Effects Of Moderate Lard Intake On Lipid And Glucose Metabolism And Its Mechanism

Objective: Obesity,non-alcoholic fatty liver disease and type 2 diabetes have become major public health problems for humans and companion animals(canine and feline)worldwide.Our previous studies have linked lard to these metabolic diseases.However,the consumption of lard has fallen sharply in recent decades while metabolic diseases have risen.Our previous works found that moderate lard and the blend oil of lard and vegetable oil could reduce body fat deposition in mice,and has a potential regulatory effect on serum insulin level.Based on the previous findings,this study used C57BL/6J mice and db/db mice,respectively,to explore the effect of dietary lard on regulating glucose and lipid metabolism under different dietary models,and to explore its mechanism from the perspective of intestinal microbiota.Chronic diseases such as obesity,non-alcoholic fatty liver disease and type 2diabetes have become major global public health problems.Our previous study proved that moderate intake of lard and blending of lard and vegetable oil in traditional Chinese diet can significantly reduce body fat and liver fat deposition.It was also found that moderate intake of lard may have the potential to reduce insulin resistance.Combined with literature analysis,it was also found that the pork and lard based dietary pattern had the lowest risk of developing type 2 diabetes.In order to further confirm the above scientific discovery and reveal its mechanism,in this study,we used C57BL/6J mice and db/db mice as experimental animal model,simulate the Chinese habits of dietary fat,explore in different levels of fat for energy and dietary fat(50% lard,soybean oil,lard oil and soybean oil blend oil)impact on glucolipid metabolism in mice and its mechanism.Experiment 1.Effects of dietary oils in different dietary models on glucose and lipid metabolism and intestinal microflora in C57BL/6J miceMethods: Three kinds of diet patterns were designed for mice,that is low fat control diet(15% energy from fat),recommended dietary fat intake level model(25%energy from fat),Chinese residents’ current dietary fat intake level model(35%energy from fat),each diet model is divided into three different groups,that is,soybean oil,lard,and the mixture of both.A total of 135 male 6-weeks-old C57BL/6J mice were divided into 9 groups.The mice were fed the above 9 diets,respectively,and were given free water and food.Fasting body weight,fasting blood glucose,OGTT and ITT were detected during feeding.At the end of 22 nd week,the final fasting body weight and blood glucose were weighed,the serum and organs were collected.Organ index was calculated and serum biochemical parameters were detected.In 35% level group,the contents of lipid and glycogen in liver were detected,the histopathological sections of liver were stained with H&E and oil red O,the m RNA expressions in liver tissues were detected by transcriptome and RT-QPCR,the protein expressions were detected by Western blot.The pancreatic double-labeled immuno-fluorescence staining were performed to detect insulin and glucagon in islet,and pancreatic AGEs were detected by immunohistochemistry.The cecal microbiota diversity was detected by 16 S r RNA sequencing.Results:(1)In 15% level dietary patterns,serum insulin of mice was significantly lower in soybean oil group than in lard group and mixed oil group(P<0.05),in addition,dietary fat intake had no significant effect on most parameters of glucose and lipid metabolism in mice(P≥0.05).(2)In 25% level dietary patterns,serum insulin,HOMA-IR index,AUC of ITT and serum resistin were significantly(P<0.05)or extremely(P<0.01)lower in lard group than in soybean oil group.(3)In 35% level dietary patterns,in mixed oil group,the body weight,body fat mass,serum insulin,serum leptin,HOMA-IR index,AUC of ITT,liver index and liver TG of mice were significantly(P<0.05)or extremely(P<0.01)lower than soybean oil group,and the liver glycogen content was significantly higher than soybean oil group(P<0.05).(4)At the transcriptional level,the expression of genes related to glucose and lipid metabolism was significantly different between mice fed soybean oil and lard or mixed oil,which were mainly concentrated in insulin signaling,glucose metabolism,adipose acid formation and catabolism and other functions and pathways.(5)The trends of P-IRS1(Tyr895)/ IRS1 and GLUT2 protein expression in this study is soybean oil group<lard group<mixed oil group,and soybean oil group were significantly(P<0.05)or extremely(P<0.01)lower than mixed oil group,respectively.The trends of P-Akt(Ser473)/ Akt and the protein expression of PI3 K and PDK4 were soybean oil group<bend oil group<lard group,and the difference between soybean oil group and lard group was extremely significant(P<0.01).PEPCK protein expression showed the same trend,and there was significant difference between soy oil group and lard group(P<0.05).The protein expression trends of PPARγ,ACC1 and SCD1 were soybean oil group>lard group>mixed oil group,and there was extremely significant difference(P<0.01)between soybean oil group and blended oil group.CD36 protein expression showed a similar trend,and there was significant difference between soybean oil group and mixed oil group(P<0.05).GSK-3 β protein expression trend was soybean oil group>mixed oil group>lard group,and there was significant difference between soybean oil group and lard group(P<0.05).(6)In 35%level dietary patterns,the abundance of SCFAs-producing probiotics,e.g.Lactobacillus,Dubosiella and Bifidobacterium was increased in cecum of mice fed with lard or mixed oil.Long-term intake of soybean oil increased the abundance of norank＿f＿＿Muribaculaceae,unclassified＿f＿＿Lachnospiraceae,Turicibacter and other harmful species.There was a significant negative correlation(P<0.05)between the abundance of Lactobacillus with body weight,abdominal fat rate,liver fat deposition and insulin resistance,and the abundance of norank＿f＿＿Muribaculaceae had a significant positive correlation with the above phenotypic indexes(P<0.05).Experiment 2.Effects of fecal bacteria transplantation(FMT)on glucose and lipid metabolism in db/db miceMethods: Thirty db/db mice were treated with antibiotics to establish low-bacterial mice model by antibiotic solution drinking.The period of modeling was 14 days,and feces of mice were collected before and after modeling to detect microbial diversity.Thirty low-bacterial db/ db mice were divided into 3 FMT groups.The mice were transplanted daily by the fecal bacteria of mice from soybean oil,lard group and mixed oil group of 35% level dietary patterns,respectively.During the period,the body weight,fasting blood glucose,OGTT and ITT was detected.The mice were sacrificed and the samples were collected after the end of 4 weeks.The glycolipid metabolism phenotypic related parameters,liver pathology and protein expression,and intestinal microflora diversity were detected as in experiment 1.During the whole experiment,10 m/m mice were used as control,and all mice were given free maintenance diet and water.Results:(1)The species α diversity and abundance of intestinal microbiota in db/db mice treated with antibiotics were significantly decreased(P<0.01),which proved the low-bacteria mice model was established successfully.(2)FMT reconstructed the intestinal microbiota of donor mice and amplified the multiple differences of the dominant flora.The abundance of Lactobacillus in caecum of recipient mice in soybean oil group was significantly lower than that in lard group and mixed oil group,and the abundance of norank＿f＿＿Muribaculaceae was significantly higher than that in blend oil group(P<0.05).(3)FMT replicated the phenotype of donor mice,that is,compared with recipient mice of soybean oil group,glucose and lipid metabolism was improved in lard group and mixed oil group.Especially,the body weight,body fat rate,HOMA-IR index,AUC of ITT,liver fat deposition and the expression of PPARγ,ACC1,CD36 and GSK-3β in liver of the recipient mice of mixed oil group were significantly(P<0.05)or extremely(P<0.01)lower than that of soybean oil group.In addition,the above phenotypic changes were significantly negatively correlated with Lactobacillus abundance(P<0.05).(4)The content of butyric acid and lactic acid in caecum of FMT recipient mice of mixed oil group was significantly higher than that of soybean oil group(P<0.01),meanwhile,and the lactic acid content in lard group recipient mice was extremely higher than that in soybean oil group(P<0.01).Conclusion:(1)In low-fat diet(15% energy from fat),there is no significant difference in the effect of different dietary oil intake on glucose and lipid metabolism in mice,that is,under the condition of controlling the total oil intake,the selection of dietary fat type is not the key factor affecting glucose and lipid metabolism.(2)In the dietary pattern of lipid intake levels recommended by the Chinese Nutrition Society(25% energy from fat),compared with soybean oil,lard or mixed oil diet tended to improve glucose and lipid metabolism.Intake of lard was associated with a significant reduction in AUC of ITT,serum insulin and resistin levels.(3)In the dietary pattern of current oil intake level of Chinese residents(35% energy from fat),compared with soybean oil,the lard diet,especially the mixed oil diet,can significantly improve glucose and lipid metabolism,reduce body fat and liver fat deposition,decrease serum insulin level and insulin resistance,and improve insulin sensitivity.(4)The mechanism of lard-including diet pattern ameliorate glucose and lipid metabolism is through increasing abundance of intestinal Lactobacillus,Dubosiella and Bifidobacterium probiotics producing lactic acid and SCFAs in the cecum of mice,and further acting on the hepatocytes,activation of hepatic insulin receptor signaling pathways;Ultimately,improves insulin sensitivity and stimulates glucose uptake and insulin clearance,increases the synthesis of glycogen and gluconeogenesis,and reduces lipids uptake and lipids synthesis in hepatocytes.