Obesity is associated with metabolic syndrome and cardiovascular diseases, which are commonly investigated with mice models of obesogenic diets rich in fat and sucrose, in order to mimic the western diet. In obesogenic diet, casein from bovine milk protein is the sole source of dietary protein and this is a problem because casein does not have the same composition as plant and animal sources of proteins that are abundant in most human diets. The authors prepared a diet comprising a mix of proteins from different sources: plants (rice, 21.1%; soy, 6.1%; pea; 6.1%), red (beef, 13.4%), and white (chicken, 13.8% and pork 13.4%) meats, dairy products (casein, 15.4% and whey, 3.8%), egg (3.7%) and fish (cod, 3.3%). This protein mix was incorporated in low-fat low-sucrose and high-fat high-sucrose diets. Data showed the protein mix influenced the obesogenic effect of high-fat high-sucrose feeding because of an increase in fat and lean mass, and exacerbated the detrimental effect of high-fat high-sucrose feeding on glucose homeostasis by impairing insulin mTORCC1/S6K1 signaling.

Examination of bacterial composition in fecal samples revealed a diet-specific clustering of microbiota from low-fat low-sucrose and high-fat high-sucrose fed mice and, in particular, high-fat high-sucrose diet promotes the abundance of Romboutsia, Adlercreutzia, and Tyzzerella while reducing Bifidobacterium, Facecalibacterium, and a Muribaculaceae bacteria genus. Data showed that the difference in gut microbiota is independent from fat and carbohydrate in the diet. Notably, the addition of protein mix to high-fat high-sucrose diet as protein source promotes a rapid increase of Ruminiclostridium, Adlercreutzia, and Tyzzerella bacteria with a decrease in the relative abundance of Akkermansia, showing that the changes in gut microbiota composition are influenced by protein sources. Interestingly, data indicated that casein promotes Akkermansia bacteria that could have a protective effect of obesity in the mice model, while protein mix in diet promotes Tyzzerella bacteria that had been correlated with the risk of cardiovascular diseases. The involvement of alterations in gut microbiota is confirmed by fecal transplantation. Mice were colonized with microbiome from high-fat high-sucrose with casein mice and high-fat high-sucrose with protein mix mice. Results showed that microbiota from high-fat high-sucrose with protein mix fed donor mice increased adipose tissues and exacerbated glucose-stimulated insulin response with respect to ones transplanted with microbiome from donor mice fed with high-fat high-sucrose with casein. Changes in bacteria composition in recipient mice were similar, with abundance of Akkermansia in mice transplanted with the feces from mice fed with high-fat high-sucrose with casein and an abundance of genus from Lachnospiraceae, and Intestinimonas in the mice transplanted with feces from high-fat high-sucrose protein mix fed donor mice.

It was also observed a rapid increase in fecal samples of branched-chain fatty acids (BCFA) levels. High-fat high-sucrose diet and protein mix induce an increase of BCFA isobutyric and isovaleric acid that increases glucose overcoming the suppression effect of insulin, which caused mTORC1/S6K1 activation in hepatocytes. In addition, compared to casein, protein mix induced acylcarnitines increasing in the plasma, which is a hallmark of metabolic disease connecting to aberrant mitochondrial fatty acid oxidation.

Overall, these data showed that dietary protein sources have an important impact on obesity and insulin resistance, which are directly linked to changes in gut microbiota and BCFA production, leading to activation of the mTORC1/S6K1 pathway and hepatic insulin resistance. Moreover, these results showed the importance of dietary protein source in nutritional interventions for weight management or in the diet design for preventing insulin resistance in patients with diabetes.