The physiological signaling mechanisms that link glucose sensing to the electrical activity in metabolism-regulating hypothalamus are still controversial. Although ATP production was considered the main metabolic signal, recent studies show that the glucose-stimulated signaling in neurons is not totally dependent on this production. Here, we examined whether mitochondrial reactive oxygen species (mROS), which are physiologically generated depending on glucose metabolism, may act as physiological sensors to monitor the glucose-sensing response. Transient increase from 5 to 20 mmol/l glucose stimulates reactive oxygen species (ROS) generation on hypothalamic slices ex vivo, which is reversed by adding antioxidants, suggesting that hypothalamic cells generate ROS to rapidly increase glucose level. Furthermore, in vivo, data demonstrate that both the glucose-induced increased neuronal activity in arcuate nucleus and the subsequent nervous-mediated insulin release might be mimicked by the mitochondrial complex blockers antimycin and rotenone, which generate mROS. Adding antioxidants such as trolox and catalase or the uncoupler carbonyl cyanide m-chlorophenylhydrazone in order to lower mROS during glucose stimulation completely reverses both parameters. In conclusion, the results presented here clearly show that the brain glucosesensing mechanism involved mROS signaling. We propose that this mROS production plays a key role in brain metabolic signaling. Diabetes 55: 2084 -2090, 2006 E lucidating the signaling mechanisms by which cells sense nutrient or metabolic status, a vital process in energy homeostasis, is of prime importance. Glucose-sensing mechanisms have been mainly characterized in two tissues, both in the pancreas (at the ␤-cell level) and in the brain (the so-called "glucose-stimulated" or "glucose-inhibited" neurons) (1,2). The cellular and molecular mechanisms underlying such glucose responsiveness appear to share similarities in the two glucose responsive cells (i.e., transport and phosphorylation by GLUT2 and glucokinase, respectively) and the consequent closure of ATP-sensitive K ϩ channels (K ATP channels) and calcium influx (3-5). Although ATP production used to be considered the main metabolic signal, recent studies show that the glucose-excited signaling in pancreatic ␤-cells and neurons is not totally dependent on this production. Within the hypothalamus, a previous work showed that glucose challenge monitors K ATP closure independently of ATP level (6), and more recent data demonstrated that glucose-induced depolarization might occur through a new K ATP channel-independent mechanism, at least in some hypothalamic arcuate neurons (7). These studies suggest that ATP-independent intracellular signaling mechanisms leading to the stimulation of hypothalamic neurons by glucose might be present.Transient increase in glucose metabolism generates the key substrates NADH and FADH 2 for the mitochondria, and their use increases electron formation without modifying other complex constraints along the res...

By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity.

Rheumatoid factors (RF) and the disease-specific anti–citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF–generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1β ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.

OBJECTIVERecent data demonstrated that glucose sensing in different tissues is initiated by an intracellular redox signaling pathway in physiological conditions. However, the relevance of such a mechanism in metabolic disease is not known. The aim of the present study was to determine whether brain glucose hypersensitivity present in obese Zücker rats is related to an alteration in redox signaling.RESEARCH DESIGN AND METHODSBrain glucose sensing alteration was investigated in vivo through the evaluation of electrical activity in arcuate nucleus, changes in reactive oxygen species levels, and hypothalamic glucose-induced insulin secretion. In basal conditions, modifications of redox state and mitochondrial functions were assessed through oxidized glutathione, glutathione peroxidase, manganese superoxide dismutase, aconitase activities, and mitochondrial respiration.RESULTSHypothalamic hypersensitivity to glucose was characterized by enhanced electrical activity of the arcuate nucleus and increased insulin secretion at a low glucose concentration, which does not produce such an effect in normal rats. It was associated with 1) increased reactive oxygen species levels in response to this low glucose load, 2) constitutive oxidized environment coupled with lower antioxidant enzyme activity at both the cellular and mitochondrial level, and 3) overexpression of several mitochondrial subunits of the respiratory chain coupled with a global dysfunction in mitochondrial activity. Moreover, pharmacological restoration of the glutathione hypothalamic redox state by reduced glutathione infusion in the third ventricle fully reversed the cerebral hypersensitivity to glucose.CONCLUSIONSThe data demonstrated that obese Zücker rats' impaired hypothalamic regulation in terms of glucose sensing is linked to an abnormal redox signaling, which originates from mitochondria dysfunction.

Collectively, our results suggest that glucose-induced DRP1-dependent mitochondrial fission is an upstream regulator for mROS signaling, and consequently, a key mechanism in hypothalamic glucose sensing. Thus, for the first time, we demonstrate the involvement of DRP1 in physiological regulation of brain glucose-induced insulin secretion and food intake inhibition. Such involvement implies DRP1-dependent mROS production.

Diabetic pregnancy results in several metabolic and hormonal disorders, both in the embryo and the fetus of different species, including humans. Insulin is a potent modulator of brain development and is suggested to promote the differentiation and maturation of hypothalamic or related extrahypothalamic structures, which are directly involved in neural inputs to the pancreas. Because these structures are known to be specifically responsive both to insulin and glucose, we examined the effects of 48-h hyperglycemic clamps in unrestrained pregnant rats on insulin binding and glucose transporter expression in hypothalamic and extrahypothalamic-related areas of their fetal offspring. The main result was an increase in insulin binding in the ventromedial hypothalamic nucleus (VMH), the arcuate nucleus (AN), and the lateral hypothalamus (LH), and in the nucleus of the tractus solitarius (NTS) for extrahypothalamic areas (ϩ30% in the VMH, ϩ37% in the AN, ϩ25.8% in the LH, and ϩ37.3% in the NTS). The deleterious effect of brain hyperinsulinism during the late gestational stage does not seem to act through glucose transporter (GLUT) expression, inasmuch as no relationship between GLUT level and hyperinsulinism in brain areas could be observed. The specific increase in insulin binding in areas involved in the nervous control of metabolism could be a factor in the increased glucose intolerance and impairment of insulin secretion that was previously observed in the adult rats from hyperglycemic mothers. Diabetic pregnancy can lead to glucose intolerance and pancreatic dysfunction in offspring when they reach adult age (1-3). This situation seems to be mainly related to intrauterine hyperglycemia, inasmuch as mild hyperglycemia induced by glucose infusion during late gestation in normal rats is sufficient to induce persistent impairment of glucose regulation and insulin secretion in the adult offspring born of hyperglycemic mothers (4, 5). Interestingly, in this model, insulin deficiency was due to a perturbation of the control of insulin secretion by the ANS and not to an intrinsic pancreatic ␤-cell defect (5). The activity of the ANS is under the influence of hypothalamic nuclei (PVN, VMH, and LH), themselves connected to preganglionic nuclei in the brainstem (6, 7). These areas present a neuronal population able to respond electrophysiologically to insulin and glucose (8, 9). Insulin, insulin receptors, and glucose transporters are present in the brain (8, 10). The defect in insulin secretion observed in the offspring of hyperglycemic mothers could be linked to impaired glucose and/or insulin sensing in the brain. We hypothesized that fetal hyperglycemia and/or hyperinsulinemia could hamper the functional development of glucose and/or insulin-sensitive brain areas. In fact, previous studies in models of perinatal hyperinsulinism (where hypothalamic insulin binding was increased) have demon-

ObjectivesRheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding the shared epitope (SE), a 5-amino acid motive. RA is usually preceded by the emergence of anti-citrullinated protein/peptide antibodies (ACPAs). Citrulline is a neutral amino acid resulting from post-translational modification of arginine involved in peptidic bounds (arginyl residue) by PeptidylArginine Deiminases (PADs). ACPAs recognize epitopes from citrullinated human fibrin(ogen) (hFib) and can be specifically detected by the AhFibA assay. Five citrullinated peptides derived from hFib together represent almost all of the epitopes recognized by patients with ACPA-positive RA, namely: α36–50cit, α171–185cit, α501–515cit, α621–635cit, and β60–74cit. The use of antibody fine specificities as markers of clinical phenotypes has become a major challenge. Our objective was to study whether RA clinical characteristics and HLA-DRB1 genetic background were associated with a specific reactivity against the epitopes borne by the five peptides.Methods184 ACPA-positive RA patients fulfilling the 2010 ACR/EULAR criteria were studied. Patient characteristics including HLA-DRB1 genotype, were collected from their medical files. Anti-CCP2 antibodies, AhFibA, and antibodies against the five citrullinated hFib (hFib-cit) peptides were analyzed by ELISA.ResultsAnti-α505-515cit antibodies were associated with HLA-DRB1*04:01 (OR = 5.52 [2.00 – 13.64]; p = 0.0003). High level anti-α505-515cit antibodies were associated with rheumatoid nodules (OR = 2.71 [1.00 – 7.16], p= 0.044).ConclusionImmune complexes containing anti-α501-515cit antibodies and rheumatoid factors might be involved in the development of rheumatoid nodules on the HLA-DRB1*04:01 background. Apheresis of these epitope-specific antibodies might be a new therapeutic opportunity for patients with rheumatoid nodules.

Background and objectives Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients were they probably contribute to synovitis. We showed that monoclonal IgM paraproteins with RF activity from patients with mixed cryoglobulinemia enhance the FcγR-mediated proinflammatory response of macrophages to ACPA immune complexes (ACPA-IC). Our aim was to investigate the influence of IgM RF from RA patients on the FcR- and complement-dependent effects of ACPA-IC and to examine that of RA-associated IgA RF. Materials and methods M-CSF-differentiated macrophages were stimulated in a serum-free medium by ACPA-IC formed with plate-bound citrullinated fibrinogen in the absence or presence of one of 5 IgM RF or 3 IgA RF fractions purified from RA serum pools by sequential affinity chromatography. Pro-inflammatory and immunoregulatory cytokines were assayed in the culture supernatants. The expression of IgM and/or IgA receptors was analysed by flow cytometry and their contribution to TNF-α secretion prompted by RF-containing ACPA-IC was evaluated. The complement activation capacity of ACPA-IC formed in the absence or presence of IgM or IgA RF was compared by ELISA measuring deposition of the terminal C5b–9 complex. Results When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with an increase of the TNF-α:IL-10 ratio and of the IL-6 and IL-8 secretions, and a decrease of the IL-1Ra:IL 1β ratio. The participation of an IgM receptor in the IgM RF-mediated amplification of the inflammatory response of macrophages was excluded, notably as they did not express any established receptor for IgM. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC. LPS further amplified the TNF-α response of macrophages to IgM or IgA RF-containing ACPA-IC. Finally, the presence of IgM RF or of IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Conclusions Specifically using autoantibodies found in RA patients, the strong complement-dependent and FcR-mediated proinflammatory effect of macro-IC including both ACPA and IgM RF or IgA RF is established, strongly reinforcing the hypothesis of their high pathogenic potential. Simultaneous FcR triggering by these macro-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.