Calcium and 1,25-dihydroxyvitamin D3 regulation of adipokine expression

Objective: Obesity is associated with elevated oxidative stress and low‐grade systemic inflammation. We have demonstrated recently that 1α,25‐(OH)₂‐D₃ promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1α,25‐(OH)₂‐D₃ by increasing dietary calcium down‐regulates diet‐induced oxidative stress in aP2‐agouti transgenic mice. However, whether the anti‐obesity effect of dietary calcium plays a role in regulation of obesity‐associated inflammation is not clear. Research Methods and Procedures: We investigated the role of dietary calcium in the regulation of inflammatory cytokine production in aP2‐agouti transgenic mice fed low‐ and high‐calcium obesigenic diets and in the modulation of cytokine production by 1α,25‐(OH)₂‐D₃ in cultured murine and human adipocytes. Results: The high‐calcium diet inhibited the expression of pro‐inflammatory factors tumor necrosis factor α and interleukin (IL)‐6 by 64% and 51%, respectively (p < 0.001), in visceral fat, stimulated the expression of the anti‐inflammatory factors IL‐15 and adiponectin by 52% (p = 0.001) and 54% (p = 0.025), respectively, in visceral fat, and induced a 2‐fold increase in IL‐15 expression in soleus muscle (p = 0.01) compared with litter mate controls on a low‐calcium diet. 1α,25‐(OH)₂‐D₃ also markedly stimulated the expression of tumor necrosis factor α (p < 0.001) and IL‐6 (p = 0.016) in differentiated 3T3‐L1 adipocytes and increased IL‐6 (p = 0.004) and IL‐8 (p < 0.001) production in differentiated human adipocytes. These effects were blocked by calcium channel antagonism with nifedipine. Discussion: These data demonstrate that 1α,25‐(OH)₂‐D₃ favors inflammatory cytokine expression and inhibits anti‐inflammatory cytokine expression; accordingly, suppression of 1α,25‐(OH)₂‐D₃ by dietary calcium inhibits adipocyte‐derived inflammation associated with obesity.     

1,25-Dihydroxyvitamin D3 induces Ca-mediated apoptosis in adipocytes via activation of calpain and caspase-12

Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca²⁺ signal, sustained increase in intracellular Ca²⁺, triggers apoptotic cell death via activation of Ca²⁺-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is mediated through Ca²⁺ signaling. Here, we report that 1,25(OH)₂D₃ induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca²⁺-dependent calpain and Ca²⁺/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)₂D₃ induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca²⁺. The increase in Ca²⁺ was associated with induction of apoptosis and activation of μ-calpain and caspase-12. Our results demonstrate that Ca²⁺-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)₂D₃ in these cells are Ca²⁺-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

Calcium-Induced Alteration of Mitochondrial Morphology and Mitochondrial-Endoplasmic Reticulum Contacts in Rat Brown Adipocytes

Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca²⁺]_i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ^® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ^® drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca²⁺ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ^® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ^® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca²⁺ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ^® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes.Key words: Brown adipocyte, mitochondrial dynamics, calcium, endoplasmic reticulum     

Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

Uncoupling protein 2: a novel player in neuroprotection

A recent report provides exciting new evidence that suggests that uncoupling protein 2 (UCP2), a mitochondrial protein expressed in specific cells of numerous tissues, might be neuroprotective by reducing mitochondrial Ca²⁺ uptake and preventing mitochondrial accumulation of reactive oxygen species (ROS) following cerebral ischemia. The mitochondrial sequestration of Ca²⁺ and ROS, which depends on the mitochondrial membrane potential (ΔΨ_m), is a deleterious consequence of excitotoxicity. A neuroprotective role for Ucp2 is consistent with the already proposed property of this gene in mitigating cellular damage caused by ROS.     

1α,25-dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts

1α,25-Dihydroxyvitamin D₃ (1α, 25-(OH)₂D₃) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)₂D₃-induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)₂D₃, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25-(OH)₂D₃, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25-(OH)₂D₃, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25-(OH)₂D₃, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1α,25-(OH)₂D₃ amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25-(OH)₂D₃ and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley-Liss, Inc.     

Antiproliferative action of menadione and 1,25(OH)2D3 on breast cancer cells

Calcitriol or 1,25(OH)₂D₃ is a negative growth regulator of MCF-7 breast cancer cells. The growth arrest is due to apoptosis activation, which involves mitochondrial disruption. This effect is blunted in vitamin D resistant cells (MCF-7^DRes cells). Menadione (MEN), a glutathione (GSH)-depleting compound, may potentiate antitumoral effects of anticancer drugs. The aim of this study was to investigate whether MEN enhances cellular responsiveness of MCF-7 cells to 1,25(OH)₂D₃. Cells were cultured and treated with different concentrations of 1,25(OH)₂D₃ ± MEN or vehicle for 96 h. GSH levels and the activity of antioxidant enzymes were determined by spectrophotometry and ROS production by flow cytometry. Both drugs decreased growth and enhanced ROS in MCF-7 cells, obtaining the maximal effects when 1,25(OH)₂D₃ was combined with MEN (P < 0.01 vs. Control and vs. each compound alone). MCF-7^DRes cells were not responsive to 1,25(OH)₂D₃, but the cell proliferation was slightly inhibited by the combined treatment. Calcitriol and MEN separately enhanced antioxidant enzyme activities, but when they were used in combination, the effect was more pronounced (P < 0.05 vs. Control and vs. each compound alone). MEN, calcitriol and the combined treatment decreased GSH levels (P < 0.05 vs. Control). The data indicate that MEN potentiates the effect of 1,25(OH)₂D₃ on growth arrest in MCF-7 cells by oxidative stress and increases the activities of antioxidant enzymes, probably as a compensatory mechanism.     

Direct action of capsaicin in brown adipogenesis and activation of brown adipocytes

The ingestion of capsaicin, the principle pungent component of red and chili peppers, induces thermogenesis, in part, through the activation of brown adipocytes expressing genes related to mitochondrial biogenesis and uncoupling such as peroxisome proliferator‐activated receptor (Ppar) γ coactivator‐1α (Pgc‐1α) and uncoupling protein 1 (Ucp1). Capsaicin has been suggested to induce the activation of brown adipocytes, which is mediated by the stimulation of sympathetic nerves. However, capsaicin may directly affect the differentiation of brown preadipocytes, brown adipocyte function, or both, through its significant absorption. We herein demonstrated that Trpv1, a capsaicin receptor, is expressed in brown adipose tissue, and that its expression level is increased during the differentiation of HB2 brown preadipocytes. Furthermore, capsaicin induced calcium influx in brown preadipocytes. A treatment with capsaicin in the early stage of brown adipogenesis did not affect lipid accumulation or the expression levels of Fabp4 (a gene expressed in mature adipocytes), Pparγ2 (a master regulator of adipogenesis) or brown adipocyte‐selective genes. In contrast, a treatment with capsaicin in the late stage of brown adipogenesis slightly increased the expression levels of Fabp4, Pparγ2 and Pgc‐1α. Although capsaicin did not affect the basal expression level of Ucp1, Ucp1 induction by forskolin was partially inhibited by capsaicin, irrespective of the dose of capsaicin. The results of the present study suggest the direct effects of capsaicin on brown adipocytes or in the late stage of brown adipogenesis.     

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.     

The effect of extracellular calcium on colonocytes: evidence for differential responsiveness based upon degree of cell differentiation

Calcium supplementation decreases the incidence of colon cancer in animal models and may prevent colon cancer in man. Potential mechanisms include binding of mitogens and direct effects of calcium on colonic epithelial cells. In this study, the effects of extracellular calcium on epithelial cell growth and differentiation were studied in three colon carcinoma and two colonic adenoma cell lines. The characteristics studied included morphology, cell cycle kinetics, [Ca²⁺]_IC (intracellular calcium concentration), proliferation, and expression of differentiation markers such as carcinoembryonic antigen (CEA) and alkaline phosphatase (AP). Sodium butyrate (NaB) and 1,25-dihydroxyvitamin D₃ were used as controls in the latter three assays as these two agents are known differentiating agents. Alteration of [Ca⁺²]_EC (extracellular calcium concentration) did not affect carcinoembryonic antigen (CEA) or alkaline phosphatase (AP) expression. NaB enhanced the expression of AP three-fold and CEA five-fold. This effect was augmented by increasing [Ca²⁺]_EC. The exposure of cells to 1,25-(OH)₂-Vitamin D₃ increased CEA but not AP. [Ca²⁺]_IC increased in response to 1,25-(OH)₂-vitamin D₃ and NaB but not with variation in [Ca²⁺]_EC. Increased [Ca²⁺]_EC inhibited proliferation of well-differentiated cells, but had no effect on poorly-differentiated cells. Morphological studies showed that extracellular calcium was necessary for normal cell—cell interactions. These studies have demonstrated direct effects of calcium on colonic epithelial cells which may contribute to the protective effects of dietary calcium against colon cancer. Loss of responsivess to the antiprotective effects of [Ca²⁺]_EC with de-differentiation suggests that calcium supplementation may be most beneficial prior to the development of neoplastic changes in colonic epithelium.     

Lead inhibits 1,25-dihydroxyvitamin D-3 regulation of calcium metabolism in osteoblastic osteosarcoma cells (ROS 17/2.8)

We have determined the dose-response of 1,25-dihydroxyvitamin D-3 (1,25-(OH)₂D₃) on the intracellular free calcium-ion concentration ([Ca²⁺¹]_i) in the osteoblastic osteosarcoma cells, ROS 17/2.8, using ¹⁹F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorephenoxy)ethane-N, N, N′, N′-tetraacetic acid (5F-BAPTA). The dose-response demonstrated an inverted U-shaped relationship with maximal elevation of [Ca²⁺]_i at doses of 1 to 10 nM 1,25-(OH)₂D₃. At 10 nM, 1,25-(OH)₂D₃ elevated the [Ca²⁺]_i from a control level of 118±4 nM to a peak value of 237±8 nM within 40 min. 1,25-(OH)₂D₃ also increased the intial rate of Ca²⁺ influx into ROs 17/2.8 cells, measured by ⁴⁵Ca uptake, with a dose-response relationship which paralleled its effects on [Ca²⁺]_i. Treatment of ROS 17/2.8 cells with Pb²⁺ at 1 and 5 μM significantly increased [Ca²⁺]_i but significantly reduced the 1,25-(OH)₂D₃-induced elevation of [Ca²⁺]i. Simultaneous treatment of naive cells with 1,25-(OH)₂D₃ and Pb²⁺ produce little reduction of 1,25-(OH)₂D₃ and Pb²⁺ produce little reduction of 1,25-(OH)₂D₃-induced ⁴⁵Ca uptake while 40 min treatment with Pb²⁺ before addition of 1,25-(OH)₂D₃ significantly reduced the 1,25-(OH)₂D₃-induced increase in ⁴⁵Ca influx. These findings suggest that Pb²⁺ acts by inhibiting 1,25-(OH)₂D₃-activation of Ca²⁺ channels and interferes with 1,25-(OH)₂D₃ regulation of Ca²⁺ metabolism in osteoblastic bone cells.     

1α,25-Dihydroxyvitamin D₃ inhibits neutrophil recruitment in hamster model of acute lung injury

The chemokine interleukin-8 (IL-8) is involved in the pathogenesis of acute lung injury (ALI). Although several studies have reported that 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) suppresses IL-8 production in vitro and in vivo, 1α,25(OH)₂D₃ has not been demonstrated to be effective in an animal model of ALI. Here, we determined its effects of 1α,25(OH)₂D₃ in a hamster model where ALI was induced by lipopolysaccharide (LPS) inhalation. 1α,25(OH)₂D₃ inhibited neutrophil recruitment in the lung by approximately 40% without increasing plasma calcium concentration, while it did not inhibit monocyte recruitment. Our findings show that vitamin D₃ analogues may be suitable as novel anti-inflammatory agents for ALI.     

Regulation of epinasty induced by 2,4‐dichlorophenoxyacetic acid in pea and Arabidopsis plants

The herbicide 2,4‐dichlorophenoxyacetic acid (2,4‐D) causes uncontrolled cell division and malformed growth in plants, giving rise to leaf epinasty and stem curvature. In this study, mechanisms involved in the regulation of leaf epinasty induced by 2,4‐D were studied using different chemicals involved in reactive oxygen species (ROS) accumulation (diphenyleniodonium, butylated hydroxyanisole, EDTA, allopurinol), calcium channels (LaCl₃), protein phosphorylation (cantharidin, wortmannin) and ethylene emission/perception (aminoethoxyvinyl glycine, AgNO₃). The effect of these compounds on the epinasty induced by 2,4‐D was analysed in shoots and leaf strips from pea plants. For further insight into the effect of 2,4‐D, studies were also made in Arabidopsis mutants deficient in ROS production (rbohD, rbohF, xdh), ethylene (ein 3‐1, ctr 1‐1, etr 1‐1), abscisic acid (aba 3.1), and jasmonic acid (coi 1.1, jar 1.1, opr 3) pathways. The results suggest that ROS production, mainly ·OH, is essential in the development of epinasty triggered by 2,4‐D. Epinasty was also found to be regulated by Ca²⁺, protein phosphorylation and ethylene, although all these factors act downstream of ROS production. The use of Arabidopsis mutants appears to indicate that abscisic and jasmonic acid are not involved in regulating epinasty, although they could be involved in other symptoms induced by 2,4‐D.     

High extracellular Ca2+ enhances the adipocyte accumulation of bone marrow stromal cells through a decrease in cAMP

Bone marrow stromal cells (BMSCs) are common progenitors of both adipocytes and osteoblasts. We recently suggested that increased [Ca²⁺]_o caused by bone resorption might accelerate adipocyte accumulation in response to treatment with both insulin and dexamethasone. In this study, we investigated the mechanism by which high [Ca²⁺]_o enhances adipocyte accumulation.We used primary mouse BMSCs and evaluated the levels of adipocyte accumulation by measuring Oil Red O staining. CaSR agonists (both Ca²⁺ and Sr²⁺) enhanced the accumulation of adipocytes among BMSCs in response to treatment with both insulin and dexamethasone. We showed that high [Ca²⁺]_o decreases the concentration of cAMP using ELISA. Real-time RT-PCR revealed that increasing the intracellular concentration of cAMP (both chemical inducer (1 μM forskolin and 200 nM IBMX) and a cAMP analog (10 μM pCPT-cAMP)) suppressed the expression of PPARγ and C/EBPα. In addition, forskolin, IBMX, and pCPT-cAMP inhibited the enhancement in adipocyte accumulation under high [Ca²⁺]_o in BMSCs. However, this inhibited effect was not observed in BMSCs that were cultured in a basal concentration of [Ca²⁺]_o. We next observed that the accumulation of adipocytes in the of bone marrow of middle-aged mice (25–40 weeks old) is higher than that of young mice (6 weeks old) based on micro CT. ELISA results revealed that the concentration of cAMP in the bone marrow mononuclear cells of middle-aged mice is lower than that of young mice. These data suggest that increased [Ca²⁺]_o caused by bone resorption might accelerate adipocyte accumulation through CaSR following a decrease in cAMP.     

Two guard cell‐preferential MAPKs,MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)‐induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca²⁺]_cyt) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL‐induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL‐induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca²⁺]_cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca²⁺]_cyt oscillation in YEL‐induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.     

Physiological uncoupling of mitochondrial oxidative phosphorylation. Studies in different yeast species

Under non-phosphorylating conditions a high proton transmembrane gradient inhibits the rate of oxygen consumption mediated by the mitochondrial respiratory chain (state IV). Slow electron transit leads to production of reactive oxygen species (ROS) capable of participating in deleterious side reactions. In order to avoid overproducing ROS, mitochondria maintain a high rate of O₂ consumption by activating different exquisitely controlled uncoupling pathways. Different yeast species possess one or more uncoupling systems that work through one of two possible mechanisms: i) Proton sinks and ii) Non-pumping redox enzymes. Proton sinks are exemplified by mitochondrial unspecific channels (MUC) and by uncoupling proteins (UCP). Saccharomyces. cerevisiae and Debaryomyces hansenii express highly regulated MUCs. Also, a UCP was described in Yarrowia lipolytica which promotes uncoupled O₂ consumption. Non-pumping alternative oxido-reductases may substitute for a pump, as in S. cerevisiae or may coexist with a complete set of pumps as in the branched respiratory chains from Y. lipolytica or D. hansenii. In addition, pumps may suffer intrinsic uncoupling (slipping). Promising models for study are unicellular parasites which can turn off their aerobic metabolism completely. The variety of energy dissipating systems in eukaryote species is probably designed to control ROS production in the different environments where each species lives.