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Objective The etiology of chondrocyte mitochondrial dysfunction in OA is incompletely understood. OA chondrocytes are deficient in active AMPK-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), metabolic biosensors that modulate the mitochondrial biogenesis “master regulator” peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α. Moreover, PGC-1α critically mediates AMPK anti-catabolic activity in chondrocytes. Here, we tested the hypotheses that mitochondrial biogenesis is deficient in human OA chondrocytes, which functionally increases chondrocyte pro-catabolic responses, but is reversed by activation of the AMPK-SIRT1-PGC-1α pathway. Methods We studied human knee chondrocytes, human and mouse knee cartilages. We examined expression and activity (phosphorylation) of AMPKα, and SIRT1 and PGC-1α, and defined and compared mitochondrial content and functions including oxidative phosphorylation (OXPHOS) with expression of mitochondrial biogenesis factors (mitochondrial transcriptional factor A (TFAM), nuclear respiratory factors (NRFs)). Results Human knee OA chondrocytes had decreased mitochondrial biogenesis capacity, linked to reduced AMPKα activity and decreased SIRT1, PGC-1α, TFAM, and NRF1,2 expression. Human knee OA and aged mouse knee cartilages had decreased TFAM and ubiquinol-cytochrome c reductase core protein I (UQCEC1), a subunit of mitochondrial complex III, in situ. Functionally, chondrocyte TFAM knockdown inhibited mitochondrial biogenesis and enhanced pro-catabolic responses to IL-1β. Last, pharmacologic AMPK activation by A-769662 increased PGC-1α via SIRT1, and reversed impairments in mitochondrial biogenesis, OXPHOS, and intracellular ATP in human knee OA chondrocytes. Conclusions Mitochondrial biogenesis is deficient in human OA chondrocytes and this promotes chondrocyte pro-catabolic responses. Activation of the AMPK-SIRT1-PGC-1α pathway reverses these effects, mediated by TFAM, suggesting translational potential to limit OA progression.

Insulin induces and dietary n-3 PUFAs suppress hepatic de novo lipogenesis by controlling sterol-regulatory element binding protein-1 nuclear abundance (nSREBP-1). Our goal was to define the mechanisms involved in this regulatory process. Insulin treatment of rat primary hepatocytes rapidly augments nSREBP-1 and mRNA while suppressing mRNA Insig-2 but not mRNA Insig-1 . These events are preceded by rapid but transient increases in Akt and Erk phosphorylation. Removal of insulin from hepatocytes leads to a rapid decline in nSREBP-1 [half-time (T 1/2 ) z 10 h] that is abrogated by inhibitors of 26S proteasomal degradation. 22:6,n-3, the major n-3 PUFA accumulating in livers of fish oil-fed rats, suppresses hepatocyte levels of nSREBP-1, mRNA , and mRNA Insig-2 but modestly and transiently induces mRNA Insig-1 . More importantly, 22:6,n-3 accelerates the disappearance of hepatocyte nSREBP-1 (T 1/2 z 4 h) through a 26S proteasomedependent process. 22:6,n-3 has minimal effects on microsomal SREBP-1 and sterol-regulatory element binding protein cleavage-activating protein or nuclear SREBP-2. 22:6,n-3 transiently inhibits insulin-induced Akt phosphorylation but induces Erk phosphorylation. Inhibitors of Erk phosphorylation, but not overexpressed constitutively active Akt, rapidly attenuate 22:6,n-3 suppression of nSREBP-1. Thus, 22:6,n-3 suppresses hepatocyte nSREBP-1 through 26S proteasome-and Erk-dependent pathways. These studies reveal a novel mechanism for n-3 PUFA regulation of hepatocyte nSREBP-1 and lipid metabolism.-Botolin, D., Y. Wang, B. Christian, and D. B. Jump. Docosahexaneoic acid (22:6,n-3) regulates rat hepatocyte SREBP-1 nuclear abundance by Erk-and 26S proteasome-dependent pathways. J. Lipid Res. 2006. 47: 181-192. Supplementary key words sterol regulatory element binding protein-Sterol-regulatory element binding proteins (SREBP-1a, SREBP-1c, and SREBP-2) are basic helix-loop-helix-leucine zipper transcription factors that play a central role in controlling the transcription of genes involved in cholesterol and fatty acid synthesis (1). The principal mechanism for SREBP regulation of gene transcription involves the control of its nuclear abundance (nSREBP). nSREBP is regulated by two posttranslational mechanisms, proteolytic processing (1) and 26S proteasomal degradation (2). All SREBPs are synthesized as precursors (pSREBP; z125 kDa) tethered to the endoplasmic reticulum (ER) and escorted to the Golgi complex by sterol-regulatory element binding protein cleavage-activating protein (SCAP) for proteolytic processing. nSREBP is transported to the nucleus via importin-b (3), where it binds sterol-regulatory elements in promoters of specific genes, recruits coactivators to the promoter, and stimulates gene transcription (4). Phosphorylation and ubiquitination of nSREBP targets nSREBP for 26S proteasomal degradation (5). Sterols regulate nSREBP levels by controlling the proteolytic processing step, not 26S proteasomal degradation. Instead, sterols induce the ERresident proteins Insig-1 and I...

Objective AMP-activated protein kinase (AMPK) is metabolic biosensor with anti-inflammatory activities. Gout is commonly associated with excesses in soluble urate and in nutrition, both of which suppress tissue AMPK activity. Gout is driven by macrophage-mediated inflammation transduced partly by NLRP3 inflammasome activation and interleukin (IL)-1β release. Hence, we tested the hypothesis that AMPK activation limits monosodium urate (MSU) crystal-induced inflammation. Methods We studied bone marrow-derived macrophages (BMDMs) from AMPKα1 knockout and wild-type mice, and assessed the selective AMPK pharmacological activator A-769662 and a low concentration (10 nM) of colchicine. We examined phosphorylation (activation) of AMPKα Thr172, NLRP3 mRNA expression, and caspase-1 cleavage and IL-1β maturation using western blot and quantitative RT-PCR approaches. We also assessed subcutaneous murine air pouch inflammatory responses to MSU crystals in vivo. Results MSU crystals suppressed phosphorylation of AMPKα in BMDMs. Knockout of AMPKα1 enhanced, and, conversely, A-769662-inhibited MSU crystal-induced inflammatory responses including IL-1β and CXCL1 release in vitro and in vivo. A-769662 promoted AMPK-dependent macrophage anti-inflammatory M2 polarisation and inhibited NLRP3 gene expression, activation of caspase-1 and IL-1β. Colchicine, at low concentration (10 nM) achieved in gout flare prophylaxis dosing, promoted phosphorylation of AMPKα and macrophage M2 polarisation, and reduced activation of caspase-1 and release of IL-1β and CXCL1 by MSU crystals in BMDMs in vitro. Conclusions AMPK activity limits MSU crystal inflammation in vitro and in vivo, and transduces multiple anti-inflammatory effects of colchicine in macrophages. Targeting increased and sustained AMPK activation in inflammatory cells merits further investigation for enhancing efficacy of prophylaxis and treatment of gouty inflammation.

Nuclear factor-κB (NF-κB) plays a central role in pathogenesis of inflammation and cancer. Many phytochemicals including gamma-tocotrienol (γTE), a natural form of vitamin E, have been shown to inhibit NF-κB activation, but the underlying mechanism has not been identified. Here we show that γTE inhibited cytokine-triggered activation of NF-κB and its upstream regulator TGFβ-activated kinase-1 in murine RAW264.7 macrophages and primary bone marrow-derived macrophages. In these cells, γTE induced up-regulation of A20, an inhibitor of NF-κB. Knockout of A20 partially diminished γTE’s anti-NF-κB effect but γTE increased another NF-κB inhibitor Cezanne in A20−/− cells. In search of the reason for A20 upregulation, we found that γTE treatment increased phosphorylation of translation initiation factor 2 (eIF2α), IκBα and JNK, indicating induction of endoplasmic reticulum (ER) stress. LC-MS/MS analyses revealed that γTE modulated sphingolipids including enhancement of intracellular dihydroceramides, sphingoid bases in de novo synthesis of sphingolipid pathway. Chemical inhibition of de novo sphingolipid synthesis partially reversed γTE’s induction of A20 and anti-NF-κB effect. The importance of dihydroceramide increase is further supported by the observation that C8-dihydroceramide mimicked γTE in up-regulating A20, enhancing ER stress and attenuating TNF-triggered NF-κB activation. Our study identifies a novel anti-NF-κB mechanism where A20 is induced by stress-induced adaptive response as a result of modulation of sphingolipids, and demonstrates an immune-modulatory role of dihydrocermides.