Expression and regulation of GPAT isoforms in cultured human keratinocytes and rodent epidermis

Abstract

The principal function of the epidermis is to provide a protective barrier against transcutaneous water loss, which is essential for survival in a terrestrial environment. This permeability barrier is localized to the stratum corneum (SC), the outermost layer of the epidermis ( 1 ). This permeability barrier is mediated by extracellular lipids, primarily cholesterol, ceramides, and free fatty acids, which are delivered to the extracellular spaces of the SC by the secretion of lamellar body contents by differentiated keratinocytes ( 1 ). Lamellar bodies contain cholesterol, glucosylceramides, and phospholipids, and following lamellar body secretion the glucosylceramides are metabolized to ceramides, a process catalyzed by the enzyme ␤ -glucocerebrosidase, and the phospholipids are metabolized to free fatty acids, a reaction catalyzed by secretory phospholipases ( 1-4 ). The formation of lamellar bodies by keratinocytes requires the prior synthesis of suffi cient quantities of these three lipids in keratinocytes. Pertinently, disruption of the permeability barrier leads to a marked increase in epidermal cholesterol, ceramide, and fatty acid synthesis, which enhances the formation of lamellar bodies ( 1, 5 ). Conversely, inhibition of cholesterol, ceramides, or fatty acid synthesis in the epidermis impairs the formation of lamellar bodies, leading to the decreased delivery of lipid to the extracellular spaces of the SC and impaired permeability barrier function ( 6-8 ).Abstract Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPAR ␥ or PPAR ␦ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPAR ␥ and PPAR ␦ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes. -Lu, B., Y. J. Jiang, P. Kim, A. Moser, P. M. Elias, C. Grunfeld, and K. R.

Inhibited Insulin Signaling in Mouse Hepatocytes Is Associated with Increased Phosphatidic Acid but Not Diacylglycerol

“…It was proposed that because different PA-producing enzymes are located on or within specific subcellular organelles, the PA molecules produced by these enzymes might function differently because of their cellular compartmentalization (1,26,41). PA derived from GPAT (via AGPAT) is produced at the outer mitochondrial membrane, at the lipid droplet, or at the ER surface (26,42,43); the PA produced by PLD1 is present in the Golgi and the nucleus (44); and DGK produces PA at the plasma membrane, ER, and nucleus (45). However, because our previous and current data show that each of three different catalytic pathways, GPAT/AGPAT, PLD, and DGK, provides PA that inhibits insulin signaling to Akt, the ability of PA to disrupt insulin signaling suggests that the effect of the PA may be unrelated to its source.…”

Glycerol-3-phosphate Acyltransferase (GPAT)-1, but Not GPAT4, Incorporates Newly Synthesized Fatty Acids into Triacylglycerol and Diminishes Fatty Acid Oxidation

“…Gpat2 mRNA is most strongly expressed in testes and probably plays a minor role in liver, but the mRNA expression and activities in liver of the remaining GPAT isoforms are high (1,5,6,19). Although studies suggest that GPAT3 is regulated by PPAR␥ and PPAR␦ in keratinocytes (20) and by insulin-mediated phosphorylation in 3T3-L1 adipocytes (21), the regulation and specific contributions of hepatic GPAT3 to glycerolipid synthesis are unknown. The availability of Gpat1 and Gpat4 knock-out mice, however, has allowed us to directly compare them and to delineate the specific roles of each isoform.…”

“…Liver was minced and then homogenized similarly. Total membranes were isolated by centrifuging the homogenate at 100,000 ϫ g for 1 h. GPAT specific activity was assayed for 10 min at room temperature in a 200-l reaction mixture containing 75 mM Tris-HCl, pH 7.5, 4 mM MgCl 2 , 1 mg/ml BSA (essentially fatty acid-free), 1 mM dithiothreitol, 8 mM NaF, 800 M [ 3 H]glycerol 3-phosphate, and 80 M palmitoyl-CoA (20). The reaction was initiated by adding 10 -30 g of membrane protein after incubating the membrane protein on ice for 15 min in the presence or absence of 2 mM N-ethylmaleimide (NEM), which inactivates GPAT isoforms 2, 3, and 4.…”

The expression and regulation of enzymes mediating the biosynthesis of triglycerides and phospholipids in keratinocytes/epidermis

“…In contrast, under the same experimental conditions, GPAT-2 is not detected in either human keratinocytes or in mouse epidermis (but is found in mouse liver),19 ( Table 1 ). GPAT-3 and -4 are mainly localized to the upper epidermis (stratum corneum/granulosum/spinosum) and GPAT-1 is found in both the upper and lower (stratum basale) epidermis in mice 19…”