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Diet-induced obesity contributes to the development of type 2 diabetes, insulin resistance, metabolic inflammation, oxidative and endoplasmic reticulum (ER) stress. Overall, obesity is associated with deviations in the composition and functionality of the gut microbiota. There are many divergent findings regarding the link between the excessive intake of certain dietary components (i.e., fat and sugar) and obesity development. We therefore investigated the effect of specific diets, with a different content of sugar and fat, in promoting obesity and related comorbidities as well as their impact on microbial load and gut microbiota composition/diversity. C57BL/6J mice were fed either a low-sugar, low-fat control diet (CT), a high-sugar diet (HS), a high-fat, high-sugar diet (HF/HS), or a high-fat diet (HF) for 8 wk. The impact of the different diets on obesity, glucose metabolism, inflammation, and oxidative and ER stress was determined. Diet-induced changes in the gut microbiota composition and density were also analyzed. HF diet-fed mice showed the highest body weight and fat mass gains and displayed the most impaired glucose and insulin profiles. HS, HF/HS, and HF diets differently affected hepatic cholesterol content and mRNA expression of several markers associated with immune cells, inflammation, oxidative and ER stress in several organs/tissues. In addition, HF diet feeding resulted in a decreased microbial load at the end of the experiment. When analyzing the gut microbiota composition, we found that HS, HF/HS, and HF diets induced specific changes in the abundance of certain bacterial taxa. This was not associated with a specific change in systemic inflammatory markers, but HS mice exhibited higher FGF21 plasma levels compared with HF diet-fed mice. Taken together, our results highlight that dietary intake of different macronutrients distinctively impacts the development of an obese/diabetic state and the regulation of metabolic inflammation in specific organs. We propose that these differences are not only obesity-driven but that changes in the gut microbiota composition may play a key role in this context. [ABSTRACT FROM AUTHOR]