We have previously shown that activation of G protein-coupled receptor 56 (GPR56) by its endogenous ligand collagen type III (Coll III) improves pancreatic β-cell function and potentiates glucose-stimulated insulin secretion (GSIS). In the current study, alignment of Coll III and GPR56 genomic DNA revealed three complementary nucleotide regions, enabling the design of three short peptides (NQGCKL.NH2, H.LLSSR.NH2 and NH2.GYYCG.COOH). The peptides in double or triple combinations markedly increased cellular cAMP levels, enhanced GSIS, and improved cell viability both in human (EndoC βH1 cells) and rat pancreatic β-cell lines (INS-1 832/13 cells). These effects were markedly absent in GPR56-knockdown (GPR56-KD) cells, supporting receptor-dependent specificity. The peptide mixtures also counteracted cytokine- and glucotoxicity-induced cellular stress, restoring cAMP generation and insulin secretion. Comparable effects were not observed with dapagliflozin while glibenclamide decreased cell viability under similar conditions. In hepatic cell lines (HepG2, Hep1-6), peptide combinations enhanced cell survival in a GPR56-dependent manner, consistent with a broader role of GPR56 in metabolic regulation. Collectively, the data identify three short sequence collagen III-derived peptides whose biological effects are dependent on GPR56 expression and support their further investigation as tools for studying GPR56-associated cellular signaling for the future in vivo validation.
Abstract Metabolic disorders are multifactorial diseases and major global health concerns, with many individuals worldwide responding inadequately to treatments. The gut bacteriome regulates metabolism and offers therapeutic potential; however, research from North Africa remains scarce despite the growing prevalence of non-communicable diseases in the region. This study examines gut bacterial profiles of Tunisian type 2 diabetic (T2D), obese (OB), and non-diabetic (ND) individuals within the framework of personalized medicine. Ninety-five participants were enrolled [33 ND, 26 T2D, 26 T2D with obesity and cardiovascular complications (T2D_OBCV) and 10 OB]. Bacterial 16S rDNA V3–V4 regions were sequenced on Illumina MiSeq and analyzed using the QIIME 2 pipeline. Obese subjects exhibited gut dysbiosis with lower Akkermansia (P=0.02) and Ligilactobacillus (P=0.04) levels, an enrichment of Bifidobacterium (P=0.004), as well as Sutterella (P=0.03) and Erysipelatoclostridium (P=0.02). However, the gut bacteriome landscape of T2D patients reveals differences in the distribution of Shigella (P=0.01) highly abundant in the intestine, and a depletion of Clostridia vadin BB 60 group (P=0.02) and Oscillospiraceae UCG 005 (P=0.01). Noting that the gut bacteriome of T2D_OBCV patients is characterized by a depletion of Asteroleplasma (P=0.01), Oscillospiraceae UCG 005 (P=0.02), Eubacterium ruminantium group (P=0.0007) and Romboutsia (P=0.002) beside an enrichment of Fusicatenibacter (P=0.02). Gut bacteriome alterations in Tunisian T2D and obese subjects may serve as metabolic biomarkers.
Endothelial dysfunction induced by hyperglycemia is a key event in diabetic vascular complications and is closely associated with impaired PI3K/Akt/eNOS signaling. Insulin-like growth factor-binding protein 2 (IGFBP2) has been implicated in metabolic regulation and cell survival, but its role in high glucose-induced endothelial injury remains unclear. In this study, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose (HG, 33 mM) for 24 hours and then treated with recombinant human IGFBP2 (rhIGFBP2, 200 ng/mL) for 60 minutes. HG reduced Akt/eNOS phosphorylation and nitric oxide (NO) production, increased the expression of ICAM-1, VCAM-1, and SELE, promoted IL-6, IL-8, and MCP-1 secretion, and enhanced apoptosis. rhIGFBP2 attenuated these HG-induced changes by restoring PI3K/Akt/eNOS signaling and NO production, reducing inflammatory activation, and decreasing apoptotic cell percentage. The effect of rhIGFBP2 on Akt/eNOS phosphorylation and NO production was broadly comparable to that of insulin under HG conditions. HG also increased p38 MAPK phosphorylation, whereas rhIGFBP2 showed only a non-significant decreasing trend. Importantly, PI3K inhibition with LY294002 abolished rhIGFBP2-induced Akt/eNOS activation, supporting the involvement of the PI3K/Akt/eNOS pathway. These findings suggest that IGFBP2 protects against HG-induced endothelial dysfunction mainly by activating PI3K/Akt/eNOS signaling and may represent a potential mediator of endothelial protection under diabetic conditions.
Abstract The intracellular unorthodox aquaporins, AQP11 and AQP12, remain the least characterized members of the aquaporin superfamily. Mammalian and teleost AQP12 channels have recently been established as polytransporters in native membranes. They are localized intracellularly in pancreatic acinar cells and considered to play roles in zymogen granule maturation and exocytosis. In contrast with the vertebrate channels, the permeability properties and physiological roles of invertebrate AQP12-like (Aqp12L) orthologs are completely unknown. Here, we report that the Aqp12L channel from the extremophile crustacean Artemia franciscana is specifically targeted to the membrane of the yolk platelets (YPs) when heterologously expressed in frog (Xenopus laevis) oocytes, as recently reported for the vertebrate orthologs. Using YPs isolated from A. franciscana Aqp12L-expressing frog oocytes, we further show that this channel can transport water, H2O2, ammonia, and urea but not glycerol. Immunostaining of the A. franciscana ovary using a specific Aqp12L antibody revealed that the in vivo localization of the channel also occurs in the YP membranes of vitellogenic oocytes. RNAi-mediated silencing of Aqp12L substantially reduces the offspring output of both ovoviviparous and oviparous females through the impairment of oocyte vitellogenesis (oocyte growth) and the premature activation of ovarian apoptotic and atretic pathways. These findings provide the first direct evidence demonstrating that invertebrate Aqp12L is a functional polytransporter with an essential role in crustacean oogenesis.
PA2125 from Pseudomonas aeruginosa PAO1 is annotated as an aldehyde dehydrogenase (ALDH), but its substrate specificity, catalytic properties, and phylogenetic family assignment had not been established. Here, we cloned the PA2125 gene, produced and purified the recombinant PA2125 protein, and combined extensive substrate screening and steady-state kinetic characterisation with molecular dockings, molecular dynamics simulations, and phylogenetic analysis. PA2125 preferred NAD⁺ over NADP⁺, and screening of 54 aldehydes revealed a narrow substrate range centred on substituted benzaldehydes, particularly hydroxybenzaldehydes. Gentisaldehyde (GSA; 2,5-dihydroxybenzaldehyde) was the preferred substrate, with a KM of 5.3 µM, a kcat of 192 s⁻¹, and a remarkably high kcat/KM (3.54 × 10⁷ M⁻¹ s⁻¹). 3-Hydroxybenzaldehyde and 3,5-dihydroxybenzaldehyde also showed low KM values but approximately sevenfold lower catalytic efficiencies. Initial-velocity patterns supported an ordered sequential steady-state Bi Bi mechanism in which NAD⁺ binds first. Whereas GSA showed no substrate inhibition at concentrations up to 45 × KM, the other two hydroxybenzaldehydes showed pronounced total substrate inhibition-likely due to aldehyde binding at the nucleotide-binding site-at concentrations close to their KM. Docking identified plausible interactions involving unusual active-site residues, whereas molecular dynamics simulations showed that GSA more frequently populated and more readily accessed catalytically competent geometries. Phylogenetic analyses defined a distinct bacterial ALDH family, here designated ALDH33, comprising three subfamilies, with PA2125 belonging to ALDH33A. Together, these results identify GSA as the preferred in vitro substrate of PA2125, support its biochemical assignment as a gentisaldehyde dehydrogenase, and provide a foundation for investigating its physiological role in aromatic-aldehyde metabolism.
