase subtilisin/kexin type 4 (PCSK4), also known as proprotein convertase 4 (PC4), is a serine endoproteinase primarily expressed in testicular germ cells and in sperm. Inactivation of its gene in mouse causes male infertility. From studies of the biosynthesis of PCSK3/furin, its closest relative, it has been inferred that PCSK4 is synthesised in the endoplasmic reticulum as a zymogen; that it is rapidly matured by autocatalytic cleavage between the prodomain and the catalytic domain; that the cleaved prodomain remains attached to the mature enzyme; and that the enzyme is finally activated by the removal of the prodomain peptides following a secondary cleavage within the prodomain. In this study, we used human embryonic kidney 293 (HEK293) cells to study the biosynthesis of rat or human PCSK4. Our results show that the bulk of PCSK4 remains as an intracellular zymogen, presumably trapped in the endoplasmic reticulum, where it interacts with the general molecular chaperone glucose-regulated protein 78/Immunoglobulin heavy-chain binding protein (GRP78/BiP). These data suggest that, unlike other members of the convertase family, proPCSK4 cannot efficiently self-activate in somatic cells. These cells may lack the intracellular environment and the interacting molecules specific to testicular germ cells where this enzyme is normally expressed.

PCSK4-null sperm display enhanced protein tyrosine phosphorylation and ADAM2 proteolytic processing during in vitro capacitation.

OBJECTIVE: To study the molecular basis for the accelerated capacitation rate in PCSK4-null sperm. DESIGN: Comparative and controlled experimental research study. SETTING: Academic medical institute. ANIMAL(S): Male mice C57BL/6J wild-type or null congenics for the Pcsk4 allele. INTERVENTION(S): Cauda and epididymal sperm were capacitated for varying times. MAIN OUTCOME MEASURE(S): Differences in sperm protein tyrosine phosphorylation and proteolytic processing of sperm-egg ligands ADAM2 and ADAM3. RESULT(S): The PCSK4-null sperm proteins are hyper-tyrosine phosphorylated during capacitation. This hyperphosphorylation is dependent on protein kinase A (PKA), albumin, and calcium. There is also more ADAM2 proteolytic processing from a 46-kDa form of ADAM2 to a 27-kDa form in PCSK4-null sperm than in wild-type sperm. This processing is dependent on cholesterol efflux. CONCLUSION(S): Lack of PCSK4 is associated with quantitative changes in the phosphorylation and proteolysis of sperm proteins during capacitation; therefore, alterations in signal transduction and proteolytic processing during capacitation may underlie the fertilization incompetence of PCSK4-null sperm. More investigation is needed to determine how and to what extent these changes might contribute to the loss of fertilizing ability of PCSK4-null sperm.

Proprotein convertase subtilisin/kexin type 4 in mammalian fertility: a review.

BACKGROUND: Proprotein convertase subtilisin/kexin type 4 (PCSK4), also known as proprotein convertase 4, belongs to a family of endoproteinases involved in the proteolytic conversion of secretory precursor proteins to their active forms. Its amino acid sequence is highly conserved in mammals, an indication of its biological importance. METHODS: We have searched PubMed and molecular biology databases for information relating to the structure, expression and biological functions of PCSK4. RESULTS: PCSK4 is predominantly expressed in male germ cells and located on the plasma membrane overlying the acrosome of sperm. It is also present in ovary and placenta. Inactivation of its gene in mouse does not alter spermatogenesis, but renders sperm incapable of fertilizing oocytes. This incapacity results in part from sperm susceptibility to a premature acrosome reaction and their reduced ability to bind to the zona pellucida. In female mice, a lack of PCSK4 causes subfertility associated with impaired folliculogenesis. In addition, this enzyme has been shown to stimulate the invasiveness of human placental trophoblasts in culture, suggesting that it may facilitate placentation in vivo. CONCLUSIONS: PCSK4 appears to be a crucial enzyme for reproduction. Alterations of PCSK4 expression or activity could be the underlying cause of some unexplained cases of human infertility. Conversely, inactivation of this protease represents a potential strategy for non-hormonal contraception.

Plasma PCSK9 levels correlate with cholesterol in men but not in women.

Proprotein convertase subtilisin kexin-like 9 (PCSK9) is a secreted glycoprotein that negatively regulates low density lipoprotein receptor (LDLR) levels. Several single nucleotide polymorphisms (SNPs) in PCSK9 have been linked to autosomal dominant hypercholesterolemia (ADH). Conversely, hypocholesterolemia associates with both 'loss of function' nonsense and missense SNPs in PCSK9. We examined the association of plasma PCSK9 with lipoprotein parameters in 182 normolipidemics. For men (n=98) plasma PCSK9 averaged 6.08+/-1.96 microg/ml and Spearman analysis revealed significant correlation between it and total cholesterol (TC), LDLC, and TC/high density lipoprotein (HDLC) (r=0.276, 0.282, and 0.228, respectively). For women (n=84) plasma PCSK9 averaged 6.46+/-1.99 microg/ml having no correlation with TC, LDLC or TC/HDLC. The ratio of plasma PCSK9/LDLC increased in men carrying 'loss of function' PCSK9 variations. Our results suggest a gender difference in PCSK9 regulation and function with PCSK9 correlated to TC and LDLC in men but not women.

Differences of pancreatic expression of 7B2 between C57BL/6J and C3H/HeJ mice and genetic polymorphisms at its locus (Sgne1).

C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.

Sperm from mice genetically deficient for the PCSK4 proteinase exhibit accelerated capacitation, precocious acrosome reaction, reduced binding to egg zona pellucida, and impaired fertilizing ability.

The gene for proprotein convertase subtilisin/kexin-like 4 (PCSK4, previously known as PC4) is primarily transcribed in testicular spermatogenic cells. Its inactivation in mouse causes severe male subfertility. To better understand the reproductive function of PCSK4, we examined its subcellular localization in the testicular epithelium via immunohistochemistry, and on intact sperm via indirect immunofluorescence and immunoelectron microscopy. PCSK4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome. We also investigated PCSK4 relevance for sperm acquisition of fertilizing ability by comparing wild-type and PCSK4-null sperm for their abilities in capacitation, acrosome reaction, and egg binding in vitro. PCSK4-null sperm underwent capacitation at a faster rate; they were induced to acrosome react by lower concentrations of zona pellucida; and their egg-binding ability was only half that of wild-type sperm. These sperm physiologic anomalies likely contribute to the severe subfertility of PCSK4-deficient male mice.