LPL activity assay.

LPL activity was determined as described previously (44, 45). Post-heparin plasma was obtained from fasted mice 5 minutes after retro-orbital injection of 5 units per mouse of heparin. To measure total lipase activity, plasma samples were incubated with 10% Intralipid/[³H]TG emulsion (Hospira) as a substrate and human serum as the source of ApoC-III (24). The contribution of hepatic lipase was determined by including 1 M NaCl in the assay, and the values were subtracted from the total lipase activity to estimate the activity attributed to LPL. Heparin-releasable LPL activity in heart, skeletal muscle, and white adipose tissue was measured using 50–80 mg of snap-frozen tissues. The tissue was minced in 0.6 ml PBS with 2 mg/ml BSA in the presence of 5 U/ml heparin. Minced tissues were incubated for 1 hour in a 37°C shaker and subsequently centrifuged at 1,000 g for 15 minutes. The supernatant was collected for LPL assay. A 100-μl aliquot of the buffer was used for the lipase assay in combination with 100 μl of a 10% Intralipid/[³H]triolein emulsion for 1 hour at 25°C. The LPL activity measurements were normalized for the initial tissue weights.