In vitro modulation of glucagon-like peptide release by DPP-IV inhibitory polyphenol-polysaccharide conjugates of sprouted quinoa yoghurt.

Glucagon-like peptide-1 (GLP-1) is an important signal in the peripheral and neural systems, which contributes to the maintenance of glucose and energy homeostasis. In this study, 1H NMR validated polyphenols and polysaccharides extracted from sprouted quinoa yoghurt were used as isolates and conjugates to upregulate the stimulation of GLP-1 release in NCI-H716 cells. In addition, we explored their effect on proglucagon and prohormone convertase 3 mRNA expressions, HNF-3γ and CCK-2R gene protein expression, as well as cytosolic calcium release. Variations in concentration showed a dose-dependent GLP-1 stimulation, and were significantly optimized by germination. Proglucagon mRNA expression in NCI-H716 cells was upregulated, and was relatively highest with QYPSP1 treatments in a 2.68 fold. The results suggested that the conjugates had greater potential to stimulate GLP-1 release than their isolates. Sprouted quinoa yoghurt could therefore be a potential functional food useful to regulate glucose and energy homeostasis.

LDL as a therapeutic objective. / LDL como objetivo terapéutico.

The incidence of atherosclerotic cardiovascular disease has increased in the developed countries. Dyslipidemia is a primary major risk factor for atherosclerotic cardiovascular disease and LDL lowering is one of the main objectives. Although treatment goals for dyslipidemias should be personalized in every patient, statins are cost-effective in primary and secondary prevention of atherosclerotic cardiovascular disease. New treatments with higher power and greater decreases in LDL, PSCK9 inhibitors, have made a new breakthrough in atherosclerotic cardiovascular disease treatment. The 2019 guidelines for de management of dyslipidemias: lipid modification to reduce cardiovascular risk (European Society of Cardiology/European Atherosclerosis Society) with the level of evidence and the strength of the recommendations can facilitate the best decisions and benefits to our patients in clinical practice.

Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study.

Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.

Glucagon-like peptide-1 production in the GLUTag cell line is impaired by free fatty acids via endoplasmic reticulum stress.

OBJECTS: Glucagon-like peptide-1 (GLP-1) is secreted from intestinal L cells, enhances glucose-stimulated insulin secretion, and protects pancreas beta cells. However, few studies have examined hypernutrition stress in L cells and its effects on their function. Here, we demonstrated that a high-fat diet reduced glucose-stimulated secretion of GLP-1 and induced expression of an endoplasmic reticulum (ER) stress markers in the intestine of a diet-induced obesity mouse model. METHODS: To clarify whether ER stress in L cells caused the attenuation of GLP-1 secretion, we treated the mouse intestinal L cell line, GLUTag cells with palmitate or oleate. RESULTS: Palmitate, but not oleate caused ER stress and decreased the protein levels of prohormone convertase 1/3 (PC1/3), an essential enzyme in GLP-1 production. The same phenomena were observed in GLUTag cells treated with in ER stress inducer, thapsigargin. Moreover, oleate improved palmitate-induced ER stress, reduced protein and activity levels of PC1/3, and attenuated GLP-1 secretion from GLUTag cells. CONCLUSIONS/INTERPRETATION: These results suggest that the intake of abundant saturated fatty acids induces ER stress in the intestine and decreases GLP-1 production.

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2.

The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, ß-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.

Identification of a small molecule that selectively inhibits mouse PC2 over mouse PC1/3: a computational and experimental study.

The calcium-dependent serine endoproteases prohormone convertase 1/3 (PC1/3) and prohormone convertase 2 (PC2) play important roles in the homeostatic regulation of blood glucose levels, hence implicated in diabetes mellitus. Specifically, the absence of PC2 has been associated with chronic hypoglycemia. Since there is a reasonably good conservation of the catalytic domain between species translation of inhibitory effects is likely. In fact, similar results have been found using both mouse and human recombinant enzymes. Here, we employed computational structure-based approaches to screen 14,400 compounds from the Maybridge small molecule library towards mouse PC2. Our most remarkable finding was the identification of a potent and selective PC2 inhibitor. Kinetic data showed the compound to be an allosteric inhibitor. The compound identified is one of the few reported selective, small-molecule inhibitors of PC2. In addition, this new PC2 inhibitor is structurally different and of smaller size than those reported previously. This is advantageous for future studies where structural analogues can be built upon.

Decreased immunoreactivity of the polypeptide precursor pro-opiomelanocortin (POMC) and the prohormone convertase pc1/3 after chronic ethanol exposure in Sprague-Dawley rats.

BACKGROUND: The melanocortin (MC) peptides and opioid peptide ß-endorphin are cleaved from the polypeptide precursor pro-opiomelanocortin (POMC). POMC-derived peptides are generated by extensive posttranslational processing that involves several enzymes including prohormone convertase 1/3 and 2 (PC1/3 and PC2). Because ethanol (EtOH) decreases POMC mRNA levels, we determined whether the exposure to an EtOH-containing diet (ED) would significantly reduce central immunoreactivity (IR) of POMC, PC1/3, PC2, and ß-endorphin. METHODS: Male Sprague-Dawley rats were given 18 days of access to a normal rodent chow or a control diet (CD), or short-term (4 days) or long-term (18 days) access to an ED. At the end of the study, rats were perfused with 4% paraformaldehyde, and their brains were sectioned into sets for processing with POMC, PC1/3, PC2, and ß-endorphin IR. RESULTS: Rats exposed to an ED for 18 days (ED18) exhibited significant reductions of POMC and PC1/3 IR in the arcuate nucleus of the hypothalamus (Arc) relative to rats pair-fed a CD. On the other hand, rats exposed to an ED did not show any changes of central ß-endorphin or PC2 IR relative to rats pair-fed a CD, regardless of length of exposure. Because there were no differences in body weights or caloric intake between the CD and ED groups, reductions of POMC and PC1/3 IR in ED-treated rats are best explained by EtOH exposure rather than altered energy balance. CONCLUSIONS: This study shows that EtOH site-specifically reduces POMC and PC1/3 IR in rat brain. These observations are consistent with EtOH-induced reductions of α-melanocyte-stimulating hormone (α-MSH) and POMC IR that were previously reported. As MC agonists have been shown to blunt EtOH intake in rodents, exogenous MC receptor agonists, as well as targets that may increase the synthesis of endogenous α-MSH (e.g., PC1/3), may have therapeutic value for treating alcohol abuse disorders and alcoholism.

Regulation of neuropeptide processing enzymes by catecholamines in endocrine cells.

Treatment of cultured bovine adrenal chromaffin cells with the catecholamine transport blocker reserpine was shown previously to increase enkephalin levels severalfold. To explore the biochemical mechanism of this effect, we examined the effect of reserpine treatment on the activities of three different peptide precursor processing enzymes: carboxypeptidase E (CPE) and the prohormone convertases (PCs) PC1/3 and PC2. Reserpine treatment increased both CPE and PC activity in extracts of cultured chromaffin cells; total protein levels were unaltered for any enzyme. Further analysis showed that the increase in CPE activity was due to an elevated V(max), with no change in the K(m) for substrate hydrolysis or the levels of CPE mRNA. Reserpine activation of endogenous processing enzymes was also observed in extracts prepared from PC12 cells stably expressing PC1/3 or PC2. In vitro experiments using purified enzymes showed that catecholamines inhibited CPE, PC1/3, and PC2, with dopamine quinone the most potent inhibitor (IC(50) values of ∼50-500 µM); dopamine, norepinephrine, and epinephrine exhibited inhibition in the micromolar range. The inhibition of purified CPE with catecholamines was time-dependent and, for dopamine quinone, dilution-independent, suggesting covalent modification of the protein by the catecholamine. Because the catecholamine concentrations found to be inhibitory to PC1/3, PC2, and CPE are well within the physiological range found in chromaffin granules, we conclude that catecholaminergic transmitter systems have the potential to exert considerable dynamic influence over peptidergic transmitter synthesis by altering the activity of peptide processing enzymes.

Proteomic analysis of neuroendocrine peptidergic system disruption using the AtT20 pituitary cell line as a model.

Environmental pollutants may affect the activities of many cellular enzymes. The effect on the proteome of enzymatic inhibitors can be determined using two-dimensional (2D) gel electrophoresis. In neuroendocrine cells, proprotein convertases 1 and 2 (PC1 and PC2) mediate the proteolytic activation of many precursors to peptide hormones and neuropeptides. Enzymatic activities of these calcium-dependent proteinases are readily regulated by chelating agents and by heavy metals ions found in the environment. Such an inhibition could result in a potentially pathological disruption of the peptidergic system. We are interesting in finding out to what extent specific inhibition of these enzymes could affect the proteome of a neuroendocrine cell. To address this question, we used the mouse pituitary AtT20 cell line as a model. We compared the proteomic pattern of control cells to that of cells overexpressing proSAAS, a PC1-specific inhibitor. The comparison was conducted using two-dimensional (2D) gel electrophoresis, mass spectrometric identification of differing proteins and immunoblotting to confirm their identity. The 2D analysis revealed a number of alterations in the proteome of proSAAS-overexpressing cells. Mass spectrometric analysis of tryptic peptides identified two proteins found in more abundance in these cells as proSAAS and Ephrin type A receptor 2.

Evidence for proprotein convertase activity in the endoplasmic reticulum/early Golgi.

Processing of precursor proteins by the proprotein convertases is thought to occur mainly in the trans-Golgi network or post-Golgi compartments. Such cleavage is inhibited by the prosegment of the convertases. During our studies of the use of the inhibitory prosegment of PC1, we noticed that a construct containing the prosegment fused to the C-terminal secretory granule sorting domain was cleaved in the endoplasmic reticulum (ER) at a pair of basic residues, best recognized by furin and PC7. This was further confirmed when this construct was fused at the C-terminus with a KDEL ER-retention signal. This suggests that the convertases could cleave some substrates within the ER, possibly by displacing the inhibitory prosegment associated with them.