Purpose: Obesity is a primary risk factor for knee osteoarthritis (OA). However, how chondrocyte metabolism and pro-catabolic mediators change in response to excess consumption of specific macronutrients, such as fat and sugar, are not fully understood. The aim of this study was to better understand how chondrocyte metabolism changes in response to a high-fat diet (HFD) and a low-fat high-sucrose diet (LFHS), and how these changes may be associated with OA pathogenesis in mice. We hypothesized that high-fat and high-sucrose feeding would reduce cartilage proteoglycan content by inducing cellular metabolic stresses and expression of matrix proteases. To test this hypothesis, we examined OA pathology and transcriptional changes in a panel of chondrocyte OA mediators, intracellular stress mediators, and metabolic markers following 20 weeks on HFD, LFHS, or matched control low-fat low-sucrose (LFLS) diets. Methods: Male C57BL/6J mice (n = 8 per diet) were fed either HFD (60% kcal fat, 7% sucrose), LFHS (10% kcal fat, 70% kcal carbohydrate [35% sucrose]), or LFLS (10% kcal fat, 70% kcal carbohydrate [7% sucrose]) from Research Diets for 20 weeks beginning at 6 weeks of age. Knee OA histopathology was examined using a modified Mankin OA scoring system and OARSI scoring system in the left knee. mRNA and protein were isolated from right knee articular cartilage to examine chondrocyte transcriptional and protein expression changes. Gene expression changes were determined using custom qPCR arrays containing a range of metabolism and mitochondrial genes, while protein expression is being determined using selected reaction monitoring (SRM) proteomics. Results: There was no difference in the OARSI OA score among the diet groups. However, modified Mankin OA score was moderately but significantly increased in LFLS compared to HFD and LFHS groups (Fig. 1) due to greater loss of Safranin-O staining. Other sub-component scores differed among groups, with the hypertrophic chondrocyte score being lower in the LFLS group compared to HFD and LFHS and the osteophyte and tidemark duplication scores being greater in the HFD group compared to LFHS and LFLS. mRNA expression of extracellular matrix proteins and extracellular matrix proteases (Aggrecan, Col2, Col10, Adamts5, Mmp9, Mmp13) were not different between groups. Of the metabolic and cellular stress genes examined, there were multiple diet specific differences (Fig. 2). Notably, Cpt1a was significantly greater in the HFD compared to LFHS and LFLS. However, Foxo3, Sod1, and Sod2 were greater in LFHS and HFD compared to LFLS. Ampk was greater in LFLS compared to LFHS, but not HFD, with no difference between LFHS and HFD. Conclusions: Both dietary fat and insulin resistance increase the risk of OA. Therefore, we hypothesized that HFD and LFHS diets would induce OA related histological changes. Further, we hypothesized that 20 weeks of HFD and LFHS diets would induce gene expression changes in OA mediators, cellular stress mediators, and metabolic markers mirroring the histological changes. Surprisingly, the modified Mankin OA score was highest in the LFLS group, primarily due to reduced Safranin-O staining. However, the HFD and LFHS groups were also independently associated with other features of OA histopathology. These findings suggest that the varied macronutrient compositions of pro-obesogenic and control diets have independent effects on joint homeostasis. Although diet composition did not alter the expression of anabolic or catabolic extracellular matrix genes, dietary macronutrients uniquely altered the expression of metabolic and stress-response genes in cartilage. For example, HFD increased expression of Cpt1a, the mitochondrial fatty acid transporter that is the rate-limiting step in fatty acid metabolism, suggesting that a HFD increases the uptake and utilization of fatty acids in chondrocytes. In contrast, high sucrose content in carbohydrate-matched diets reduced Ampk expression, supporting the hypothesis that fat and carbohydrate macronutrients have specific effects on chondrocyte phenotypes. In conclusion, chondrocytes appear metabolically flexible in response to high-fat and high-sucrose diets in independent manners related to OA pathology. These findings illustrate the importance of considering dietary macronutrient composition when designing studies and interpreting the effect of diet-induced obesity on knee OA in mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)