Mimosine-induced apoptosis in C6 glioma cells requires the release of mitochondria-derived reactive oxygen species and p38, JNK activation.

Growth-inhibitory effects of mimosine, a plant amino acid, on rat C6 glioma cells were analyzed. Mimosine markedly inhibited proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent manner. Mimosine-mediated apoptosis was accompanied by promoting reactive oxygen species (ROS) generation in mitochondria, and by decreased mitochondrial membrane potential (Δψ), and release of cytochrome c from mitochondria, followed by caspase 3 activation. Furthermore, mimosine increased the phosphorylation level of c-Jun-N-terminal protein kinase and p38, which was the downstream effect of ROS accumulation. Mimosine was confirmed to show profound effects on apoptosis of C6 glioma cells by ROS-regulated mitochondria pathway, and these results bear on the hypothesized potential for mimosine as promising agents in the treatment of malignant gliomas.

The membrane topological analysis of 3ß-hydroxysteroid-Delta24 reductase (DHCR24) on endoplasmic reticulum.

DHCR24 encodes 3ß-hydroxysteroid-Δ24 reductase, catalyzing the conversion of desmosterol to cholesterol. Our previous study demonstrated that DHCR24 exerts an anti-apoptotic function as a reactive oxygen species (ROS) scavenger, for which it needs its FAD-binding domain. The membrane topology of DHCR24 on endoplasmic reticulum (ER) and the functional significance of its FAD-binding domain are not completely understood. Based on the structure predicted by bioinformatics, we studied the membrane topology of DHCR24 in murine neuroblastoma cells (N2A), using the fluorescent protease protection (FPP) technique. We showed that full-length DHCR24 is localized to the membrane of ER, whereas the predicted transmembrane (TM) domain-deleted DHCR24 mutation is localized to the cytoplasm. The change of DHCR24 localization suggests that the N-terminal TM domain is essential for the ER membrane targeting of DHCR24. The FPP assay demonstrated the membrane topology of DHCR24 with an N-terminal luminal/C-terminal cytoplasmic orientation. Measurement of intracellular ROS using H(2)DCFDA revealed that the ROS levels of cells infected by plasmids driving expression of full-length DHCR24 or the TM domain-deleted DHCR24 mutation after H(2)O(2) exposure were lower than those of control cells, suggesting that the ER membrane targeting of DHCR24 is not required for its enzymatic ROS scavenging activity. Confocal fluorescence microscopy revealed that the DHCR24-overexpressed cells were protected from apoptosis in response to oxidative stress, which was accompanied by a decrease in DHCR24 content on the ER and activation of caspase-3, suggesting that the anti-apoptotic function of DHCR24 is associated with its cleavage by caspase.

3ß-Hydroxysterol-Delta24 reductase plays an important role in long bone growth by protecting chondrocytes from reactive oxygen species.

Desmosterolosis is an autosomal recessive disease caused by mutations in the 3ß-hydroxysterol-Delta24 reductase (DHCR24) gene, with severe developmental anomalies including short limbs. We utilized DHCR24 knockout (KO) mice to study the underlying bone pathology. Because the KO mice died within a few hours after birth, we cultured metatarsal bones from newborn mice. The growth of bones from KO mice was significantly retarded after 1 week of culture. Absence of proliferating chondrocytes in the growth plate and abnormal hypertrophy of prehypertrophic chondrocytes were observed in the bones from KO mice. Hypertrophic differentiation was evidenced by higher expression of Indian hedgehog, alkaline phosphatase, and matrix metalloproteinase 13. Since elevated levels of reactive oxygen species (ROS) during chondrogenesis are known to inhibit proliferation and to initiate chondrocyte hypertrophy in the growth plate, and since DHCR24 acts as a potent ROS scavenger, we hypothesized that the abnormal chondrocyte proliferation and differentiation in KO mice were due to decreased ROS scavenging activity. Treatment with an antioxidant, N-acetyl cysteine, could correct the abnormalities observed in the bones from KO mice. Treatment of bones from wild-type mice with U18666A, a chemical inhibitor of DHCR24, resulted in short broad bones with a disrupted proliferating zone. Treatment of ATDC cells with hydrogen peroxide (H(2)O(2)) induced hypertrophic changes as evidenced by the expression of the marker genes specific for hypertrophic chondrocyte differentiation. H(2)O(2)-induced hypertrophic change was prevented by adenoviral delivery of DHCR24. Induction of chondrocyte differentiation in ATDC cells by insulin was associated with increased ROS production that was markedly enhanced by treatment of ATDC5 cells with DHCR24 siRNA. This is the first demonstration that DHCR24 plays an important role in long bone growth by protecting chondrocytes from ROS.

Native low density lipoprotein induces pancreatic ß cell apoptosis through generating excess reactive oxygen species.

BACKGROUND: The growing evidences demonstrated hyperlipidemia in obesity and type 2 diabetes is characterized by high levels of free fatty acids, low-density lipoprotein (LDL), triglyceride, and cholesterol. METHOD AND RESULTS: We investigated the effect of LDL particles (LDLs) loading on MIN6 cells derived from pancreatic ß cells. Exposure of MIN6 cells to LDLs induced apoptosis in dose and time-dependent manner, demonstrated by the TUNEL in situ apoptotic assay. The immunocytochemical analysis and Western blotting revealed that the LDLs-induced apoptosis is associated with the activation of caspase 3 and upregulation of p53. The intracellular concentration of Reactive Oxygen Species (ROS) measured by use of DCFHDA was significantly increased after loading LDLs, demonstrating the induced apoptosis by LDLs loading was mediated through oxidative stress. Addition of reduced form of Glutathione (GSH) in the medium rescued MIN6 cells from apoptosis. The Cellular cholesterol level was increased significantly after LDLs loading, suggesting that the excess cholesterol induced by LDLs loading might contribute to the apoptosis in MIN6s. Agarose electrophoresis demonstrated that the LDLs added to the medium were not oxidized. CONCLUSION: Taken together, these results demonstrate that the LDLs loading can induce apoptosis of MIN6 cells mediated by the excess cellular cholesterol and generation of oxidative stress.

Cholesterol induces pancreatic ß cell apoptosis through oxidative stress pathway.

Type 2 diabetes is often associated with high blood cholesterol. Here, we investigated the effect of cholesterol loading on MIN6 cells derived from pancreatic ß cells. Exposure of MIN6 cells to cholesterol-induced apoptosis in time- and dose-dependent manner. Treatment with methyl-ß-cyclodextrin that removes cholesterol from plasma membrane prevented the cells from cholesterol-induced apoptosis. Western blot analysis revealed that the levels of phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and c-Jun N-terminal kinases (P-JNK) were significantly increased after the cholesterol loading, suggesting that the stress-activated protein kinase signaling was stimulated. A specific p38 inhibitor rescued MIN6 cells from cholesterol-induced apoptosis, while JNK inhibitor failed, suggesting the importance of activation of p38 MAPK signaling in response to cholesterol. The expression of Bip and CHOP, the endoplasmic reticulum (ER) stress markers, remained unaffected, indicating that the ER stress may not be involved in the cytotoxicity of cholesterol on the ΜΙΝ6 cells. The intracellular concentration of reactive oxygen species measured by use of 2',7'-dichlorofluorescin diacetate was significantly increased after cholesterol loading, demonstrating the induced apoptosis was mediated through oxidative stress. Addition of reduced form of glutathione in the medium rescued MIN6 cells from apoptosis induced by cholesterol loading. Taken together, these results demonstrate that the free cholesterol loading can induce apoptosis of MIN6 cells mediated by oxidative stress and the activation of p38 MAPK signaling.

Thyroid-hormone-dependent activation of the phosphoinositide 3-kinase/Akt cascade requires Src and enhances neuronal survival.

We have reported previously a non-genomic action of T3 (3,3',5-tri-iodothyronine), which stimulates the PI3K (phosphoinositide 3-kinase)/Akt pathway via p85alpha, the regulatory subunit of PI3K, in human skin fibroblasts. The aim of the present study was to elucidate the mechanism by which T3 activates PI3K, and to investigate the physiological role of this T3 action in neuronal cells. We found that T3 activates PI3K/Akt through Src. First, T3 rapidly induced the activation of Src and Akt in N2a cells expressing TRalpha1 (thyroid hormone receptor alpha1; N2aTRalpha), and both were attenuated by either the addition of a Src inhibitor or Src siRNA. In contrast, a PI3K inhibitor could only block the activation of Akt. Secondly, T3 enhanced TRalpha1-p85alpha-Src complex formation, which was also abrogated by a Src inhibitor. The activation of Src and PI3K/Akt contributes to the anti-apoptotic effect of T3 in N2aTRalpha cells. Moreover, it was also observed in primary cerebral cortical neurons that T3 induced the activation of PI3K/Akt and suppressed serum-deprivation-induced apoptosis. Together, the findings of the present study demonstrate a novel non-genomic action of T3 on neuronal cell survival, and provide new insights into the mechanism underlying this action, which involves Src activation and TRalpha1-p85alpha-Src complex formation.

Requirement of DHCR24 for postnatal development of epidermis and hair follicles in mice.

Desmosterolosis is an autosomal recessive disorder with severe developmental anomalies due to mutations in the DHCR24 gene, encoding an enzyme to convert desmosterol to cholesterol. We reported that DHCR24 [knockout (KO)] mice were born with wrinkleless taut skin and with impaired development of epidermis. In this study, we investigated the postnatal development of epidermis and hair follicle in the skin of KO mice grafted to the nude mice. Skin graft was required since the KO mice die within few hours after birth. Forty days after the skin graft, epidermis from the KO mice revealed the characteristic phenotype observed at birth. Furthermore, the number of hair follicles in the skin graft from KO mice to the nude mice was significantly less and development was delayed than that from control. These findings implicate that DHCR24 plays important roles for normal development of epidermis and hair follicle even in postnatal life.

3beta-Hydroxysteroid-delta24 reductase is a hydrogen peroxide scavenger, protecting cells from oxidative stress-induced apoptosis.

3beta-Hydroxysteroid-Delta24 reductase (DHCR24) is an endoplasmic reticulum-resident, multifunctional enzyme that possesses antiapoptotic and cholesterol-synthesizing activities. To clarify the molecular basis of the former activity, we investigated the effects of hydrogen peroxide (H(2)O(2)) on embryonic fibroblasts prepared from DHCR24-knockout mice (DHCR24(-/-) mouse embryonic fibroblasts). H(2)O(2) exposure rapidly induced apoptosis, which was associated with sustained activation of apoptosis signal-regulating kinase-1 and stress-activated protein kinases, such as p38 MAPK and c-Jun N-terminal kinase. Complementation of the mouse embryonic fibroblasts by adenovirus expressing DHCR24 attenuated the H(2)O(2)-induced kinase activation and apoptosis. Concomitantly, intracellular generation of reactive oxygen species (ROS) in response to H(2)O(2) was also diminished by the adenovirus, suggesting a ROS-scavenging activity of DHCR24. Such antiapoptotic effects of DHCR24 were duplicated in pheochromocytoma PC12 cells infected with adenovirus. In addition, it was found that DHCR24 exerted cytoprotective effects in the tunicamycin-induced endoplasmic reticulum stress by eliminating ROS. Finally, using in vitro-synthesized and purified proteins, DHCR24 and its C-terminal deletion mutant were found to exhibit high H(2)O(2)-scavenging activity, whereas the N-terminal deletion mutant lost such activity. These results demonstrate that DHCR24 can directly scavenge H(2)O(2), thereby protecting cells from oxidative stress-induced apoptosis.

Thyroid hormone activates adenosine 5'-monophosphate-activated protein kinase via intracellular calcium mobilization and activation of calcium/calmodulin-dependent protein kinase kinase-beta.

AMP-activated protein kinase (AMPK) is a key regulator of glucose and fatty acid homeostasis. In muscle cells, AMPK stimulates mitochondrial fatty acid oxidation and ATP production. The thyroid hormone T3 increases cellular oxygen consumption and is considered to be a major regulator of mitochondrial activities. In this study, we examined the possible involvement of AMPK in the stimulatory action of T3 on mitochondria. Treatment of C2C12 myoblasts with T3 rapidly led to phosphorylation of AMPK. Acetyl-coenzyme A carboxylase, a direct target of AMPK, was also phosphorylated after T3 treatment. Similar results were obtained with 3T3-L1, FRTL-5, and HeLa cells. Stable expression of T3 receptor (TR)-alpha or TRbeta in Neuro2a cells enhanced this effect of T3, indicating the involvement of TRs. Because HeLa cells express only Ca2+/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta), one of two known AMPK kinases, it was suggested that the effect of T3 is mediated by CaMKKbeta. Indeed, experiments using a CaMKK inhibitor, STO-609, and an isoform-specific small interfering RNA demonstrated the CaMKKbeta-dependent phosphorylation of AMPK. Furthermore, T3 was found to rapidly induce intracellular Ca2+ mobilization in HeLa cells, and a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suppressed T3- as well as ionomycin-dependent phosphorylation of AMPK. In addition, T3-dependent oxidation of palmitic acids was attenuated by BAPTA, STO-609, and the small interfering RNA for CaMKKbeta, indicating that T3-induced activation of AMPK leads to increased fatty acid oxidation. These results demonstrate that T3 nontranscriptionally activates AMPK via intracellular Ca2+ mobilization and CaMKKbeta activation, thereby stimulating mitochondrial fatty acid oxidation.

Long-term amiodarone treatment causes cardioselective hypothyroid-like alteration in gene expression profile.

The long-term cardiac effects of amiodarone resemble many aspects of hypothyroidism. The anti-arrhythmic potential of amiodarone may therefore be the result of a drug-induced, local hypothyroid-like condition. To investigate this controversial issue, we compared gene expression profiles in the hearts of rats treated with amiodarone with those of rats with hypothyroidism. Wistar male rats were assigned to 3 groups (n=6-8): Control, systemic hypothyroidism (hypothyroidism) and amiodarone treatment (amiodarone, 150 mg/kg/day, p.o., 4 weeks). Electrocardiogram (ECG) recordings, gene profiling by DNA microarray and Northern blotting were carried out. Amiodarone, like hypothyroidism, caused significant prolongation of RR and QT intervals in ECGs. Microarray analysis of 8435 genes in the left ventricular myocardium revealed a significant similarity in expression profiles between hypothyroidism and amiodarone (R=0.63, p<0.00001). The gene expression profiles of hypothyroidism and amiodarone showed closer correlation when top 100 up-regulated and 100 down-regulated genes in hypothyroidism (total 200 genes) were analyzed (R=0.78, p<0.00001). Northern blots of left ventricular myocardium showed a parallel decrease in mRNAs for myosin heavy chain (MHC)-alpha and a parallel increase for myosin heavy chain (MHC)-beta in hypothyroidism and amiodarone. In the liver and pituitary, in contrast, Northern blots showed quite different changes in the transcripts of the representative T3-responsive genes in the hypothyroidism and amiodarone. In conclusion, long-term treatment with amiodarone causes cardioselective hypothyroid-like alterations in gene expression profiles. The potent anti-arrhythmic activity of amiodarone may be attributable, in part at least, to this unique transcriptional remodeling.

Insulin-like growth factor-I activation of Akt survival cascade in neuronal cells requires the presence of its cognate receptor in caveolae.

Insulin-like growth factor-I (IGF-I) plays important roles in survival of neurons. Caveolae, cholesterol-rich microdomains of plasma membrane, act as platforms for some neurotrophic factors. In this study, we examined a possible role of caveolae in IGF-I signal transduction in pheochromocytoma PC12 cells. IGF-I treatment attenuated serum withdrawal-induced apoptosis, which was reversed by treatment with methyl-beta-cyclodextrin (CD) that removes cholesterol from plasma membrane. Immunocytochemical and subcellular fractionation analyses revealed that IGF-I receptor (IGF-IR) was colocalized with caveolin-1, a major protein component in caveolae, and that CD treatment reduced IGF-IR contents in caveolae. Consistent with these findings, IGF-I phosphorylation of insulin receptor substrate-1 and Akt was impaired, and cholesterol supply restored the IGF-I action. Furthermore, experiments using small interfering RNA revealed that the reduction of caveolin-1 expression impaired the IGF-I action. In addition, the colocalization of IGF-IR with caveolin-1, and the caveolae-dependent IGF-I action were duplicated in primary culture of rat cerebellar granule neurons. These results demonstrate that the presence of IGF-IR in caveolae is required for the neuroprotective action of IGF-I.

A case with isolated growth hormone deficiency caused by compound heterozygous mutations in GH-1: a novel missense mutation in the initiation codon and a 7.6kb deletion.

OBJECTIVE: To characterize the cause of a sporadic isolated growth hormone deficiency in a single patient. METHODS: Genomic DNA was extracted from blood samples of the patient and his family. Exons and exon-intron junctions of the GH-1 gene were amplified by PCR and sequenced. To characterize possible GH-1 deletions we performed Southern blot analysis and PCR-restriction fragment length analyses. RESULTS: An adenine to guanine mutation at the first nucleotide of the initiation codon (Met [ATG](-26)Val [GTG]) of the GH-1 gene was identified in the patient and the mother. A 7.6kb GH-1 deletion was identified in the patient, the brother and the father. CONCLUSION: The patient was a compound heterozygote for an allele bearing a Met(-26)Val missense mutation inherited from his mother and an allele containing deletion of the entire GH-1 gene inherited from his father. The present missense mutation has not been described previously. Attention should be paid to the heterozygous gene deletion that is difficult to detect by PCR-based genetic analysis. The patient responded to GH replacement therapy fairly well, without developing anti-hGH antibody.

Parathyroid hormone activates phosphoinositide 3-kinase-Akt-Bad cascade in osteoblast-like cells.

To understand the molecular basis underlying the anabolic action of parathyroid hormone (PTH) on bone, the anti-apoptotic action of PTH on osteoblast-like cells was investigated. Since Akt is a key protein kinase for cell survival, we focused on a possible involvement of Akt in the anti-apoptotic action of PTH. Human osteoblast-like MG-63 cells cultured without serum were treated with PTH. Western blot analysis revealed that PTH rapidly phosphorylated Akt and induced its nuclear translocation. The phosphorylation of pro-apoptotic protein Bad was also increased by PTH, leading to its inactivation. The PTH-dependent activation of Akt was also detected in other osteoblastic cell lines, SaOS-2 and ROS 17/2.8. The pretreatment of MG-63 cells with either one of inhibitors for phosphoinositide 3-kinase (PI3K), wortmannin or LY294002 prevented Akt and Bad phosphorylation. Furthermore, co-immunoprecipitation analysis revealed that PTH receptor (PTH-1R) directly interacted with p85, a regulatory subunit of PI3K, in a PTH-dependent manner. Serum withdrawal induced the apoptosis of MG-63 cells, and PTH prevented the apoptosis, which was inhibited by PI3K inhibitors. These results demonstrate the presence of a novel PTH/PTH receptor signaling cascade consisting of PTH-1R, PI3K, Akt and Bad and that this cascade can work as an anti-apoptotic signaling pathway in osteoblast-like cells.

Molecular cloning of prostaglandin EP3 receptors from canine sensory ganglia and their facilitatory action on bradykinin-induced mobilization of intracellular calcium.

We previously demonstrated that the activation of prostaglandin E-prostanoid-3 (EP3) receptor sensitized the canine nociceptor response to bradykinin (BK). To elucidate the molecular mechanism for this sensitization, we cloned two cDNAs encoding EP3s with different C-terminals, from canine dorsal root ganglia, and established the transformed cell lines stably expressing them. In both transformants, EP3 agonist did not increase intracellular cAMP levels, but it attenuated forskolin-dependent cAMP accumulation in a pertussis toxin (PTX)-sensitive manner and increased intracellular calcium levels in a PTX-resistant manner, indicating that both EP3s can couple with Gi and Gq, but not with Gs proteins. As the nociceptor response to BK is mediated by BK B2 receptor, it was transfected into the transformants and the effects of EP3 agonist on BK-dependent calcium mobilization were investigated. When BK was applied twice with a 6-min interval, the second response was markedly attenuated. Pre-treatment with EP3 agonist had no effect on the initial response, but restored the second response in a PTX-sensitive manner. A protein kinase A inhibitor mimicked the effect of EP3 agonist. These results demonstrate that the activation of EP3 restores the response to BK by attenuating the desensitization of BK B2 receptor activity via Gi protein.

Thyroid hormone mediated changes in gene expression can be initiated by cytosolic action of the thyroid hormone receptor beta through the phosphatidylinositol 3-kinase pathway.

Thyroid hormone (TH) action is mediated principally through binding of the hormone ligand, 3,3,5-triiodothyronine (T3), to TH receptors (TRs). This hormone-receptor interaction recruits other proteins to form complexes that regulate gene expression by binding to DNA sequences in the promoter of target genes. We recently described an extranuclear mechanism of TH action that consists of the association of TH-liganded TRbeta with p85alpha [regulatory subunit of phosphatidylinositol 3-kinase (PI3K)] in the cytosol and subsequent activation of the PI3K, generating phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]. This initiates the activation of a signaling cascade by phosphorylation of Akt, mammalian target of rapamycin (mTOR) and its substrate p70(S6K), leading to the stimulation of ZAKI-4alpha synthesis, a calcineurin inhibitor. Furthermore, we found that this same mechanism leads to induction of the transcription factor hypoxia-inducible factor (HIF-1alpha), and its target genes, glucose transporter (GLUT)1, platelet-type phosphofructokinase (PFKP), and monocarboxylate transporter (MCT) 4. These genes are of special interest, because their products have important roles in cellular glucose metabolism, from glucose uptake (GLUT1) to glycolysis (PFKP) and lactate export (MCT4). These results demonstrate that the TH-TRbeta complex can exert a non-genomic action in the cytosol leading to changes in gene expression by direct (HIF-1alpha) and indirect (ZAKI-4alpha, GLUT1, PFKP) means.

Differential expression of cyclin-dependent kinase inhibitors, p27Kip1 and p57Kip2, by corticotropin in rat adrenal cortex.

An important role for the cyclin-dependent kinase inhibitors (CDKIs), p27Kip1 and p57Kip2, in the proliferation and differentiation of adrenal cells has been suggested by their knockout mice, which display adrenal hyperplasia. Adrenal development and function are primarily regulated by ACTH. In the present study, we investigated the effects of ACTH on the expression of p27Kip1, p57Kip2 and proliferating cell nuclear antigen (PCNA) in rat adrenals. Male Wistar rats were treated with dexamethasone (Dex) to inhibit endogenous ACTH secretion. ACTH was then administered to the rats, and the adrenals were examined by Western blot and immunohistochemical analyses. Dex treatment induced shrinkage of adrenals where no PCNA-expressing cells were detected, but most of the cells expressed p27Kip1. Subsequent ACTH treatment resulted in the marked suppression of p27Kip1 expression, specifically in adrenocortical cells at 12 h after the stimulus. At 48 h, the p27Kip1 suppression still continued in the cortex, while the PCNA-expressing cells appeared mainly around the zona glomerulosa and increased at 72 h. At this time, the p27Kip1-expressing cells also appeared in the same zone. In contrast to p27Kip1, the expression of p57Kip2 was not detected in the Dex-treated adrenal. However, its expression was markedly induced by ACTH in the zona glomerulosa at 48 and 72 h. The results demonstrate that the primary site for mitogenic action of ACTH in rat adrenocortex is the zona glomerulosa, and that ACTH modulates proliferation of adrenocortical cells by regulating p27Kip1 and p57Kip2 expression in a time- and site-specific manner.

DHCR24-knockout embryonic fibroblasts are susceptible to serum withdrawal-induced apoptosis because of dysfunction of caveolae and insulin-Akt-Bad signaling.

The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24(-/-) mice. Both DHCR24(-/-) and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24(-/-) MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.

Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC.

Fenofibrate improves endothelial function by lipid-lowering and anti-inflammatory effects. Additionally, fenofibrate has been demonstrated to upregulate endothelial nitric oxide synthase (eNOS). AMP-activated protein kinase (AMPK) has been reported to phosphorylate eNOS at Ser-1177 and stimulate vascular endothelium-derived nitric oxide (NO) production. We report here that fenofibrate activates AMPK and increases eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase. Fenofibrate simultaneously increased eNOS phosphorylation and NO production. Inhibitors of protein kinase A and phosphatidylinositol 3-kinase failed to suppress the fenofibrate-induced eNOS phosphorylation. Neither bezafibrate nor WY-14643 activated AMPK in HUVEC. Furthermore, fenofibrate activated AMPK without requiring any transcriptional activities. These results indicate that fenofibrate stimulates eNOS phosphorylation and NO production through AMPK activation, which is suggested to be a novel characteristic of this agonist and unrelated to its effects on peroxisome proliferator-activated receptor alpha.