Five-week-old C57BL/6 mice were placed on either an HF diet representing 60% of total dietary kcal or an LF (lean) diet with 10% of kilocalories derived from fat. The HF diet has been shown to cause hyperglycemia and insulin resistance in this strain of mice [29
]. After 8 weeks, mice on the HF diet weighed 29% more than those on the lean diet (30.9 ± 0.9 g versus 23.9 ± 0.6 g; P
< 0.0001; Figure ). Fasting blood glucose levels were in the diabetic range only for the HF group (182.3 ± 10.5 mg/dl; Figure ), confirming a dysmetabolic state associated with the HF diet.
Figure 1 High-fat diet promotes weight gain and hyperglycemia. Five-week-old male C57BL/6 mice were placed on either a high-fat (60% kcal) or low-fat (10% kcal) diet for 2 months before meniscal-ligamentous injury (MLI). Body weight (a) and blood glucose levels (more ...)
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 ). 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 ). 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 ) 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.
Figure 2 Osteoarthritic changes were more pronounced in high-fat (HF) diet-treated mice. (a) Histologic sections of injured and sham joints at 1-, 2-, 3-, and 4-month time points after meniscal-ligamentous injury (MLI) were stained with Alcian blue. Representative (more ...)
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 ). 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 ). 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 ). 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 ). 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 ). 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 ). 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.
Figure 3 Differing weight gain but similar hyperglycemia in short- and long-term high-fat (HF) diets. Five-week-old male C57BL/6 mice were given either a high-fat (60% kcal) or low-fat (10% kcal) diet for 3 months before meniscal-ligamentous injury (MLI). After (more ...)
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 ). 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 ). 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 ). 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.
Figure 4 OA progression after meniscal-ligamentous injury MLI is comparable in mice on either short- or long-term HF diets. Injured and sham joints at 3 months after MLI were harvested from the lean, short-term HF, and long-term HF groups. (a) Histologic slides (more ...)
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 ).
Figure 5 Micro-CT analysis of knee joints after meniscal-ligamentous injury (MLI) as a function of diet. (a) Knee-bone volumes of MLI and sham-treated limbs at 3 months in HF, Lean to HF, and Lean diet groups were analyzed with micro-CT. (b) Three-dimensional (more ...)
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 ). 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 ). 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 ). Interestingly, 30% of sham knees from HF-diet mice contained small osteophytes (Figure ), 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 ). 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.
Figure 6 Osteophyte analysis of knee joints after meniscal-ligamentous injury (MLI) as a function of diet. (a) A representative osteophyte in a histologic section from an MLI-treated knee of an HF mouse is shown (boxed area). (b) Average osteophyte diameters on (more ...)