Comorbidity of osteoporosis and Alzheimer's disease: Is `AKT `-ing on cellular glucose uptake the missing link?

Osteoporosis and Alzheimer's disease (AD) are both degenerative diseases. Osteoporosis often proceeds cognitive deficits, and multiple studies have revealed common triggers that lead to energy deficits in brain and bone. Risk factors for osteoporosis and AD, such as obesity, type 2 diabetes, aging, chemotherapy, vitamin deficiency, alcohol abuse, and apolipoprotein Eε4 and/or Il-6 gene variants, reduce cellular glucose uptake, and protective factors, such as estrogen, insulin, exercise, mammalian target of rapamycin inhibitors, hydrogen sulfide, and most phytochemicals, increase uptake. Glucose uptake is a fine-tuned process that depends on an abundance of glucose transporters (Gluts) on the cell surface. Gluts are stored in vesicles under the plasma membrane, and protective factors cause these vesicles to fuse with the membrane, resulting in presentation of Gluts on the cell surface. This translocation depends mainly on AKT kinase signaling and can be affected by a range of factors. Reduced AKT kinase signaling results in intracellular glucose deprivation, which causes endoplasmic reticulum stress and iron depletion, leading to activation of HIF-1α, the transcription factor necessary for higher Glut expression. The link between diseases and aging is a topic of growing interest. Here, we show that diseases that affect the same biochemical pathways tend to co-occur, which may explain why osteoporosis and/or diabetes are often associated with AD.

Activation of the aryl hydrocarbon receptor by clozapine induces preadipocyte differentiation and contributes to endothelial dysfunction.

BACKGROUND: The superior therapeutic benefit of clozapine is often associated with metabolic disruptions as obesity, insulin resistance, tachycardia, higher blood pressure, and even hypertension. AIMS: These adverse vascular/ metabolic events under clozapine are similar to those caused by polycyclic aromatic hydrocarbons (PAHs), and clozapine shows structural similarity to well-known ligands of the aryl hydrocarbon receptor (AhR). Therefore, we speculated that the side effects caused by clozapine might rely on AhR signaling. METHODS: We examined clozapine-induced AhR activation by luciferase reporter assays in hepatoma HepG2 cells and we proved upregulation of the prototypical AhR target gene Cyp1A1 by realtime-PCR (RT-PCR) analysis and enzyme activity. Next we studied the physiological role of AhR in clozapine's effects on human preadipocyte differentiation and on vasodilatation by myography in wild-type and AhR-/- mice. RESULTS: In contrast to other antipsychotic drugs (APDs), clozapine triggered AhR activation and Cyp1A1 expression in HepG2 cells and adipocytes. Clozapine induced adipogenesis via AhR signaling. After PGF2α-induced constriction of mouse aortic rings, clozapine strongly reduced the maximal vasorelaxation under acetylcholine in rings from wild-type mice, but only slightly in rings from AhR-/- mice. The reduction was also prevented by pretreatment with the AhR antagonist CH-223191. CONCLUSION: Identification of clozapine as a ligand for the AhR opens new perspectives to explain common clozapine therapy-associated adverse effects at the molecular level.

Aldosterone resistance in kidney transplantation is in part induced by a down-regulation of mineralocorticoid receptor expression.

BACKGROUND: After renal transplantation immunosuppressive drugs-like cyclosporin A (CsA) and FK506 induce either hypoaldosteronism or pseudo-hypoaldosteronism presenting with hyperkalemia and metabolic acidosis. We investigated the relationship between renal allograft function under CsA therapy and plasma aldosterone concentration, potassium- and water homeostasis and mineralocorticoid receptor (MR) expression level in peripheral leukocytes. METHODS: We studied 21 renal transplant patients under CsA therapy and 12 healthy controls. Transplant recipients were studied before and under fludrocortisone treatment. Using quantitative reverse-phase polymerase chain reaction (RT-PCR) specific for the MR, we analyzed the level of expression of MR in peripheral leukocytes. RESULTS: In acidotic transplant recipients (HCO(3) 18.5 +/- 1.2 mM) renal function was only slightly impaired with 2.0 +/- 0.2 mg creatinine/dL when compared with 1.8 +/- 0.3 mg/dL (ns) in non-acidotic patients (HCO(3) 23.0 +/- 2.8 mM). Mean plasma aldosterone levels in renal transplant recipients did not differ from control levels (150 +/- 33 pg/mL vs. 148 +/- 33 pg/mL, ns). In contrast, the expression level of MR in peripheral leukocytes of renal transplant recipients treated with CsA was significantly decreased when compared with healthy controls without renal disease (120 +/- 78 vs. 423 +/- 73 RNA molecules/0.5 microg total RNA, p < 0.01). The level of expression of MR in renal transplant recipients did not differ between acidotic patients and non-acidotic patients (ns). The application of fludrocortisone reversed hyperkalemia and metabolic acidosis without significant effect on MR expression. CONCLUSIONS: The present data demonstrate that hyperkalemia and metabolic acidosis following CsA treatment in kidney transplantation might be associated with a down-regulation of MR expression on peripheral leukocytes. Electrolyte imbalance is reversible on application of fludrocortisone. This observation supports fludrocortisone treatment in transplant patients with severe electrolyte disturbances.

Decreased mineralocorticoid receptor expression in blood cells of kidney transplant recipients undergoing immunosuppressive treatment: cost efficient determination by quantitative PCR.

AIMS: Electrolyte imbalances caused by impaired ion transport are a frequent side effect of immunosuppressive treatment in renal transplant recipients. Clinical symptoms resemble features of hypoaldosteronism, although concentrations of aldosterone are in the normal range. Because immunosuppression might affect the hormone receptor status of cells, mineralocorticoid receptor (hMR) expression by peripheral blood leucocytes (PBL) was studied in these patients. METHODS: Twenty one renal transplant recipients being treated with cyclosporine A and 19 healthy controls were tested. hMR expression was quantified by means of competitive reverse transcription polymerase chain reaction (cRT-PCR) and compared with receptor binding studies with subsequent Scatchard plot analysis carried out previously on 20 renal transplant recipients and 25 controls. Advantages of PCR were summarised and compared with Scatchard plot analysis. RESULTS: Cyclosporine A caused a 37% decrease in hMR molecules on PBL in 75% of renal transplant recipients, and this effect was attributable to the downregulation of hMR transcription. PCR was 99% specific for the detection of hMR in PBL and highly reproducible. CONCLUSIONS: Decreases in hMR protein and RNA in PBL of transplant recipients revealed an inhibitory effect of cyclosporine A on hMR transcription. Because hMR acts as a transcription factor, the expression of several genes involved in electrolyte homeostasis is affected, leading to signs of nephrotoxicity that require therapeutic adjustments. Because of the small volume of blood, the assay can be repeated during treatment and is therefore useful for measuring treatment outcomes. Lower costs and the absence of radioactive challenge are further advantages of the PCR method.

Effects of the genetic polymorphisms of the renin-angiotensin system on focal segmental glomerulosclerosis.

BACKGROUND/AIMS: We analyzed the influence of angiotensin-converting enzyme (ACE) I/D, angiotensinogen (AGT) M235T and angiotensin-II-type-1 receptor (AT1R) A1166C genetic polymorphisms on the clinical course of focal segmental glomerulosclerosis (FSGS). METHODS: This study consisted of 71 patients with nephrotic syndrome due to biopsy proven FSGS and 100 healthy controls. According to the slope of the reciprocal serum creatinine (1/Cr, >or= or <-0.1 dl x mg(-1) x year(-1)) patients were classified into group A (slow progressors, n = 50) and group B (fast progressors, n = 21). Genotyping was performed using polymerase chain reaction (PCR). RESULTS: There were no relevant differences in the allele frequencies of the investigated polymorphisms between patients with FSGS and controls. Patients carrying the T- allele of the AGT polymorphism required a larger number of antihypertensive agents (MM: 1.35 +/- 1.0 vs. MT/TT: 2.0 +/- 1.2, p < 0.05). The ACE-ID/DD genotypes were more frequently found in patients with fast progression (group A: II: 38.0%, ID/DD: 62.0% vs. group B: II: 14.3%, ID/DD: 85.7%, p < 0.05). The AT1R-A1166C polymorphism was not associated with any of the parameters studied. CONCLUSION: The course of FSGS is in part genetically determined by polymorphisms of the renin-angiotensin-system. The ACE-I/D polymorphism was shown to be a risk factor of progression of renal disease and the AGT-M235T polymorphism was associated with the severity of arterial hypertension.

Increased apoptosis of neutrophils in a case of clozapine-induced agranulocytosis - a case report.

A 45-year-old female suffering from severe chronic schizophrenia of the paranoid type did not respond to typical antipsychotics. Five weeks after starting therapy with clozapine, she developed a clozapine-induced agranulocytosis (CA). Discontinuation of clozapine and treatment with granulocyte colony-stimulating factor (G-CSF) led to normalization of blood neutrophil counts within three weeks. This report suggests enhanced apoptosis of blood neutrophils during the acute phase of CA resulting from enhanced expression of the pro-apoptotic proteins Bax and Bik and from a decrease of the anti-apoptotic BCl-X(L) mRNA. The time course of decline and recovery of neutrophilic cells, as well as the release pattern of endogenous G-CSF, resembles those of chemotherapy-induced neutropenia. The kinetics of CD 34-positive cells mimics that of cytotoxic progenitor cell mobilization, e. g., after cytostatic drug administration. Our findings argue against the hypothesis that clozapine-mediated inhibition of G-CSF or granulocyte-macrophage colony-stimulating factor (GM-CSF) release is involved in CA development. Because clozapine-induced cell death mainly affects the neutrophil lineage, the elucidation of the exact mechanism of CA may open new perspectives for the treatment of psychiatric and possibly hematological disorders.

Inducible nitric oxide synthase-derived nitric oxide in gene regulation, cell death and cell survival.

Studies from many laboratories have demonstrated the complex role of NO in inflammatory processes. Prolonged exposure to NO shifts the cellular redox potential to a more oxidized state and this is critically regulated by intracellular levels of reduced glutathione. NO-mediated stress will alter gene expression patterns, and the number of genes known to be involved is steadily increasing. Indeed, due to its S-nitrosating activity in the presence of oxygen, NO can modify the activity of transcription factors containing zinc finger motifs or cysteines within the DNA-binding domain. In addition, we are faced with not only NO acting as a powerful inducer of apoptosis or of necrosis in some cells, but also representing an equally powerful protection from cell death in many instances. Some of these apparent discrepancies may be explained by different capacities of cells to cope with the stress of NO exposure. Here, we review our findings on the complex impact of NO on transcriptional regulation of genes, cell death and cell survival. These NO-mediated actions will contribute to a better understanding of the impact of inducible nitric oxide synthase (iNOS) enzyme activity during inflammatory reactions.

Differential apoptotic response of human melanoma cells to 1 alpha,25-dihydroxyvitamin D3 and its analogues.

Pleiotropic actions of the biologically active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (VD), include antiproliferative effects in both normal human melanocytes and malignant melanoma cell lines. In this study the actions of VD and its low calcemic analogues EB1089 and CB1093, have been examined in two human melanoma cell lines MeWo and WM1341. Both cell lines express similar amounts of vitamin D receptor mRNA and show functional gene regulatory effects in response to VD and its analogues. VD, EB1089 and CB1093 induced apoptosis only in WM1341 cells and not in MeWo cells, even though both cell lines responded well to etoposide, a strong inducer of apoptosis. Additionally, these results were confirmed by analysis of cell morphology. Interestingly in WM1341 cells, CB1093 was found to be more potent in inducing apoptosis than EB1089 and the natural hormone. Moreover, CB1093 appeared to induce apoptosis at a relatively low concentration of 0.1 nM, whereas greater than tenfold higher concentrations of VD and EB1089 were needed to obtain comparable effects. These observations highlight CB1093 as a promising drug for a future treatment against specific types of melanoma.

Aberrant timing in epidermal expression of inducible nitric oxide synthase after UV irradiation in cutaneous lupus erythematosus.

Photosensitivity is a main criterion for the diagnosis of systemic lupus erythematosus (LE), and ultraviolet (UV) irradiation plays a key role in the pathogenesis of cutaneous LE. Patients with a tentative diagnosis of LE are routinely tested for skin lesion development after experimental UV irradiation, providing an ideal opportunity to evaluate early, preclinical events involved in the pathogenesis of LE. Several reports have shown expression of the cytokine-inducible nitric oxide synthase (iNOS) in autoimmune diseases. Therefore, we investigated the role of iNOS expression at mRNA and protein level in the pathogenesis of LE lesions. Skin biopsies from patients with different subtypes of LE were examined, and iNOS expression was found in six of 18 biopsies from cutaneous LE patients and two of three biopsies from systemic LE patients. In biopsies taken 4-20 d after UV irradiation, epidermal iNOS expression was seen in all patients (n = 10) after UVB and in four of 10 patients provoked by UVA. In healthy controls (n = 8) epidermal iNOS expression was detected 24 h after UV irradiation, persisting for another day before subsiding on day 3. In LE patients (n = 8) the exact reverse situation was seen: an iNOS-specific signal was undetectable in keratinocytes for 2 d after UV irradiation, but became positive on day 3 and persisted for up to 25 d in the evolving skin lesions. Our findings demonstrate a time-restricted, UV-induced iNOS expression in human skin; moreover, the results indicate that both the kinetics of iNOS induction as well as the time span of local iNOS expression may be critical to the development of cutaneous LE lesions.

Nitric oxide-induced expression of C-reactive protein in islet cells as a very early marker for islet stress in the rat pancreas.

In searches for marker molecules specifically expressed in nitric oxide-treated islet cells as a means to recognize early events in islet destruction, we now establish the presence of neo-C-reactive protein (neoCRP) in rat islet cells as early as 2 hr after treatment. We detected this altered molecular form of the acute-phase-reactant C-reactive protein (CRP) using immunocytochemistry with an anti-neoCRP-specific monoclonal antibody as well as reverse transcription-polymerase chain reaction with CRP-specific primers and in situ hybridization to demonstrate the presence of CRP-specific mRNA. After induction of a generalized inflammatory reaction in rats with heat-inactivated Corynebacterium parvum in vivo, neoCRP expression in islets is also found and within the pancreas restricted to pancreatic islet cells only. Our findings suggest an early heat-shock-like expression of this molecule in response to local nitrite oxide production or to exogeneously added nitric oxide in islet cells.

Nitric oxide: cytotoxicity versus cytoprotection--how, why, when, and where?

Nitric oxide (NO) has been found to play an important role as a signal molecule in many parts of the organism as well as a cytotoxic effector molecule of the nonspecific immune response. It appears paradoxical that NO on one side acts as a physiological intercellular messenger and on the other side may display cytotoxic activity in vivo. To make things even more complicated, cytoprotective properties of NO are also described. We here review the current understanding of cytotoxic versus cytoprotective effects of NO in mammalian cells and try to highlight the janus-faced properties of this important small molecule.

Endothelial cells as cytotoxic effector cells: cytokine-activated rat islet endothelial cells lyse syngeneic islet cells via nitric oxide.

In vivo, each beta cell is located in proximity to at least one capillary islet endothelial cell. Rat aorta and islet endothelial cells can be activated in vitro to express inducible nitric oxide synthase by a cytokine mixture of tumour necrosis factor-alpha, gamma-interferon, and interleukin-1 beta and to produce high concentrations of nitric oxide. We have performed co-culture experiments with rat islet endothelial cells together with isolated syngeneic islet cells at low target:effector ratios with or without previous cytokine challenge of endothelial cultures. Co-cultures were always free of exogenous cytokines, which were removed prior to addition of islet cells. We found that pre-activated, in contrast to resident islet endothelial cells, at a target:effector ratio as low as 1:1 almost completely lysed syngeneic beta and non-beta cells with 24 h of co-culture. Lysis by pre-activated islet endothelial cells was found to be preceded by DNA damage found in 46% of islet cells after 8 h of co-culture with pre-activated vs 7% with resting islet endothelial cells. Lysis was blocked to control levels in the presence of the nitric oxide synthase inhibitor NG-methyl-L-arginine. With the results presented here, we demonstrate for the first time, that activated endothelial lining cells can express effector cell activity and thus can contribute to local tissue destruction, especially in organs that are densely capillarized such as pancreatic islets.

A proinflammatory activity of interleukin 8 in human skin: expression of the inducible nitric oxide synthase in psoriatic lesions and cultured keratinocytes.

Psoriasis is a common chronic skin disease mediated by cellular immune mechanisms and characterized by an intense neutrophil cell infiltrate and proliferative activation of epidermal keratinocytes. We have previously described the expression of the inducible nitric oxide synthase (iNOS) in epidermal keratinocytes of psoriatic skin lesions. In this study, the role of iNOS in psoriatic inflammation was explored ex vivo in psoriatic skin biopsies and in vitro in primary cultures of human keratinocytes. Messenger RNA for the iNOS enzyme (iNOS mRNA) was detected by reverse transcriptase polymerase chain reaction in skin biopsies from patients with psoriasis, but not in skin specimens from patients with atopic eczema or from healthy volunteers. As demonstrated by in situ hybridization and immunohistochemistry, expression of iNOS mRNA and its gene product was localized to the epidermal keratinocytes of psoriatic skin lesions. In situ hybridization further revealed a complete colocalization of mRNA expression for iNOS with interleukin (IL) 8 receptor-specific mRNA either in the basal germinative cell layer or at focal sites of ongoing neutrophil inflammation in suprabasal cell layers. Because psoriatic keratinocytes have previously been shown to express mRNA transcripts for IL-8, it seemed reasonable to hypothesize that iNOS expression could be induced in an autocrine loop by IL-8. This hypothesis was substantiated by our in vitro experiments showing that a combination of IL-8 and interferon gamma induces the expression of iNOS-specific mRNA and of the functional enzyme in cultured human keratinocytes. These results suggest an important role for iNOS in concert with IL-8 and its receptor early during the formation of psoriatic lesions.

Nitric oxide induces apoptosis in mouse thymocytes.

Nitric oxide (NO) produced at high concentrations by the inducible NO synthase is an important effector molecule involved in immune regulation and defense. We have examined whether NO represents a signal for triggering apoptosis in thymocytes. Freshly isolated thymocytes were incubated with different chemical NO donors for various intervals. Apoptosis was determined by detection of DNA strand breaks with in situ nick translation. All NO donors induced thymocyte apoptosis with 30% positive thymocytes vs 10% in controls after 8 h. Apoptosis was prevented by addition of ZnSO4. Short-term pre-exposure to NO resulted in protection from apoptosis induced by glucocorticoids comparable with the protective effect of heat shock. Flow cytometry revealed that NO treatment as well as heat shock or dexamethasone incubation is accompanied by reduction in the CD4+ CD8+ thymocyte subpopulation. Apoptosis induction was accompanied by increased expression of p53, as detected by PCR analysis 2 h after NO donor addition. In vivo treatment of mice with endotoxin results in increased thymic apoptosis. Focal apoptosis was found to occur in close proximity to blood vessels 18 h after LPS treatment. Capillary endothelium and dendritic cells adjacent to apoptotic foci were found to stain strongly for inducible NO synthase expression. Furthermore, in an in vitro experiment using cocultures of thymocytes with LPS/cytokine-activated endothelial cells expressing inducible NO synthase, a significantly increased rate of thymocyte apoptosis was found, and this could be prevented completely by inhibiting NO production. Addition of dexamethasone to these cocultures did not lead to a further increase in the percentage of apoptotic thymocytes, underlining the protective effect of NO on dexamethasone-induced apoptosis.

Inducible nitric oxide synthase and its product nitric oxide, a small molecule with complex biological activities.

More and more attention is paid to the radical nitric oxide which is now known to be part of the mammalian physiology and immune system. Nitric oxide is synthesized by one of the most complicated and fascinating enzyme families identified so far. Inducible nitric oxide synthesis after appropriate stimuli has regulatory, cytostatic and/or toxic consequences and may play an important role in the pathophysiology of several diseases.