Selvaraj, R. | Search bioRxiv

bioRxiv2021

Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes

Hepatic steatosis is defined by accumulation of neutral lipids in lipid droplets (LDs) including triacylglycerol (TG) and steryl esters. Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze synthesis of TG in hepatocytes. TG formed through either DGAT1 or DGAT2 appears to be preferentially directed to distinct intercellular fates, such as fatty acid production for oxidation or very-low density lipoprotein assembly, respectively. Because of the preferential use of TG generated by DGAT1 and DGAT2, we hypothesized that targeting/association of lipolytic machinery to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Inhibition of DGAT1 or DGAT2 in human hepatoma cells (Huh7) incubated with oleic acid resulted in only a small change in TG accretion suggesting that the two DGATs can compensate for each other in fatty acid esterification. This compensation was not accompanied by changes in DGAT1 or DGAT2 mRNA expression. DGAT1 inhibition (TG synthesized by DGAT2) resulted in large LDs, whereas DGAT2 inhibition (TG synthesized by DGAT1) caused the accumulation of numerous small LDs. Oleic acid treatment increased mRNA and protein expression of the LD-associated protein PLIN2 but not PLIN5 or the lipase ATGL and its activator ABHD5/CGI-58. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, ABHD5/CGI-58, PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL localized preferentially to DGAT1-made LDs rather than to DGAT2-made LDs, and TG in these LDs was preferentially used for fatty acid (FA) oxidation. A combination of DGAT2 inhibitor and the pan lipase inhibitor E600 resulted in large LDs, suggesting that the small size of DGAT1-made LDs is due to a lipolytic process.

cell biology↗

bioRxiv2020

Oral immunization of broilers with chitosan nano-encapsulated extracellular and cell wall proteins of necrotic enteritis-induced Clostridium perfringens

Currently, there is no commercial vaccine to control Clostridium perfringens (CP) or necrotic enteritis - a clinically and economically devastating disease. Two chitosan-nanoparticle encapsulated CP (CNP-CP) vaccines using extracellular proteins (ECP) and cell wall proteins (CWP) were synthesized: a CNP-CP ECP + CWP vaccine (SC vaccine) and a CNP-CP ECP + CWP surface-adsorbed CWP vaccine (SCC vaccine). The experiment comprised a complete randomized design of 3 treatments replicated 5 times: SC, SCC and nonimmunized control. Broilers orally gavaged with SC or SCC vaccine were primed and boosted with 70g dose at 3- and 14-days post-hatch (dph). SDS-PAGE analysis revealed bands at 54.7 and 84.7 kDa in the ECP and 17 bands for CWP fraction. There were no differences in body weight gain, feed conversion ratio and mortality rate between treatments. At 17dph, serum of birds in the SC and SCC groups had higher neutralizing antibodies (P<0.01) compared to the control group. At 17dph, there was an increase in bile anti-ECP IgA levels in the SC vaccinated birds and a non-significant increase in SCC vaccinated birds compared to control. At 17dph, bile specific anti-CP CWP IgA levels were greater (P<0.05) in both immunized groups compared to control. At 17 and 21dph, serum anti-ECP and anti-CP CWP IgY levels in SC and SCC birds were comparable to the control. At 21dph, CD4+/CD8+ T-cell ratio in SC and SCC vaccinated groups were elevated (P [≤] 0.01) compared to control. At 17dph, SC and SCC vaccinated birds had a significant reduction (P [≤] 0.001) in -toxin levels in caecal contents compared to control. Caecal -toxin levels remained reduced at 21dph (P < 0.05) in SC birds and numerically reduced in SCC vaccinated birds compared to control. Jejunal CP load in SCC birds was significantly reduced by 1.4 Log10 copy numbers of CP/g compared to control and no differences were observed in liver CP load between immunized and non-immunized birds. SC and SCC immunization did not alter TNF-, IL-10, iNOS, IL-6 or IL-17 mRNA expression. At 17 and 21dph, SC and SCC immunized birds demonstrated greater sera bactericidal activity compared to control. CNP-SC and CNP-SCC immunization induced specific immune response against C. perfringens and reduced CP colonization via oral route of administration.

immunology↗