Loss of Monoacylglycerol O-Acyltransferase 2 can be Compensated for by Diacylglycerol O-Acyltransferases 1 and 2 in High-Fat Diet-Induced Obesity and Mammary Cancer Development

Background: Dietary fat absorption involves the re-esterication of digested triacylglycerol in the enterocytes, it is a biological process catalyzed by monoacylglycerol O-acyltransferase 2 (MOGAT2, aka MGAT2), which is highly expressed in the small intestine. A previous study showed that the loss of the Mogat2 gene can prevent high-fat diet-induced obesity in mice. Obesity is associated with an increased risk of several types of cancer including postmenopausal breast cancer. Methods: We collected 147 patients with triple negative breast adenocarcinoma to explore the relationship between the expression of MOGAT2 and patient overall survival. And we generated a Mogat2-decient mouse mammary tumor model by crossing Mogat2-decient mice with MMTV-PyMT mice to examine the effect of losing MOGAT2 in vivo. Results: Our founding suggest that obesity was induced by a relatively high-fat diet (37% of calories from fat) in the mice with or without Mogat2 knockout. Mammary tumor development was deteriorated by a relatively high-fat diet regardless of Mogat2 deciency. As a compensation mechanism, upregulation of diacylglycerol O-acyltransferases 1 and 2 (Dgat1 and Dgat2) in the Mogat2 decient mice was found. Conclusions: Elevated expression of MOGAT2 in triple negative breast adenocarcinoma predicts poorer patient overall survival. With the compensation of Dgat1 and Dgat2, Mogat2 deciency alone cannot prevent fat diet-induced obesity, nor prevent mammary tumor development in a mouse model.


Background
Breast cancer is the most commonly diagnosed cancer and the second most common cause of cancerinduced death in women worldwide (1). Over 600,000 premenopausal and 1.4 million postmenopausal breast cancer cases were diagnosed around the world in 2018 (2). Relatedly, obesity poses an increased risk in developing breast cancer, particularly in postmenopausal women (1). Sustained weight loss can signi cantly reduce breast cancer risk (3). However, the impact of genetically preventing obesity on breast cancer risk is unclear.
Triacylglycerols, the bulk of dietary fat, are absorbed in the intestine and readily stored as body fat (4).
The hydrolysis of triacylglycerols to 2-monoacylglycerol and fatty acids are primarily catalyzed by pancreatic lipase in the intestinal lumen, where these hydrolysis products are taken up by enterocytes and resynthesized into triacylglycerols via the monoacylglycerol pathway (5). The monoacylglycerol pathway is a process catalyzed by the acyl-CoA: monoacylglycerol acyltransferase (MOGAT) family which includes MOGAT1, MOGAT2, and MOGAT3, and accounts for 70-80% of triglyceride re-synthesis in the intestine (6, 7). Another pathway involved in triglyceride synthesis is the glycerol 3-phosphate pathway (8). Among the three MOGAT enzymes, only MOGAT2 (also known as MGAT2) is highly expressed in the intestine of both mice and humans (9)(10)(11)(12).
It has been reported that a de ciency in MOGAT2 protects mice from obesity and associated metabolic disorders induced by a high fat diet (13,14). MOGAT2 de ciency also protects genetically obese Agouti mice from excess weight gain on a chow diet (4). Diacylglycerol (DAG) is usually acylated by the acyl coenzyme A: diacylglycerol acyltransferase (DGAT) to re-synthesize triacylglycerol (TAG). Importantly, enzymes DGAT1 (15) and DGAT2 have been shown to acylate MAG to form DAG, and MOGAT2 and MOGAT3 have the capacity to acylate DAG to form TAG (16,17).
To elucidate the role of MOGAT2 in breast cancer progression, especially under obese conditions, we evaluated the prognostic value of MOGAT2 expression in triple negative breast adenocarcinoma, and generated a MOGAT2-de cient mouse mammary tumor model by crossing MOGAT2-de cient mice with MMTV-PyMT mice.

Methods
Mice and animal care FVB/N-Tg (MMTV-PyMT) 634Mul mice and B6.129S4-Mogat2 tm1Far /J (MOGAT2 −/− ) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed under speci c pathogen-free conditions in the Animal Center of Guangdong Pharmaceutical University. All animal experiments were performed in accordance with institutional guidelines and were approved by Sun Yat-sen University Cancer Center's Institutional Animal Care and Usage Committee, with the ethical approval number L102012017000G. The control group were fed by a normal diet consisting of 4% fat, 65% carbohydrates and 20% protein (weight ratio). And 10% of the total calories were from fat. The experimental group were fed by a relatively high-fat diet consisting of 20% sugar, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, 10% casein, 0.6% calcium hydrogen phosphate, 0.4% stone powder, 0.4% premix, and a 52.2% basal diet (weight ratio). And 37% of the total calories were from fat. The normal diet and the relatively high fat diet were purchased from the Experimental Medicine Animals Center (Foshan, China).
All animals were killed by inhalation of carbon dioxide. Putting the animals into a closed transparent container, carbon dioxide is injected into the container at a rate of 30% per minute, until the animals die.

Histological examination of murine tissues
Whole-mount carmine alum staining was performed according to the standard protocol (18). The inguinal mammary glands from 5-week-old F2 Mogat2 +/+ PyMT and Mogat2 −/− PyMT female mice were xed overnight in Carnoy's solution (75% absolute ethanol [EtOH] and 25% glacial acetic acid [Sigma-Aldrich]). Following xation, the glands were washed with 70% ethanol for 30 mins, gradually rinsed in water and stained with carmine alum (Sigma-Aldrich) for at least two days. To examine the lung metastasis foci, tissues were xed in Bouin's solution (Sigma-Aldrich) for two days, and the number of metastatic lesions larger than 0.5 mm in diameter were counted macroscopically.

RT-PCR
At 98 days old, the F2 Mogat2 +/+ PyMT and Mogat2 −/− PyMT female mice were euthanized for tissue harvesting. Mammary tumors, stomach, small intestine and colorectal tissues were collected and stored in RNAstore reagent (TIANGEN) for RNA isolation.
Total RNA was extracted from mice tissues using Trizol reagent (Invitrogen, CA, USA) and subjected to reverse transcription using a PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara), followed by reactions using the Roche LightCycler 480 System (Roche). Beta-actin served as the normalization genes for these studies. The relative expression levels of the target genes were calculated using the 2 − ΔΔCt method. The qPCR primers were obtained from PrimerBank (19). The IHC scores for MOGAT2 (Cat#ab62526, Abcam) in the breast cancer tissues were calculated by two independent pathologists. For each tumor, two scores were given. One for the percentage of positively stained tumor cells, and another for staining intensity. The percentage of positive cells were categorized as: no staining = 0, 1-10% of stained cells = 1, 11-50% = 2, 51-80% = 3 and 81-100% = 4. Cytoplasmic staining intensity was categorized as: no staining = 0, weak staining = 1, moderate staining = 2 and strong staining = 3. The proportion and intensity were then multiplied to produce a total score ranging from 0 to 12. A cut-off value of 4 was the optimal balance between sensitivity and speci city with the patients divided into high-level (n = 51) and low-level (n = 96) groups.

Statistical analysis
Statistical analyses were performed using GraphPad Prism v7.0 software and the data presented as mean ± SEM. The correlations between MOGAT2 expression and overall survival, mice lacking palpable tumors and the mice's age were analyzed with Kaplan-Meier survival curves and the log-rank test. The differences between the groups were compared using Student's t-test, and ANOVA for multiple comparisons. P-values were used to denote statistical signi cance.

Results
Elevated MOGAT2 expression predicts poorer prognosis in breast cancer patients IHC staining showed that MOGAT2 was localized in the cytoplasm of breast cancer cells (Fig. 1A). Survival analysis revealed that elevated expression of MOGAT2 correlated with poorer overall survival in patients with triple negative breast adenocarcinoma (Fig. 1B). This suggests that depletion of MOGAT2 might be bene cial to the host in animal models.
A relatively high fat diet promotes obesity regardless of MOGAT2 de ciency Previous studies (13,14,20) reported that MOGAT2 ablation protected mice from high-fat diet induced obesity (60% of calories from fat). In our study, in the mice fed with a relatively high fat diet (37% calories from fat) between 5 and 12 weeks old, obesity was observed regardless of MOGAT2 de ciency (Fig. 2).

MOGAT2 depletion does not inhibit mammary tumorigenesis or metastasis
To determine whether loss of MOGAT2 in MMTV-PyMT mice affects the development of multifocal dysplastic lesions, we collected the right fourth (inguinal) mammary gland from virgin female To assess MOGAT2's depletion effect on mammary tumor progression, the number of tumor-affected mammary glands and total tumor volume were monitored by weekly palpation. The results showed there was no statistically signi cant difference between the two groups ( Fig. 4A and 4B).
The effect of MOGAT2 ablation on the number of metastases was also determined. Female Mogat2 +/+ PyMT and Mogat2 −/− PyMT mice were euthanized at 16 weeks old, the lungs were xed in Bouin's solution (Fig. 4C), and the metastases quanti ed (Fig. 4D). No statistical signi cant differences were found between the two groups.
Almost all MMTV-PyMT mice developed lung metastases, and all died of lung metastasis within several weeks of development. To observe the effect of MOGAT2 depletion on survival, we collected the survival time of the two groups of mice and found that MOGAT2 depletion did not signi cantly in uence overall survival (P = 0.977) (Fig. 5A).

A high fat diet accelerates MMTV-PyMT mammary tumor incidence and growth regardless of MOGAT2 depletion
Previous studies (13,14,20) reported that MOGAT2 ablation protects mice from high-fat diet (60% of calories from fat) induced obesity. We investigated the effect of MOGAT2's depletion on mammary tumorigenesis of MMTV-PyMT mice fed with a relatively high fat diet (37% of calories from fat). Female Mogat2 +/+ PyMT and Mogat2 −/− PyMT mice were produced by mating MMTV-PyMT males with Mogat2 −/ − females. Both of them were fed with a relatively high fat diet, commencing at 5 weeks old which continued for 12 weeks. Compared with normal diet-fed Mogat2 +/+ PyMT mice, a relatively high fat diet signi cantly accelerated mammary tumor incidence and growth regardless of MOGAT2 depletion (Fig. 5B  and 5C).

DGAT1 and DGAT2 act as functional substitutes in MOGAT2 −/− PyMT mice
To investigate the mechanism of functional cover-up upon MOGAT2 depletion in mice, we collected the mammary tumors, stomach, small intestine and colorectal tissues of the mice with or without MOGAT2 depletion, and examined the RNA levels of MOGAT1, MOGAT2, DGAT1 and DGAT2 by qPCR (Fig. 6).
Compared with the high fat diet-fed MOGAT2 +/+ PyMT mice, a signi cant reduction in MOGAT2 was evident in the high fat diet-fed MOGAT2 −/− PyMT mice, while MOGAT1 expression was unaltered (Fig. 6B).
However, DGAT1 and DGAT2 were signi cantly upregulated in the gastrointestinal tract in Mogat2 −/ − PyMT mice ( Fig. 6C and D), suggesting that both DGAT1 and DGAT2 play some role along with MOGAT2 which promote obesity.

Discussion
Previous studies (13,14) showed that MOGAT2 ablation protects mice from high fat diet induced obesity. In our study, a relatively high fat diet (37% of calories from fat) promoted obesity and subsequent mammary tumor formation and metastasis regardless of Mogat2 depletion. This discrepancy however, may be the result of a lower amount of fat in the food: 60% versus 37%.
In terms of dietary habits, most countries in Asia have a relatively low total fat intake (< 30% total energy, TE), such as 20% TE for China (21), 22.5% TE for India (22), 21.1% TE for South Korea (23) and 23.3-26.3% TE for Japan (24), whereas, most western countries have a relatively higher total fat intake (> 30% TE), with 34% TE for the USA (25), 37.6% TE for Germany (26) and 38.2% TE for France (27). A systematic review of data from 40 countries found that the total fat intake ranged from 11.1 to 46.2 % TE (25). Therefore, the relatively high fat diet (37% of calories from fat) used in our study is more in line with human dietary habits compared with the higher fat diet (60% of calories from fat) used in other studies (4,13,14).
Our ndings about the upregulation of Dgat1 and Dgat2 expression upon Mogat2 depletion suggest that genetic depletion of MOGAT2 alone is insu cient in preventing obesity and mammary tumor formation. DGAT1 and DGAT2 are abundantly expressed in small intestine (28, 29) and mediate the nal step in TAG re-synthesis during dietary fat absorption. Previous studies have shown that DGAT1 (15) and DGAT2 have the capacity to acylate MAG to form DAG (16). These results imply that DGAT1 and DGAT2 can act as functional substitutes in Mogat2 −/− PyMT mice.

Conclusions
In summary, elevated expression of MOGAT2 in triple negative breast cancer correlates with poorer patient overall survival. With the compensation of DGAT1 and DGAT2, MOGAT2 depletion alone, cannot prevent fat food-induced obesity, and has no effect on mammary tumor initiation, growth or metastasis. Targeting multiple key enzymes in fat metabolism might generate better outcomes in controlling obesity and probably in inhibiting mammary tumor development as well. Availability of data and materials

Abbreviations
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.      (d) The level of Dgat2 mRNA had a similar overexpression pattern with that of Dgat1. Data represent the mean ± SEM, NS = non-signi cant, *P < 0.05, **P < 0.01 and ***P < 0.001.