Arunabh Bhattacharya

Dietary n‐3 Fatty Acids Decrease Osteoclastogenesis and Loss of Bone Mass in Ovariectomized Mice

The mechanisms of action of dietary fish oil (FO) on osteoporosis are not fully understood. This study showed FO decreased bone loss in ovariectomized mice because of inhibition of osteoclastogenesis. This finding supports a beneficial effect of FO on the attenuation of osteoporosis.

Increased superoxide in vivo accelerates age-associated muscle atrophy through mitochondrial dysfunction and neuromuscular junction degeneration

Oxidative stress has been implicated in the etiology of age‐related muscle loss (sarcopenia). However, the underlying mechanisms by which oxidative stress contributes to sarcopenia have not been thoroughly investigated. To directly examine the role of chronic oxidative stress in vivo, we used a mouse model that lacks the antioxidant enzyme CuZnSOD (Sodl). Sod1−/− mice are characterized by high levels of oxidative damage and an acceleration of sarcopenia. In the present study, we demonstrate that muscle atrophy in Sod1−/− mice is accompanied by a progressive decline in mitochondrial bioenergetic function and an elevation of mitochondrial generation of reactive oxygen species. In addition, Sod1−/− muscle exhibits a more rapid induction of mitochondrial‐mediated apoptosis and loss of myonuclei. Furthermore, aged Sod1−/− mice show a striking increase in muscle mitochondrial content near the neuromuscular junctions (NMJs). Despite the increase in content, the function of mitochondria is significantly impaired, with increased denervated NMJs and fragmentation of acetylcholine receptors. As a consequence, contractile force in aged Sod1−/− muscles is greatly diminished. Collectively, we show that Sod1−/− mice display characteristics of normal aging muscle in an accelerated manner and propose that the superoxide‐induced NMJ degeneration and mitochondrial dysfunction are potential mechanisms of sarcopenia.—Jang, Y. C., Lustgarten, M. S., Liu, Y., Muller, F. L., Bhattacharya, A., Liang, H., Salmon, A. B., Brooks, S. V., Larkin, L., Hayworth, C. R., Richardson, A., and Van Remmen, H. Increased superoxide in vivo accelerates age‐associated muscle atrophy through mitochondrial dysfunction and neuro‐muscular junction degeneration. FASEB J. 24, 1376–1390 (2010). www.fasebj.org

High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates

Despite the considerable interest in superoxide as a potential cause of pathology, the mechanisms of its deleterious production by mitochondria remain poorly understood. Previous studies in purified mitochondria have found that the highest rates of superoxide production are observed with succinate-driven reverse-electron transfer through complex I, although the physiological importance of this pathway is disputed because it necessitates high concentrations of succinate and is thought not to occur when NAD is in the reduced state. However, very few studies have examined the rates of superoxide production with mitochondria respiring on both NADH-linked (e.g. glutamate) and complex II-linked substrates. In the present study, we find that the rates of superoxide production (measured indirectly as H2O2) with glutamate+succinate (approximately 1100 pmol of H2O2 x min(-1) x mg(-1)) were unexpectedly much higher than with succinate (approximately 400 pmol of H2O2 x min(-1) x mg(-1)) or glutamate (approximately 80 pmol of H2O2 x min(-1) x mg(-1)) alone. Superoxide production with glutamate+succinate remained high even at low substrate concentrations (<1 mM), was decreased by rotenone and was completely eliminated by FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), indicating that it must in large part originate from reverse-electron transfer through complex I. Similar results were obtained when glutamate was replaced with pyruvate, alpha-ketoglutarate or palmitoyl carnitine. In contrast, superoxide production was consistently lowered by the addition of malate (malate+succinate approximately 30 pmol of H2O2 x min(-1) x mg(-1)). We propose that the inhibitory action of malate on superoxide production can be explained by oxaloacetate inhibition of complex II. In summary, the present results indicate that reverse-electron transfer-mediated superoxide production can occur under physiologically realistic substrate conditions and suggest that oxaloacetate inhibition of complex II may be an adaptive mechanism to minimize this.

Docosahexaenoic acid is more potent inhibitor of osteoclast differentiation in RAW 264.7 cells than eicosapentaenoic acid

Fish oil rich in n−3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protects inflammation induced bone loss in chronic inflammatory diseases like rheumatoid arthritis, periodontitis, and osteoporosis. EPA and DHA differentially regulate functional parameters and gene expression in different cell types. One of the risk factors for bone loss in inflammatory bone diseases is the elevation of bone‐resorbing osteoclasts and a very few studies so far have indicated that attenuation of osteoclastogenesis might be one of the mechanisms by which n−3 PUFA exert its effect on bone loss protection. However, the precise mechanism underlying this process remains unclear. Receptor activator of NF‐κB ligand (RANKL) is known to be the most critical mediator of osteoclastogenesis. Therefore, in this study, we examined the differential effect of EPA and DHA on RANKL‐stimulated osteoclastogenesis and RANKL signaling using a murine monocytic cell line RAW 264.7. DHA was found to inhibit osteoclast differentiation, activation and function more potently than EPA. The differential potential also closely correlated with the inhibition of osteoclast‐specific genes like tartrate resistant acid phosphatase, cathepsin K, calcitonin receptor, matrix metalloproteinase‐9 expression and osteoclast‐specific transcription factor, c‐Fos, as well as osteotropic proinflammatory cytokine, TNF‐α to a greater extent with DHA than EPA. Further, pretreatment of RAW 264.7 cells with DHA also showed significantly reduced activation of NF‐κB and p38MAPK than EPA. Our findings suggest that DHA may be much more effective than EPA in alleviating RANKL induced proinflammatory cytokine production, intracellular signaling activation, thereby decreasing osteoclast activation and bone resorption. J. Cell. Physiol. 214:201–209, 2008.

Conjugated linoleic acid inhibits osteoclast differentiation of RAW264.7 cells by modulating RANKL signaling

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis, periodontitis, and osteoporosis. Inflammation-induced bone loss of this sort results from increased numbers of bone-resorbing osteoclasts. Numerous studies have indicated that conjugated linoleic acid (CLA) positively influences calcium and bone metabolism. Gene-deletion studies have shown that receptor activator of nuclear factor-κB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. In this report, we examine the ability of CLA to suppress RANKL signaling and osteoclastogenesis in RAW264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated nuclear factor-κB (NF-κB), and preexposure of the cells to CLA significantly suppressed RANKL-induced NF-κB activation, including phosphorylation of I-κBα, degradation of I-κBα, and nuclear translocation of p65. RANKL induced osteoclastogenesis in these monocytic cells, and CLA inhibited RANKL-induced tumor necrosis factor-α production and osteoclast differentiation, including osteoclast-specific genes such as tartrate-resistant acid phosphatase, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9 expression and osteoclast-specific transcription factors such as c-Fos, nuclear factor of activated T-cells expression, and bone resorption pit formation. CLA also inhibited RANKL-induced activation of mitogen-activated protein kinase p38 but had little effect on c-Jun N-terminal kinase activation. Collectively, these data demonstrate for the first time that CLA inhibits osteoclastogenesis by modulating RANKL signaling. Thus, CLA may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption by excessive osteoclastogenesis.

Effect of Dietary n-3 and n-6 Oils with and without Food Restriction on Activity of Antioxidant Enzymes and Lipid Peroxidation in Livers of Cyclophosphamide Treated Autoimmune-Prone NZB/W Female Mice

Objective: Cyclophosphamide (CTX), an alkylating agent, is extensively used in the treatment of lupus nephritis, but its administration has been associated with free radical mediated oxidative stress. The present study was designed to investigate the effect of dietary corn oil (CO), fish oil (FO) and food restriction (FR) on the activities of hepatic antioxidant enzymes, fatty acid composition and lipid peroxidation following CTX administration in autoimmune-prone NZB/W female mice. Methods: Autoimmune-prone NZB/W female mice were fed either ad libitum (AL) or food restricted (60% of AL intake), semipurified diets containing 5% CO or 5% FO supplemented with equal levels of antioxidants and injected with either phosphate buffered saline (PBS), or CTX (50 mg/kg body weight) every 10 days. Proteinuria was measured biweekly. The treatment was stopped at 10 months and diets were continued until the mice were killed at 12 months. Fatty acid composition, activity of antioxidant enzymes and lipid peroxidation were analyzed in liver homogenates, and anti-DNA antibodies were analyzed in the serum. Results: Mice in the FO/AL dietary group exhibited significantly higher liver catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities compared to the CO/AL dietary group. CTX significantly decreased SOD and GSH-Px activity in the FO/AL group and CAT and GSH-Px in the CO/AL group. In AL fed mice given CTX, activities of CAT, GSH-Px and GST were significantly higher in mice fed FO diets than in mice fed CO diets. FR increased the activity of enzymes in both the CO and FO diet groups. In FR mice, CTX decreased CAT and GSH-Px activity in both the CO and FO dietary groups, but glutathione S-transferase (GST) only in the CO group. The decrease in SOD activity was not significant in either of the restricted groups. CTX significantly increased generation of thiobarbituric acid reactive substances (TBARS) in both AL groups. FR significantly decreased lipid peroxidation in both the CO and FO groups, with or without CTX. CTX decreased serum anti-DNA antibody levels in both the CO and FO dietary groups. FR also decreased antibody titer in both the CO and FO dietary groups, and it was decreased further with CTX treatment. FO fed animals had higher levels of n-3 fatty acids, whereas CO fed animals had high levels of n-6 fatty acids. CTX significantly increased 20:4 and decreased 18:1 in CO/AL fed animals, whereas it increased 18:1 and decreased 22:6 in FO/AL fed animals. Conclusions: Results obtained in the present study suggests that FO and, more significantly, FO combined with FR can have a beneficial effect in hepatic tissues subjected to CTX induced oxidative stress by regulating the activity of antioxidant enzymes. In addition, the study also indicates that n-3 and n-6 dietary lipids are susceptible to lipid peroxidation, particularly in the presence of a prooxidant like CTX, and that FR is beneficial in decreasing lipid peroxidation. The study also suggests that FO and CTX can have additive effects in preventing kidney disease in NZB/W mice.

Omega-3 Fatty Acid Effect on Alveolar Bone Loss in Rats

Gingival inflammation and alveolar bone resorption are hallmarks of adult periodontitis, elicited in response to oral micro-organisms such as Porphyromonas gingivalis. We hypothesized that omega (ω)-3 fatty acids (FA) dietary supplementation would modulate inflammatory reactions leading to periodontal disease in infected rats. Rats were fed fish oil (ω-3 FA) or corn oil (n-6 FA) diets for 22 weeks and were infected with P. gingivalis. Rats on the ω-3 FA diet exhibited elevated serum levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), documenting diet-induced changes. PCR analyses demonstrated that rats were orally colonized by P. gingivalis; increased IgG antibody levels substantiated this infection. P. gingivalis-infected rats treated with ω-3 FA had significantly less alveolar bone resorption. These results demonstrated the effectiveness of an ω-3 FA-supplemented diet in modulating alveolar bone resorption following P. gingivalis infection, and supported that ω-3 FA may be a useful adjunct in the treatment of periodontal disease. Abbreviations: PUFA, polyunsaturated fatty acid; EPA, eicosapentanoic acid; DHA, docosahexanoic acid; and PCR, polymerase chain-reaction.

Docosahexaenoic Acid-Enriched Fish Oil Attenuates Kidney Disease and Prolongs Median and Maximal Life Span of Autoimmune Lupus-Prone Mice

The therapeutic efficacy of individual components of fish oils (FOs) in various human inflammatory diseases still remains unresolved, possibly due to low levels of n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) or lower ratio of DHA to EPA. Because FO enriched with DHA (FO-DHA) or EPA (FO-EPA) has become available recently, we investigated their efficacy on survival and inflammatory kidney disease in a well-established animal model of human systemic lupus erythematosus. Results show for the first time that FO-DHA dramatically extends both the median (658 d) and maximal (848 d) life span of (NZB × NZW)F1 (B × W) mice. In contrast, FO-EPA fed mice had a median and maximal life span of ∼384 and 500 d, respectively. Investigations into possible survival mechanisms revealed that FO-DHA (versus FO-EPA) lowers serum anti-dsDNA Abs, IgG deposition in kidneys, and proteinuria. Further, FO-DHA lowered LPS-mediated increases in serum IL-18 levels and caspase-1–dependent cleavage of pro–IL-18 to mature IL-18 in kidneys. Moreover, FO-DHA suppressed LPS-mediated PI3K, Akt, and NF-κB activations in kidney. These data indicate that DHA, but not EPA, is the most potent n-3 fatty acid that suppresses glomerulonephritis and extends life span of systemic lupus erythematosus-prone short-lived B × W mice, possibly via inhibition of IL-18 induction and IL-18–dependent signaling.

Endogenous n-3 fatty acids protect ovariectomy induced bone loss by attenuating osteoclastogenesis

Beneficial effects of n‐3 fatty acids (FA) on bone mineral density (BMD) have been reported in mice, rats and human beings, but the precise mechanisms involved have not been described. This study used the Fat‐1 mouse, a transgenic model that synthesizes n‐3 FA from n‐6 FA to directly determine if outcome of bone health were correlated with n‐3 FA. Ovariectomized (Ovx) and sham operated wild‐type (WT) and Fat‐1 mice were fed an AIN‐93M diet containing 10% corn oil for 24 weeks. BMD was analysed by dual energy x‐ray absorptiometry. Fat‐1 Ovx mice exhibited significantly lower level of osteotropic factors like receptor activator of NF‐κB ligand and tartrate‐resistant acid phosphatase (TRAP)5b in serum and higher BMD in distal femoral metaphysis, proximal tibial metaphysis, femoral diaphysis and lumbar vertebra as compared to WT Ovx mice. LPS‐stimulated bone marrow (BM) cells from Fat‐1 Ovx mice produced significantly lower level of pro‐inflammatory cytokines like tumour necrosis factor‐α, interleukin (IL)‐1‐β, IL‐6 and higher level of anti‐inflammatory cytokines like IL‐10, IFN‐γ and higher level of nitric oxide as compared to BM cells from WT Ovx mice. LPS‐stimulated COX‐II activity as well as NF‐κB activation in BM cells from Fat‐1 Ovx mice was significantly less as compared to BM cells from WT Ovx mice. Furthermore, Fat‐1 BM cells generated significantly less number of TRAP osteoclast‐like cells as compared to WT BM cells. In conclusion, we offer further insight into the mechanisms involved in preventing the BMD loss in Ovx mice by n‐3 FA using a Fat‐1 transgenic mouse model.

PGC-1α protects neurons and alters disease progression in an amyotrophic lateral sclerosis mouse model

Introduction: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. We sought to determine whether peroxisome proliferator–activated receptor γ coactivator 1α (PGC‐1α) would have a beneficial effect on this disease. Methods: PGC‐1α transgenic mice were crossed with SOD1 mutant G93A DL mice. Results: We observed a moderate but non‐significant increase in average lifespan in PGC‐1α/G93A DL mice, as compared with G93A DL mice (292 ± 3 days vs. 274 ± 7 days). Although the onset of ALS was not altered, progression of the disease was significantly slower (∽34% increase in duration) in the PGC‐1α/G93A DL mice. These mice also exhibited markedly improved performance on the rotarod test, and the improved motor activity was associated with a decreased loss of motor neurons and less degeneration of neuromuscular junctions. Conclusion: A sustained level of excitatory amino acid transporter protein 2 (EAAT2) in astrocytes of the PGC‐1α/G93A DL mice may contribute to neuronal protection. Muscle Nerve 2011