Effects of exercise training on adipogenesis of stromal-vascular fraction cells in rat epididymal white adipose tissue.

AIM: Previous studies have shown that exercise training reduced white adipose tissue (WAT) mass compared to that in sedentary controls, and that the smaller mass contained fewer adipocytes. However, the effect of exercise training on adipogenesis is not completely clear. Therefore, we re-examined the effect of exercise training on adipocyte numbers in WAT and, if such an effect was found tested the adipogenic responses of stromal-vascular fraction (SVF) cells containing adipose tissue-derived stem cells (ADSC) in epididymal WAT from exercise-trained (TR) rats. METHODS: Wistar male rats were divided into two groups: control (C) and TR. The TR rats were subjected to exercise on a treadmill for 9 weeks. SVF cells containing ADSC were separated from epididymal WAT by centrifugation. Expression of adipocyte differentiation-related genes and adipogenesis of SVF cells were examined. RESULTS: In SVF cells of TR rats, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and that of PPARγ target lipogenic genes was dramatically downregulated, whereas that of preadipocyte factor-1 gene was significantly upregulated. Lipid accumulation in SVF cells of TR rats after the induction of adipocyte differentiation was significantly suppressed in comparison with that of C rats. Moreover, increased expression of hypoxia-inducible factor-1α (HIF-1α) protein was observed in SVF cells of TR rats. Pre-treatment of YC-1, a potent HIF-1α inhibitor, in SVF cells of TR rats restored adipogenesis. CONCLUSION: These results suggest that exercise training suppresses the ability of SVF cells to differentiate into adipocytes, and that underlying mechanisms involve the upregulation of HIF-1α expression.

Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice.

We found that intraperitoneal injection of organic acids, such as propionic and lactic acid, are able to develop writhing responses in mice similarly as that of acetic acid. These acid-induced writhing reactions were significantly attenuated by capsazepine, a VR1 receptor-specific antagonist, but the phenylbenzoquinone-induced one was not, suggesting that the acids but not phenylbenzoquinone activate the VR1 receptor, which is involved in polymodal pain perception. Hoe 140, a bradykinin B2 receptor antagonist, also suppressed the acid-induced writhing response. Furthermore, these writhing responses were significantly suppressed after neonatal treatment with capsaicin, which treatment is known to destroy peripheral sensory afferent C-fibers. Capsazepine and Hoe 140 did not further attenuate the already reduced writhing responses of capsaicin-treated mice, suggesting that the acids stimulate the VR1 and the bradykinin B2 receptor in the pathway comprising sensory afferent C-fibers. On the other hand, indomethacin further significantly suppressed the writhing number of the capsaicin-treated animals, suggesting that the acid-induced pain perception requires prostanoid receptors not only in the pathway via capsaicin-sensitive C-fibers but also in other sensory pathways. These results provide the first evidence for the involvement of the vanilloid receptor in the acid-induced inflammatory pain perception via sensory C-fibers in addition to the known mediators bradykinin, neurokinins, and prostanoids.

Major roles of prostanoid receptors IP and EP(3) in endotoxin-induced enhancement of pain perception.

To know the roles of prostaglandin I (IP) and prostaglandin E (EP) receptors in pain perception, we compared the acetic acid-induced writhing response in mice deficient in prostaglandin receptors, i.e. IP, EP(1,) EP(2,) EP(3,) or EP(4,) with or without lipopolysaccharide (LPS) pretreatment. Without LPS pretreatment, IP-receptor deficient mice showed a significantly smaller number of responses, as previously reported, whereas mice deficient in any of the EP-receptor subtypes showed a number of writhings similar to those of wild-type mice. When mice were pretreated with LPS for 24 hr to induce cyclooxygenase-2 expression, the wild-type as well as EP(1)-, EP(2)-, or EP(4)-receptor-deficient mice showed a similar enhanced writhing response, whereas IP- and EP(3)-receptor-deficient mice had a significantly less enhanced number of writhings. These results indicate that IP and EP(3) are the major prostaglandin receptors mediating the enhanced acetic acid-induced writhing response in mice pre-exposed to LPS, i.e. in endotoxin-enhanced inflammatory nociception.

Strenuous endurance training in humans reduces oxidative stress following exhausting exercise.

The aim of this study was to evaluate whether high-intensity endurance training would alleviate exercise-induced oxidative stress. Nine untrained male subjects (aged 19-21 years) participated in a 12-week training programme, and performed an acute period of exhausting exercise on a cycle ergometer before and after training. The training programme consisted of running at 80% maximal exercise heart rate for 60 min.day-1, 5 days.week-1 for 12 weeks. Blood samples were collected at rest and immediately after exhausting exercise for measurements of indices of oxidative stress, and antioxidant enzyme activities [superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT)] in the erythrocytes. Maximal oxygen uptake (VO2max) increased significantly (P < 0.001) after training, indicating an improvement in aerobic capacity. A period of exhausting exercise caused an increase (P < 0.01) in the ability to produce neutrophil superoxide anion (O2.-) both before and after endurance training, but the magnitude of the increase was smaller after training (P < 0.05). There was a significant increase in lipid peroxidation in the erythrocyte membrane, but not in oxidative protein, after exhausting exercise, however training attenuated this effect. At rest, SOD and GPX activities were increased after training. However, there was no evidence that exhausting exercise enhanced the levels of any antioxidant enzyme activity. The CAT activity was unchanged either by training or by exhausting exercise. These results indicate that high-intensity endurance training can elevate antioxidant enzyme activities in erythrocytes, and decrease neutrophil O2.- production in response to exhausting exercise. Furthermore, this up-regulation in antioxidant defences was accompanied by a reduction in exercise-induced lipid peroxidation in erythrocyte membrane.

Regulation of TNFalpha and interleukin-10 production by prostaglandins I(2) and E(2): studies with prostaglandin receptor-deficient mice and prostaglandin E-receptor subtype-selective synthetic agonists.

To know which receptors of prostaglandins are involved in the regulation of TNFalpha and interleukin 10 (IL-10) production, we examined the production of these cytokines in murine peritoneal macrophages stimulated with zymosan. The presence of PGE(2) or the PGI(2) analog carbacyclin in the medium reduced the TNFalpha production to one-half, whereas IL-10 production increased several fold; and indomethacin caused the reverse effects, suggesting that endogenous prostaglandins may have a regulatory effect on the cytokine production. Among prostaglandin E (EP) receptor-selective synthetic agonists, EP2 and EP4 agonists caused down-regulation of the zymosan-induced TNFalpha production, but up-regulation on the IL-10 production; while EP1 and EP3 agonists showed no effect. Macrophages harvested from prostaglandin I (IP) receptor-deficient mice showed the up- and down-regulatory effects on the cytokine production by the EP2 and EP4 agonists or PGE(2), but no effect was obtained by carbacyclin. On the contrary, macrophages from EP2-deficient mice showed the effect by PGE(2), carbacyclin, and the EP4 agonist, but not by the EP2 agonist; and the cells from EP4-deficient mice showed the effect by PGE(2), carbacyclin, and EP2 agonist, but not by the EP4 agonist. These functional effects of prostaglandins well accorded with the mRNA expression of TNFalpha and IL-10 when such expression was examined by the RT-PCR method. The peritoneal macrophages from normal mice expressed IP, EP2, and EP4 receptors, but not EP1 and EP3, when examined by RT-PCR. Thus the results suggest that PGI(2) and PGE(2) generated simultaneously with cytokines by macrophages treated with zymosan may influence the cytokine production through IP, EP2, and EP4 receptors.

Evidence for bradykinin mediation of carrageenin-induced inflammatory pain: a study using kininogen-deficient Brown Norway Katholiek rats.

Inflammatory pain was induced following an intradermal injection of carrageenin into rat paws, and the hyperalgesia was measured in terms of withdrawal time following thermal pain stimulation of the inflamed paw. This hyperalgesia was significantly less in kininogen-deficient Brown Norway (B/N)-Katholiek rats, which also showed less swelling in carrageenin-induced paw edema, than in normal B/N-Kitasato rats at 1 approximately 4 hr after the carrageenin injection (at the early phase). However, 24 hr after the injection, hyperalgesia and the swelling volume of the kininogen-deficient rats were almost the same as those in normal rats. The bradykinin B2 receptor antagonist FR173657, (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide, attenuated the carrageenin-induced swelling and hyperalgesia of the normal rats at the early phase to almost the levels of the B/N-Katholiek rats. Pretreatment with indomethacin, a cyclooxygenase inhibitor, also inhibited the carrageenin-induced responses significantly in normal rats. These results indicate that bradykinin, acting on the B2 receptor, is the main mediator at the early phase of inflammatory pain of carrageenin edema and that prostaglandins, produced by cyclooxygenase, potentiate the effects of bradykinin.

Intracellular Ca2+ increase in neuro-2A cells and rat astrocytes following stimulation of bradykinin B2 receptor.

Murine neuroblastoma cell line Neuro-2A cells and rat brain astrocytes showed a dose-dependent increase in intracellular Ca2+ in response to bradykinin, when assessed by a single cell image analyzing system. The Ca2+ increase in Neuro-2A cells by bradykinin was also examined by a suspension fluorescent assay using fura-2 loading. The Ca2+ increase in both cases was suppressed by a bradykinin B2 receptor antagonist, Hoe 140, but not by a B1 receptor antagonist, des-Arg-Hoe 140, suggesting that the effect occurred via specific B2 receptor activation. RT-PCR for bradykinin B2 receptor mRNA showed that both Neuro-2A cells and the astrocytes expressed B2 receptor mRNA. Binding of [3H]bradykinin to Neuro-2A cells was assessed, and a specific binding constant of 0.75 nM was determined. Furthermore, the increase in [Ca2+]i by bradykinin could be caused by a release of Ca2+ from storage sites in the endoplasmic reticulum, since thapsigargin and U-73122 attenuated the effect of bradykinin in Neuro-2A as well as in astrocytes. These results indicate that both astrocytes and neuroblastoma Neuro-2A cells stimulated by bradykinin could express a bradykinin B2 receptor-mediated intracellular Ca2+ increase leading to signal transduction.

Inhibition of adipocyte lipolysis by papaverine: papaverine can inhibit the redistribution of hormone-sensitive lipase.

Papaverine, despite being a potent phosphodiesterase inhibitor, actually blocks adipocyte lipolysis. The present study was designed to clarify the mechanism of the inhibitory effect of papaverine on lipolysis. Lipolysis, stimulated by either 10 microM isoproterenol or 5 mM dibutyryl cAMP, was significantly inhibited by papaverine (100 microM and above). Papaverine, however, did not affect the isoproterenol-induced increase in the protein kinase A (A-kinase) activity ratio. In cell-free extract from non-stimulated adipocytes, cAMP-stimulated A-kinase activities were almost completely blocked by H-89, a potent inhibitor of A-kinase, but not by papaverine. Thus, the inhibitory effect of papaverine on lipolysis could be responsible for a deficit in step(s) distal to A-kinase activity. Hormone-sensitive lipase activities in the infranatant fraction of centrifuged homogenates of cells, which were maximally stimulated with isoproterenol were significantly reduced. This result indicates that hormone-sensitive lipase redistributes from cytosol to its substrate in lipolytically stimulated cells. Papaverine completely blocked the isoproterenol-induced decrease in lipase activity in the infranatant fraction. These results suggest that papaverine blocks lipolysis through its inhibitory effect on the redistribution of hormone-sensitive lipase.

Intrinsic prostacyclin contributes to exudation induced by bradykinin or carrageenin: a study on the paw edema induced in IP-receptor-deficient mice.

To prove that prostaglandin I2 (PGI2) is a major prostaglandin involved in bradykinin-induced exudation, we examined carrageenin- or bradykinin-induced paw edema in prostacyclin receptor-deficient mice (IPKO). Paw volume of wild-type mice (IPWT) increased gradually 5-6 hr after the carrageenin injection in a similar manner as in ICR mice, but the swelling in IPKO mice was significantly smaller (about 60% of the IPWT volume). Indomethacin, at 10 mg/kg, suppressed the swelling of the IPWT paw to the level of the non-pretreated IPKO, which was not affected by indomethacin, confirming the previous result that PGI2 is a major prostaglandin involved in the swelling. The paw edema of IPWT and IPKO was significantly attenuated by the nonpeptide bradykinin B2-receptor antagonist FR173657, at 30 mg/kg, to the same level of swelling, indicating kinin involvement. Injection of bradykinin (1.2 nmole) into the paw caused rapid edema, which peaked around 15 min in both mice. However, the edema induced in IPKO was smaller and almost at the same level as that elicited in the indomethacin-treated IPWT, suggesting that edema induced by bradykinin includes the intrinsic effect of PGI2. Concomitant injection of carbacyclin with bradykinin caused enhancement of edema in IPWT mice but not in IPKO mice, indicating that intrinsic PGI2 could cause enhancement of bradykinin- or even carrageenin-induced edema formation. These results clearly demonstrate that bradykinin released by carrageenin may be a key mediator to induce PGI2 formation, and both autacoids work together to induce enhanced inflammatory exudation.

Effects of swimming training on three superoxide dismutase isoenzymes in mouse tissues.

The purpose of the present study was to investigate the effects of swimming training on the changes in three superoxide dismutase (SOD) isoenzymes in mice. The trained mice underwent a 6-wk swimming program (1 h/day, 5 days/wk) in water at 35-36 degrees C. Immunoreactive extracellular SOD (EC-SOD), copper- and zinc-containing SOD (CuZn-SOD), and manganese-containing SOD (Mn-SOD) contents and their mRNA abundance were determined in serum, heart, lung, liver, kidney, and gastrocnemius muscle. EC-SOD content in liver and kidney was significantly increased with training. After training, CuZn-SOD content rose significantly only in kidney but decreased significantly in heart, lung, and liver. Mn-SOD content showed a significant increase in lung, kidney, and skeletal muscle but a significant decrease in liver. In most tissues, however, the changes in SOD isoenzyme contents were not concomitant with those in their mRNA levels. The results obtained thus suggest that, except for kidney, the responses in mouse tissues of three SOD isoenzymes (protein levels and mRNA abundance) to swimming training are different and that kidney may be one of the most sensitive organs to adapt to oxidative stress during physical training, although the mechanism remains vague.

Examination of signal transduction pathway of stimulated B1 and B2 kinin receptors; MAP kinase pathway to AP-1 translocation in HEK 293 cells.

B1 or B2 kinin receptor-overexpressing HEK293 cells were stimulated with des-Arg9-BK or BK, respectively. Each agonist induced translocation of AP-1 into the nuclear fraction as well as activation of MAP kinases in each cells. MAP kinase inhibitor PD98059 suppressed translocation of AP-1 and agonist-induced MAP kinase activation in both cells. These results indicate that stimulation of B1 or B2 receptor expresses a feature of the signal transduction pathway of MAP kinase activation to translocation of AP-1. This signal transduction pathway of HEK cells through B1 and B2 receptors may be similar in response to respective agonists.

FR190997, a novel bradykinin B2 agonist, expresses longer action than bradykinin in paw edema formation and hypotensive response.

Biological actions of a novel non-peptide B2 receptor agonist, FR190997, were examined by comparing them with those of bradykinin. The paw edema was induced by subcutaneous injection of 30 microl of solution of bradykinin (0.3, 0.6, and 1.2 nmol) or FR190997 (0.1, 0.3, and 0.9 nmol) into the right hind paw of ICR male mice. Bradykinin caused a dose-dependent edema formation, which peaked at 15 min and ceased after 150 min. FR190997 also formed a dose-dependent edema, peaking at 15-30 min with a slight delay compared to bradykinin and this response continued over 200 min. The edema formed by bradykinin or FR190997 was inhibited by pretreatment with HOE140 (1 mg/kg) injected intraperitoneally 30 min before the injection of each agonist. A novel non-peptide B2 antagonist, FR173657 (30 mg/kg, i.p. 30 min before the agonist), also diminished these responses by bradykinin and FR190997 dose-dependently. Indomethacin (10 mg/kg, i.p. 30 min before) inhibited the response to FR190997, suggesting that release of prostaglandins induced by the B2 agonistic action might be involved in this inflammatory process induced by FR190997. The hypotensive action of FR190997 was also examined. Intravenously injected FR190997 caused the systemic hypotensive response in Sprague-Dawley male rats anesthetized with pentobarbital. The potency of FR190997 was weaker than that of bradykinin, when compared with the maximal hypotension. Duration of the hypotensive response of FR190997 was significantly longer than that of bradykinin. These results indicate that FR190997 has the B2 agonistic action similar to bradykinin and is also a good tool for in vivo examination of the B2 receptor.

Unloading does not increase brown-adipose-tissue activity in rat pups.

Our previous study has shown that chronic hindlimb suspension leads to an increase in both the thermogenic capacity and the activity in brown adipose tissue (BAT) of adult rats (Yamashita, H., Ohira, Y., Wakatsuki, T., Yamamoto, M., Kizaki, T., Oh-ishi, S., Sato, Y. and Ohno, H. (1995). J. Appl. Physiol. 78: 384-387). In order to examine if unloading also increases the BAT activity in rat pups, the hindlimbs in the suspended pups were unloaded by tail suspension beginning on postnatal day 4 and suspended until day 21. The thermogenic activity (which was assessed by guanosine 5'-diphosphate binding to BAT mitochondria) was markedly lower in 21-day-old suspended pups than in 21-day-old control pups, although there was no difference in uncoupling protein 1 (UCP1) content or UCP1 mRNA expression in the BAT mitochondrial fraction between both pups. Likewise, there was no disparity in either adrenal or thymus mass between the control and suspended pups throughout the experiment. These results suggest that, in contrast to adult rats, chronic hindlimb suspension leads to a decrease in the thermogenic activity in BAT of rat pups possibly for reason that pups are less susceptible to the stress of unloading.

Mouse paw edema induced by a novel bradykinin agonist and its inhibition by B2-antagonists.

A novel non-peptide bradykinin B2-receptor agonist, FR190997 (8-[2,6-dichloro-3-[N-[(E)4-(N-methylcarbamoyl)cinnamidoacetyl+ ++]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinolin e), induced dose-dependent and longer-lasting swelling than bradykinin in the mouse paw. The swelling, peaking around 30 min, was suppressed dose-dependently by intraperitoneal administration of FR173657, a novel non-peptide B2-receptor antagonist. A known B2-antagonist, Hoe 140, also significantly suppressed this edema. The result indicates that the novel B2-agonist FR190997, being more stable than bradykinin, could induce plasma exudation locally in mice via the B2-receptor as a substitute for bradykinin.

Agonist stimulation of B1 and B2 kinin receptors causes activation of the MAP kinase signaling pathway, resulting in the translocation of AP-1 in HEK 293 cells.

In response to bradykinin, phosphorylated MAP kinases (ERK-1 and ERK-2) were abundantly increased in HEK 293 cells, which overexpress the rat B2 kinin receptor. In a similar way des-Arg9-bradykinin stimulation of B1 kinin receptor-overexpressing HEK 293 cells caused activation of the same species of MAP kinase. Furthermore, nuclear translocation of transcription factor AP-1 was also found in the cells after stimulation with either agonist. PD98059, a MAP kinase kinase (MEK-1) inhibitor, blocked the agonist-induced AP-1 translocation as well as the phosphorylation of the MAP kinases. This communication provides the first evidence for both B1 and B2 kinin receptors mediating the MAP kinase signaling pathway to activate AP-1.

Tissue distribution of immunoreactive mouse extracellular superoxide dismutase.

Protein content and mRNA expression of extracellular superoxide dismutase (EC-SOD) were investigated in 16 mouse tissues. We developed a double-antibody sandwich ELISA using the affinity-purified IgG against native mouse EC-SOD. EC-SOD could be detected in all of the tissues examined (lung, kidney, testis, brown fat, liver, adrenal gland, pancreas, colon, white fat, thymus, stomach, spleen, heart, skeletal muscle, ileum, and brain, in decreasing order of content measured as microg/g wet tissue). Lung showed a markedly higher value of EC-SOD than other tissues. Interestingly, white fat had a high content of EC-SOD in terms of micrograms per milligram protein, which corresponded to that of lung. Kidney showed the strongest expression of EC-SOD mRNA. Relatively strong expression of the mRNA was observed in lung, white fat, adrenal gland, brown fat, and testis. Heart and brain showed only weak signals, and no such expression could be detected in either digestive organs or skeletal muscle. Immunohistochemically, EC-SOD was localized mainly to connective tissues and vascular walls in the tissues examined. Deep staining in the cytosol was observed in the cortical tubular cells of kidney. These results suggest that EC-SOD is distributed systemically in mice and that the physiological importance of this enzyme may be a compensatory adaptation to oxidative stress, particularly in lung and kidney.

Induction of cyclooxygenase-2 causes an enhancement of writhing response in mice.

Pretreatment of mice with lipopolysaccharide for 16 h enhanced the number of acetic acid-induced writhing reactions by 2 to 3-fold. In the peritoneal exudates at 10 min after acetic acid injection, 6-keto-prostaglandin F1alpha was detected as a major prostanoid, and this level increased by several-fold by the pretreatment with lipopolysaccharide. The writhing reaction and the prostaglandin formation were almost completely suppressed by indomethacin. However, the lipopolysaccharide-induced enhancement of writhing reaction and an increment of 6-keto-prostaglandin F1alpha level were diminished by the administration of cyclooxygenase-2-selective inhibitors, such as NS-398, nimesulide, or L-745337, to a level similar to the mice that did not receive lipopolysaccharide. Cyclooxygenase-2 protein in the exudates became detectable at 5-48 h after the lipopolysaccharide-pretreatment. These results suggest that the increased prostaglandin production by cyclooxygenase-2 could be responsible for enhancement of the acetic acid-induced writhing reaction by lipopolysaccharide pretreatment.

An increase in basal glucocorticoid concentration with age induces suppressor macrophages with high-density Fc gamma RII/III.

Ageing is usually accompanied by a decline in immune and neuroendocrine functions. To elucidate the mechanisms underlying age-related immunosuppression, the functions and surface phenotypes of peritoneal cells in the monocyte/macrophage lineage from old mice were investigated. The role of glucocorticoids (GC) in the immunomodulation was also examined. Proliferative responses of spleen cells from control mice stimulated with concanavalin A (Con A) were significantly suppressed by adding peritoneal exudate cells from old mice. Flow cytometry analysis revealed that the proportion of MAC-1+ cells with a high density of type II or type III receptor for the Fc portion of IgG (Fc gamma RII/IIIbright cells) was increased markedly in the periotoneal exudate cells from old mice. The prominent suppressor activity for Con A responses of control spleen cells was found in the Fc gamma RII/IIIbright cells, whereas MAC-1+ cells with a low density of Fc gamma RII/III (Fc gamma RII/IIIdull cells) did not suppress the Con A responses. On the other hand, both the basal corticosterone concentrations in serum and the mRNA expression for GC receptor in peritoneal exudate cells increased significantly in old mice. Furthermore, the proportion of Fc gamma RII/IIIbright cells in peritoneal exudate cells from old mice was normalized on administration of the GC antagonist RU 38,486 (mifepristone). These results suggest that the increase in basal corticosterone concentrations in old mice induces the generation of Fc gamma RII/IIIbright suppressor cells, possibly leading to the immune-suppressive state.

Effect of running training on brown adipose tissue activity in rats: a reevaluation.

The effect was investigated of running training on the thermogenic activity of brown adipose tissue (BAT) in rats. The exercised rats were trained on a rodent treadmill for 5 days per week and a total of 9 weeks. After the training, a significantly lower rate of increase in body weight was found, suggesting some training effect, whereas the training failed to induce a decrease in BAT mass. As previously reported (Yamashita, Yamamoto et al., 1993), there was also a markedly lower expression of uncoupling protein (UCP) mRNA in BAT from trained rats; nevertheless, no definite effect of the running training was noted on either UCP content or guanosine 5'-diphosphate binding in the mitochondria recovered from BAT. The results obtained suggest that running training has no overt effect on the thermogenic activity of BAT in rats.