Hypertriglyceridemia is a hallmark of many disorders, including metabolic syndrome, diabetes, atherosclerosis and obesity. A well-known cause is the deficiency of lipoprotein lipase (LPL), a key enzyme in plasma triglyceride hydrolysis. Mice carrying the combined lipase deficiency (cld) mutation show severe hypertriglyceridemia owing to a decrease in the activity of LPL and a related enzyme, hepatic lipase (HL), caused by impaired maturation of nascent LPL and hepatic lipase polypeptides in the endoplasmic reticulum (ER). Here we identify the gene containing the cld mutation as Tmem112 and rename it Lmf1 (Lipase maturation factor 1). Lmf1 encodes a transmembrane protein with an evolutionarily conserved domain of unknown function that localizes to the ER. A human subject homozygous for a deleterious mutation in LMF1 also shows combined lipase deficiency with concomitant hypertriglyceridemia and associated disorders. Thus, through its profound effect on lipase activity, LMF1 emerges as an important candidate gene in hypertriglyceridemia.

Triacylglycerol (TG) hydrolase activities were characterized in myocytes isolated from rat hearts. Acid hydrolase activity with a pH optimum of 5 could be measured in myocyte homogenates, and the subcellular distribution suggested that this activity originated in lysosomes. Lipoprotein lipase (LPL) was also present in myocyte homogenates, as evidenced by TG hydrolase activity that was stimulated by serum and apolipoprotein CII, and inhibited by apolipoprotein CIII2, high ionic strength (NaCl and MgCl2, I = 1 M) and antibodies to LPL. Serum-independent neutral (pH 7.5) TG hydrolase activity was less sensitive to inhibition by 1 M-NaCl, by antibodies to LPL and by preincubation at 40 degrees C than was serum-stimulated hydrolase activity. Furthermore, there were modest but significant differences in the subcellular distribution of the serum-independent and serum-stimulated hydrolase activities. Hydrolase activities in myocyte homogenates could be solubilized by 7.2 mM-deoxycholate. Acid hydrolase activity was recovered in the unbound fraction after heparin-Sepharose chromatography, whereas LPL was bound to the affinity column and was eluted by 0.9-1.2 M-NaCl. Approximately one-third of the serum-independent TG hydrolase activity was not bound to the heparin-Sepharose affinity column. This unbound TG hydrolase activity had a pH optimum of 7 and was stimulated by 50 mM-MgCl2, but not by serum and was resistant to inhibition by high ionic strength (1 M-NaCl), to preincubation at 40 degrees C for 2 h, and by antibodies to LPL. It is concluded that, in addition to an acid lysosomal TG hydrolase and LPL, myocytes from rat heart contain a serum-independent TG hydrolase with unique characteristics.

OBJECTIVETo identify metabolic derangements contributing to diabetes susceptibility in the leptin receptor–deficient obese C57BLKS/J-db/db (BKS-db) mouse strain.RESEARCH DESIGN AND METHODSYoung BKS-db mice were used to identify metabolic pathways contributing to the development of diabetes. Using the diabetes-resistant B6-db strain as a comparison, in vivo and in vitro approaches were applied to identify metabolic and molecular differences between the two strains.RESULTSDespite higher plasma insulin levels, BKS-db mice exhibit lower lipogenic gene expression, rate of lipogenesis, hepatic triglyceride and glycogen content, and impaired insulin suppression of gluconeogenic genes. Hepatic insulin receptor substrate (IRS)-1 and IRS-2 expression and insulin-stimulated Akt-phosphorylation are decreased in BKS-db primary hepatocytes. Hyperinsulinemic-euglycemic clamp studies indicate that in contrast to hepatic insulin resistance, skeletal muscle is more insulin sensitive in BKS-db than in B6-db mice. We also demonstrate that elevated plasma triglyceride levels in BKS-db mice are associated with reduced triglyceride clearance due to lower lipase activities.CONCLUSIONSOur study demonstrates the presence of metabolic derangements in BKS-db before the onset of β-cell failure and identifies early hepatic insulin resistance as a component of the BKS-db phenotype. We propose that defects in hepatic insulin signaling contribute to the development of diabetes in the BKS-db mouse strain.

The maturation of lipoprotein lipase (LPL) into a catalytically active enzyme was believed to occur only after its transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To test this hypothesis, LPL located in these two subcellular compartments was separated and compared. Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. The latter forms were further separated by ␤-ricin chromatography and were found to have comparable activities per unit of LPL mass. Thus, LPL must reach a functional conformation in the ER. Active LPL, regardless of its cellular location, exhibited the expected dimer conformation. However, inactive LPL, found only in the ER, was highly aggregated. Kinetic analysis indicated a concurrent formation of LPL dimer and aggregate and indicated that the two forms have dissimilar fates. Whereas the dimer remained stable even when confined to the ER, the aggregate was degraded. Degradation rates were not affected by proteasomal or lysosomal inhibitors but were markedly reduced by ATP depletion. Lowering the redox potential in the ER by dithiothreitol caused the dimer to associate with calnexin, BiP, and protein-disulfide isomerase to form large, inactive complexes; dithiothreitol removal induced complex dissociation with restoration of the functional LPL dimer. In contrast, the LPL aggregate was only poorly associated with ER chaperones, appearing to be trapped in an irreversible, inactive conformation destined for ER degradation.