Osteonecrosis of the Jaw (ONJ) in Osteoporosis Patients: Report of Delayed Diagnosis of a Multisite Case and Commentary about Risks Coming from a Restricted ONJ Definition.

Osteonecrosis of the jaws (ONJ) in osteoporosis patients has been defined as rare, but the number of reported cases is increasing. We report a case of delayed ONJ diagnosis in a patient, who was being treated with alendronate, developing bone alterations both in maxilla and in mandible. Underestimation of ONJ incidence and missed or delayed ONJ diagnosis in osteoporosis patients might derive from lack of awareness of health providers as well as from an ONJ definition that is too restricted. The more recent definition of medication-related osteonecrosis of the jaws (MRONJ) released in 2014 by the American Association of Oral Maxillofacial Surgeons (AAOMS) accept fistula, besides bone exposure, as a major sign of disease, but it seems to be insufficient since it excludes all cases of ONJ disease without bone exposure. A new MRONJ definition is needed to avoid missing or delayed diagnosis.

Physical activity and dietary habits during pregnancy: effects on glucose tolerance.

OBJECTIVES: We designed this study to assess the potential effects of physical activity and dietary habits on glucose tolerance during pregnancy. METHODS: This is an observational study involving 268 women who underwent a 50-g oral glucose challenge test (GCT) at 27±6.9 week of gestation. Plasma glucose level at 1-h GCT ≥ 140 mg/dl was used to define abnormal glucose tolerance (AGT). Physical activity was evaluated using the short form of the International Physical Activity Questionnaire (IPAQ), while for dietary habits we used a food frequency questionnaire linked to a computerised program. RESULTS: One hundred five women had AGT (AGT+) and 163 had normal glucose tolerance (AGT−). There was no difference between the two groups in demographic and clinical data, with the exception of pre-pregnancy BMI and weight gain both higher in AGT+ women. Also, all parameters referring to physical activity energy and diet (Kcal and diet components) were not statistically different between the two groups. After a multivariate analysis, only pre-pregnancy BMI (F-value 9.264, p=0.002) remained an independent predictor of 1-h plasma glucose. CONCLUSIONS: Our study suggests that high pre-pregnancy BMI confers a substantially high risk of AGT, independently of lifestyle during pregnancy.

Optimizing management of metabolic syndrome to reduce risk: focus on life-style.

The prevalence of metabolic syndrome (MS) is increasing all over the world and its incidence is expected to rise in the next years. Although genetic predisposition appears to play an important role in the regulation of metabolic parameters and in particular of body weight, the rapid increase in the prevalence of obesity and MS suggests that ecological factors (social, economic, cultural and physical environment) are promoting those conditions in susceptible individuals. People with MS are at increased risk of type 2 diabetes and cardiovascular disease and therefore they represent a priority target for preventive strategies. Life-style modifications based on healthy diet and increased physical activity are an effective preventing and therapeutic approach. Unfortunately, implementation of life-style modification and maintenance of effects is a difficult task both at personal and social level, thus drug therapy can be taken into account.

Modulation of palmitic acid-induced cell death by ergothioneine: evidence of an anti-inflammatory action.

Inflammation and reactive oxygen species have been implicated in pathogenesis of vascular diabetic complications. However, treatment with classic free-radical scavengers and antioxidants has not been yet proved to reduce the risk of developing such complications. In search of more effective treatment we have tested the protective role of Ergothioneine (EGT), in vitro, on C2C12 cells model on FFA-induced lipotoxicity. Cells were incubated for 24 h in the presence of palmitic acid (PA) (250, 500, 750, 1000 microM), added as pro-oxidant compound, with or without 24-h pre-treatment with EGT. Cells were assessed for cell viability and MAPKs expression by Western Blot. Pre-treatment with EGT resulted in greater cell viability at each PA concentration (EGT 500 microM: 5, 16, 17, 23% and EGT 1000 microM: 9, 18, 21 and 25%). In response to PA exposure, p38 and JNK activity increased significantly while EGT prevented such activation. Moreover the analysis of the IL-6 production reveal that EGT is also able to exert anti-inflammatory action inhibiting the PA IL-6 modulation (P < 0.001). In conclusion, these results indicate that 1. EGT has a protective role on PA-induced cell death, possibly via 2. reduced activity of MAPKs cascade having also 3. an anti-inflammatory action exerted on the IL-6 modulation.

High insulin levels impair intracellular receptor trafficking in human cultured myoblasts.

Chronic hyperinsulinemia is both a marker and a cause for insulin resistance. This study analyzes the effect of long-term exposure to high insulin levels on insulin-insulin receptor metabolism in human myoblasts. Cells were grown in the presence of low (107 pM, SkMC-L) or high (1430 pM, SkMC-H) insulin concentrations. Insulin receptor (IR) phosphorylation, IR internalization, dissociation and recycling, as well as insulin degradation have been investigated. Basal IR phosphorylation was higher in SkMC-H than in SkMC-L (P<0.01) but after acute insulin stimulation (10nM insulin for 10 min), IR phosphorylation increased (P<0.01) in SkMC-L, but not in SkMC-H. Chronic hyperinsulinism significantly decreased insulin-IR complex internalization (P<0.01). Nevertheless the t(1/2) value of receptor internalization was similar in both cells. Intracellular dissociation of insulin-IR complex was slightly but significantly lower in SkMC-H than in SkMC-L. Finally, SkMC-H showed a complete, but significantly delayed recycling of IR to plasma membrane (t(1/2)=20 min versus SkMC-L t(1/2)=7 min). The time course of intracellular degradation measured by HPLC, showed whenever studied, significantly (P<0.01) higher levels of intracellular intact insulin in cells exposed to high insulin concentrations. Nevertheless, the patterns of insulin degradation were over-imposable between SkMC-H and SkMC-L. In summary, continuous exposure of cultured myoblasts to high insulin levels induces subtle derangements of intracellular receptor trafficking and insulin degradation. These alterations may contribute to the insulin resistance of hyperinsulinemic states such as obesity and Type 2 Diabetes.

Early impairment of beta-cell function and insulin sensitivity characterizes normotolerant Caucasian women with previous gestational diabetes.

BACKGROUND AND AIMS: Women with previous gestational diabetes (pGDM) are at high risk of developing type 2 diabetes mellitus. The aim of this study was to evaluate insulin action and insulin secretion in women with pGDM. METHODS AND RESULTS: One hundred and fifty-three pGDM women and 45 with normal glucose tolerance during pregnancy (controls) were studied 1-3years after delivery. Insulin sensitivity (ISI) and beta-cell secretory capacity (beta-index) were derived from 75-g OGTT. Disposition Index was calculated as the product of beta-index and ISI. One hundred and twenty-two pGDM were normotolerant (NGT) and 31 had impaired glucose regulation (IGR) i.e. impaired glucose tolerance and/or impaired fasting glucose. NGT-pGDM, as compared to controls, had significant impairment in insulin action (ISI: 5.46+/-2.81 vs. 7.38+/-3.68, P<0.01) and insulin secretion (beta-index: 4.68+/-1.01 vs. 5.24+/-0.82 pmol/min/m(2); P<0.01). A further impairment was apparent in IGR-pGDM for beta-index (4.16+/-1.09; P<0.05). The disposition index was reduced in NGT-pGDM as compared to controls (33.9%) and further reduced in IGR-pGDM (28.6%, vs. NGT-pGDM; ANOVA P<0.001). In women of normal weight, ISI and beta-index were significantly (P<0.01) impaired in NGT-pGDM compared to controls and further reduced in IGR-pGDM, although a more pronounced defect in insulin secretion was apparent in these women (beta-index: 4.02+/-0.9; P<0.05). CONCLUSIONS: Normotolerant women with pGDM show both impairment in insulin secretion and action irrespective of body weight. A more pronounced defect in insulin secretion seems to characterize normal weight women while a more prominent defect in insulin action is found in overweight women.

Molecular effects of fermented papaya preparation on oxidative damage, MAP Kinase activation and modulation of the benzo[a]pyrene mediated genotoxicity.

The involvement of oxidative and nitrosative stress mechanisms in several biological and pathological processes including aging, cancer, cardiovascular and neurodegenerative diseases has continued to fuel suggestions that processes can potentially be modulated by treatment with free-radical scavengers and antioxidant. The fermented papaya preparation (FPP) derived from Carica papaya Linn was investigated for its ability to modulate oxidative DNA damage due to H2O2 in rat pheochromocytoma (PC12) cells and protection of brain oxidative damage in hypertensive rats. Cells pre-treated with FPP (50 microg/ml) prior to incubation with H2O2 had significantly increased viability and sustenance of morphology and shape. The human hepatoma (HepG2) cells exposed to H2O2 (50 microM) showed an olive tail moment of 10.56 +/- 1.44 compared to 1.37 +/- 0.29 of the solvent control. A significant reduction (P < or = 0.05) of DNA damage was observed at concentrations > or = 10 microg/ml FPP, with 50 microg/ml FPP reducing the genotoxic effect of H2O2 by about 1.5-fold compared to only H2O2 exposed cells.

Modulation of hydrogen peroxide-induced DNA damage, MAPKs activation and cell death in PC12 by ergothioneine.

BACKGROUND & AIMS: Ergothioneine (EGT) is a natural occurring compound, synthesized by soil bacteria in fungal substrates, exhibiting antioxidant functions in many cell models. The aim of this study was to assess the effect of EGT in the prevention of H2O2-dependent cell death and oxidative damage on a model of neural cell derived from rat pheocromocytoma, the PC12. METHODS: The ability of EGT was tested by the 3 (4,5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay and Comet assay. H2O2 insult was challenged with increasing concentration of antioxidant using two different incubation periods: 1 and 23 h of EGT pre-treatment followed by 23 and 1 h of H2O2, respectively, for both the MTT and the Comet assay data. CONCLUSION: The pre-treatment for 23 h with EGT, 250 microM and 1mM, followed by 1h of H2O2 incubation at the concentration of 250 and 500 microM, resulted in increased cell viability (P < 0.001) compared to the H2O2 cell batch. This correlated with a decrease in DNA damage as visualized by the Comet assay. Moreover, protein analysis reveals that in the presence of 250 microM of H2O2, EGT acted as a p38-MAPK and Akt specific inhibitor. EGT may play a protective role in rescuing cells from stress-induced apoptosis, likely by activating an intracellular antioxidant pathway involving p38 MAPK genes cascade.

Continually high insulin levels impair Akt phosphorylation and glucose transport in human myoblasts.

Chronic hyperinsulinemia is both a marker and a cause for insulin resistance. This study analyzes the effect of long-term exposure to high insulin levels on the insulin-signaling pathway and glucose transport in cultured human myoblasts. Human myoblasts were grown in the presence of low (107 pmol/L, SkMC-L) or high (1430 pmol/L, SkMC-H) insulin concentrations for 3 weeks. Glucose transport, insulin receptor (IR), and IR substrate 1 (IRS1) phosphorylation, phosphatidylinositol 3'-kinase (PI3K) activity, as well as Akt-Ser473 phosphorylation have been investigated at the end of the incubation period and after a further short-term insulin stimulation. At the end of the incubation period, IR, IRS1, p85/PI3K, Akt, and GLUT4 protein expression levels were similar in both culture conditions. Basal glucose transport was similar in SkMC-L and SkMC-H, but after short-term insulin stimulation significantly increased (P < .01) only in SkMC-L. IR binding was down-regulated in SkMC-H (P < .01), but IR and IRS1 tyrosine phosphorylation and PI3K activity were significantly higher (P < .01) in SkMC-H than SkMC-L. Despite increased PI3K activation, Akt-Ser473 phosphorylation was similar in SkMC-L and SkMC-H. After a short-term insulin stimulation (10 nmol/L insulin for 10 minutes), IR and IRS1 tyrosine phosphorylation, PI3K activation, and Akt-Ser473 phosphorylation significantly increased (P < .01 and P < .05 for Akt) in SkMC-L but not in SkMC-H. Serine phosphorylation of IRS1 was similar in SkMC-L and SkMC-H. Moreover, in the SkMC-H, insulin stimulation was associated with the inhibition of IRS1 tyrosine dephosphorylation (P < .05). In summary, continuous exposure of cultured myoblasts to high insulin levels induces a persistent up-regulation of IR, IRS1, and PI3K activity associated with the demodulation of insulin signaling. Moreover, the impairment of the insulin-signaling steps between PI3K and Akt is concomitant with the desensitization of glucose transport. These alterations may contribute to the derangement insulin-signaling pathway states of hyperinsulinemia such as obesity and type 2 diabetes.

Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody: possible role of the protein in cytoskeleton rearrangements during differentiation.

Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.

Effects of prolonged in vitro exposure to sulphonylureas on the function and survival of human islets.

The direct effects of prolonged exposure to sulphonylureas on the function and survival of human islets are unknown. This study assessed the insulin content, glucose-stimulated insulin release, islet cell apoptosis, and mRNA expression of insulin and GLUT-1 in isolated human islets cultured in the presence of therapeutical concentrations of glimepiride (10 microM), glibenclamide (10 microM), or chlorpropamide (600 microM). Islets were prepared by collagenase digestion and density gradient purification from 18 multiorgan donors and were then exposed for 24 h to the different sulphonylureas. Insulin content decreased significantly following culture with any sulphonylurea compound. In response to an acute challenge with 3.3 and 16.7 mM glucose, insulin release from the control islets accounted for 1.9 +/- 0.5% and 4.9 +/- 1.7% of total insulin content (P<.01), respectively. Glucose responsiveness was preserved in islets precultured in the presence of glimepiride, whereas high glucose level did not elicit any significant increase of insulin secretion from islets preincubated with glibenclamide or chlorpropamide. These alterations were reverted by an additional 48-h incubation in drug-free conditions. The amount of apoptotic cells did not differ significantly among the experimental groups. Quantitative RT-PCR studies showed that, compared with the control islets, cells preincubated with glibenclamide or chlorpropamide had an increased expression of insulin mRNA, with no change in the expression of GLUT-1. In conclusion, prolonged exposure of human islets to different sulphonylureas causes different disturbances of islet cell function, with glimepiride showing milder effects, as compared with chlorpropamide and glibenclamide.

Intraocular delivery of BDNF following visual cortex lesion upregulates cytosolic branched chain aminotransferase (BCATc) in the rat dorsal lateral geniculate nucleus.

Visual cortex ablation in newborn rats determines the almost complete degeneration of neurons in the dorsal lateral geniculate nucleus (dLGN), as a consequence of the axotomy of the geniculo-cortical fibres. Death of dLGN neurons is massive and rapid, and occurs by apoptosis. We recently showed that exogenous administration of the neurotrophin brain-derived neurotrophic factor (BDNF) in the eye prevents the degeneration of dLGN neurons occurring after visual cortex lesion in newborn rats. To elucidate the molecular mechanisms of BDNF-mediated neuroprotection, we sought to identify novel genes regulated by BDNF in the rat dLGN after visual cortex lesion. By using mRNA fingerprinting, we isolated a cDNA fragment upregulated in the dLGN of lesioned rats treated with BDNF. This cDNA fragment shared 100% homology with the rat cytosolic branched chain aminotransferase (BCATc), a key enzyme of glutamate metabolism. Quantitative reverse transcription-polymerase chain reaction and in situ hybridization confirmed that BCATc mRNA is markedly overexpressed by exogenous supply of BDNF to axotomized dLGNs. Immunohistochemical analysis showed that upregulation of BCATc in the dLGN of lesioned rats treated with BDNF takes place in astrocytes. These results suggest that modulation of glutamate metabolism by astrocytes might play an important role in BDNF-mediated survival of axotomized dLGN neurons.

Identification of cathepsin K as a novel marker of adiposity in white adipose tissue.

In obesity, adipocytes undergo dramatic morphological and molecular changes associated with alterations in their gene expression profile. To identify genes differentially modulated in white adipose tissue (WAT) of obese db/db mice compared to wild type (wt) mice, we utilized RNA fingerprinting. Among the 52 candidates that we identified, we focused here on cathepsin K (ctsk), a cysteine protease, prevalently localized in lysosomes and involved in bone extracellular matrix degradation. In db/db mice, WAT ctsk mRNA was elevated 5.9-fold, as were Mitf and TFE3 (2- and 3.3-fold respectively), two transcription factors involved in ctsk induction in osteoclasts. Moreover, the level of WAT ctsk mRNA was increased in other obese models including A(y), fat, and tubby (2.8-, 3.2-, and 4.9-fold respectively) and decreased in mice undergoing weight loss. Despite the ubiquitous distribution of the ctsk transcript, we demonstrated that the obesity related increase is specific to the adipocytes. Further, in vitro experiments proved that the abundance of ctsk transcript increases upon adipose conversion of the established cell line of preadipocytes 3T3-F442A. In addition, ctsk gene expression was examined in adipose tissue of 21 lean and obese male subjects and significant correlations with BMI (r = 0.54, P = 0.012) and plasma leptin levels (r = 0.54, P = 0.015) were found. In conclusion, the WAT of obese db/db mice exhibits a different expression profile from that of the wt mice, and cathepsin K can be considered a novel marker of obesity and a target for the inhibition of adipose mass growth.

Insulin permeability across an in vitro dynamic model of endothelium.

PURPOSE: Endothelium insulin permeability was investigated using in vitro, dynamic culture of endothelial cells. METHODS: Endothelial cells were cultured in a hollow fiber apparatus and continuously exposed to a flow. Transendothelial electrical resistance and permeability to [14C]sucrose and [14C]inulin were used to monitor the integrity of the endothelial monolayer. RESULTS: Under these experimental conditions, measurements of insulin permeability, investigated at increasing hormone concentrations, suggested that the predominant transendothelial insulin fluxes were attributable to bidirectional convective transport rather than to a saturable transport mechanism, in agreement with in vivo experiment results published earlier. Analytical determinations of insulin catabolism demonstrated a low percent of insulin degradation by the endothelium, leading to production of insulin metabolites qualitatively identical to those produced by human monocytes. CONCLUSIONS: The findings of this paper indicated that (a) insulin crosses the endothelial monolayer by paracellular "leak" and endothelial insulin receptors have a minor (if any) role in insulin transport; (b) degradation of the hormone by BAEC is minimal; (c) the in vitro, dynamic culture of endothelial cells presented here should represent a valuable transport model system to study permeability mechanisms of insulin and many other drugs.

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFalpha.

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor alpha (TNFalpha) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) A(y), ob/ob and goldthioglucose-treated mice (10-, 8-, and 7-fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFalpha in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFalpha in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFalpha function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFalpha is an important signal for this regulation.