Hepatocyte-specific Expression of Human Carboxylesterase 2 Attenuates Non-alcoholic Steatohepatitis in Mice

Human carboxylesterase 2 (CES2) has triacylglycerol hydrolase (TGH) activities and plays an important role in lipolysis. In this study, we aim to determine the role of human CES2 in the progression or reversal of steatohepatitis in diet-induced or genetically obese mice. High-fat/high-cholesterol/high-fructose (HFCF) diet-fed C57BL/6 mice or db/db mice were i.v. injected with an adeno-associated virus expressing human CES2 under the control of an albumin promoter. Human CES2 protected against HFCF diet-induced non-alcoholic fatty liver disease (NAFLD) in C57BL/6J mice and reversed steatohepatitis in db/db mice. Human CES2 also improved glucose tolerance and insulin sensitivity. Mechanistically, human CES2 reduced hepatic triglyceride and free fatty acid levels by inducing lipolysis and fatty acid oxidation and inhibiting lipogenesis via suppression of sterol regulatory element-binding protein 1. Furthermore, human CES2 overexpression improved mitochondrial respiration and glycolytic function, and inhibited gluconeogenesis, lipid peroxidation, apoptosis and inflammation. Our data suggest that hepatocyte-specific expression of human CES2 prevents and reverses steatohepatitis. Targeting hepatic CES2 may be an attractive strategy for treatment of NAFLD.

High-level soluble expression and enzymatic characterization of Burkholderia sp. lipase with steryl ester hydrolysis activity in E. coli

Background Burkholderia cepacia lipase is an important industrial biocatalyst for biodiesel production and chiral pharmaceutical synthesis. Heterologous soluble expression of lipase lipA gene from B. cepacia in Escherichia coli highly depends on co-expression of its cognate foldase gene, lipB. However, the interaction between recombinant lipase LipA and chaperonin LipB is rather complicated and confusing. In this research, various systems of lipA/lipB co-expression combinations are investigated to obtain high-level soluble expression of lipA, respectively. Results The best co-expression combination system for lipA and lipB is E. coli Origami 2 (DE3)/pETDuet-lipB(MCS1)/lipA(MCS2). The soluble expression level of lipA is 100.4 U/OD600 towards 4-nitrophenyl laurate hydrolysis. The recombinant LipA can be rapidly isolated from cell-free supernatant of recombinant E. coli lysate using HisTrap HP affinity chromatography column, and the lipA/LipB complex is obtained. Enzymatic characterization analysis shows that the purified LipA is a mesothermal and alkaline enzyme. LipA displays preference for medium-chain-length acyl groups (C10-C12) and sn-1,3 regioselectivity. Besides triacylglycerol hydrolase activity (EC. 3.1.1.3), LipA also displays steryl ester hydrolase activity (EC. 3.1.1.13). The specific activity of LipA towards 4-nitrophenyl decanoate and cholesterol linoleate are 638.9 U/mg and 1111.5 mU/mg, respectively. Conclusions Host strain E. coli Origami 2 (DE3), lipB locus at MCS1 on the dual expression cassette plasmid pETDuet, and low-temperature induction contribute to the soluble expression of lipA. Recombinant LipA displays both triacylglycerol hydrolase activity and steryl ester hydrolase activity.

Altered Lipid Droplet Dynamics in Hepatocytes Lacking Triacylglycerol Hydrolase Expression

Lipid droplets (LDs) form from the endoplasmic reticulum (ER) and grow in size by obtaining triacylglycerols (TG). Triacylglycerol hydrolase (TGH), a lipase residing in the ER, is involved in the mobilization of TG stored in LDs for the secretion of very-low-density lipoproteins. In this study, we investigated TGH-mediated changes in cytosolic LD dynamics. We have found that TGH deficiency resulted in decreased size and increased number of LDs in hepatocytes. Using fluorescent fatty acid analogues to trace LD formation, we observed that TGH deficiency did not affect the formation of nascent LDs on the ER. However, the rate of lipid transfer into preformed LDs was significantly slower in the absence of TGH. Absence of TGH expression resulted in increased levels of membrane diacylglycerol and augmented phospholipid synthesis, which may be responsible for the delayed lipid transfer. Therefore, altered maturation (growth) rather than nascent formation (de novo synthesis) may be responsible for the observed morphological changes of LDs in TGH-deficient hepatocytes.

Amino acid-sensing mTOR signaling is involved in modulation of lipolysis by chronic insulin treatment in adipocytes

Chronically high insulin levels and increased circulating free fatty acids released from adipose tissue through lipolysis are two features associated with insulin resistance. The relationship between chronic insulin exposure and adipocyte lipolysis has been unclear. In the present study we found that chronic insulin exposure in 3T3-L1 adipocytes, as well as in mouse primary adipocytes, increased basal lipolysis rates. This effect of insulin on lipolysis was only observed when the mammalian target of rapamycin (mTOR) pathway was inhibited by rapamycin in the adipocytes. In addition, amino acid deprivation in adipocytes phenocopied the effect of rapamycin in permitting the stimulation of lipolysis by chronic insulin exposure. The phosphatidylinositol 3-kinase-Akt pathway does not appear to be involved in this insulin effect. Furthermore, we found that triacylglycerol hydrolase (TGH) activity was required for the stimulation of lipolysis by combined exposure to insulin and rapamycin. Therefore, we propose that nutrient sufficiency, mediated by an mTOR pathway, suppresses TGH-dependent lipolysis stimulated by chronic insulin exposure in adipocytes.

Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY

ABSTRACT PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(ΔPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipYmar, in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipYmar in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipYmar was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(ΔPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(ΔPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(ΔPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.