An in vivo and in vitro Investigation of Glycerol Utilization in Intestinal Triacylglycerol Metabolism.

Overproduction of intestinal lipoproteins contribute to postprandial hypertriacylglycerolaemia. Understanding the mechanisms that modulate chylomicron (CM) production in health and disease may lead to prevention and improvement of treatments for atherosclerosis. It is well known that following the hydrolysis of dietary triacylglycerol (TAG) in the intestine, glycerol is taken up by the luminal side of absorptive cells. However, it has recently been shown that in humans, endogenously derived glycerol tracer (administered intravenously) also appears to be incorporated into exogenous (dietary derived) lipoprotein particles. This was investigated further utilising stable isotopic techniques in human volunteers and by an in vitro model utilising the Caco2 cell line. Phospholipids (PL) synthesis was also measured in the Caco2 cells. Five healthy subjects (age 50-65, BMI21-24) were fed identical high fat meals every 2 hours for 11 hours. During this time, two different tracers, one administered intravenously (2H5-glycerol) and another given orally (13C3-glycerol) as part of a high fat meal were used to measure the incorporation of endogenous and exogenous glycerol into CM or very low density lipoproteins (VLDL). 2H5-glycerol was detected in VLDL and CM derived TAG, confirming endogenous glycerol could be incorporated into TAG derived from enterocytes. 13C3-glycerol was found to be predominantly associated with CM. There was no significant difference in the proportion of oral or intravenous tracer used for TAG synthesis. VLDL1 production rate (PR) was significantly higher than VLDL2 PR; CM1 PR was significantly higher than CM2 PR. The incorporation of 13C3-labelled glycerol into TAG and PL by Caco2 cells was measured by the addition of a trace quantity of labelled C3-glycerol to either the apical or basolateral side of cells. As different culture conditions might affect lipoprotein synthesis and secretion, the effect of culturing in different media concentrations of glucose and insulin was also investigated. Incubation with labelled 13C3-glycerol on either the apical or the basolateral surface of the cells resulted in the detection of glycerol enriched TAG and PL in the basolateral media and the cells themselves. Because the enrichment of basolateral media TAG and PL was higher than that in the cells, the presence of unlabelled cellular storage pools was proposed. Insulin had no effect on the synthesis and secretion or cellular enrichment of TAG and PL, but reduced enrichment of TAG and PL in the media. This suggests that insulin increased the incorporation of newly synthesised (labelled) TAG and PL into the cellular storage pool and reduced the amount in the export pool. This study clearly demonstrates that both exogenous and endogenous glycerol can be used to synthesise TAG in enterocytes in both an in vivo and in an in vitro model. The study also confirms the presence of intracellular storage pools of TAG in enterocytes and that enterocytes can regulate CM synthesis and secretion using these pools. Endogenous substrates may have an important role in the overproduction of CM and further studies are needed to understand their role in postprandial hypertriacylglycerolaemia

An in vivo and in vitro Investigation of Glycerol Utilization in Intestinal Triacylglycerol Metabolism.

Overproduction of intestinal lipoproteins contribute to postprandial hypertriacylglycerolaemia. Understanding the mechanisms that modulate chylomicron (CM) production in health and disease may lead to prevention and improvement of treatments for atherosclerosis. It is well known that following the hydrolysis of dietary triacylglycerol (TAG) in the intestine, glycerol is taken up by the luminal side of absorptive cells. However, it has recently been shown that in humans, endogenously derived glycerol tracer (administered intravenously) also appears to be incorporated into exogenous (dietary derived) lipoprotein particles. This was investigated further utilising stable isotopic techniques in human volunteers and by an in vitro model utilising the Caco2 cell line. Phospholipids (PL) synthesis was also measured in the Caco2 cells. Five healthy subjects (age 50-65, BMI21-24) were fed identical high fat meals every 2 hours for 11 hours. During this time, two different tracers, one administered intravenously (2H5-glycerol) and another given orally (13C3-glycerol) as part of a high fat meal were used to measure the incorporation of endogenous and exogenous glycerol into CM or very low density lipoproteins (VLDL). 2H5-glycerol was detected in VLDL and CM derived TAG, confirming endogenous glycerol could be incorporated into TAG derived from enterocytes. 13C3-glycerol was found to be predominantly associated with CM. There was no significant difference in the proportion of oral or intravenous tracer used for TAG synthesis. VLDL1 production rate (PR) was significantly higher than VLDL2 PR; CM1 PR was significantly higher than CM2 PR. The incorporation of 13C3-labelled glycerol into TAG and PL by Caco2 cells was measured by the addition of a trace quantity of labelled C3-glycerol to either the apical or basolateral side of cells. As different culture conditions might affect lipoprotein synthesis and secretion, the effect of culturing in different media concentrations of glucose and insulin was also investigated. Incubation with labelled 13C3-glycerol on either the apical or the basolateral surface of the cells resulted in the detection of glycerol enriched TAG and PL in the basolateral media and the cells themselves. Because the enrichment of basolateral media TAG and PL was higher than that in the cells, the presence of unlabelled cellular storage pools was proposed. Insulin had no effect on the synthesis and secretion or cellular enrichment of TAG and PL, but reduced enrichment of TAG and PL in the media. This suggests that insulin increased the incorporation of newly synthesised (labelled) TAG and PL into the cellular storage pool and reduced the amount in the export pool. This study clearly demonstrates that both exogenous and endogenous glycerol can be used to synthesise TAG in enterocytes in both an in vivo and in an in vitro model. The study also confirms the presence of intracellular storage pools of TAG in enterocytes and that enterocytes can regulate CM synthesis and secretion using these pools. Endogenous substrates may have an important role in the overproduction of CM and further studies are needed to understand their role in postprandial hypertriacylglycerolaemia

Prandial Hypertriglyceridemia in Metabolic Syndrome is due to an Overproduction of both Chylomicron and VLDL Triacylglycerol

Abstract The aim was to determine whether fed very low density lipoprotein (VLDL) and chylomicron (CM) triacylglycerol (TAG) production rates are elevated in metabolic syndrome (MetS). Eight men with MetS (BMI 29.7± 1.1) and 8 lean age matched healthy men (BMI 23.1± 0.4) were studied using a frequent feeding protocol. After 4 hours of feeding an intravenous bolus of 2 H 5 -glycerol was administered to label VLDL1, VLDL2 and CM TAG. 13 C glycerol tripalmitin was administered orally as an independent measure of CM TAG metabolism. Hepatic and intestinal lipoproteins were separated by an immunoaffinity method. In MetS, fed TAG and the increment in TAG from fasting to feeding were higher (p=0.03, p<0.05 respectively) than in lean men. Fed CM, VLDL1 and VLDL2 TAG pool sizes were higher (p=0.006, p=0.03, p<0.02) and CM, VLDL1 and VLDL2 TAG production rates were higher (p<0.002, p<0.05, p=0.06) than in lean men. VLDL1, VLDL2 and CM TAG clearance rates were not different between groups. In conclusion prandial hypertriglyceridemia in men with MetS was due to an increased production rate of both VLDL and CM TAG. Since both groups received identical meals this suggests that in MetS the intestine is synthesising more TAG de novo for export in CMs.

A novel method for measuring intestinal and hepatic triacylglycerol kinetics

This study aimed to 1) develop a method that completely separated hepatic (VLDL1, VLDL2) and intestinal [chylomicron (CM)] lipoproteins and 2) use the method to measure triacylglycerol (TAG) kinetics in these lipoproteins in the fed and fasting state in healthy subjects, using intravenous [²H₅]glycerol as the tracer. An immunoaffinity method that completely separated hepatic and intestinal particles using sequential binding to three antibodies to apolipoprotein B-100 (apoB-100) was established and validated. Six healthy volunteers were studied in a fasted and continuous feeding study (study 1). Five additional healthy volunteers were studied in a continuous feeding study that included an oral [¹³C₃]glycerol tripalmitin tracer (study 2). In both studies, an intravenous bolus of [²H₅]glycerol was administered to label TAG in hepatic and intestinal lipoproteins. In both feeding studies there was sufficient incorporation of the [²H₅]glycerol tracer into the exogenous lipoproteins to enable isotopic enrichment to be measured. In study 2, the oral tracer enrichment in VLDL1 was <5% of CM enrichment 150 min after tracer administration, demonstrating negligible contamination of VLDL1 with apoB-48. Western blotting showed no detectable apoB-100 in CMs. VLDL1 and VLDL2 TAG fractional catabolic rate (FCR) did not differ between feeding and fasting (study 1). There was no difference between CM and VLDL1 TAG FCR in both fed studies. In fed study 2, 47% of the total TAG production rate (CM + VLDL1) was from CM. This methodology may be a useful tool for understanding the abnormalities in postprandial TAG kinetics in metabolic syndrome and type 2 diabetes