Lard-Blended Vegetable Oil Diet Alleviates Metabolic Syndrome in Mice


 Background: Our previous work has suggested that the balance fatty acid diet, blending lard with soybean oil, could reduce adipose tissue accumulation by decreasing adipogenesis and lipogenesis while increasing the hydrolysis of triglycerides. The aim of present study is to investigate whether the fat reducing function of balanced fatty acid diet extended to sunflower oil, and explore its effects on liver lipids metabolism and insulin resistance.Methods: 50 mice were divided into 5 groups, fed with lard, sunflower oil, soybean oil, mixture of lard and sunflower oil, and mixture of lard and soybean oil for 12 weeks separately.Results: Results showed that a mixture of lard and vegetable oil (sunflower oil and soybean oil), particularly a mixture of lard and soybean oil decreased body weight, body fat rate, liver triglyceride level compared to lard, sunflower oil, and soybean oil, a mixed oil also decreased serum triglyceride and free fatty acid levels compared to lard diet. Further analysis indicated that activation of the AMPK pathway contributes to these observed phenotypes, and co-upregulated of glucagon and GLP-1 in mice fed with mixture of lard and soybean oil may contribute to improved lipids metabolism. Conclusion: Moderate lard intake—blended lard with vegetable oil especially soybean oil—has the potential to alleviate metabolic syndrome via activating AMPK pathway and co-upregulated of glucagon and GLP-1.


Introduction
Since 1950s, SFA was regard negative for lipid metabolism, consumption of SFA be correlated with cardiovascular disease 1 . Thus, energy derived from fat limited range from 20-30% with saturated fatty acid (SFA) limited to less than 10% in American and Chinese dietary guideline from the rst edition 2 .
In recent years, the consumption of saturated fatty acid (SFA) and their replacement with unsaturated fatty acids (UFA) have triggered a growing number of disputes 3,4 . Most recent meta-analyses of randomized trials and observational studies found no bene cial effects of reducing SFA intake on cardiovascular disease, and instead found protective effects against stroke 5 . These contradictions result may contribute to neglect of possible maximal extent, over the maximal extent, has adverse effects on individual, but under the premise of not exceeding the maximum, decreasing consumption of SFA, even replace SFA with UFA may be not good for health. For majority of the American population, consumption of SFA far exceed recommendation 6-8 , it is necessary to reduce the intake of SFA. For most of Chinese, SFA energy is less than 10% 9 , it is worth exploring whether it is more healthy to replace SFA with UFA.
Lard primarily comprises of SFA and monounsaturated fatty acid (MUFA). Soybean oil and sun ower oil are rich in polyunsaturated fatty acid (PUFA); soybean oil has a higher n-3 PUFA content than sun ower oil 10 . Soybean oil and sun ower oil is widely consumed all over the world. Previous studies have mostly focused on fatty acids or a single oil such as animal-derived fat and vegetable oils, or a mixture of various vegetable oils [11][12][13] . According to dietary guideline, SFA should limited to 10%, and acceptable ranges of n-3 PUFA and n-6 PUFA are 0.5-2.0% and 2.5-10%, respectively 14 . In addition, previous studies used diets containing fat energy of up to 40%, even reaching up to 60%, which all simulated the fat consumption in the Mediterranean and America that range from 27-48% [15][16][17] . However, in the Eastern diet, particularly the Chinese diet, fat energy is usually lower than 40%. Some studies even reported a fat energy content of 22% in 1992, and 32.9% in 2012 18 both in China, while a 27% fat energy in west India 19 . In our previous study, we mixed lard with vegetable oil to make SFA: MUFA: PUFA close to 1:1:1, we have found that lard blended with soybean oil has anti-obesity effects under 25% fat energy by decreasing adipogenesis and lipogenesis while increasing the hydrolysis of triglycerides in adipose tissues 20 , but the effects disappeared under 35% fat energy 21 . In the present study, we aimed to investigate whether the anti-obesity effect of balanced fatty acid diet extended to sun ower oil under 30% fat energy, and explore its effects on liver lipids metabolism and serum hormones.

Animals, diet and experimental design
50 six-weeks-old male C57BL/6J mice were purchased from the Hunan Silaike Laboratory Animal Co., Ltd. (Changsha, China). The mice were housed in collective polypropylene cages inside an isolated room with controlled temperature (22±2℃) and humidity (65% ± 5%), and a 12-h light/dark cycle. They received an ad libitum supply of water and feeds/diet during the entire experimental period. After one week of acclimatization, they were randomly divided into ve groups, 2 mice per cage, and fed with lard, sun ower oil (SFO), mixture of lard and sun ower oil (L-SFO), soybean oil (SBO), and mixture of lard and soybean oil (L-SBO) each group for 12 weeks. The compositions of the diets are listed in Supplementary   Table S1. After the experiment, all mice were weighed, then anesthetized with iso urane, collected blood, and euthanatized by cervical dislocation. All the experimental protocols were approved by the Ethics Committee of Hunan Agricultural University, China (No. 43321543).

Sample collection and preparation
Blood samples were collected overnight at 4°C through the retro-orbital plexus of the mice. The sera were separated by centrifugation at 3500g for 10 min at 4°C and were then immediately stored at −80° C until analyses. The liver, epididymal and perirenal adipose tissues were collected and weighed. The liver and epididymal adipose tissues were cut into ve parts and washed with a physiologic saline solution. The left lobe of the liver was xed in optimal cutting temperature compound, one part of was epididymal adipose tissues xed in 10% neutral buffered formalin, whereas the remaining parts were stored at -80°C immediately until analyses.

Histological analysis
Epididymal adipose tissues were xed in 10% neutral buffered formalin, embedded in para n, sectioned, and stained with hematoxylin and eosin (H&E) for histological analyses. The sections (6-mm thick) of the left lateral lobe of the frozen liver were stained with Oil Red O (Sigma, USA) for 20 min, and counterstained with hematoxylin for 1 min. The stained areas were observed using Olympus photomicroscope (Olympus Inc., Tokyo, Japan) at a magni cation of 400× for epididymal adipose tissue, while at 200× for the liver.

Western blot analysis
Proteins were extracted from liver by lysis and homogenization using radio-immunoprecipitation assay buffer (Beijing Solarbio Science & Technology Co. Ltd. Beijing China) at 4°C. The total protein concentrations were measured by the bicinchoninic acid (BCA) method using a protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene di uoride membranes. The membranes were blocked using a buffer composed of 5% non-fat dry milk and tris-buffered saline tween 20 (TBST) for 1 h at 4°C and were incubated at 4°C overnight with appropriate antibodies, including hormone sensitive lipase (HSL; Cell Signaling Technology, Inc. USA), Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK; Proteintech, Inc. USA), phosphorylated AMPK Thr 172 (p-AMPK; Proteintech, Inc. USA), carnitine palmitoyl transferase 1 (CPT-1; Proteintech, Inc. USA), and β-actin (Proteintech, Inc. USA). The membranes were washed with TBST, incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 1 h at 25℃, washed with TBST buffer, and nally incubated with an enhanced chemiluminescence labeling kit (ECL; Nanjing KeyGen Biotech. Co. Ltd., China). The intensities of the bands were quanti ed by AlphaEase FC software (Alpha Innotech Co., USA).
2.6. Measurement of serum hormones and liver enzyme related lipids metabolism Serum adiponectin was tested by using ELISA assay kits purchased from Elabscience Biotechnology Co.,

Statistical analysis
The results were expressed as the mean ± standard error of the mean (SEM). The statistical signi cance of the mean differences among the groups was carried out via one-way analysis of variance (ANOVA) and least signi cant difference test. A p value < 0.05 was considered statistically signi cant. Graphical data presentations were created using GraphPad Prism version 7 (Graph Pad Software, San Diego, CA, USA).
The dominant fatty acid in both SFO and SBO was linoleic acid (C18:2 and C18:1, respectively). However, SBO has higher linoleic acid (

Mixture of lard and vegetable oil reduced body weight
No signi cant difference in the initial body weight was observed among the ve groups ( Fig. 1. A), at the end of the experiment, body weight of the L-SFO group was lower than that of Lard (p<0.05) and SFO groups (p<0.05), body weight of the L-SBO group was lower than that of Lard (p<0.05) and SBO groups (p<0.05), and there was little difference of body weight between L-SFO and L-SBO ( Fig. 1. B). During the 12 weeks of the experiment, the average food intake and energy intake among groups were no signi cant difference ( Fig. 1. C, D), this result indicated that the change of body weight was not contribute to food intake.

Mixture of lard and vegetable oil reduced body fat accumulation
White adipose tissue weight including epididymal fat weight and perirenal fat weight were measured. The result showed that epididymal fat weight of the L-SFO group was lower than that of Lard (p<0.05) and SFO groups, epididymal fat weight of the L-SBO group was lower than that of Lard (p<0.05) and SBO groups (p<0.05) (Fig. 2. A). Similarly, perirenal fat weight of the L-SFO group was lower than that of Lard and SFO groups, perirenal fat weight of the L-SBO group was lower than that of Lard (p<0.05) and SBO groups (p<0.05) (Fig. 2. B). Consistent with epididymal fat weight, body fat rate of the L-SFO group was lower than that in Lard (p<0.05) and SFO groups, body fat rate of the L-SBO group was lower than that in Lard (p<0.05) and SBO groups (p<0.05) (Fig. 2. C). In addition, it was observed that cross-sectional areas of adipocytes in L-SFO group and L-SBO groups were smaller than Lard, SFO, and SBO groups from the HE staining sections (Fig. 2. D). Between the two mixed oil groups, body fat accumulation of the L-SBO group showed a decreasing trend compared to the L-SFO group (Fig. 2. A-D).

Mixture of lard and vegetable oil reduced serum lipids accumulation
Serum TC, TG and FFA levels of Lard group were highest among groups, especially serum TG and FFA of Lard group showed signi cantly higher than other four groups (Fig. 3. A-C). Serum TC level of the L-SFO group was lower than that of Lard (p<0.05) and SFO groups, serum TC level of the L-SBO group was lower than that of Lard (p<0.05) and SBO groups (p<0.05) (Fig. 2. A). Serum TG level of L-SFO and L-SBO groups were lower than that of SFO and SBO groups, respectively, but there was no signi cant difference (Fig. 2. B). Serum FFA level of the L-SFO group was lower than that of Lard (p<0.05) and SFO groups (p<0.05), serum FFA level of the L-SBO group was lower than that of the Lard (p<0.05), but little change compared to the SBO group ( Fig. 2. C). Serum GLU among groups no signi cant difference, but that of Lard and L-SBO groups showed a decreasing trend compared to other three groups (Fig. 2. D).

Mixture of lard and vegetable oil improved liver and kidney function
Liver weight of the L-SFO group was lower than that of Lard (p<0.05) and SFO groups (p<0.05), liver weight of the L-SBO group was lower than that of Lard (p<0.05) and SBO groups (p<0.05) (Fig. 4. A).
Similarly, ALT activity of the L-SFO group was lower than that of Lard (p<0.05) and SFO groups, ALT activity of the L-SBO group was lower than that of Lard and SBO groups (p<0.05) (Fig. 4. B). AST activity and UA content of ve groups were no signi cant difference ( Fig. 4. C, D). UREA level of the SBO group was signi cantly higher than that of other four groups (Fig. 4. E). This result illustrated that mixed oil could improve liver and kidney function compared to single oil.

Mixture of lard and vegetable oil reduced liver lipids accumulation
It was observed that Oil red O intensity in L-SFO group and L-SBO groups were less than Lard, SFO, and SBO groups from the Oil red O staining sections (Fig. 5. A). Liver TG also be tested, result showed that liver TG level of the L-SFO group was lower than that in Lard and SFO (p<0.05) groups, body fat rate of the L-SBO group was lower than that in Lard (p<0.05) and SBO groups (p<0.05) (Fig. 2. C) (Fig. 5. B).

Mixture of lard and vegetable oil activated AMPK pathway in liver
To further understand the effects of the different types of dietary oils on lipid metabolism, the protein expressions of HSL, CPT-1α, AMPK, p-AMPK, and FAS in the liver were analyzed. HSL protein expressions of SFO and SBO groups were the lowest among ve groups, HSL protein expression of L-SFO group was 2.47 folds higher than that of SFO group, HSL protein expression of L-SBO group was 2.8 folds higher than that of SBO group (Fig. 6. A), ELISA analysis also showed that HSL activity of L-SBO group signi cantly higher than SBO group (Fig. 6. D). CPT-1α protein expression of L-SFO group was 1.87 and 1.55 folds higher than that of SFO and Lard groups, respectively, CPT-1α protein expression of L-SBO group was 1.96 and 2 folds higher than that of SBO and Lard groups, respectively (Fig. 6. B), ELISA analysis also showed that CPT-1α activity of L-SBO group signi cantly higher than SBO and Lard groups (Fig. 6. E). Ratio of p-AMPK/AMPK of L-SFO group was 2.02 and 2.26 folds higher than that of SFO and Lard groups, respectively, Ratio of p-AMPK/AMPK of L-SBO group was 1.91 and 1.66 folds higher than that of SBO and Lard groups, respectively ( Fig. 6. C). There were no signi cantly changes of FAS activity among Lard, SBO, and L-SBO groups (Figure 6. F). This result showed that mixture of lard and vegetable oil (sun ower oil or soybean oil) could activate AMPK, increase HSL and CPT-1α protein expressions.

Mixture of lard and soybean oil co-upregulated GLP-1 and glucagon
There were no signi cant changes of resistin and adiponectin concentrations among ve groups ( Fig. 7.A, B). PAI-1 concentration of SFO group was higher than Lard (p<0.05), L-SFO, SBO (p<0.05), and L-SBO groups (p<0.05) (Fig. 7. C). GLP-1 and glucagon concentrations of L-SBO group were signi cantly higher than other four groups ( Fig. 7. D, E), GLP-1 and glucagon concentrations of SFO and SBO groups were lowest among ve groups ( Fig. 7. D, E). Correlation heatmap analysis revealed that liver TG, serum TC, body fat rate, and body weight were signi cantly negative correlated with GLP-1 and glucagon, respectively, body weight and GLU were positive correlated with resistin ( Fig. 7. F)

Discussion
Randomized trial and rodent experiments have shown that SFA rich diet induce body fat, serum lipids, and liver lipids accumulation [22][23][24][25] . However, discordant results also be reported 26,27 . Our present study also found that SFA rich diet (lard) induce body fat (p>0.05) and serum lipids (p<0.05) accumulation than PUFA rich diet (sun ower oil or soybean oil), but liver lipids accumulation higher in PUFA rich diet (sun ower oil (p>0.05) or soybean oil) than SFA rich diet (lard), especially in soybean oil diet. Deol et al. showed that soybean oil diet induces greater weight gain, adiposity, and fatty liver than coconut oil diet that rich in SFA 26 . Liver damage caused by dietary cholesterol in mice was strongly enhanced by a high fat diet containing soybean oil, but not by a lard-based high fat diet, soybean oil-based diet enhanced cholesterol-induced mitochondrial damage and ampli ed the ensuing oxidative stress 27 . Di Rienzi proved that toxicity of soybean oil fatty acid inhibits growth of Lactobacilli, bene cial members of the small intestinal microbiota 28 . Jurgoński also found that cecal butyrate level higher in lard diet rats than soybean oil diet rats, bene t gut metabolism 22 . Studies showed that the abundance of short chain fatty acid bacteria such as Bi dobacterium, Enterococcus and Allobaculum increased with high lard diet [29][30][31] . Thus, it is speculated that different fat/oil might exert different effects in different tissues, mix lard with vegetable oil (a balance fatty acid diet) bene t for lipids metabolism from multiple metabolic pathways.
In the next step, we will explore effect of lard and soybean oil diet on gut microbiota and metabolite change in gut and liver.
Our previous study (under 25% fat energy) found that a mixture of lard and soybean oil could reduce body fat accumulation compared to lard or soybean oil 20 , it was also proved in present study (under 30% fat energy), and can be extended to sun ower oil. However, under 35% fat energy, the function cannot be speci cally lard, beef tallow, sun ower oil, and soybean oil, can induce obesity 34 . A high-fat diet with 42% energy for 12 weeks also showed that rats fed with lard and olive oil were the most obese, having no signi cant differences between these diets 35 . The contradictory results may be attributed to the different level of fat energy, but the mechanism worth further exploration.
In the present study, mixture of lard and vegetable oil (sun ower oil or soybean oil) activated AMPK compared to single oil diet, and mixture of lard and soybean oil increased serum GLP-1 and glucagon levels. AMPK plays a key role in regulating energy metabolism 36 . Liver AMPK can decrease the rate of hepatic lipogenesis. Its phosphorylation leads to the inhibition of fatty acid biosynthesis via phosphorylation of acetyl Co-A carboxylase (ACC), thus affecting malonyl-CoA content that synthesis catalyzed by ACC 37 . The activity of CPT-1α can be regulated by malonyl-CoA 38 , besides, activation of AMPK also can induce HSL and inhibit FAS expression 39 . HSL is a key enzyme that catalyzes the ratelimiting step of adipose tissue lipolysis from TG to FFA 40 . A previous study showed that administration of a speci c HSL inhibitor can reduce the serum FFA levels in mice, rats, and dogs, demonstrating its role in vivo 41 . Thus, the expression of HSL is closely associated with FFA content. HSL protein expressions were higher in lard, L-SFO, and L-SBO groups, bene t serum TG lipolysis to FFA, however, serum FFA cannot be oxidized due to inactivation of the AMPK and lower expression of CPT-1α, it also explains why serum FFA levels in the mice fed with lard were signi cantly higher than those fed with other oils. Interestingly, the mixture of lard and vegetable oil not only induced the expression of HSL protein but also activated AMPK pathway, thereby increasing the expression of CPT-1α proteins. This, subsequently, reduced fatty acid synthesis, induced higher levels of CPT-1 proteins, and, ultimately, strengthened fatty acid oxidation, indicating that the energy expenditure was higher. Palmitoleic acid has been reported to improve metabolic functions by increasing the AMPK phosphorylation in the fatty liver induced by highfat-diet 42 .
Fasted GLU levels among groups were no signi cant different, even though GLU of mice fed with lard and mixture of lard and soybean oil were lower than other three groups. Wang

Conclusions
In conclusion, the present study fully proved that a mixture of lard and sun ower oil or soybean oil (a balanced fatty acid diet) bene t to improve metabolic disorder in mice. Mixed oil activating AMPK pathway, promoting TG hydrolysis and FFA oxidation, and a mixture of lard and soybean oil also can coupregulating GLP-1 and glucagon that bene t for lipids metabolism. Extending from those results, the present ndings provide a caution and much worth consideration against the current of replacing SFA with UFA.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1.docx