From masochistic enzymology to mechanistic physiology and disease

Abstract

The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. However, the regulation of PC biosynthesis was poorly understood at that time. When I started my lab at the University of British Columbia in the 1970s, this was the focus of my research. This article provides my reflections on these studies that began with enzymology and the use of cultured mammalian cells, and progressed to utilize the techniques of molecular biology and gene-targeted mice. The research in my lab and others demonstrated that the regulated and rate-limiting step in the choline pathway for PC biosynthesis was catalyzed by CTP:phosphocholine cytidylyltransferase. This enzyme is regulated by its movement from a soluble form (largely in the nucleus) to a membrane-associated form where the enzyme becomes activated. Gene targeting in mice subsequently demonstrated that this gene is essential for development of mouse embryos. The other mammalian pathway for PC biosynthesis is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) that converts phosphatidylethanolamine to PC. Understanding of the regulation and function of the integral membrane protein PEMT was improved when the enzyme was purified (a masochistic endeavor) in 1987, leading to the cloning of the Pemt cDNA. Generation of knock-out mice that lacked PEMT showed that they were protected from atherosclerosis, diet-induced obesity, and insulin resistance. The protection from atherosclerosis appears to be due to decreased secretion of lipoproteins from the liver. We continue to investigate the mechanism(s) by which Pemt^-/- mice are protected from weight gain and insulin resistance.

Keywords: choline; insulin resistance; lipid; lipoprotein; lysosomal storage disease; membrane enzyme; phosphatidylcholine; phosphatidylethanolamine.

Figure 1.

Charles C. Sweeley in 1990.

Figure 2.

Konrad Bloch, 1965.

Figure 3.

Pathway for the biosynthesis of phosphatidylcholine as first described by Kennedy and co-workers in the mid-1950s.

Figure 4.

Eugene Kennedy and Dennis Vance, 2011.

Figure 5.

Pathway for the conversion of PE to PC, showing the methylation status of the ethanolamine headgroup (above) in each case. PMME, phosphatidylmonomethylethanolamine; PDME, phosphatidyldimethylethanolamine; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine.

Figure 6.

Livers from mice fed a choline-deficient diet for 3 days. Pemt^+/+, mice that express phosphatidylethanolamine N-methyltransferase. Pemt^−/−, mice in which the gene encoding phosphatidylethanolamine N-methyltransferase has been deleted. Reprinted from Vance, D. E. (2013) Biochim. Biophys. Acta - Molecular and Cell Biology of Lipids, Physiological roles of phosphatidylethanolamine N-methyltransferase, Volume 1831, Issue 3, 626–632 (47). Copyright (2013), with permission from Elsevier.

Figure 7.

Sections from the aortic roots of mice fed a cholesterol-enriched diet. Ldlr^−/− mice lack the low-density lipoprotein receptor and so are susceptible to atherosclerosis; Ldlr^−/−/Pemt^−/− mice lack both the low-density lipoprotein receptor and phosphatidylethanolamine N-methyltransferase. Samples were stained with oil red O (red) and hematoxylin (light blue). Reprinted from Vance, D. E. (2013) Biochim. Biophys. Acta - Molecular and Cell Biology of Lipids, Physiological roles of phosphatidylethanolamine N-methyltransferase, Volume 1831, Issue 3, 626–632 (47). Copyright (2013), with permission from Elsevier.

Figure 8.

The connections between PEMT and a high-fat diet. Shown are mice that either express PEMT (WT) or lack PEMT (KO) that were fed a high-fat diet for 10 weeks. Reprinted from Vance, D. E. (2013) Biochim. Biophys. Acta - Molecular and Cell Biology of Lipids, Physiological roles of phosphatidylethanolamine N-methyltransferase, Volume 1831, Issue 3, 626–632 (47). Copyright (2013), with permission from Elsevier.