Digestion and absorption of long-chain TAG are highly efficient processes with >90% of dietary TAG from each meal being hydrolyzed and absorbed by the intestine. Because of the fundamental importance of efficient dietary lipid absorption, redundancy exists in the repertoire of enzymes capable of hydrolyzing lipids in the gut, thereby ensuring availability of these nutrients. The purpose of this study was to determine how PTL and CEL contribute to dietary lipid absorption in vivo. Results showed that PTL and CEL both participate in lipid absorption and may serve a mutually compensatory role. Importantly, inactivation of either the PTL or CEL gene alone had minimal effects on TAG absorption, whereas disruption of both genes resulted in significant reduction of TAG absorption. Likewise, both PTL and CEL appear to participate in dietary retinyl ester hydrolysis and absorption. The decrease in retinyl palmitate absorption observed in PTL−/− mice but not in CEL−/− mice may imply a more prominent role for PTL in retinyl ester digestion. Nevertheless, CEL is also capable of catalyzing retinyl ester hydrolysis and mediating retinol absorption, as mice with both PTL and CEL deficiency absorbed significantly less retinyl ester than PTL-deficient mice. The difference in retinyl ester absorption efficiency between PTL−/− and PTL−/−,CEL−/− mice indicates a more avid retinyl ester hydrolytic activity of PTL than CEL in vivo, which was not previously appreciated from in vitro enzyme assays. Alternatively, this difference may reflect the abundance of PTL in the digestive tract compared with CEL and that total PTL deficiency results in insufficient enzyme activity in the intestinal lumen for retinyl ester hydrolysis.
An important observation made in the current study is that although PTL and CEL deficiency dramatically reduced dietary TAG and retinyl ester absorption, a substantial amount (~50 − 60%) of these lipids was still absorbed in PTL−/−,CEL−/−
mice. Therefore, at least one other lipolytic enzyme must be present in the intestinal lumen to catalyze TAG and retinyl ester hydrolysis in the absence of PTL and CEL. A likely candidate is PTLRP2, another enzyme secreted by the pancreas with avid in vitro
TAG hydrolytic activity (15
). The pancreas expresses PTLRP2 before birth, and its expression level persists at a lower level into adulthood (16
). In contrast, the pancreas does not express PTL until near the suckling/weaning transition period (17
). It is noteworthy that suckling PTLRP2
−/− mice have steatorrhea and fat malabsorption with major increases of undigested and partially digested TAG in the feces (18
). In addition, weight gain of suckling PTLRP2
−/− mice is reduced compared with that of wild type mice, which is consistent with their diminished ability to acquire calories from dietary fat. Those data clearly documented that in the absence of PTL, PTLRP2 is capable of catalyzing TAG digestion and mediating fat absorption in vivo
. In the current study we showed no significant difference in the expression level of PTLRP2 between wild type, PTL
−/−, and PTL
−/− mice (). Thus, the low level of PTLRP2 expression in the digestive tract may be responsible for the luminal lipolytic activity that facilitates TAG digestion and absorption in the PTL
−/− mice. The results showing dramatic decrease of fat absorption efficiency in colipase knock-out mice (19
) but marginal decrease observed in PTL
−/− mice together with the requirement of colipase for both PTL and PTLRP2 activity are supportive of this possibility. Mice with defective expression of PTL, CEL, and PTLRP2 are now being generated to test this hypothesis. Additionally, a phospholipase B residing in brush border membranes was also shown to hydrolyze retinyl esters in vitro
). Thus, this enzyme may also participate in retinyl ester hydrolysis and absorption, particularly in the [PTL
Our previous study showing that PTL deficiency reduces cholesterol absorption efficiency was confirmed and extended in the current study, with results showing additional reduction of cholesterol absorption efficiency in mice lacking both PTL and CEL. The reduced cholesterol absorption efficiency observed in these animals is consistent with our previous hypothesis that efficient TAG digestion in the proximal intestine is necessary for cholesterol partitioning to micelles for subsequent absorption by enterocytes. The deficiency in PTL alone or in combination with CEL delays TAG hydrolysis to the distal intestine (1
) where the cholesterol is less efficiently taken up (4
). The lower fasting cholesterol levels observed in PTL
−/− and PTL
−/− mice consuming the Western-type diet () is consistent with the reduced cholesterol absorption efficiency in these animals.
In contrast to the digestion and absorption of TAG, retinyl ester, and unesterified cholesterol, intestinal absorption of cholesteryl esters was dramatically reduced in CEL−/− mice but was unaffected in PTL−/− mice. Thus, PTL failed to compensate for the lack of CEL in cholesteryl ester digestion in the intestinal lumen. Inactivation of the PTL gene in CEL-deficient mice also did not cause additional inhibition of cholesteryl ester absorption compared with that observed in CEL−/− mice. Taken together these results indicated that CEL is the only enzyme in the digestive tract responsible for cholesteryl ester hydrolysis. The difference between cholesteryl ester and retinyl ester absorption in these mouse models illustrated their unique metabolic fate, suggesting that studies monitoring dietary retinyl ester metabolism as a surrogate for cholesteryl ester metabolism should take into consideration whether they are substrates for these and possibly other lipolytic enzymes.
Finally, the results of this study also revealed that the reduction of dietary TAG absorption due to PTL and CEL inactivation significantly reduced susceptibility to high fat/ high cholesterol diet-induced obesity. This observation has direct clinical implications. Currently, the only strategy to reduce obesity through inhibition of dietary lipid absorption is via tetrahydrolipstatin (Orlistat) therapy (21
). Tetrahydrolipstatin inhibits all lipolytic enzyme activities in the digestive tract (22
); therefore, its use needs to be titrated carefully to avoid the unpleasant adverse effects of fecal spotting (24
). In this study we showed that inactivation of both PTL and CEL without the inhibition of other lipolytic enzymes in the digestive tract is sufficient to provide resistance to diet-induced obesity. Thus, the development of pharmacological strategies aimed at specific inhibition of PTL and CEL may be warranted to suppress diet-induced obesity without the adverse consequences of fat-soluble vitamin deficiency or steatorrhea that culminate upon inhibition of all lipolytic enzymes in the digestive tract.