- Email: [email protected]
Adenosine triphosphate citrate lyase in cartilage homeostasis and osteoarthritis
S64
Abstracts / Osteoarthritis and Cartilage 25 (2017) S8eS75
Table 2 OR (95% CI) of incident hand OA in persons with MetS at baseline.
MetS (3 or more components) Central obesity Elevated blood pressure or anti-hypertensive treatment Elevated fasting glucosis or anti-diabetic treatment Elevated triglycerides Low HDL or lipid-lowering treatment
Adjusted for age and sex
Adjusted for age, sex and BMI
0.98 (0.66e1.46) 1.42 (0.97e2.09) 1.24 (0.85e1.82)
0.91 (0.58e1.44) 1.68 (1.00e2.83) 1.23 (0.82e1.83)
0.95 (0.63e1.44)
0.91 (0.59e1.41)
1.01 (0.68e1.49) 0.57 (0.38e0.84)
0.98 (0.66e1.47) 0.53 (0.35e0.80)
87 OBESITY-RELATED SYSTEMIC INFLAMMATION AND KNEE SYNOVITIS
Table 1 Participant characteristics from baseline by tertiles of HMW adiponectin. Tertile 1
Tertile 2
Tertile 2
Mean Age (years ±SD) % women Mean weight (KG (range)) Physical activity (Mean PASE score (±SD)) % non-smoKer Median serum HMW adiponectin (pg/ml)
60 (7.6) 59 91 (52e153) 185 (90.1)
61 (7.7) 59 86 (49e150) 182 (90.3)
63 (7.8) 59 81 (46e141) 180 (86.2)
60 3168.06
59 7267.47
58 13508.32
Table 2 Association of serum HMW adiponectin tertiles and site-specific MRI based Knee synovitis.
D. Misra y, S. Sheehy y, S. Jafarzadeh y, T. Neogi y, M. Nevitt z, C.E. Lewis x, J. Torner k, D.T. Felson y , The Multicenter Osteoarthritis Studyy. y Boston Univ. Sch. of Med., Boston, MA, USA; z UCSF Sch. of Med., San Francisco, CA, USA; x UAB Sch. of Med., Birmingham, AL, USA; k Univ. of Iowa, Coll. of Publ. Hlth., Iowa City, IA, USA Purpose: Obesity, a major risk factor for knee osteoarthritis (OA), is a state of systemic inflammation through elaboration of inflammatory cytokines (adipokines) from adipose tissue. Whether obesity-related systemic inflammation leads to local inflammation i.e., synovitis, a common feature of OA, is not known but merits investigation to provide insight into inflammatory mechanisms through which obesity might lead to knee OA. In prior work, we found an inverse relation of systemic leptin (proinflammatory) level with knee synovitis. Leptin however is one of many adipokines. The most abundant adipokine is adiponectin which negatively correlates with obesity and obesity-related systemic inflammation (anti-inflammatory). High molecular weight (HMW) adiponectin is suggested to be a better predictor of metabolic parameters than total adiponectin and has not been examined in relation to knee OA. Thus, to understand the effect of adipokines on synovitis in OA, in this study we examined the relation of serum HMW adiponectin to MRI-based knee synovitis in community dwelling-older adults at risk for knee OA. Methods: We recruited participants from Multicenter Osteoarthritis (MOST) study, a longitudinal cohort of older adults, where serum HMW adiponectin is available in a subset of participants from baseline. Knee MRI (fat-suppressed (FS) fast spin-echo intermediate-weighted (IW) sequences) for assessment of synovitis is read at multiple time-points. In this study, person-level synovitis was defined by WORMS score 1 on knee MRI from 3 specific sites which represent distinct pathologies: 1) Infrapatellar fat pad (IF); 2) Intercondylar region (IC); and 3) whole knee Effusion (EFF). We examined the cross-sectional and longitudinal relation of sex-specific tertiles of log of serum HMW adiponectin from baseline with site specific prevalent (existing) and incident (new-onset) knee synovitis from baseline and 60 month visit, respectively, using logistic regression. In the multivariable analyses, we adjusted for age, sex, physical activity (PASE score) and smoking status. We additionally adjusted for body weight at baseline as a proxy for mechanical loading effect in sensitivity analyses. Results: Among 1,442 participants at baseline, synovitis was present in 283 at IF, 631 at IC and 688 with EFF. Participant characteristics from baseline by HMW adiponectin tertiles is presented in Table 1, with expected decrease in body weight and PASE score with increasing HMW adiponectin tertiles. In the cross-sectional analyses, we found significant increase in the risk of IC synovitis in tertile (highest) 3 compared to tertile 1 (Table 2), which was attenuated on adjusting for body weight. In the longitudinal analyses, the effect estimates indicated increased risk of synovitis in tertiles 2 and 3 compared to tertile 1 with IF and EFF synovitis but results did not reach statistical significance, likely due to few cases of incident synovitis (Table 2). Results did not significantly change upon additionally adjusting for body weight. Conclusions: Elevated systemic levels of HMW adiponectin may be associated with increased prevalence and/or incidence of knee synovitis in OA. Larger studies are needed to corroborate our findings.
Characteristics
Sex-specific log of serum HMW adiponectin tertiles
Tertile 1 (Ref) Tertile 2 Tertile 3 (highest) Tertile 1 (Ref) Tertile 2 Tertile 3 (highest) Tertile 1 (Ref) Tertile 2 Tertile 3 (highest)
Tertile 1 (Ref) Tertile 2 Tertile 3 (highest) Tertile 1 (Ref) Tertile 2 Tertile 3 (highest) Tertile 1 (Ref) Tertile 2 Tertile 3 (highest)
n/N (%)
Adjusted* OR (95% CI)
Cross-sectional analysis Infrapatellar synovitis 94/480 (20) 1.0 96/482 (20) 1.03 (0.75e1.42) 93/480 (19) 1.01 (0.73e1.40) Intercondylar synovitis 201/480 (42) 1.0 199/482 (41) 0.98 (0.53e1.19) 231/480 (48) 1.31 (1.01e1.70) Effusion synovitis 230/480 (48) 1.0 227/482 (47) 0.98 (0.76e1.26) 211/480 (44) 0.87 (0.68e1.13) Longitudinal analysis Infrapatellar synovitis 2/335 1.0 3/367 1.81 (0.30e11.18) 3/348 2.46 (0.39e15.50) Intercondylar synovitis 9/335 1.0 7/367 0.82 (0.26e2.26) 8/348 1.14 (0.43e3.09) Effusion synovitis 22/335 1.0 28/367 1.27 (0.71e2.28) 27/348 1.39 (0.77e2.53)
*
Adjusted for age, sex, physical activity (PASE score), education, smoking
88 ADENOSINE TRIPHOSPHATE CITRATE HOMEOSTASIS AND OSTEOARTHRITIS
LYASE
IN
CARTILAGE
L.-Y. Chen y, R. Liu-Bryan z. y VAMC, San Diego, CA, USA; z VAMC/UCSD, San Diego, CA, USA Purpose: Adenosine triphosphate citrate lyase (ACLY) is a key enzyme that generates acetyl-CoA in the nucleocytosolic compartment from mitochondrial-derived citrate for fatty acid and cholesterol biosynthesis as well as histone acetylation. It plays an important role linking glucose metabolism to chromatin modification and global transcription. Since metabolic risk factors are shown to associate with cartilage degradation, we investigated the function of ACLY in articular chondrocytes and tested the hypothesis that ACLY dysregulation leads to cartilage matrix degradation. Methods: Primary human knee chondrocytes isolated from both normal and osteoarthritic (OA) donors were subjected to Western blot analysis for phosphorylation of ACLY (T447 þ S451, indicating ACLY activity) and expression of ACLY. Human knee articular chondrocytes (passage 1) were cultured under conditions of glucose starvation and re-feeding, or either treated with ACLY inhibitor hydroxycitrate (HC) or transfected with ACLY siRNA. In some experiments, the cells were also treated with IL-1b. Cytosolic acetyl-CoA levels were measured by fluorometric quantification. Expression of anabolic genes (SOX9, Col2a,
Abstracts / Osteoarthritis and Cartilage 25 (2017) S8eS75
aggrecan) and catabolic genes (MMP3 and MMP13) were assessed by RT-PCR. Generation of nitric oxide (NO) and release of MMP3 and MMP13 were determined from the conditioned media by Griess reaction and ELISA analysis, respectively. Phosphorylation and expression of ACLY, and AMP-activated protein kinase alpha (p-Thr172, indicating activity of metabolic energy sensor AMPK), acetyl-CoA carboxylase which is a key enzyme in fatty acid synthesis (p-Ser79, indicating inhibition of activity), p65 NF-kB (p-Ser536, indicating NF-kB activation), mTOR and LC3B (involved in autophagy) were examined by Western blot analysis. Total acetylated histone H3 (Ac-H3), or specific Ac-H3K9 and Ac-H3K27 (both indicating transcriptional activation) were also assessed. Results: Glucose starvation of chondrocytes for 5 hours led to drastic loss of cytosolic acetyl-CoA, phosphorylation and expression of ACLY and histone H3 acetylation (total Ac-H3, as well as Ac-H3K9, Ac-H3K27), correlated with increased phosphorylation of AMPKa. This effect was reversed soon after re-feeding the cells with glucose, but only when ACLY was present. These results suggest that glucose availability in chondrocytes influenced histone acetylation in an ACLY-dependent manner. Primary human knee OA chondrocytes or cultured human knee chondrocytes (passage 1) treated with IL-1b exhibited increased phosphorylation of ACLY, correlated with increased levels of Ac-H3K9 and Ac-H3K27 and decreased phosphorylation of AMPKa. Treatment of chondrocytes with ACLY inhibitor HC resulted in increased basal levels of mRNA expression of SOX9, Col2a and aggrecan. In addition, HC inhibited IL-1b-induced increased phosphorylation of ACLY and p65 NFkB and prevented IL-1b-induced de-phosphorylation of AMPKa. In parallel, HC inhibited not only mRNA expression of iNOS, MMP3 and MMP13, but also release of NO, MMP3 and MMP13 induced by IL-1b. Similar results were observed in ACLY knockdown chondrocytes. These data indicate that excessive ACLY expression and activity in chondrocytes contributed to inflammation-mediated matrix degradation. Inhibition of ACLY in chondrocytes resulted in increased phosphorylation of ACC1, implying that fatty acid synthesis was inhibited. Interestingly, inhibition of ACLY in chondrocytes also resulted in increased autophagy, evidenced by reduced phosphorylation of mTOR and increased transition of LC3B-I to LC3B-II. Conclusions: Human knee OA chondrocytes appear to have increased ACLY activity. Glucose and inflammatory cytokine IL-1b modulate ACLY activity in chondrocytes. Inhibition of excessive ACLY activity not only inhibited excessive catabolic responses and promoted autophagy but also limited fatty acid synthesis in chondrocytes, possibly through activation of AMPK and histone modification. Given that metabolic syndrome is linked to OA, ACLY could be an attractive novel target for OA. 89 HIGH FAT AND HIGH SUCROSE DIETS IN MICE HAVE INDEPENDENT EFFECTS ON OSTEOARTHRITIS PATHOLOGY AND CHONDROCYTE METABOLIC STRESS E.L. Donovan, E. Barboza, M. Kinter, T.M. Griffin. Oklahoma Med. Res. Fndn., Oklahoma City, OK, USA 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 ¼ 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
S65
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 SafraninO 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.
Abstracts / Osteoarthritis and Cartilage 25 (2017) S8eS75
Table 2 OR (95% CI) of incident hand OA in persons with MetS at baseline.
MetS (3 or more components) Central obesity Elevated blood pressure or anti-hypertensive treatment Elevated fasting glucosis or anti-diabetic treatment Elevated triglycerides Low HDL or lipid-lowering treatment
Adjusted for age and sex
Adjusted for age, sex and BMI
0.98 (0.66e1.46) 1.42 (0.97e2.09) 1.24 (0.85e1.82)
0.91 (0.58e1.44) 1.68 (1.00e2.83) 1.23 (0.82e1.83)
0.95 (0.63e1.44)
0.91 (0.59e1.41)
1.01 (0.68e1.49) 0.57 (0.38e0.84)
0.98 (0.66e1.47) 0.53 (0.35e0.80)
87 OBESITY-RELATED SYSTEMIC INFLAMMATION AND KNEE SYNOVITIS
Table 1 Participant characteristics from baseline by tertiles of HMW adiponectin. Tertile 1
Tertile 2
Tertile 2
Mean Age (years ±SD) % women Mean weight (KG (range)) Physical activity (Mean PASE score (±SD)) % non-smoKer Median serum HMW adiponectin (pg/ml)
60 (7.6) 59 91 (52e153) 185 (90.1)
61 (7.7) 59 86 (49e150) 182 (90.3)
63 (7.8) 59 81 (46e141) 180 (86.2)
60 3168.06
59 7267.47
58 13508.32
Table 2 Association of serum HMW adiponectin tertiles and site-specific MRI based Knee synovitis.
D. Misra y, S. Sheehy y, S. Jafarzadeh y, T. Neogi y, M. Nevitt z, C.E. Lewis x, J. Torner k, D.T. Felson y , The Multicenter Osteoarthritis Studyy. y Boston Univ. Sch. of Med., Boston, MA, USA; z UCSF Sch. of Med., San Francisco, CA, USA; x UAB Sch. of Med., Birmingham, AL, USA; k Univ. of Iowa, Coll. of Publ. Hlth., Iowa City, IA, USA Purpose: Obesity, a major risk factor for knee osteoarthritis (OA), is a state of systemic inflammation through elaboration of inflammatory cytokines (adipokines) from adipose tissue. Whether obesity-related systemic inflammation leads to local inflammation i.e., synovitis, a common feature of OA, is not known but merits investigation to provide insight into inflammatory mechanisms through which obesity might lead to knee OA. In prior work, we found an inverse relation of systemic leptin (proinflammatory) level with knee synovitis. Leptin however is one of many adipokines. The most abundant adipokine is adiponectin which negatively correlates with obesity and obesity-related systemic inflammation (anti-inflammatory). High molecular weight (HMW) adiponectin is suggested to be a better predictor of metabolic parameters than total adiponectin and has not been examined in relation to knee OA. Thus, to understand the effect of adipokines on synovitis in OA, in this study we examined the relation of serum HMW adiponectin to MRI-based knee synovitis in community dwelling-older adults at risk for knee OA. Methods: We recruited participants from Multicenter Osteoarthritis (MOST) study, a longitudinal cohort of older adults, where serum HMW adiponectin is available in a subset of participants from baseline. Knee MRI (fat-suppressed (FS) fast spin-echo intermediate-weighted (IW) sequences) for assessment of synovitis is read at multiple time-points. In this study, person-level synovitis was defined by WORMS score 1 on knee MRI from 3 specific sites which represent distinct pathologies: 1) Infrapatellar fat pad (IF); 2) Intercondylar region (IC); and 3) whole knee Effusion (EFF). We examined the cross-sectional and longitudinal relation of sex-specific tertiles of log of serum HMW adiponectin from baseline with site specific prevalent (existing) and incident (new-onset) knee synovitis from baseline and 60 month visit, respectively, using logistic regression. In the multivariable analyses, we adjusted for age, sex, physical activity (PASE score) and smoking status. We additionally adjusted for body weight at baseline as a proxy for mechanical loading effect in sensitivity analyses. Results: Among 1,442 participants at baseline, synovitis was present in 283 at IF, 631 at IC and 688 with EFF. Participant characteristics from baseline by HMW adiponectin tertiles is presented in Table 1, with expected decrease in body weight and PASE score with increasing HMW adiponectin tertiles. In the cross-sectional analyses, we found significant increase in the risk of IC synovitis in tertile (highest) 3 compared to tertile 1 (Table 2), which was attenuated on adjusting for body weight. In the longitudinal analyses, the effect estimates indicated increased risk of synovitis in tertiles 2 and 3 compared to tertile 1 with IF and EFF synovitis but results did not reach statistical significance, likely due to few cases of incident synovitis (Table 2). Results did not significantly change upon additionally adjusting for body weight. Conclusions: Elevated systemic levels of HMW adiponectin may be associated with increased prevalence and/or incidence of knee synovitis in OA. Larger studies are needed to corroborate our findings.
Characteristics
Sex-specific log of serum HMW adiponectin tertiles
Tertile 1 (Ref) Tertile 2 Tertile 3 (highest) Tertile 1 (Ref) Tertile 2 Tertile 3 (highest) Tertile 1 (Ref) Tertile 2 Tertile 3 (highest)
Tertile 1 (Ref) Tertile 2 Tertile 3 (highest) Tertile 1 (Ref) Tertile 2 Tertile 3 (highest) Tertile 1 (Ref) Tertile 2 Tertile 3 (highest)
n/N (%)
Adjusted* OR (95% CI)
Cross-sectional analysis Infrapatellar synovitis 94/480 (20) 1.0 96/482 (20) 1.03 (0.75e1.42) 93/480 (19) 1.01 (0.73e1.40) Intercondylar synovitis 201/480 (42) 1.0 199/482 (41) 0.98 (0.53e1.19) 231/480 (48) 1.31 (1.01e1.70) Effusion synovitis 230/480 (48) 1.0 227/482 (47) 0.98 (0.76e1.26) 211/480 (44) 0.87 (0.68e1.13) Longitudinal analysis Infrapatellar synovitis 2/335 1.0 3/367 1.81 (0.30e11.18) 3/348 2.46 (0.39e15.50) Intercondylar synovitis 9/335 1.0 7/367 0.82 (0.26e2.26) 8/348 1.14 (0.43e3.09) Effusion synovitis 22/335 1.0 28/367 1.27 (0.71e2.28) 27/348 1.39 (0.77e2.53)
*
Adjusted for age, sex, physical activity (PASE score), education, smoking
88 ADENOSINE TRIPHOSPHATE CITRATE HOMEOSTASIS AND OSTEOARTHRITIS
LYASE
IN
CARTILAGE
L.-Y. Chen y, R. Liu-Bryan z. y VAMC, San Diego, CA, USA; z VAMC/UCSD, San Diego, CA, USA Purpose: Adenosine triphosphate citrate lyase (ACLY) is a key enzyme that generates acetyl-CoA in the nucleocytosolic compartment from mitochondrial-derived citrate for fatty acid and cholesterol biosynthesis as well as histone acetylation. It plays an important role linking glucose metabolism to chromatin modification and global transcription. Since metabolic risk factors are shown to associate with cartilage degradation, we investigated the function of ACLY in articular chondrocytes and tested the hypothesis that ACLY dysregulation leads to cartilage matrix degradation. Methods: Primary human knee chondrocytes isolated from both normal and osteoarthritic (OA) donors were subjected to Western blot analysis for phosphorylation of ACLY (T447 þ S451, indicating ACLY activity) and expression of ACLY. Human knee articular chondrocytes (passage 1) were cultured under conditions of glucose starvation and re-feeding, or either treated with ACLY inhibitor hydroxycitrate (HC) or transfected with ACLY siRNA. In some experiments, the cells were also treated with IL-1b. Cytosolic acetyl-CoA levels were measured by fluorometric quantification. Expression of anabolic genes (SOX9, Col2a,
Abstracts / Osteoarthritis and Cartilage 25 (2017) S8eS75
aggrecan) and catabolic genes (MMP3 and MMP13) were assessed by RT-PCR. Generation of nitric oxide (NO) and release of MMP3 and MMP13 were determined from the conditioned media by Griess reaction and ELISA analysis, respectively. Phosphorylation and expression of ACLY, and AMP-activated protein kinase alpha (p-Thr172, indicating activity of metabolic energy sensor AMPK), acetyl-CoA carboxylase which is a key enzyme in fatty acid synthesis (p-Ser79, indicating inhibition of activity), p65 NF-kB (p-Ser536, indicating NF-kB activation), mTOR and LC3B (involved in autophagy) were examined by Western blot analysis. Total acetylated histone H3 (Ac-H3), or specific Ac-H3K9 and Ac-H3K27 (both indicating transcriptional activation) were also assessed. Results: Glucose starvation of chondrocytes for 5 hours led to drastic loss of cytosolic acetyl-CoA, phosphorylation and expression of ACLY and histone H3 acetylation (total Ac-H3, as well as Ac-H3K9, Ac-H3K27), correlated with increased phosphorylation of AMPKa. This effect was reversed soon after re-feeding the cells with glucose, but only when ACLY was present. These results suggest that glucose availability in chondrocytes influenced histone acetylation in an ACLY-dependent manner. Primary human knee OA chondrocytes or cultured human knee chondrocytes (passage 1) treated with IL-1b exhibited increased phosphorylation of ACLY, correlated with increased levels of Ac-H3K9 and Ac-H3K27 and decreased phosphorylation of AMPKa. Treatment of chondrocytes with ACLY inhibitor HC resulted in increased basal levels of mRNA expression of SOX9, Col2a and aggrecan. In addition, HC inhibited IL-1b-induced increased phosphorylation of ACLY and p65 NFkB and prevented IL-1b-induced de-phosphorylation of AMPKa. In parallel, HC inhibited not only mRNA expression of iNOS, MMP3 and MMP13, but also release of NO, MMP3 and MMP13 induced by IL-1b. Similar results were observed in ACLY knockdown chondrocytes. These data indicate that excessive ACLY expression and activity in chondrocytes contributed to inflammation-mediated matrix degradation. Inhibition of ACLY in chondrocytes resulted in increased phosphorylation of ACC1, implying that fatty acid synthesis was inhibited. Interestingly, inhibition of ACLY in chondrocytes also resulted in increased autophagy, evidenced by reduced phosphorylation of mTOR and increased transition of LC3B-I to LC3B-II. Conclusions: Human knee OA chondrocytes appear to have increased ACLY activity. Glucose and inflammatory cytokine IL-1b modulate ACLY activity in chondrocytes. Inhibition of excessive ACLY activity not only inhibited excessive catabolic responses and promoted autophagy but also limited fatty acid synthesis in chondrocytes, possibly through activation of AMPK and histone modification. Given that metabolic syndrome is linked to OA, ACLY could be an attractive novel target for OA. 89 HIGH FAT AND HIGH SUCROSE DIETS IN MICE HAVE INDEPENDENT EFFECTS ON OSTEOARTHRITIS PATHOLOGY AND CHONDROCYTE METABOLIC STRESS E.L. Donovan, E. Barboza, M. Kinter, T.M. Griffin. Oklahoma Med. Res. Fndn., Oklahoma City, OK, USA 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 ¼ 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
S65
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 SafraninO 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.