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PubMed2024

Lipid-lowering drugs and risk of rapid renal function decline: a mendelian randomization study.

BACKGROUND: Chronic kidney disease (CKD) patients face the risk of rapid kidney function decline leading to adverse outcomes like dialysis and mortality. Lipid metabolism might contribute to acute kidney function decline in CKD patients. Here, we utilized the Mendelian Randomization approach to investigate potential causal relationships between drug target-mediated lipid phenotypes and rapid renal function decline. METHODS: In this study, we utilized two methodologies: summarized data-based Mendelian randomization (SMR) and inverse variance-weighted Mendelian randomization (IVW-MR), to approximate exposure to lipid-lowering drugs. This entailed leveraging expression quantitative trait loci (eQTL) for drug target genes and genetic variants proximal to drug target gene regions, which encode proteins associated with low-density lipoprotein (LDL) cholesterol, as identified in genome-wide association studies. The objective was to investigate causal associations with the progression of rapid kidney function decline. RESULTS: The SMR analysis revealed a potential association between high expression of PCSK9 and rapid kidney function decline (OR = 1.11, 95% CI= [1.001-1.23]; p = 0.044). Similarly, IVW-MR analysis demonstrated a negative association between LDL cholesterol mediated by HMGCR and kidney function decline (OR = 0.74, 95% CI = 0.60-0.90; p = 0.003). CONCLUSION: Genetically predicted inhibition of HMGCR is linked with the progression of kidney function decline, while genetically predicted PCSK9 inhibition is negatively associated with kidney function decline. Future research should incorporate clinical trials to validate the relevance of PCSK9 in preventing kidney function decline.

Mendelian Randomization Analysis↗
PubMed2004

Biosynthesis and secretion of insect lipoprotein: involvement of furin in cleavage of the apoB homolog, apolipophorin-II/I.

The biosynthesis of neutral fat-transporting lipoproteins involves the lipidation of their nonexchangeable apolipoprotein. In contrast to its mammalian homolog apolipoprotein B, however, insect apolipophorin-II/I (apoLp-II/I) is cleaved posttranslationally at a consensus substrate sequence for furin, resulting in the appearance of two apolipoproteins in insect lipoprotein. To characterize the cleavage process, a truncated cDNA encoding the N-terminal 38% of Locusta migratoria apoLp-II/I, including the cleavage site, was expressed in insect Sf9 cells. This resulted in the secretion of correctly processed apoLp-II and truncated apoLp-I. The cleavage could be impaired by the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (decRVKRcmk) as well as by mutagenesis of the consensus substrate sequence for furin, as indicated by the secretion of uncleaved apoLp-II/I-38. Treatment of L. migratoria fat body, the physiological site of lipoprotein biosynthesis, with decRVKRcmk similarly resulted in the secretion of uncleaved apoLp-II/I, which was integrated in lipoprotein particles of buoyant density identical to wild-type high density lipophorin (HDLp). These results show that apoLp-II/I is posttranslationally cleaved by an insect furin and that biosynthesis and secretion of HDLp can occur independent of this processing step. Structure modeling indicates that the cleavage of apoLp-II/I represents a molecular adaptation in homologous apolipoprotein structures. We propose that cleavage enables specific features of insect lipoproteins, such as low density lipoprotein formation, endocytic recycling, and involvement in coagulation.

Amino Acid Chloromethyl Ketones↗
PubMed2005

Saturated fat-induced changes in Sf 60-400 particle composition reduces uptake of LDL by HepG2 cells.

The ability of human postprandial triacylglycerol-rich lipoproteins (TRLs), isolated after meals enriched in saturated fatty acids (SFAs), n-6 PUFAs, and MUFAs, to inhibit the uptake of 125I-labeled LDL by the LDL receptor was investigated in HepG2 cells. Addition of TRLs resulted in a dose-dependent inhibition of heparin-releasable binding, cell-associated radioactivity, and degradation products of 125I-labeled LDL (P < 0.001). SFA-rich Svedberg flotation rate (Sf) 60-400 resulted in significantly greater inhibition of cell-associated radioactivity than PUFA-rich particles (P = 0.016) and total uptake of 125I-labeled LDL compared with PUFA- and MUFA-rich particles (P < 0.02). Normalization of the apolipoprotein (apo)E but not apoC-III content of the TRLs removed the effect of meal fatty acid composition, and addition of an anti-apoE antibody reversed the inhibitory effect of TRLs on the total uptake of 125I-labeled LDL. Real time RT-PCR showed that the SFA-rich Sf 60-400 increased the expression of genes involved in hepatic lipid synthesis (P < 0.05) and decreased the expression of the LDL receptor-related protein 1 compared with MUFAs (P = 0.008). In conclusion, these findings suggest an alternative or additional mechanism whereby acute fat ingestion can influence LDL clearance via competitive apoE-dependent effects of TRL on the LDL receptor.

Adult↗
PubMed2002

Oligonucleotide microarray analysis of intact human pancreatic islets: identification of glucose-responsive genes and a highly regulated TGFbeta signaling pathway.

Human pancreatic islets are a major focus of diabetes research due to their key role in glucose homeostasis and their potential for transplantation in the treatment of type 1 diabetes. Currently, no comprehensive analysis of baseline or glucose-stimulated islet gene expression is available. Using oligonucleotide microarrays we analyzed isolated intact human islets incubated at low and high glucose. We identified approximately 6000 islet genes, several with clinical implications, as well as a number of glucose-regulated genes. Interestingly, two transforming growth factor beta (TGFbeta) superfamily members were highly regulated by glucose. One of them, PDF, was found to have a very high expression level compared to other TGFbeta superfamily members. Quantitative reverse transcriptase polymerase chain reaction confirmed these results and demonstrated that the highly expressed PDF was approximately 10-fold down- regulated by glucose while other TGFbeta superfamily members and target genes were up-regulated. These results suggest that a highly regulated TGFbeta signaling cascade exists in human islets, and that PDF may play a central role in islet biology. Since TGFbeta is involved in differentiation and immune modulation, this novel pathway may link glucose metabolism, immune response and development of human islets. We report here the first gene expression profile of intact human islets. These and similar analyses will provide better understanding of human islet biology and enhance the development of novel diabetes therapies.

Amyloid↗
PubMed2004

Molecular cloning and developmental expression of corticotropin-releasing factor in the chicken.

We have characterized the structure of the chicken corticotropin-releasing factor (CRF) gene through cDNA cloning and genomic sequence analysis, and we analyzed the expression of CRF mRNA and peptide in the diencephalon of the chick throughout embryonic development. The structure of the chicken CRF gene is similar to other vertebrate CRF genes and contains two exons and a single intron. The primary structure of the mature chicken CRF peptide is identical to human and rat CRF. This is the first archosaurian CRF gene to be characterized. We used RIAs to analyze CRF peptide content in the diencephalon and the median eminence and plasma corticosterone during the last week of embryonic development. We also developed a semiquantitative RT-PCR method to analyze the expression of CRF mRNA during the same period. CRF peptide content in the diencephalon increased, whereas peptide content in the ME decreased just before hatching, suggesting that release and biosynthesis are coupled. Plasma corticosterone concentration significantly increased between embryonic d 20 and the first day post hatch. By contrast, CRF mRNA levels in the diencephalon decreased just before hatching. Changes in CRF production just before hatching may be causally related to the regulation of the thyroid and interrenal axes at this stage of chicken development.

Aging↗
PubMed1982

Proopiocortin-converting enzyme activity in bovine neurosecretory granules.

Neurosecretory granules (NSGs) from neural lobes of bovine pituitary glands were isolated in a highly purified form by metrizamide-sucrose gradient centrifugation. The purified NSGs were lysed and centrifuged, and the supernatants were further fractionated by gel filtration on Sephadex G-75. Proopiocortin-converting enzyme activity was assayed by incubation of [3H]arginine- or [3H]phenylalanine-labeled toad proopiocortin with NSG supernatant fractions. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea gel electrophoresis. The optimum pH for the enzyme-mediated conversion was around pH 5.0. Conversion of toad proopiocortin by NSG converting enzyme activity was inhibited by leupeptin, antipain, p-chloromercuribenzoate, and pepstatin A, but not by diisopropyl fluorophosphate, EDTA, or N-alpha-p-tosyl-L-lysine-chloromethyl ketone HCl. The results suggest that the proopiocortin-converting enzyme activity in bovine neurosecretory granules is due to an acid-thiol protease which may contain secondary hydrophobic binding sites that are involved in substrate recognition.

Adrenocorticotropic Hormone↗
PubMed1983

Characterization of proopiocortin converting activity in rat anterior pituitary secretory granules.

Lysates from purified secretory granules of rat anterior pituitary glands were incubated with [3H]phenylalanine or [3H]arginine-labeled toad proopiocortin. The processed products formed were identified by immunoprecipitation with ACTH and beta-endorphin antisera, and by comigration with known markers on acid-urea polyacrylamide gels. Proopiocortin was cleaved by the secretory granule lysate primarily to 21,000 mol wt ACTH, 13,000 mol wt ACTH, 16,000 mol wt NH2-terminal glycopeptide, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin. Characterization of the anterior pituitary proopicortin-converting activity shows that it: (1) cleaves specifically at the peptide bond on the carboxy side of the lysine-arginine residues of proopiocortin, (2) has a pH optimum in the acidic range, (3) is present in membrane and soluble fractions of the granule lysate, and (4) is inhibited by leupeptin, pepstatin A, and 2,2' dithiodipyridine, but not by p-chloromercuribenzoate, diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, L-1-tosylamide-2-phenylethyl-chloromethyl ketone, or EDTA.

Adrenocorticotropic Hormone↗
PubMed1984

In vitro processing of proopiocortin by membrane-associated and soluble converting enzyme activities from rat intermediate lobe secretory granules.

Secretory granules (SGs) from rat intermediate lobes (IL) were isolated in a highly purified form by differential centrifugation, followed by sucrose density gradient centrifugation. The purified IL-SGs were lysed by freezing and thawing. The granule lysate was then centrifuged to generate membrane and soluble fractions. Proopiocortin -converting enzyme (PCE) activity was assayed by incubation of [3H]arginine- or [3H] phenylalanine-labeled toad proopiocortin with the total granule lysate, the membrane, or the soluble fraction at pH 5.0. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea-gel electrophoresis. The PCE activity in rat IL-SG lysate cleaved proopiocortin to 21,000 mol wt ACTH, 21,000 mol wt ACTH/lipotropin (LPH), 13,000 mol wt ACTH, beta LPH, beta-endorphin-like peptides, and alpha MSH-like peptides, similar to those synthesized by the toad intermediate lobe in situ. Treatment of the PCE cleavage products with carboxypeptidase B resulted in the liberation of free arginine. This observation together with the nature of the products formed suggest that the PCE activity cleaved at pairs of basic residues of proopiocortin , yielding one or more products that terminated with an arginine or an arginine-lysine. PCE activity was found in membrane and soluble granule fractions, and both activities were inhibited by leupeptin, p-chloromercuribenzoate, dithiodipyridine, and pepstatin A, but not by chloroquine or N-alpha-p-tosyl-L-lysine-chloromethylketone HCl. Diisopropyl fluorophosphate and other thiol protease reagents (p-chloromercuriphenyl sulfonic acid, iodoacetic acid, and HgCl2) had a small inhibitory effect. The products formed by PCE activities in the membrane and soluble fractions were similar to those cleaved by the total granule lysate. The membrane fraction primarily cleaved proopiocortin between ACTH and beta LPH to form 21,000 (21 K) mol wt ACTH and beta-LPH, similar to the first processing step in the IL in situ. The soluble fraction, however, showed a greater tendency to cleave proopiocortin between the 16 K N-terminal glycopeptide and ACTH, to yield twice as much 21 K ACTH/LPH product as the membrane fraction. The membrane-associated PCE activity was found to be easily solubilized by extraction with high salt (1 M NaCl), suggesting that it is not an integral granule membrane protein.

Adrenocorticotropic Hormone↗
PubMed1991

Identification of a somatostatin-14-generating propeptide converting enzyme as a member of the kex2/furin/PC family.

A propeptide converting enzyme capable of producing somatostatin-14 has been identified and partially characterized from anglerfish pancreatic islets. Results from N-terminal protein sequence analysis indicate that this enzyme is a member of the kex2/furin/PC family. This observation provides strong corroborative evidence that the kex2/furin/PC protease family is involved in propeptide conversion. Comparison of the obtained protein sequence with the cDNA sequence of mammalian PC2 also suggests that the active enzyme is derived from a precursor by cleavage at a site containing four consecutive basic amino acids.

Amino Acid Sequence↗
PubMed1994

Characterization of the effects of retinoic acid on vasoactive intestinal polypeptide gene expression in neuroblastoma cells.

The neuropeptide vasoactive intestinal polypeptide (VIP) has a broad range of functions, and its expression has been correlated with neuronal differentiation. Here we present data on the effects of retinoic acid (RA), a known modulator of neuronal differentiation, on VIP gene expression in the human neuroblastoma cell line NB-1. Morphological data, surprisingly, indicate that these cells are not differentiated concomitant with the increase in VIP gene expression. RA was found to exert a concentration-dependent induction of peptides derived from the VIP precursor molecule, prepro-VIP. The effect at both the messenger RNA (mRNA) level, evaluated by Northern blots, and the peptide level, measured by RIAs, was found to be slow and long lasting. No changes in the processing of prepro-VIP were observed using gel chromatography and RIAs specific for various prepro-VIP sequences. Also, the expression of mRNA for the prohormone-processing enzyme PC2, present in these cells, was not altered by RA. The lag period preceding the increase in VIP mRNA led to experiments with the translational inhibitor cycloheximide showing an indirect effect of RA on VIP mRNA expression. Northern blots revealed that at least three mRNAs encoding RA receptor were expressed and rapidly induced by RA in the cells, thus making them possible candidates for the intermediate protein(s) required from the induction of VIP gene expression.

Aspartic Acid Endopeptidases↗
PubMed1995

Distribution of the Kexin family proteases in pancreatic islets: PACE4C is specifically expressed in B cells of pancreatic islets.

The distribution of Kexin family proteases in adult rat pancreatic islets was investigated by immunohistochemical means using a series of specific antibodies specific for PC1, PC2, PC6, Furin, PACE4A and a recently identified member of the Kexin family, PACE4C. PACE4C expression was limited to B cells of the pancreatic islets. PC2 was found in A and in some D cells more than in B cells and PC1 was evident only in B cells. Furin and PC6 were weakly and evenly expressed in the entire islet. PACE4A was hardly found in the islets. These findings indicated that individual Kexin family proteases are uniquely distributed in the islets and suggested that these proteases share roles in these cells as follows: PC2 is involved in the peptide hormone precursor processing in A cells and in D cells, and PACE4C, PC1 and PC2 (mainly PACE4C and PC1) are responsible for the processing event(s) specific to B cells.

Animals↗
PubMed1996

Immunological identification and localization of yeast aspartic protease 3-like prohormone-processing enzymes in mammalian brain and pituitary.

The novel aspartic proteases, yeast aspartic protease 3 and the mammalian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites. We show that an antibody against yeast aspartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on Western blot, indicating structural homology between these two enzymes, but not with other aspartic proteases, such as renin or cathepsin D. A PCE-sized anti-YAP3p-immunoreactive band was detected on Western blots of bovine intermediate lobe where PCE activity has been found. YAP3p antiserum also cross-reacted with a protein of approximately 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituitary secretory granules. Distribution studies showed the presence of anti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich brain regions, including the mouse arcuate nucleus and hippocampus and the rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, and reticular nucleus. In the bovine intermediate pituitary, a subpopulation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to contain POMC messenger RNA (mRNA). Only a subpopulation of cells was immunopositive for anti-YAP3p in bovine anterior pituitary, and most of these cells were identified by double immunostaining with ACTH antiserum as corticotrophs. In situ hybridization in combination with immunocytochemistry provided evidence for the localization of arginine vasopressin mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cells in the rat cortex and hippocampus. These results support the hypothesis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.

Animals↗
PubMed1996

Neural and glial-mediated effects of growth factors acting via tyrosine kinase receptors on luteinizing hormone-releasing hormone neurons.

It is becoming increasingly evident that the secretory activity of LHRH neurons is regulated not only by transsynaptic inputs but also by trophic molecules of glial and neuronal origin. The present experiments were undertaken to gain insights into the potential cell-cell mechanisms by which basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha), two growth factors produced in the hypothalamus, may affect LHRH neuronal function. Northern blot analysis showed that the LHRH-producing cell line GT1-7 contains the messenger RNA (mRNA) encoding the type 1 fibroblast growth factor receptor (FGFR-1) but not that encoding the epidermal growth factor (EGF) receptors, which mediates the biological actions of both TGF alpha and EGF. Ligand-induced receptor phosphorylation experiments demonstrated that GT1-7 cells possess biologically active FGFR-1s but not EGF receptors. Exposure of the cells to bFGF resulted not only in FGFR-1 tyrosine phosphorylation, but also in tyrosine phosphorylation of phospholipase C gamma, one of the initial enzymes in the intracellular signaling cascade initiated by FGFR activation. GT1-7 cells proliferated in response to this activation. Despite the presence of biologically active receptors, bFGF did not significantly stimulate release of the mature LHRH decapeptide. Instead, bFGF increased the steady-state levels of the mRNA encoding the LHRH precursor processing endoprotease PC2, with a time course comparable to that of phorbol esters, suggesting that, as shown in the companion paper, the actions of the growth factor on LHRH neurons involve facilitation of the initial step in LHRH prohormone processing. The increase in PC2 gene expression was not accompanied by changes in LHRH mRNA levels. Unlike these direct actions of bFGF on GT-1 cells, TGF alpha appears to act indirectly via astroglial intermediacy. Exposure of GT1-7 cells to TGF alpha or EGF failed to affect several parameters of cellular activity including LHRH release, LHRH and PC2 mRNA levels, and cell proliferation. In contrast, astrocyte culture medium conditioned by treatment with TGF alpha led to sustained stimulation of LHRH release with no changes in LHRH gene expression and a transient increase in PC2 mRNA levels. Although no definitive evidence for the presence of FGFR-1 in normal LHRH neurons could be obtained by either double immunohistochemistry or double in situ hybridization procedures, fetal LHRH neurons in primary culture responded to bFGF with neurite outgrowth. Thus, normal LHRH neurons may have an FGFR-1 content too low for detection by regular histochemical procedures, and/or detectable expression of the receptor may be confined to a much earlier developmental stage. The mitogenic effect of bFGF on GT1-7 cells supports this possibility and suggests a role for FGF in the cell proliferation events that precede acquisition of the LHRH neuronal phenotype. It appears that once this phenotype is established, bFGF may promote the differentiation of LHRH neurons. The results also suggest that the secretory capacity of LHRH neurons develops under a dual trophic influence, one on peptide processing exerted directly by bFGF on early neurons, and another on LHRH release, exerted by TGF alpha via the intermediacy of astroglial cells.

Animals↗
PubMed2006

Genetics of obesity in humans.

Considerable attention has focused on deciphering the hypothalamic pathways that mediate the behavioral and metabolic effects of leptin. We and others have identified several single gene defects that disrupt the molecules in the leptin-melanocortin pathway causing severe obesity in humans. In this review, we consider these human monogenic obesity syndromes and discuss how far the characterization of these patients has informed our understanding of the physiological role of leptin and the melanocortins in the regulation of human body weight and neuroendocrine function.

Body Weight↗
PubMed1990

The yeast KEX-2-processing endoprotease is active in the Golgi apparatus of transfected NIH 3T3 fibroblasts.

Proteolytic processing of polyprotein precursors at pairs of basic amino acids is a prerequisite for the generation of bioactive peptide hormones. While the mammalian endoproteases responsible for these cleavages are yet to be identified, this function has been unequivocally assigned in yeast to the product of the KEX-2 gene. To study the molecular mechanisms involved in polyprotein processing, we have transfected the yeast KEX-2 gene into mouse NIH 3T3 fibroblasts and established a new cell line (called 2N-DK) where the KEX-2 endoprotease is permanently expressed. Immunofluorescence studies show that the KEX-2 enzyme is retained within the Golgi of the 2N-DK cells. The evidence for this cellular location is supported by measurement of intracellular and extracellular KEX-2 enzyme activity. In this permanently transfected cell line, KEX-2 activity is exclusively intracellular, in contrast to the situation previously described in transiently infected cell lines, where extracellular KEX-2 activity was detected. Furthermore, infection of 2N-DK cells with a recombinant retrovirus expressing a cDNA coding for porcine proopiomelanocortin (POMC) resulted in the synthesis of POMC and its efficient processing into beta-lipotropin and beta-endorphin, two of its physiologically authentic maturation products. These results suggest that in the fibroblast cell line 2N-DK, proteolytic processing of POMC by KEX-2 endoprotease occurs in the Golgi apparatus.

Amino Acid Sequence↗
PubMed2004

Monogenic human obesity syndromes.

Over the past decade, we have witnessed a major increase in the scale of scientific activity devoted to the study of energy balance and obesity. This explosion of interest has, to a large extent, been driven by the identification of genes responsible for murine obesity syndromes and the novel physiological pathways revealed by those genetic discoveries. We and others recently have identified several single-gene defects causing severe human obesity. Many of these defects have occurred in molecules identical or similar to those identified as a cause of obesity in rodents. This chapter will consider the human monogenic obesity syndromes that have been characterized to date and discuss how far such observations support the physiological role of these molecules in the regulation of human body weight and neuroendocrine function.

Energy Metabolism↗
PubMed1993

Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment.

One of the Golgi compartments of Saccharomyces cerevisiae is defined by the presence of a specific endoproteinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi compartment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.

Carboxypeptidases↗