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Journal of Endocrinology & Metabolism

Short Communication

Volume 000, Number 000, September 2024, pages 000-000

A Comparison Between Canonical and Alternative Pathways in Triglyceride Synthesis

Because energy sources are not always available from the environment, mammals have evolved the ability to store and transport energy as triglycerides (TGs) [1-3]. TG are ideal for energy storage because they serve as stores of highly reduced carbon, high energy per unit mass, and do not require water for their storage, as opposed to glycogen, which is stored in hydrated form [4, 5].

Chemically, TG are neutral lipids composed of a glycerol backbone conjugated to three fatty acyl chains, hence the name “triglyceride”. TG synthesis occurs in the endoplasmic reticulum (ER) membrane of cells by bonding three fatty acid molecules to a glycerol molecule. In humans, many types of cells have the capacity of TG synthesis, but the liver and white adipose tissue (WAT) are especially critical [6]. In the liver, TG synthesis primarily occurs in the fasting state, while in WAT, it happens predominantly in the fed state [1, 7].

In the liver, during fasting, TG are synthesized from fatty acids, which are from two sources: either the liver, which converts glucose into fatty acids, or WAT, which breaks down stored TG into fatty acids, which are then secreted into the circulation and, in turn, taken up by the liver. The newly synthesized TGs are then packaged by the liver into TG-rich lipoproteins called very low-density lipoprotein (VLDL). VLDL particles are secreted directly into blood, where TGs are delivered to peripheral tissues, such as the heart and muscle to generate energy during fasting through the process of β-oxidation [1].

In WAT, after food intake, dietary fats in the intestine are broken down into fatty acids, which are packaged into chylomicrons (CM). CM, high in TG, are then secreted into the bloodstream to meet lipoprotein lipase (LPL) in the capillaries of adipose tissues. LPL hydrolyzes TG into fatty acids, which are taken up by adipocytes to synthesize into TG for long-term energy storage. In adipocytes, TGs are stored in organelles called lipid droplets (LDs) [8, 9].

The last step of TG synthesis in humans is carried out by diacylglycerol O-acyltransferase 1 (DGAT1) and diacylglycerol O-acyltransferase 2 (DGAT2), which catalyze the covalent addition of a fatty acyl chain to diacylglycerol (DAG). DGAT1 and DGAT2 catalyze the same chemical reaction, but they are genetically unrelated. DGAT1 is part of the membrane bound O-acyl transferase (MBOAT) protein family, and DGAT2 is a member of the diacyl-glycerol acyltransferase (DAGAT) family [10-13]. Mouse gain- and loss-of-function studies showed that the two proteins have important, non-redundant roles in both the liver and WAT [13-20].

The canonical TG synthesis pathway from fatty acyl-CoA and DAGTs has been clearly defined. Recently a new alternative TG synthesis pathway has been discovered [21, 22], and it is reported that transmembrane protein 68 (TMEM68, also named DIESL) and thioredoxin-related transmembrane protein 1 (TMX1), within the ER, carry out cell-autonomous alternative TG synthesis. TMEM68 functions as a 1,2-diacylglycerol O-acyltransferase, and TMX1 potently inhibits TMEM68. It is proposed that TMEM68 converts endogenous acyl chains into TG, a process that requires regulations of TMX1, and that TMX1-TMEM68 may enable the supply of fatty acids to mitochondria during times of lipid or nutrient scarcity [21].

TG synthesis in the body is tightly and accurately regulated during the fed-fast cycle. We then asked the following questions: 1) During the fed-fast cycle, how are the two pathways regulated? 2) Does the body use the two pathways similarly or differently in tissues including the liver and WAT? To answer the questions, we examined mRNA levels of these genes in the liver and fat during the fed-fast cycle, and in other conditions, including aging and high-fat diet (HFD) treatment.

Mice were housed at 22 - 24 °C with a 14-h light, 10-h dark cycle and provided with ad libitum water and a chow diet (6% calories from fat, 8664; Harlan Teklad, Indianapolis, IN) unless otherwise indicated. Eight-week-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were treated with 20-h fasting with fed mice as controls. Young (3-month-old) and old (20-month-old) C57BL/6J mice were purchased from the Jackson Laboratory. To examine nutritional stimulation, 10 4-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were placed on either a chow diet or a high-fat, high-sucrose diet (58% kcal from fat, 26% kcal from sucrose, D-12331; Research Diets, New Brunswick, NJ) for 3 months. At International Mouse Phenotyping Consortium (IMPC), the mouse strain is C57BL/6NTac. All animal protocols were approved by the Animal Care and Use Committee of Wayne State University.

Dissected tissues were immediately placed into RNAlater solution (Ambion, Austin, TX) for subsequent RNA extraction. Total RNA was isolated from tissues with RNeasy Tissue Mini Kit with deoxyribonuclease treatment (Qiagen, Valencia, CA). One microgram of RNA was reverse transcribed to cDNA using random hexamers (Superscript; Ambion). Relative expression levels were calculated, and β-actin was used as an internal control. Primer sequences for mouse Dgat1 were forward: 5′-TCCGTCCAGGGTGGTAGTG-3′; reverse: 5′-TGAACAAAGAATCTTGCAGACGA-3′. Primer sequences for mouse Dgat2 were forward: 5′-GCGCTACTTCCGAGACTACTT-3′; reverse: 5′-GGGCCTTATGCCAGGAAACT-3′. Primer sequences for mouse Tmem68 were forward: TGTGGGATGGTGCAAGGAAAA-3′; reverse: 5′-CAGCTACTACTCGGCAAGTTC-3′. Primer sequences for mouse Tmx1 were forward: 5′-CACTTGGGGCGTCTTATGGTT-3′; reverse: 5′-CCAGTTCTCATCGGTGAGGAC-3′. Primer sequences for mouse β-actin were forward: 5′-GTGACGTTGACATCCGTAAAGA-3′; reverse: 5′-GCCGGACTCATCGTACTCC-3. All methods were carried out in accordance with relevant guidelines and regulations.

Data are expressed as the mean ± SEM (standard error of the mean). Statistical significance was tested with unpaired two-tailed Student’s t-test unless otherwise indicated. One-way analysis of variance (ANOVA) was used to compare the means of three groups, e.g., hepatic expression levels of the genes in fed, fasting, re-fed mice. Prism Graphpad was used to draw figures. The differences were considered statistically significant if P < 0.05.

Because TG synthesis is tightly and accurately orchestrated by the fed-fasting cycle, we hypothesized that expression of the TG synthesis genes is altered by treatments of fasting and refeeding. The liver is a central organ that is involved in TG synthesis, and thus we first checked the expression of these genes in the liver. The mice were treated with ad libitum feeding, 20-h fasting, and 4-h refeeding following the fasting. qPCR analysis was then performed to examine mRNA expressions. Fasting increased liver Dgat1 expression at least two-fold, and 4 h after refeeding the expression returned to the basal level (Fig. 1), consistent with previous results [14]. In other words, expression of liver Dgat1 was significantly reduced 4 h after refeeding, compared to fasted mice. Dgat2 showed a similar pattern to that of Dgat1, that is, fasting upregulated its expression, and re-feeding significantly reduced its expression. These results suggested that both Dgat1 and Dgat2 were upregulated during fasting in the liver, while in a fed state, their expressions were reduced.

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Figure 1. Liver expression of Dgat1, Dgat2, Tmem68, and Tmx1. Hepatic mRNA levels of (a) Dgat1, (b) Dgat2, (c) Tmem68, and (d) Tmx1 in mice with ad libitum feeding, 20-h fasting, and 4-h refeeding following the fasting (n = 4, 5, 3, for the fed, fasting, and re-fed groups, respectively). Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns: non-significant; SEM: standard error of the mean.

Both fasting and refeeding did not significantly alter the expression of Tmem68. However, fasting significantly increased the expression of Tmx1, and 4 h following the refeeding, its expression returned to the basal level. Thus, the hepatic expression of Tmx1 was increased by fasting, but repressed by feeding (Fig. 1). Because Tmx1 is a potent inhibitor of Tmem68, the enzyme for TG synthesis, these results are consistent with a notion that fasting suppressed the alternative TG synthesis pathway but upregulated the canonical pathway.

WAT is a critical organ for postprandial TG synthesis. Following a meal, TG synthesis is increased in WAT, while fasting induces lipolysis leading to release of fatty acids into the circulation. We, therefore, examined the expression of TG synthesis pathways in WAT.

Fasting dramatically suppressed the expression of Dgat2 (more than 80%), while feeding dramatically increased its expression (Fig. 2). This is consistent with the role of Dgat2 in WAT. Both fasting and re-feeding did not alter the expression of Dgat1, consistently with the notion that Dgat2, but not Dgat1, is physiologically relevant in WAT.

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Figure 2. Expression of Dgat1, Dgat2, Tmem68, and Tmx1 in white adipose tissue (WAT). WAT mRNA levels of (a) Dgat1, (b) Dgat2, (c) Tmem68, and (d) Tmx1 in mice with ad libitum feeding, 20-h fasting, and 4-h refeeding following the fasting (n = 4, 5, 3, for the fed, fasting, and re-fed groups, respectively). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are represented as mean ± SEM. ns: non-significant; SEM: standard error of the mean.

Both fasting and refeeding did not significantly alter the expression of Tmem68. However, fasting significantly increased the expression of Tmx1 (Fig. 2). Thus, this result is consistent with a notion that fasting downregulates, and feeding upregulates the alternative TG synthesis pathway. In other words, it is likely that in WAT, both canonical and alternative pathways are induced by feeding and suppressed by fasting, consistently with their physiological roles.

Excess TG synthesis contributes to obesity and metabolic disease. To study this, we subjected mice to HFD for 3 months and analyzed gene expression in both the liver and WAT. In the liver, HFD treatment did not significantly alter the expression of any of these genes (data not shown). In WAT, HFD dramatically reduced the expression of Dgat1 (85% reduced) and Dgat2 (60%) (Fig. 3). The expression levels of Tmem68 and Tmx1 in WAT remained unaffected by the HFD.

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Figure 3. Expression of Dgat1, Dgat2, Tmem68, and Tmx1 in white adipose tissue (WAT). WAT mRNA levels of (a) Dgat1, (b) Dgat2, (c) Tmem68, and (d) Tmx1 in mice on chow and 3-month HFD (n = 5 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are represented as mean ± SEM. ns: non-significant; SEM: standard error of the mean; HFD: high-fat diet.

Aging and TG metabolism are closely related [23], and therefore we examined the expression of these genes in young vs. aged mice. It is commonly believed that 3-month-old mice are considered young, and 20-month is considered old mice [24]. Thus, we compared the expression of these genes in 3-month vs. 20-month-old mice. In the liver, there were no significant differences between young and old mice for all these genes (data not shown). In WAT, aging significantly reduced the expression of Dgat1 and Dgat2, while expression of Tmem68 and Tmx1 was not changed (Fig. 4).

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Figure 4. Expression of Dgat1, Dgat2, Tmem68, and Tmx1 in white adipose tissue (WAT). WAT mRNA levels of (a) Dgat1, (b) Dgat2, (c) Tmem68, and (d) Tmx1 in young and old mice. Young and old mice that are 3 and 20 months old, respectively (n = 5 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are represented as mean ± SEM. ns: non-significant; SEM: standard error of the mean.

Gene deletion has become a powerful way to examine gene functions. Brummelkamp’s group reported that Tmem68 KO mice exhibited increased respiratory exchange ratio in response to fasting, indicating an attenuated switch from carbohydrate to lipid oxidation [21]. The International Mouse Phenotypic Consortium (IMPC) produces whole body KO mouse lines for community use and delivers broad comprehensive phenotyping data and the KO lines as an international resource enabling functional analyses with genome-wide coverage [25, 26]. Tmem68 KO mice have been generated and phenotyped by IMPC (Tmem68^{tm1a(EUCOMM)Wtsi}). In male Tmem68 KO mice generated by IMPC, plasma aspartate aminotransferase (AST) levels were significantly increased (Fig. 5), suggesting liver phenotypes. Plasma glucose levels were reduced (Fig. 5), suggesting that Tmem68 is involved in glucose homeostasis. Therefore, Tmem68 KO mice generated by IMPC independently show consistent metabolism phenotypes.

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Figure 5. Plasma AST and glucose levels in Tmem68 KO mice. (a) plasma AST and (b) glucose levels in WT and Tmem68 KO mice. Data were obtained from the International Mouse Phenotyping Consortium (IMPC). #P < 0.01. Data are represented as mean ± SEM. SEM: standard error of the mean; AST: aspartate aminotransferase; WT: wild-type; KO: knockout.

TG metabolism has profound implications for human health and disease [27, 28]. As a standard component of the vascular system, TGs, in the form of lipoproteins, are continuously being circulated in the bloodstream to be metabolized to provide a source of energy for oxidative tissues or saved in WAT, according to the body’s needs [1, 3]. High TG levels in the blood (hypertriglyceridemia) are associated with a collection of disorders known as metabolic syndrome, increasing the risk of developing diabetes, stroke, or heart disease [29-31]. Therefore, TG metabolism, i.e., its synthesis (lipogenesis) and breaking down (lipolysis) is accurately and carefully orchestrated during the fed-fast cycle.

In the current study, we examined how the two pathways, the canonical and alternatively TG synthesis pathways, are regulated, at the mRNA levels, throughout the fed-fast cycle. We found Tmem68 mRNA levels are relatively stable, without statistically significant changes by fed and fasting treatments. In contrast, Tmx1, the Tmem68 inhibitor, is induced by fasting in both the liver and WAT. Thus, it is likely that the alternative pathway is regulated mainly by altering the level of Tmx1.

Additionally, in the liver, during fasting, the canonical pathway is upregulated, while the alternative pathway is suppressed. These results support the notion that during fasting, the liver relies more on the canonical pathway to generate TG (Fig. 6). In contrast, in WAT, fasting dramatically reduces the expression of Dgat2, while feeding increases it, which is consistent with its role in TG synthesis in WAT. Additionally, fasting modestly increases the expression of Tmx1. These results are consistent with a notion that both pathways are needed for TG synthesis in WAT (Fig. 7).

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Figure 6. A model of how canonical and alternative pathways work during the fed-fasting cycle in the liver. DG: diacylglycerol; TG: triglyceride.

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Figure 7. A model of how canonical and alternative pathways work during the fed-fasting cycle in white adipose tissue. DG: diacylglycerol; TG: triglyceride.

Moreover, we found that HFD treatment does not significantly change levels of Tmem68 and Tmx1 in the liver. In contrast, in WAT, HFD dramatically suppressed the levels of Dgat1 and Dgat2. This seems to suggest that the body downregulates the two TG synthetases as a compensatory mechanism in response to obesity. Interestingly, aging also downregulates the expression of Dgat1 and Dgat2 in WAT, but the implications of these aging-associated changes are not clear.

The current study has the following limitations. One limitation is that we examined the mRNA levels of these genes, but not the protein levels, which may have varied half-lives. Therefore, in some cases, the mRNA levels do not reflect the relative or absolute protein abundance. Another limitation is that for enzymes, their activity is more important than their mRNA expression levels and protein abundance. Thus, an ideal readout would be enzyme activity, such as for Tmem68. However, comparing the mRNA levels of genes involved in the two pathways can be a starting point to address these questions. To the best of our knowledge, this study is the first attempt to compare and contrast these two pathways. Future studies ideally would need to examine the abundance of the proteins in the two pathways during the fed-fast cycle, enzymatic activities of the enzymes, and metabolite flux analysis in the relevant KO mice.

The Tmem68 KO mice showed phenotypes of changes in fasting glucose and AST levels, but not in TG levels and adiposity. Because the regulation of TG synthesis is sensitive to nutrition states, it is possible that the phenotypes such as TG levels, fatty acid levels, and body composition, can only be revealed when the KO mice are challenged with fasting, refeeding, and HFD. The opposite to TG synthesis is fatty acid oxidation, which primarily happens during fasting. Fasting activates a series of mechanisms to increase fatty acid oxidation and ATP production. For example, in mitochondria, these mechanisms include increased expression of the mitochondrial NAD transporter [32], activation of sirtuin 3 [33], and the activation of mitochondrial NAD kinase [34-36]. Thus, another question to address is how the expression of TG synthetase genes and that of fatty acid oxidation genes are coordinated under various physiological and pathological conditions.

In summary, the study explores TG metabolism, with a focus on synthesis pathways in the liver and WAT, comparing the canonical and the newly discovered alternative pathways. The study investigates gene expression changes during the fed-fast cycle, fasting, refeeding, HFD treatments, and aging. Our results are consistent with a notion that fasting upregulates the canonical pathway in the liver, while in WAT, feeding upregulates both pathways, and that HFD and aging downregulate the canonical pathway in WAT. Tmem68 KO mice exhibit liver and glucose homeostasis phenotypes. We showed that the regulation at the mRNA level of the newly identified alternative TG synthesis pathway is likely through Tmx1, but not Tmem68, during the fed-fast cycle. Our results are consistent with the notion that during fasting, the liver relies more on the canonical pathway than the alternative pathway. In WAT, there is a possibility that both pathways are upregulated and are needed following feeding.

Acknowledgments

None to declare.

Financial Disclosure

The research was supported in part by Wayne State University (RZ) and by National Institutes of Health (NIH) grants DK132065 (to RZ and KZ).

Conflict of Interest

The authors declare no conflict of interest.

Informed Content

Not applicable.

Author Contributions

RZ conceived the study, analyzed the data, and wrote the manuscript. ZF and QC performed the experiments. KZ analyzed the data and revised the manuscript.

Data Availability

All data supporting the findings of this study are available within the article.

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