Diacylglycerol production induced by growth hormone in Ob1771 preadipocytes arises from phosphatidylcholine breakdown

Growth Hormone has recently been shown to stimulate the formation of diacylglycerol in Ob1771 mouse preadipocyte cells without increasing inositol lipid turnover. Addition of growth hormone to Ob1771 cells prelabelled with [3H]glycerol or [3H]choline led to a rapid, transient and stoechiometric formation of labelled diacylglycerol and phosphocholine, respectively. In contrast, no change was observed in the level of choline and phosphatidic acid whereas the release of water-soluble metabolites in [3H]ethanolamine prelabelled cells exposed to growth hormone was hardly detectable. Stimulation by growth hormone of cells prelabelled with (2-palmitoyl 9, 10 [3H])phosphatidylcholine also induced the production of labelled diacyglycerol. Pertussis toxin abolished both diacylglycerol and phosphocholine formation induced by growth hormone. It is concluded that growth hormone mediates diacylglycerol production in Ob1771 cells by means of phosphatidylcholine breakdown involving a phospholipase C which is likely coupled to the growth hormone receptor via a pertussis toxin-sensitive G-protein.     

Genetic and developmental regulation of the lipoprotein lipase gene: loci both distal and proximal to the lipoprotein lipase structural gene control enzyme expression

We report here a study of the developmental and genetic control of tissue-specific expression of lipoprotein lipase, the enzyme responsible for hydrolysis of triglycerides in chylomicrons and very low density lipoproteins. Lipoprotein lipase (LPL) mRNA is present in a wide variety of adult rat and mouse tissues examined, albeit at very different levels. A remarkable increase in the levels of LPL mRNA occurs in heart over a period of several weeks following birth, closely paralleling developmental changes in lipase activity and myocardial beta-oxidation capacity. Large increases in LPL mRNA also occur during differentiation of 3T3L1 cells to adipocytes. As previously reported, at least two separate genetic loci control the tissue-specific expression of LPL activity in mice. One of the loci, controlling LPL activity in heart, is associated with an alteration in LPL mRNA size, while the other, controlling LPL activity in adipose tissue, appears to affect the translation or post-translational expression of LPL. To examine whether these genetic variations are due to mutations of the LPL structural locus, we mapped the LPL gene to a region of mouse chromosome 8 using restriction fragment-length polymorphisms and analysis of hamster-mouse somatic cell hybrids. This region is homologous to the region of human chromosome 8 which contains the human LPL gene as judged by the conservation of linked genetic markers. Genetic variations affecting LPL expression in heart cosegregated with the LPL gene, while variations affecting LPL expression in adipose tissue did not. Furthermore, Southern blotting analysis indicates that LPL is encoded by a single gene and, thus, the genetic differences are not a consequence of independent regulation of two separate genes in the two tissues. These results suggest the existence of cis-acting elements for LPL gene expression that operate in heart but not adipose tissue. Our results also indicate that two genetic mutations resulting in deficiencies of LPL in mice, the W mutation on chromosome 5 and the cld mutation on mouse chromosome 17, do not involve the LPL structural gene locus. Finally, we show that the gene for hepatic lipase, a member of a gene family with LPL, is unlinked to the gene for LPL. This indicates that combined deficiencies of LPL and hepatic lipase, observed in humans as well as in certain mutant strains of mice, do not result from focal disruptions of a cluster of lipase genes.     

Linkage of low-density lipoprotein size to the lipoprotein lipase gene in heterozygous lipoprotein lipase deficiency.

Small low-density lipoprotein (LDL) particles are a genetically influenced coronary disease risk factor. Lipoprotein lipase (LpL) is a rate-limiting enzyme in the formation of LDL particles. The current study examined genetic linkage of LDL particle size to the LpL gene in five families with structural mutations in the LpL gene. LDL particle size was smaller among the heterozygous subjects, compared with controls. Among heterozygous subjects, 44% were classified as affected by LDL subclass phenotype B, compared with 8% of normal family members. Plasma triglyceride levels were significantly higher, and high-density lipoprotein cholesterol (HDL-C) levels were lower, in heterozygous subjects, compared with normal subjects, after age and sex adjustment. A highly significant LOD score of 6.24 at straight theta=0 was obtained for linkage of LDL particle size to the LpL gene, after adjustment of LDL particle size for within-genotype variance resulting from triglyceride and HDL-C. Failure to adjust for this variance led to only a modest positive LOD score of 1.54 at straight theta=0. Classifying small LDL particles as a qualitative trait (LDL subclass phenotype B) provided only suggestive evidence for linkage to the LpL gene (LOD=1. 65 at straight theta=0). Thus, use of the quantitative trait adjusted for within-genotype variance, resulting from physiologic covariates, was crucial for detection of significant evidence of linkage in this study. These results indicate that heterozygous LpL deficiency may be one cause of small LDL particles and may provide a potential mechanism for the increase in coronary disease seen in heterozygous LpL deficiency. This study also demonstrates a successful strategy of genotypic specific adjustment of complex traits in mapping a quantitative trait locus.     

High-level expression of the bovine growth hormone gene in heterologous mammalian cells

The gene coding for bovine growth hormone (bGH) was isolated from a lambda-phage library constructed using bovine pituitary DNA partially digested with MboI. Expression of this gene transfected into mouse and monkey cells was studied. CV-1 monkey cells transfected with simian virus 40 (SV40) vectors containing the intact bGH gene, including the putative promoter region, did not express bGH. However, replacement of the bGH promoter with the mouse metallothionein-I (MT) promoter resulted in high-level synthesis and secretion of bGH. These results show that the bGH promoter functions poorly in CV-1 cells but CV-1 cells process and translate the bGH mRNA accurately. The MT-bGH chimeric gene was used to establish permanent bGH-secreting mouse C127 cell lines using the 69% transforming fragment of bovine papilloma virus (BPV) as the vector. One such cell line produced high levels of bGH and secreted it into the medium efficiently. Secreted bGH is processed accurately and is bioactive as judged by its ability to bind to rabbit liver membrane preparations.     

Expression of lipoprotein lipase gene in combined lipase deficiency

The expression of the gene for lipoprotein lipase (LPL) was studied in brown adipose tissue and the liver of combined lipase deficient (cld/cld) and unaffected mice. The mRNA specific for LPL was detected in both animals. Although the size of LPL mRNA in cld mice was similar to that of unaffected mice, the mRNA concentration in affected animals was higher than in unaffected animals. We also studied the LPL gene mutation in cld mice by Southern blot analysis. No restriction fragment length polymorphisms were observed after digestion with 16 endonucleases. These data indicate that there is no gene insertion or deletion, but do not exclude the possibility of point mutation in the LPL structural gene. However, the present results agree with the hypothesis that the genetic defect in cld is not due to a mutation in the LPL structural gene, but instead involves the defective post-translational processing of LPL or defective cellular function affecting transport and secretion of this enzyme group.     

The in vitro effect of growth hormone on adipose tissue lipoprotein lipase in rats

The $\text{in vitro}$ effect of growth hormone on adipose tissue lipoprotein lipase was studied in rats. Epididymal adipose tissue was incubated with human growth hormone in the presence of heparin. Growth hormone at a concentration of 0.1 μg per ml decreased by approximately 20% (p<0.005) the heparin-releasable lipoprotein lipase in rat adipose tissue. Discussion was focussed on the reciprocal changes caused by growth hormone of the activities of lipoprotein lipase and hormone-sensitive lipase in the rat adipose tissue.     

Cardiac-specific knock-out of lipoprotein lipase alters plasma lipoprotein triglyceride metabolism and cardiac gene expression

Fatty acids are the primary energy source for the heart. The heart acquires fatty acids associated with albumin or derived from lipoprotein lipase (LpL)-mediated hydrolysis of lipoprotein triglyceride (TG). We generated heart-specific LpL knock-out mice (hLpL0) to determine whether cardiac LpL modulates the actions of peroxisome proliferator-activated receptors and affects whole body lipid metabolism. Male hLpL0 mice had significantly elevated plasma TG levels and decreased clearance of postprandial lipids despite normal postheparin plasma LpL activity. Very large density lipoprotein-TG uptake was decreased by 72% in hLpL0 hearts. However, heart uptake of albumin-bound free fatty acids was not altered. Northern blot analysis revealed a decrease in the expression of peroxisome proliferator-activated receptor alpha-response genes involved in fatty acid beta-oxidation. Surprisingly, the expression of glucose transporters 1 and 4 and insulin receptor substrate 2 was increased and that of pyruvate dehydrogenase kinase 4 and insulin receptor substrate 1 was reduced. Basal glucose uptake was increased markedly in hLpL0 hearts. Thus, the loss of LpL in the heart leads to defective plasma metabolism of TG. Moreover, fatty acids derived from lipoprotein TG and not just albumin-associated fatty acids are important for cardiac lipid metabolism and gene regulation.