FormalPara Core Messages

  • The final reaction of triglyceride synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2.

  • Despite their ability to similarly catalyze TG synthesis, DGAT1 and DGAT2 belong to two separate gene families, share neither DNA nor protein sequence homology and differ in their biochemical, cellular, and physiological functions.

  • DGAT2 only catalyzes the synthesis of TG whereas DGAT1 also catalyzes the synthesis of diacyalglcyerols, retinyl esters, and wax esters.

  • DGAT1 functions primarily as a wax synthase and an acyl-CoA:retinol acyltransferase (ARAT) to regulate normal fur lipid composition and retinoid homeostasis, respectively.

  • DGAT2 functions as a TG-synthesizing enzyme in murine skin to regulate acylaceramides, which are required for normal barrier function.

Introduction

In this chapter, we will discuss the two enzymes known to synthesize triacylglycerol (or triglyceride, TG) and their role in murine skin and fur physiology. However, it is imperative to note that these enzymes were named based on the initial discovery that they possessed the ability to synthesize TG. Moreover, it is likely that the additional enzymatic activities of one of the enzymes play more important roles in regulating lipid homeostasis in the skin than its ability to synthesize TG.

Triglyceride Synthesis

Two major pathways for TG biosynthesis, elucidated in the 1950s and 1960s are known as the monoacylglycerol pathway (Yen et al. 2008) and the glycerol phosphate pathway, or the Kennedy pathway (Kennedy 1957; Fig. 16.1). The monoacylglycerol pathway is the dominant pathway in cell types such as enterocytes, hepatocytes, and adipocytes that participate in the reesterification of hydrolyzed dietary fats, whereas the glycerol phosphate pathway is present in most cells types (Kayden et al. 1967). In the final reaction of both pathways, diacylglycerol and a fatty acyl-CoA, the “active” form of fatty acids (Coleman et al. 2002), are covalently bound to form TG. This reaction is catalyzed by the acyl-CoA:diacylglycerol acyltransferase (DGAT, E.C.2.3.1.20) enyzmes DGAT1 and DGAT2.

Fig. 16.1
figure 1

Acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes and triacylglycerol (or triglyceride) biosynthesis. There are two major pathways for triacylglycerol synthesis: the glycerol phosphate pathway (or Kennedy pathway) and the monoacylglycerol pathway. DGAT enzymes are involved the final reaction of both pathways by catalyzing an ester linkage between the free hydroxyl group of diacylglycerol and a fatty acyl-CoA (FA-CoA; the active form of fatty acids) to form triacylglycerol. MGAT acyl CoA:monoacylglycerol acyltransferase

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Both DGAT1 and DGAT2 reside in the endoplasmic reticulum, which is believed to be the main site of newly synthesized TG (Weiss et al. 1960). TGs synthesized by DGAT enzymes are then either stored in cytosolic lipid droplets or, in some organs such as the liver and small intestine, secreted as components of lipoproteins. Although several models have emerged (Guo et al. 2009; Walther and Farese 2009) the exact mechanism(s) by which TGs are deposited into lipid droplets remains unclear.

DGAT Enzymes

DGAT1

Despite their ability to similarly catalyze TG synthesis, DGAT1 and DGAT2 belong to two separate gene families, share neither DNA nor protein sequence homology and differ in their biochemical, cellular, and physiological functions (Yen et al. 2008). DGAT1 belongs to a large family of membrane-bound O-acyltransferases known as the MBOAT family. The genes encoding human and murine DGAT1 were identified by their sequence homology to acyl-CoA:cholesterol acyltransferase enzymes but were shown to encode a protein having DGAT activity (Cases et al. 1998). In humans, the DGAT1 gene is located on chromosome 8 and comprises 17 exons spanning 10.62 kb. In most species, the encoded protein has a predicated molecular weight of ~ 55 kDa and consists of about 500 amino acids. In humans, DGAT1 is ubiquitously expressed, with the highest mRNA expression in tissues that make considerable amounts of TG, such as the small intestine, liver, and adipose tissue (Cases et al. 1998). In mice, the expression pattern is similar to that of humans, except in that mRNA levels are relatively low in liver (Cases et al. 1998). As for the mRNA expression of DGAT specifically in the skin, it is scarcely detectable in the epidermis or dermis of neonatal mice (Stone et al. 2004). Although it is expressed at very low levels in the epidermis of adult mice, it is highly expressed in sebaceous glands (Chen et al. 2002a).

Regarding biochemical function, DGAT1 not only catalyzes the synthesis of TGs, but also the synthesis of diacyalglcyerols, retinyl esters, and wax esters via its acyl-CoA:monoacylglycerol acyltransferase (MGAT), acyl-CoA:retinol acyltransferase (ARAT) and wax synthase activities, respectively (Yen et al. 2005a). In effect, studies in vivo show that its ARAT and wax synthase activities play an important role in skin and hair physiology (Chen et al. 2002a; Shih et al. 2009). Furthermore, DGAT1 functions as such a potent ARAT that it may have been named differently had it been initially identified to possess this activity (Yen et al. 2005a).

DGAT2

DGAT2 belongs to the DGAT2/MGAT gene family (Cases et al. 2001). In addition to DGAT2 and MGATs 1, 2, and 3 (Yen et al. 2002; Yen and Farese 2003; Cheng et al. 2003), this family also consists of wax monoester synthases (Cheng and Russell 2004; Turkish et al. 2005) and a multifunctional O-acyltransferase (Yen et al. 2005b). Interestingly, this multifunctional enzyme catalyzes the synthesis of diacyalglcyerols, retinyl esters, and wax esters (Yen et al. 2005b) similarly to the multiple enzymatic activities of DGAT1.The fact that members of the DGAT2/MGAT gene family comprise enzymes that possess wax synthase activity highlights the overlapping similarities between the gene families of DGAT1 and DGAT2.

In humans, the DGAT2 gene is located on chromosome 11 and comprises eight exons spanning 42.03 kb. In most species, the encoded protein has a predicated molecular weight ranging from 40 to 44 kDa and consists of 350–400 amino acids. Like DGAT1, DGAT2 is expressed in most mammalian tissues, with the highest mRNA levels in organs that make considerable amounts of TG (small intestine, liver, and adipose tissue; Cases et al. 2001). In mice, DGAT2 mRNA is more widely expressed than humans with less variation in levels across tissues (Cases et al. 2001). As for the mRNA expression of DGAT2 specifically in the skin, it is expressed at higher levels in the epidermis than dermis of neonatal mice (Stone et al. 2004). In the epidermis of adult mice, DGAT2 mRNA expression is much more robust than DGAT1 and the majority of its expression is accounted for in the basal layer (Stone et al. 2004).

DGAT-Deficient Mice

Understanding of the physiologic function(s) of DGAT enzymes chiefly arose from genetically modified mice. The generation of mice lacking Dgat1 (Dgat1 −/−) and Dgat2 (Dgat2 −/−) provided the opportunity to study the importance of TG synthesis in systemic- and tissue-specific metabolic homeostasis. Importantly, observations from these mice demonstrate the distinct biochemical functions of DGAT1 and DGAT2, which are mirrored in their divergent physiological roles. Moreover, mRNA expression is differentially regulated under various metabolic conditions and the absence of one DGAT enzyme is not compensated by the other in either DGAT-deficient mouse model.

Dgat1 −/− mice are viable and have been studied extensively. These mice exhibit multiple phenotypes that include extended longevity (Streeper 2012), resistance to diet-induced obesity (due to increased energy expenditure) and glucose intolerance (Smith et al. 2000), enhanced insulin and leptin sensitivity (Chen et al. 2002b), impaired mammary gland development (Cases et al. 2004), and skin and fur abnormalities (Chen et al. 2002a). DGAT2-deficient mice, on the-other-hand, have been studied significantly less as homozygous DGAT2-deficient mice survive only a few hours after birth (Stone et al. 2004) and heterozygous DGAT2-deficient mice, although viable, exhibit few detectable phenotypes (unpublished). The early mortality of Dgat2 −/− mice is contributed to by insufficient substrates required for energy metabolism and impaired permeability barrier function of the skin. Together, these findings suggest that DGAT2 is the dominant DGAT enzyme in regulating TG homeostasis.

Skin and Fur Abnormalities in Dgat1-Deficient Mice

Although Dgat1 −/− mice have been primarily studied in regards to whole-body energy metabolism, important observations have been made in respect to DGAT1 function in skin and fur (Chen et al. 2002a; Shih et al. 2009). Adult Dgat1 −/− mice develop dry fur and hair loss, which are associated with atrophic sebaceous glands and fur lipid abnormalities. Young Dgat1 −/− mice exhibit normal fur appearance at weaning. However, after puberty (6–8 weeks of age) the fur of Dgat1 −/− mice appears dry and less sheen compared to that of wild-type mice and subsequent hair loss is observed (Chen et al. 2002a). The hair loss phenotype is more prominent in young males than in females (Chen et al. 2002a) but is similar regardless of gender as it worsens with age (unpublished).

In a wild-type mouse, the sheen appearance of the fur is due to lipids (sebum) secreted from the sebaceous glands associated with hair follicles. In 6-week-old Dgat1 −/− mice, the sebaceous glands are normal in appearance and located in a typical association with normal hair follicles. However, in 3-month-old Dgat1 −/− mice, sebaceous glands are atrophied along the ventral and dorsal surfaces and few are associated with hair follicles. Although fur lipids from both wild-type and Dgat1 −/− mice are composed of similar levels of sterol esters, free cholesterol, and surprisingly, triglycerides, Dgat1 −/− mice lack several fur lipids, with the most prominent of these being type II wax diesters. Like the hair loss phenotype, the difference in fur lipid composition is also age-dependent where the differences are less striking in young mice but more pronounced in adults. Of note, the fur of heterozygous (Dgat1 +/−) mice is normal in appearance throughout their lifespan.

As a result of atrophied sebaceous glands and decreased fur lipids, Dgat1 −/− mice exhibit impaired water repulsion and hypothermia following water immersion. The fur of Dgat1 −/− mice retains significantly more water compared to wild-type controls, which are almost completely dry, 5 min after water immersion (Chen et al. 2002a). Consequently, Dgat1 −/− mice become hypothermic and this persists for an hour following immersion.

Farese and colleagues found the skin and fur phenotypes of Dgat1 −/− mice to be unexpectedly modulated by the adipocyte-secreted hormone, leptin (Chen et al. 2002a). Leptin is a peptide hormone known to reduce food intake and enhance energy expenditure through its receptors in the central nervous system (specifically in the hypothalamus; Morton and Schwartz 2011) but also functions to regulate energy metabolism directly through its receptors in peripheral tissues, including the skin (Frank et al. 2000; Poeggeler et al. 2010). When DGAT1 deficiency is introduced into mice with a spontaneous mutant in the leptin gene (Dgat1 −/− ob/ob), the hair loss, water repulsion, and sebaceous gland abnormalities are almost absent. However, it is important to note that the sebaceous gland size and fur lipid levels are slightly decreased in Dgat1 −/− ob/ob mice compared to controls (Dgat1 +/+  ob/ob). Following 2 weeks of peripheral or central leptin infusion, the size of sebaceous glands and fur lipid levels are slightly reduced in controls but both are markedly reduced in Dgat1 −/− ob/ob mice. Furthermore, these changes are reverted to pretreatment states 2 weeks after leptin administration is withdrawn. Since both peripheral and central administrations of leptin have similar effects on sebaceous glands and fur lipid composition, it is reasonable that these observations can be mediated entirely through the hypothalamus. One plausible mechanism by which leptin centrally modulates the skin and fur phenotypes of Dgat1 −/− mice is by activation of the sympathetic nervous system to suppress the expression of DGAT2. Indeed, mice deficient in DGAT1 are known to exhibit enhanced leptin sensitivity (Chen et al. 2002b) and DGAT2 mRNA is significantly decreased in the skin of these mice (Chen et al. 2002a). Moreover, compared to wild-type controls, DGAT2 mRNA expression is increased at similar levels in the skin of leptin-deficient mice despite the presence or absence of DGAT1.

Upon further investigation of the hair loss observed in Dgat1 −/− mice, Farese and colleagues discovered this phenotype to also be, in part, due to impaired retinoid (retinol and its derivatives) homeostasis and that the ARAT activity of DGAT1 plays an important role in murine skin (Shih et al. 2009). As mentioned above, DGAT1 not only catalyzes the synthesis of TGs, but also functions as a potent ARAT synthesizing retinyl esters through the esterification of retinol (vitamin A) with fatty acyl-CoA substrates (Yen et al. 2005a). Retinol is obtained through the diet and mostly stored in the form of retinyl esters in the cytosolic lipid droplets of cells providing then a local source of retinol. Retinol is the precursor for retinoic acids (all-trans and 9-cis-retinoic acid), which are ligands for nuclear hormone receptors (retinoic acid receptor and retinoid-x receptor) and are known to affect sebaceous gland function (Zouboulis et al. 1991; Strauss et al. 1987) and hair growth (Bazzano et al. 1993). In whole skin of Dgat1 −/− mice compared to controls, ARAT activity is significantly reduced by ~ 90 % and levels of unesterified retinol and all-trans retinoic acid are increased by ~ 22 and 40 %, respectively (Shih et al. 2009). Studies from Dgat1 −/− mice suggest that when dietary retinol is sufficient, DGAT1 deficiency results in retinoic acid toxicity in the skin, which subsequently leads to cyclical hair loss. Consistent with retinoic acid toxicity, genes regulated by the retinoic acid receptor are also elevated in the skin of these mice. When dietary retinol is deficient however, neither retinoic acid toxicity nor hair loss is observed in Dgat1 −/− mice. Moreover, mice deficient of DGAT1 specifically in the skin exhibit cyclical hair loss and increased expression of retinoic acid receptor target genes indicating that dysregulation of retinoid homeostasis is due loss of the enzyme in the skin rather than systemic changes in metabolism (Shih et al. 2009). Collectively, these findings reveal DGAT1 to be a major ARAT in murine skin and therefore essential for maintaining retinoid homeostasis and preventing retinoic acid toxicity-induced hair loss. Of note, it remains to be determined if the sebaceous gland and fur lipid phenotypes of Dgat1 −/− mice are also due to the dysregulation of retinoid homeostasis.

Similarities Between Dgat1- and Scd1-Deficient Mice

The pleiotropic phenotype of Dgat1 −/− mice is remarkably similar to mice that lack stearoyl-CoA desaturase1 (Scd1 −/−), an enzyme that catalyzes the synthesis of the delta-9-monounsaturated fatty acids (described in Chapter “Stearoyl-CoA Desaturases are Regulators of Lipid Metabolism in Skin” this volume). Like Dgat1 −/− mice, Scd1 −/− mice exhibit increased energy expenditure, resistance to diet-induced obesity and glucose intolerance, enhanced insulin and leptin sensitivity, sebaceous gland atrophy, and hair loss (Ntambi and Miyazaki 2003). Interestingly, mice deficient of SCD1 specifically in the skin (SKO) show increased energy expenditure and are protected from diet-induced obesity, recapitulating the global SCD1 deficiency phenotype. Furthermore, thorough analysis of the skin of SKO mice show reduced mRNA expression of enzymes involved in TG synthesis, including Dgat1, increased mRNA expression of retinoic acid regulated genes (Chapter “Stearoyl-CoA Desaturases are Regulators of Lipid Metabolism in Skin” this volume), and a robust increase in retinol and retinoic acid (Flowers et al. 2011). Given the similarities of altered epidermal retinoid metabolism and energy balance observed in both the global DGAT1- and skin-specific SCD1-deficent mice, it is possible that the skin-specific deletion of Dgat1 could also result in increased energy expenditure and protection from diet-induced obesity. In effect, unpublished data suggest that the deletion of Dgat1 specifically in the skin results in an increase in heat loss, energy expenditure and decreased adiposity on a regular chow diet. Collectively, one can speculate that a reduction of ARAT activity in the skin and subsequent retinoic acid toxicity results in a loss of insulating factors (fur and skin/fur lipids) and consequently increases thermogenesis in order to maintain normal core body temperature. This in-turn results in an increase in energy expenditure and therefore a reduction in adiposity. Further experimentation is required to determine if skin-specific Dgat1-deficient mice are protected from diet-induced obesity and if this effect is lost by a high-fat, retinol-deficient diet.

Skin Abnormalities in Dgat2-Deficent Mice

Studies from Dgat2 −/− mice show that DGAT2 also plays an important role in the skin. As mentioned previously, Dgat2 −/− mice survive only a few hours after birth which is contributed to, in part, by severe skin abnormalities (Stone et al. 2004). The skin of newborn Dgat2 −/− mice is shiny, lacks elasticity, and exhibits impaired permeability barrier function leading to increased transepidermal water loss and rapid dehydration. While there is no evidence of abnormal epidermal differentiation, the structure of the skin from neonatal Dgat2 −/− mice exhibits thinning of the epidermis, compact hyperkeratosis of affected stratum corneum and effacement of the epidermal rete ridges/papillary projections which leads to a flattened dermal–epidermial interface. In normal skin barrier function, lamellar bodies are secreted from keratinocytes in the stratum spinosum/granulosum into the stratum corneum, resulting in the formation of an impermeable, lipid-containing membrane that serves as a water barrier. In the epidermis of Dgat2 −/− mice, examination by electron microscopy revealed a typical number of lamellar bodies but a reduction in the lamellar body content and the number of lamellar membranes in the stratum corneum extracellular space. Further analysis of the skin of Dgat2 −/− mice showed a 96 % decrease in TG content while other lipids were similar to those of control mice. Assessment of the composition of these lipids revealed a significant reduction (> 90 %) in linoleic acid containing TGs and free fatty acids. Also observed was a 60 % decrease in skin levels of acyl-ceramide, a skin lipid also composed of linoleic acid, which is thought to be required for the maintenance of the skin permeability barrier. Skin grafting experiments also suggest that the skin defects in Dgat2 −/− mice are partially due to a lack of the enzyme systemically in addition to the loss specifically in the skin. Skin from Dgat2 −/− and control mice grafted onto athymic nude mice showed similar fur development and transepidermal water loss 3 weeks following transplantation. Importantly, plasma levels of TG and free fatty acids were reduced by 64 and 80 %, respectively. Taken together, decreased triglyceride synthesis, via DGAT2 deficiency, leads to a dysregulation of systemic and epidermal lipid metabolism resulting in abnormal lamellar body secretory system, impaired skin permeability barrier function, and ultimately rapid dehydration.

Similarities Between Dgat2- and Scd2-Deficient Mice

Interestingly, the skin phenotype of Dgat2 −/− mice is similar to mice that lack SCD2, an isoform of SCD that is important in lipid synthesis in early development and is required for survival (Miyazaki et al. 2005). Scd2 −/− neonatal mice exhibit increased transepidermal water loss that associated with a reduction of linoleic acid incorporation into acyl-aceramides. Futhermore, only ~ 30 % of Scd2 −/− mice survive to adulthood. One can speculate the possibility that SCD2 provides DGAT2 with monounsaturated fatty acids required for TG biosynthesis during early skin development and the maintenance of skin permeability barrier function.

In summary, studies from DGAT-deficient mice reveal that the enzymatic activities of DGAT1 and DGAT2 play important roles in regulating murine skin and fur physiology. Although DGAT1 and DGAT2 were initially discovered to possess diacylglycerol acyltransferase activity, it is likely that the additional enzymatic activities of DGAT1 may be dominant. At least in murine skin, it seems as though DGAT1 primarily functions as a wax synthase and an ARAT to regulate normal fur lipid composition and retinoid homeostasis whereas DGAT2 functions as a TG synthesizing enzyme to regulate acylaceramides, which are required for normal barrier function. Since the expression profile of DGAT1 and DGAT2 are similar to that of humans, it is likely that DGATs also play an important role in regulating lipid metabolism in the skin of humans.