Increased dietary fat, as consumed in Western diets, can contribute to both obesity and the metabolic dysfunction that is associated with insulin resistance/type 2 diabetes. Surplus dietary fat is deposited in adipose tissue as well as in skeletal muscle, heart, and liver. As this fat burden increases, adverse effects on systemic metabolism increase [11]. The current model of insulin resistance of obesity as a proinflammatory state is based in part on the observation that adipose tissue in obese individuals and animal models contains increased numbers of activated macrophages that release proinflammatory cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor-α [12,13]. These cytokines act both locally on adipocytes and systemically to impair insulin action in insulin-target tissues [11,14,15]. Metabolism of the abundant circulating free fatty acids in the high-fat diet can also lead to oxidative stress in cells, formation of lipid peroxides, and subsequent oxidative damage to DNA, RNA, and proteins [16-18]. Oxidative stress has been more specifically shown to inhibit insulin-receptor signaling pathways in cells, at least in part through activation of the stress kinases JNK and p38 and to impair mitochondrial function [19,20]. The net effect of the proinflammatory state and tissue oxidative stress is a systemic metabolic dysfunction that defines the metabolic syndrome and type 2 diabetes that are associated with high-fat diets and obesity.

Progression to severe OA is typically observed within 5 months by using the meniscal-ligamentous injury (MLI) technique [25]. With the objective to determine whether the HF diet with its associated type 2 diabetic phenotype accelerates OA progression, mice from each dietary group (n = 4 to 9) were killed at monthly intervals out to 4 months after MLI surgery. Both groups gained weight over this 4-month period, with the HF group becoming markedly obese, with a peak weight of 45.9 ± 2.1 g compared with only 29.9 ± 1.8 g (P < 0.0001) for the lean group (Figure ​(Figure1a).1a). As expected, the HF group was diabetic (fasting blood glucose greater than 125 mg/dl), with peak levels of 288.8 ± 21.4 mg/dl at 4 months (Figure ​(Figure1b).1b). Histologic analysis of the articular cartilage at the 4-month time point revealed increased fibrillation, clefting, and decreased proteoglycan staining in response to MLI. These changes were more pronounced in the HF group (P < 0.01; Figure ​Figure2a)2a) with major loss of cartilage. In some cases, this loss progressed nearly to the subchondral bone. No significant cartilage loss was observed in sham-operated knees of either HF or lean groups, with only occasional superficial zone fibrillation.

A progressive worsening of OA from 2 to 4 months was demonstrated in both diet groups, with the score for OA severity being significantly higher for the HF group at each of these time points (Figure ​(Figure2b).2b). Data for lean mice at month 1 indicate an unexpectedly advanced stage of OA for that time point. The explanation for this aberrant data point is not clear. With the OARSI score, the HF group on average had 25% to 50% loss of articular cartilage at 2 months (score of 4), whereas the lean group had less than 25% loss of articular cartilage at this same time point (score of 3; P < 0.01). As expected, the sham controls had scores of 1 or less, representing only occasional superficial zone fibrillation, even at 4 months (data not shown).

Histomorphometric analysis of the articular cartilage in the lean and HF groups over the 4-month time course confirmed the accelerated loss of articular cartilage in the HF diet group. Articular cartilage thickness on the tibial plateau progressively decreased in both groups but was approximately 40% to 50% thinner in the HF group between 2 and 4 months after MLI (Figure ​(Figure2c).2c). Similarly, total articular cartilage area, representing the combined cartilage areas of the femur and tibia, decreased progressively with the most marked difference between lean and HF diets being observed at the 4-month time point (Figure ​(Figure2d).2d). At this time point, cartilage area in the HF group had decreased by an additional 35% relative to lean controls (P < 0.01).

In the preceding experiments, the HF-fed mice at the later time points were markedly obese in consequence of a 6-month diet protocol that included 2 months of the HF diet before the initiation of OA with MLI. To determine whether this marked increase in body weight was primarily responsible for the accelerated progression of OA with HF feeding, two HF diet protocols were compared. One group of mice (n = 6) was fed an HF diet for 3 months before MLI to establish obesity before initiation of OA. In a second group (n = 8), an HF diet was begun only at the initiation of OA. As a result of beginning the HF diet at the time of MLI, this group of mice demonstrated only a modest increase in body weight relative to the lean diet controls (n = 7) (Figure ​(Figure3a).3a). This contrasts with the long-term HF diet group, which again attained a marked obese body weight that was 80% above that of lean mouse controls. Despite the modest weight gain, the short-term HF group displayed metabolic dysfunction, as indicated by fasting hyperglycemia at 1-, 2-, and 3-month time points, although levels were less than those of the long-term HF group. Values at 3 months are shown (Figure ​(Figure3b).3b). An additional marker for a dysmetabolic state, hemoglobin A1c, a measure of average blood glucose levels over the preceding 3-month period [30], was nearly equally elevated in the two HF groups (Figure ​(Figure3c).3c). Note that the absolute hemoglobin A1c values were less than those of humans, for whom this immunoassay-based test is designed. These results indicate that the short-term HF diet produced metabolic dysfunction without requiring marked obesity or the extended 3-month period on the HF diet before the MLI surgery.

Histologic analysis of the progression of OA at 3 months after MLI confirmed that a long-term HF diet was again associated with accelerated OA progression, with major loss of cartilage relative to the lean controls. In some cases, this loss progressed nearly to the subchondral bone. Importantly, the short-term HF diet group had an equally accelerated progression of OA when compared with the long-term HF group (Figure ​(Figure4a).4a). No significant cartilage loss was observed in sham-operated knees of either HF or lean groups, with only occasional superficial zone fibrillation. These observations were confirmed by comparably higher osteoarthritis grades for the HF groups relative to the lean controls, as assessed by the OARSI scoring method (P < 0.05; Figure ​Figure4b).4b). It should be noted that the accelerated cartilage degeneration occurred in the short-term HF group despite the absence of marked obesity. In agreement with the OARSI scoring and supporting the idea that marked obesity is not required for acceleration of cartilage degeneration in mice fed an HF diet, tibial plateau articular cartilage thicknesses were 25% to 50% lower in the two HF groups compared with the lean group (Figure ​(Figure4c).4c). Cartilage-area determination supported the thickness data by demonstrating similar trends toward decreased area in both HF diet groups relative to mice fed the lean diet (data not shown). These results indicate that accelerated progression of OA in mice on an HF diet is associated with a diabetic metabolic phenotype, does not correlate with degree of weight gain, and does not require prolonged exposure to the HF diet.

Quantitative micro-CT analysis of knee joints at the 3-month time point showed increased bone volume in the MLI knees relative to sham-treated controls, consistent with OA-like subchondral sclerosis and joint/periarticular mineralization. Only differences in the HF-diet groups, however, were statistically significant (P < 0.01; Figure ​Figure5a5a).

Regarding enhanced joint and periarticular mineralization specifically, 3-dimensional reconstructions of the raw micro-CT data revealed a more progressive meniscal calcification and a suggestion of increased presence of osteophytes in both high-HF groups relative to the lean diet controls (Figure ​(Figure5b).5b). To investigate the possibility that HF diets promote the formation of osteophytes, all histologic slides were evaluated for the presence and size of osteophytes on the tibial surface. The blinded analysis revealed osteophytes in essentially all MLI-injured knees from all diet groups (Figure ​(Figure6a).6a). Importantly, not only was the size of the osteophytes greater in both HF-diet groups (P < 0.01), but they also were of comparable size in the short-term and long-term HF groups (Figure ​(Figure6b).6b). Interestingly, 30% of sham knees from HF-diet mice contained small osteophytes (Figure ​(Figure6c),6c), and another 20% showed early changes suggestive of osteophytes. This was distinct from sham knees from the lean mice, which did not display osteophytes, with early changes evident only in less than 20% of knees (Figure ​(Figure6d).6d). As with progression of cartilage degeneration and loss, more-aggressive osteophyte formation in MLI-injured knees from HF-diet mice is associated with a diabetic metabolic phenotype but does not correlate with degree of weight gain.

The relative contributions to OA of biomechanical factors resulting from increased body mass versus metabolic dysfunction in the obese and type 2 diabetic population have not been resolved. In humans, a strong correlation has been shown between elevated body mass index (BMI) and OA [21-23]. Biomechanical factors related to this increased body mass have been argued to make critical contributions to OA of the knee [34,35]. As for metabolic factors, correlations exist between diabetic parameters (hyperglycemia, hyperinsulinemia) and OA [36-39]. Another strong argument for systemic metabolic factors contributing to OA in the obese and diabetic population comes from the increased incidence of OA in the hand, a site not susceptible to the biomechanical overload present in the weight-bearing joints of an obese individual [37,38]. Logically, detrimental effects of body mass on OA would be a function of both magnitude of body-mass increase and length of exposure to this condition after initiation of OA. Our short-term HF diet group weighed the same as lean controls at the time of injury and gained approximately 6 g over the following 3-month period. In contrast, the long-term HF-diet group weighed more than 11 g more than controls at the time of knee injury and increased the differential to more than 20 g by 3 months. Despite this obvious difference in duration and degree of exposure to increased body mass, the two diet groups had comparable acceleration of OA progression. From our results, we conclude that weight gain does not correlate with progression of OA in our HF-diet mouse model. Silberberg and Silberberg [40] came to a similar conclusion that increased body weight in response to HF diets does not correlate with the incidence of joint disease in mice. Recently, Griffin et al. [41] reported the effect of HF diet-induced obesity on spontaneous OA of aging in female mice at 54 weeks of age and concluded that weight gain and loss of Safranin-O cartilage staining or OA severity score, but not cartilage degeneration, were correlated. Their histologic data suggested a modest OA progression with little loss of cartilage. Perhaps the early stages of OA initiation and progression are more strongly influenced by body mass. As cartilage degeneration becomes more severe, as in our MLI model, a high-fat diet continues to accelerate progression, and in this phase of the disease, the associated metabolic dysfunction becomes more important.

Silberberg and co-workers [42] did not investigate the metabolic dysfunction in their mouse models as a result of HF diets, but they did observe that the C57BL/6 strain but not the DBA strain was susceptible to increased OA of aging when on HF diets, despite weight gain in both strains. Sokoloff et al. [43,44] also observed that OA in the DBA strain was not altered by diet, despite weight gain. Recently, Griffin et al. [45] reported that markedly obese leptin-deficient mice do not have an increased incidence of OA. These observations provide further evidence that factors other than increased body mass are critical to the changes in OA in C57BL/6 mice. Interestingly, C57BL/6 mice are well known for their susceptibility to diabetic changes (hyperinsulinemia, hyperglycemia) when placed on HF diets, whereas DBA mice are resistant to these metabolic changes [29,46]. Although no cause and effect has been established, these observations show an association between development of a diabetic phenotype and accelerated progression of OA in the C57BL/6 mouse model.

In the mouse, metabolic changes in response to HF diets appear to be important in mediating osteoarthritic pathogenesis. Saturated fat diets derived from animal fat (lard) are most effective in mediating OA changes [31,44]. They are also more effective than dietary sugar in generating obesity and diabetes in experimental mice [47]. N-3 polyunsaturated fat (PUFA), such as linolenic acid, decreases the detrimental effects of the lard diet [48] and decrease expression and activity of proteoglycan-degrading enzymes and expression of inflammatory cytokines in articular chondrocyte cultures [49,50]. As a potential mechanism of action, the n-3 or omega-3 PUFAs that are found in fish oils have been shown to have anti-inflammatory effects with multiple mechanisms of action being proposed [51]. Importantly, one PUFA, eicosapentaenoic acid, has recently been shown to prevent and reverse insulin resistance in mice fed an HF diet [52]. The mechanism of this latter effect has been proposed to be suppression of adipose tissue inflammation [52]. These results suggest that HF diets mediate their effects on both OA and the diabetic phenotype, at least in part through inflammatory effects.