SummaryCellular senescence is a state of stable proliferation arrest of cells. The senescence pathway has many beneficial effects and is seen to be activated in damaged/stressed cells, as well as during embryonic development and wound healing. However, the persistence and accumulation of senescent cells in various tissues can also impair function and have been implicated in the pathogenesis of many age‐related diseases. Osteoarthritis (OA), a severely debilitating chronic condition characterized by progressive tissue remodeling and loss of joint function, is the most prevalent disease of the synovial joints, and increasing age is the primary OA risk factor. The profile of inflammatory and catabolic mediators present during the pathogenesis of OA is strikingly similar to the secretory profile observed in ‘classical’ senescent cells. During OA, chondrocytes (the sole cell type present within articular cartilage) exhibit increased levels of various senescence markers, such as senescence‐associated beta‐galactosidase (SAβGal) activity, telomere attrition, and accumulation of p16ink4a. This suggests the hypothesis that senescence of cells within joint tissues may play a pathological role in the causation of OA. In this review, we discuss the mechanisms by which senescent cells may predispose synovial joints to the development and/or progression of OA, as well as touching upon various epigenetic alterations associated with both OA and senescence.

The activation of phosphoinositide 3-kinase (PI3K) by insulin represents a key signalling event in the hormonal stimulation of diverse cellular responses including glucose transport and glycogen synthesis. The activation of PI3K increases the production of 4,5 trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4 bisphosphate [PtdIns(3,4)P 2 ]), which act as important signalling intermediates in the downstream activation of the serine/threonine kinase, Protein Kinase B (PKB/Akt). Activation of PKB depends upon its phosphorylation on two key amino acid residues, Thr 308 and Ser 473, with full activation requiring the phosphorylation of both [1]. The Nterminal domain of PKB contains a pleckstrin homology (PH) domain, which is thought to be critical in allowing the kinase to interact with 3-phospho- Diabetologia (2001) Abstract Aims/hypothesis. Increased cellular production of ceramide has been implicated in the pathogenesis of insulin resistance and in the impaired utilisation of glucose. In this study we have used L6 muscle cells to investigate the mechanism by which the short-chain ceramide analogue, C 2 -ceramide, promotes a loss in insulin sensitivity leading to a reduction in insulin stimulated glucose transport and glycogen synthesis. Method. L6 muscle cells were pre-incubated with C 2 -ceramide and the effects of insulin on glucose transport, glycogen synthesis and the activities of key molecules involved in proximal insulin signalling determined.Results. Incubation of L6 muscle cells with ceramide (100 mmol/l) for 2 h led to a complete loss of insulinstimulated glucose transport and glycogen synthesis. This inhibition was not due to impaired insulin receptor substrate 1 phosphorylation or a loss in phosphoinositide 3-kinase activation but was caused by a failure to activate protein kinase B. This defect could not be attributed to inhibition of 3-phosphoinositidedependent kinase-1, or to impaired binding of phosphatidylinositol 3,4,5 triphosphate (PtdIns(3,4,5)P 3 ) to the PH domain of protein kinase B, but results from the inability to recruit protein kinase B to the plasma membrane. Expression of a membrane-targetted protein kinase B led to its constitutive activation and an increase in glucose transport that was not inhibited by ceramide. Conclusions/interpretation. These findings suggest that a defect in protein kinase B recruitment underpins the ceramide-induced loss in insulin sensitivity of key cell responses such as glucose transport and glycogen synthesis in L6 cells. They also suggest that a stimulated rise in PtdIns(3,4,5)P 3 is necessary but not sufficient for protein kinase B activation in this system. [Diabetologia (2001) 44: 173±183]

Leptin acts as a pro-inflammatory adipokine with a catabolic role on cartilage metabolism via the upregulation of proteolytic enzymes and acts synergistically with other pro-inflammatory stimuli. This suggests that the infrapatellar fat pad and other WAT in arthritic joints are local producers of leptin, which may contribute to the inflammatory and degenerative processes in cartilage catabolism, providing a mechanistic link between obesity and osteoarthritis.

The serine/threonine kinase protein kinase B (PKB/ Akt) has been shown to play a crucial role in the control of diverse and important cellular functions such as cell survival and glycogen metabolism. There is also convincing evidence that PKB plays a role in the insulin-mediated regulation of glucose transport. Furthermore, states of cellular insulin resistance have been shown to involve impaired PKB activation, and this usually coincides with a loss of glucose transport activation. However, evidence to the contrary is also available, and the role of PKB in the control of glucose transport remains controversial. Here we provide an overview of recent findings, discuss the potential importance of PKB in the regulation of glucose transport and metabolism, and comment on future directions. ß

Elevated non-esterified fatty acids, triglyceride, diacylglycerol, and ceramide have all been associated with insulin resistance in muscle. We set out to investigate the role of intramyocellular lipid metabolites in the induction of insulin resistance in human primary myoblast cultures. Muscle cells were subjected to adenovirus-mediated expression of perilipin or incubated with fatty acids for 18 h, prior to insulin stimulation and measurement of lipid metabolites and rates of glycogen synthesis. Adenovirus-driven perilipin expression lead to significant accumulation of triacylglycerol in myoblasts, without any detectable effect on insulin sensitivity, as judged by the ability of insulin to stimulate glycogen synthesis. Similarly, incubation of cells with the monounsaturated fatty acid oleate resulted in triacylglycerol accumulation without inhibiting insulin action. By contrast, the saturated fatty acid palmitate induced insulin resistance. Palmitate treatment caused less accumulation of triacylglycerol than did oleate but also induced significant accumulation of both diacylglycerol and ceramide. Insulin resistance was also caused by cell-permeable analogues of ceramide, and palmitate-induced resistance was blocked in the presence of inhibitors of de novo ceramide synthesis. Oleate co-incubation completely prevented the insulin resistance induced by palmitate. Our data are consistent with ceramide being the agent responsible for insulin resistance caused by palmitate exposure. Furthermore, the triacylglycerol derived from oleate was able to exert a protective role in sequestering palmitate, thus preventing its conversion to ceramide.Obesity exerts a major impact on insulin resistance and is closely associated with type 2 diabetes (1). Two-thirds of individuals diagnosed with type 2 diabetes are overweight, and almost half are clinically obese (2), furthermore the risk of developing diabetes increases dramatically with the degree of obesity. The relationship between obesity and insulin resistance has led to the hypothesis that ectopic storage of fat in non-adipose tissues such as skeletal muscle under conditions of lipid oversupply is causal in the development of obesity-associated insulin resistance, and elevated intramyocellular triacylglycerol (IMTG) 3 levels have been identified as a potential link between increased non-esterified fatty acid (NEFA) availability and insulin resistance. The molecular mechanism behind the coupling of IMTG content and insulin resistance is unclear, although it appears unlikely that a neutral lipid metabolite such as triacylglycerol (TG) directly impairs insulin action. Indeed, there is evidence that IMTG stores are elevated not only in insulin resistance but also in insulin-sensitive athletes (3). Hence it is generally believed that IMTG represents a surrogate for other lipid metabolites in muscle. This includes an increasing body of evidence that lipid intermediates such as diacylglycerol (DAG) and ceramides have direct effects on insulin signaling and glucose utilization.DAG...

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has emerged as a major regulator of cellular functions and has been implicated in several pathologies involving remodeling of extracellular matrix (ECM). The end stage of inflammatory joint diseases is characterized by excessive ECM catabolism, and in this study we assess the role of PI3K signaling in the induction of collagenolytic matrix metalloproteinases (MMPs) in human chondrocytes. We used the most potent cytokine stimulus reported to promote cartilage ECM catabolism, namely interleukin-1 (IL-1) in combination with oncostatin M (OSM). Both OSM and IL-6 (in the presence of its soluble receptor), but not IL-1 nor leukemia inhibitory factor, induced Akt phosphorylation in human chondrocytes. Inhibition of PI3K signaling using LY294002 blocked IL-1؉OSM-mediated Akt phosphorylation, induction of MMP-1 and MMP-13, and cartilage collagenolysis. To further explore the role of downstream substrates within the PI3K pathway, complementary use of small molecule inhibitors and specific small interfering RNAs demonstrated that the PI3K subunit p110␣ and Akt1 were required for MMP-1 mRNA induction. MMP-13 induction was also reduced by loss of function of these molecules and by a lack of p110␦, 3-phosphoinositide-dependent kinase-1 or Akt3. We therefore propose that the activities of specific elements of the PI3K signaling pathway, including Akt, are necessary for the synergistic induction of MMP-1 and MMP-13 and the cartilage breakdown stimulated by IL-1؉OSM. Our data provide new insight into the mechanism of synergy between IL-1 and OSM and highlight new therapeutic targets for inflammatory joint diseases that aim to repress the expression of collagenases. Phosphatidylinositol 3-kinase (PI3K)3 comprises a group of lipid kinases that catalyze the phosphorylation of the 3-position of the inositol moiety of phosphoinositides at the cell membrane (1). Activation of class I PI3K signaling plays a vital role in the response of many cytokine and growth factor receptors to control cellular processes such as growth, survival, proliferation, differentiation, and migration (1). Activity of the downstream serine/threonine kinase, Akt (protein kinase B), is of key importance for these events. This PI3K subgroup is further subdivided: class IA comprises heterodimers consisting of a p110 catalytic subunit (␣, ␤, or ␦ isoforms), closely associated with a p50, p55, or p85 regulatory subunit, which is responsible for recruitment to tyrosine kinase-associated receptors activated by cytokines and growth factors. In class IB enzymes, usually activated by G-protein-coupled receptors, the catalytic subunit is p110␥, and the regulatory subunit is p84 or p101 (1). Akt is a pivotal kinase in PI3K signaling, and mechanisms exist to limit Akt activation. Positive or negative regulation of PI3K/Akt signaling enables functional differences between receptors which, for example, determine the outcome of T cell activation (2). There are three mammalian Akt family members involved in a plethora ...

The recently cloned amino acid transporter SAT2 is ubiquitously expressed and confers Na+-dependent transport of short-chain neutral amino acids, characteristics of the functionally defined System A transporter. Here we report the presence of SAT2 mRNA and protein in both skeletal muscle and adipocytes, and the characterization of polyclonal antibodies directed against this transporter. SAT2 protein was present in both plasma-membrane and internal-membrane fractions derived from rat skeletal muscle and adipose tissue, L6 myotubes and 3T3-L1 adipocytes, having a localization similar to that of the glucose transporter GLUT4. Moreover, consistent with the adaptive up-regulation of System A activity following chronic amino acid deprivation, a time-dependent increase in SAT2 protein abundance was observed in amino-acid-deprived L6 myotubes and 3T3-L1 adipocytes. These studies provide the first evidence regarding the subcellular distribution and adaptive up-regulation of SAT2 protein and the characterization of molecular probes for this physiologically important transporter, the function of which is altered in several disease states.

a b s t r a c t a r t i c l e i n f oHistone deacetylases (HDACs) regulate the acetylation of histones in the control of gene expression. Many non-histone proteins are also targeted for acetylation, including TGF-β signalling pathway components such as Smad2, Smad3 and Smad7. Our studies in mouse C3H10T1/2 fibroblasts suggested that a number of TGF-β-induced genes that regulate matrix turnover are selectively regulated by HDACs. Blockade of HDAC activity with trichostatin A (TSA) abrogated the induction of a disintegrin and metalloproteinase 12 (Adam12) and tissue inhibitor of metalloproteinases-1 (Timp-1) genes by TGF-β, whereas plasminogen activator inhibitor-1 (Pai-1) expression was unaffected. Analysis of the activation of cell signalling pathways demonstrated that TGF-β induced robust ERK and PI3K activation with delayed kinetics compared to the phosphorylation of Smads. The TGF-β induction of Adam12 and Timp-1 was dependent on such non-Smad signalling pathways and, importantly, HDAC inhibitors completely blocked their activation without affecting Smad signalling. Analysis of TGF-β-induced Adam12 and Timp-1 expression and ERK/PI3K signalling in the presence of semiselective HDAC inhibitors valproic acid, MS-275 and apicidin implicated a role for class I HDACs. Furthermore, depletion of HDAC3 by RNA interference significantly down-regulated TGF-β-induced Adam12 and Timp-1 expression without modulating Pai-1 expression. Correlating with the effect of HDAC inhibitors, depletion of HDAC3 also blocked the activation of ERK and PI3K by TGF-β. Collectively, these data confirm that HDACs, and in particular HDAC3, are required for activation of the ERK and PI3K signalling pathways by TGF-β and for the subsequent gene induction dependent on these signalling pathways.