Loss of hypothalamic Furin affects POMC to proACTH cleavage and feeding behavior in high-fat diet-fed mice.

OBJECTIVE: The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. METHODS: POMC-Cre+/- mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. RESULTS: In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. CONCLUSIONS: These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.

Mitochondrial Dynamics and Microglia as New Targets in Metabolism Regulation.

Energy homeostasis regulation is essential for the maintenance of life. Neuronal hypothalamic populations are involved in the regulation of energy balance. In order play this role, they require energy: mitochondria, indeed, have a key role in ensuring a constant energy supply to neurons. Mitochondria are cellular organelles that are involved in dynamic processes; their dysfunction has been associated with many diseases, such as obesity and type 2 diabetes, indicating their importance in cellular metabolism and bioenergetics. Food intake excess can induce mitochondrial dysfunction with consequent production of reactive oxygen species (ROS) and oxidative stress. Several studies have shown the involvement of mitochondrial dynamics in the modulation of releasing agouti-related protein (AgRP) and proopiomelanocortin (POMC) neuronal activity, although the mechanisms are still unclear. However, recent studies have shown that changes in mitochondrial metabolism, such as in inflammation, can contribute also to the activation of the microglial system in several diseases, especially degenerative diseases. This review is aimed to summarize the link between mitochondrial dynamics and hypothalamic neurons in the regulation of glucose and energy homeostasis. Furthermore, we focus on the importance of microglia activation in the pathogenesis of many diseases, such as obesity, and on the relationship with mitochondrial dynamics, although this process is still largely unknown.

Seasonal consumption of polyphenol-rich fruits affects the hypothalamic leptin signaling system in a photoperiod-dependent mode.

Leptin has a central role in the maintenance of energy homeostasis, and its sensitivity is influenced by both the photoperiod and dietary polyphenols. The aim of this study was to investigate the effect of seasonal consumption of polyphenol-rich fruits on the hypothalamic leptin signaling system in non-obese and obese animals placed under different photoperiods. Non-obese and diet-induced obese male Fischer 344 rats were placed under either a short-day (SD) or long-day (LD) photoperiod and were supplemented with either 100 mg/kg of lyophilized red grapes or cherries. In non-obese animals, both fruits reduced energy balance independent of the photoperiod to which they were placed. However, the hypothalamic gene expression of Pomc was significantly up-regulated only in the SD photoperiod. In contrast, in obese animals only cherry significantly decreased the energy balance, although both fruits were able to counteract the diet-induced increase in hypothalamic AgRP mRNA levels when consumed during the SD photoperiod. In conclusion, the consumption of rich-polyphenol fruits may increase leptin sensitivity through the modulation of the hypothalamic leptin signal pathway mainly when consumed in the SD photoperiod. Therefore, fruit seasonality should be considered, as it can influence energy homeostasis and obesity.

Blackcurrant Extract Ameliorates Hyperglycemia in Type 2 Diabetic Mice in Association with Increased Basal Secretion of Glucagon-Like Peptide-1 and Activation of AMP-Activated Protein Kinase.

Blackcurrants are berries that contain high levels of anthocyanins, particularly delphinidin 3-rutinoside (D3R). Several studies have reported that the consumption of blackcurrant extract (BCE) lowers blood glucose levels and ameliorates glucose tolerance, but the mechanism underlying this effect remains unclear. Glucagon-like peptide-1 (GLP-1) and AMP-activated protein kinase (AMPK) are considered one of the most significant molecular targets for the prevention and treatment of type 2 diabetes. In this study, we showed that dietary BCE significantly reduced blood glucose concentration and improved glucose tolerance in type 2 diabetic mice (KK-Ay). The basal GLP-1 concentration in plasma was significantly increased in the BCE group accompanied by upregulation of prohormone convertase 1/3 (PC1/3), the enzyme that processes intestinal proglucagon. Moreover, the level of phospho-AMPKα protein in skeletal muscle was significantly increased in the BCE group, and this was increase accompanied by significant upregulation of glucose transporter 4 (Glut4) proteins in the plasma membrane of BCE group. In conclusion, dietary BCE significantly reduced blood glucose concentration and improved glucose tolerance in association with increased basal GLP-1 concentration in plasma, upregulation of PC1/3 expression, and translocation of Glut4 to the plasma membrane of skeletal muscle in type 2 diabetic mice; furthermore, these effects were accompanied by activation of AMPK. Our findings demonstrated that D3R-rich BCE may help prevent diabetes and allow the dosages of diabetes drugs to be reduced.

Genetic Variants of LDLR and PCSK9 Associated with Variations in Response to Antihypercholesterolemic Effects of Armolipid Plus with Berberine.

BACKGROUND: Armolipid Plus (AP) is a nutraceutical that contains policosanol, fermented rice with red yeast, berberine, coenzyme Q10, folic acid, and astaxanthin. It has been shown to be effective in reducing plasma LDL cholesterol (LDLc) levels. In the multicenter randomized trial NCT01562080, there was large interindividual variability in the plasma LDLc response to AP supplementation. We hypothesized that the variability in LDLc response to AP supplementation may be linked to LDLR and PCSK9 polymorphisms. MATERIAL AND METHODS: We sequenced the LDLR 3' and 5' untranslated regions (UTR) and the PCSK9 5' UTR of 102 participants with moderate hypercholesterolemia in trial NCT01562080. In this trial, 50 individuals were treated with AP supplementation and the rest with placebo. RESULTS: Multiple linear regression analysis, using the response of LDLc levels to AP as the dependent variable, revealed that polymorphisms rs2149041 (c.-3383C>G) in the PCSK9 5' UTR and rs14158 (c.*52G>A) in the LDLR 3' UTR explained 14.1% and 6.4%, respectively, of the variability after adjusting for gender, age, and BMI of individuals. Combining polymorphisms rs2149041 and rs14158 explained 20.5% of this variability (p < 0.004). CONCLUSIONS: Three polymorphisms in the 3' UTR region of LDLR, c.*52G>A, c.*504G>A, and c.*773A>G, and two at the 5' UTR region of PCSK9, c.-3383C>G and c.-2063A>G, were associated with response to AP. These results could explain the variability observed in the response to berberine among people with moderate hypercholesterolemia, and they may be useful in identifying patients who could potentially benefit from supplementation with AP.

Polydatin ameliorates lipid and glucose metabolism in type 2 diabetes mellitus by downregulating proprotein convertase subtilisin/kexin type 9 (PCSK9).

BACKGROUND: Abnormalities in lipid and glucose metabolism are constantly observed in type 2 diabetes. However, these abnormalities can be ameliorated by polydatin. Considering the important role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in metabolic diseases, we explore the possible mechanism of polydatin on lipid and glucose metabolism through its effects on PCSK9. METHODS: An insulin-resistant HepG2 cell model induced by palmitic acid (PA) and a db/db mice model were used to clarify the role of polydatin on lipid and glucose metabolism. RESULTS: In insulin-resistant HepG2 cells, polydatin upregulated the protein levels of LDLR and GCK but repressed PCSK9 protein expression, besides, polydatin also inhibited the combination between PCSK9 and LDLR. Knockdown and overexpression experiments indicated that polydatin regulated LDLR and GCK expressions through PCSK9. In the db/db mice model, we found that polydatin markedly enhanced GCK and LDLR protein levels, and inhibited PCSK9 expression in the liver. Molecular docking assay was further performed to analyze the possible binding mode between polydatin and the PCSK9 crystal structure (PDB code: 2p4e), which indicated that steady hydrogen bonds formed between polydatin and PCSK9. CONCLUSIONS: Our study indicates that polydatin ameliorates lipid and glucose metabolism in type 2 diabetes mellitus by downregulating PCSK9.

The PCSK6 gene is associated with handedness, the autism spectrum, and magical ideation in a non-clinical population.

Common polymorphisms in the gene PCSK6, whose protein product mediates the development of brain and body asymmetry through the NODAL pathway, have recently been associated with handedness in three studies, making it a key candidate gene for understanding the developmental and expression of human lateralization. We tested the hypothesis that the PCSK6 VNTR polymorphism rs1053972 influences the expression of handedness and aspects of dimensional schizotypy and autism. For a sample of 709 healthy individuals, rs1053972 genotype was significantly associated with categorical measures of handedness, and with dimensional handedness in subsets of the population with high schizotypy and magical ideation or a lack of strong right-handedness. Both findings showed evidence of stronger or exclusive effects among females, compared to males. Genotypes of PCSK6 also showed significant sex-limited associations with magical ideation, a component of positive schizotypal cognition measured using the Schizotypal Personality Questionnaire, and total autism score, measured using the Autism Spectrum Quotient. These results partially replicate previous studies on effects of PCSK6 rs1053972 genetic variation on handedness phenotypes, link the PCSK6 gene with the dimensional expression of neurodevelopmental conditions in healthy individuals, and show that associations of this gene with handedness and psychological phenotypes exhibit evidence of sex-limited effects.

Naturally-occurring TGR5 agonists modulating glucagon-like peptide-1 biosynthesis and secretion.

Selective GLP-1 secretagogues represent a novel potential therapy for type 2 diabetes mellitus. This study examined the GLP-1 secretory activity of the ethnomedicinal plant, Fagonia cretica, which is postulated to possess anti-diabetic activity. After extraction and fractionation extracts and purified compounds were tested for GLP-1 and GIP secretory activity in pGIP/neo STC-1 cells. Intracellular levels of incretin hormones and their gene expression were also determined. Crude F. cretica extracts stimulated both GLP-1 and GIP secretion, increased cellular hormone content, and upregulated gene expression of proglucagon, GIP and prohormone convertase. However, ethyl acetate partitioning significantly enriched GLP-1 secretory activity and this fraction underwent bioactivity-guided fractionation. Three isolated compounds were potent and selective GLP-1 secretagogues: quinovic acid (QA) and two QA derivatives, QA-3ß-O-ß-D-glycopyranoside and QA-3ß-O-ß-D-glucopyranosyl-(28→1)-ß-D-glucopyranosyl ester. All QA compounds activated the TGR5 receptor and increased intracellular incretin levels and gene expression. QA derivatives were more potent GLP-1 secretagogues than QA. This is the first time that QA and its naturally-occurring derivatives have been shown to activate TGR5 and stimulate GLP-1 secretion. These data provide a plausible mechanism for the ethnomedicinal use of F. cretica and may assist in the ongoing development of selective GLP-1 agonists.

PCSK9 and its modulation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a newly-recognized protein, plays a key role in regulating cholesterol homeostasis. PCSK9 reduces hepatic low-density lipoprotein receptors (LDLRs) thereby increasing LDL-cholesterol (LDL-C). Recently, biologic and genetic research proposed several approaches to inhibit or reduce PCSK9 to improve lipid profile and cardiovascular performance in patients with dyslipidemia, particularly hypercholesterolemia. Of note, PCSK9 is a secreted protein under tight control by multiple modulators. Therefore, elucidating the factors that influence PCSK9 would enhance our understanding of PCSK9 and potentially day-to-day management of these patients at high cardiovascular risk. This review will focus on genetic variants, physiologic processes, pharmacologic agents and pathologic conditions related to PCSK9 in order to assess current and future therapeutic strategies targeting this molecule.

[Cloning, expression, purification of spinach carboxyl-terminal processing protease of D1 protein with hydrolysis activity and preparation of polyclonal antibody].

Carboxyl-terminal processing protease of D1 protein (CtpA) catalyzes carboxyl terminal processing of the D1 protein of photosystem II, which is essential for the assembly of a manganese cluster and consequent light-mediated water oxidation. It is a target for the discovery of wide-spectrum herbicide. We amplified the CtpA gene from spinach cDNA with standard PCR method and constructed it into pET-28a vector to generate a recombinant expression plasmid. Recombinant CtpA fusion protein with His-tag was expressed as soluble protein in Escherichia coli BL21(DE3) after induction with 0.1 mmol/L IPTG at 8 degrees C for 72 h. We purified the CtpA protein with the Ni-NTA affinity chromatography and Superdex 75 gel filtration chromatography respectively, and verified the protein by SDS-PAGE and Western blotting with anti-his antibody. Hydrolysis activity of CtpA was assayed by HPLC method with a synthetic 24-mer oligopeptide corresponding to carboxyl terminal of precursor D1 protein, and gave a total activity of 1.10 nmol/(mg x min). We used the purified CtpA protein as antigen to immune rabbit for the production of polyclonal antibody, and prepared antibody with high specificity and sensitivity. The results obtained in this paper provided the feasibility of high-throughput screening of lead compounds for the protease as inhibitors and mechanism analysis of CtpA enzyme.

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.