Ca2+/Calmodulin induces translocation of membrane-associated TSC2 to the nucleus where it suppresses CYP24A1 expression.

Tuberous sclerosis complex 2 (TSC2) is a tumor-suppressor protein. A loss of TSC2 function induces hyperactivation of mechanistic target of rapamycin (mTOR). The C-terminal region of TSC2 contains a calmodulin (CaM) binding region and the CaM-TSC2 interaction contributes to proper mTOR activity. However, other downstream signaling pathways/effectors activated by the CaM-TSC2 complex have not been fully elucidated. In this study, we found that activation of Ca2+/CaM signaling resulted in the translocation of membrane-associated TSC2 to the nucleus and suppressed the transcriptional activity of the vitamin D receptor (VDR). TSC2 was released from the membrane in an activated CaM-dependent state in rat brain and HeLa cells. It subsequently formed a transcriptional complex to partially suppress the transcription of CYP24A1, a well-known VDR target gene. These data suggest, in part, that TSC2 attenuates VDR-associated transcriptional regulation via Ca2+/CaM signaling.

Beneficial Effect of Dietary Fiber on Hyperuricemia in Rats and Humans: A Review.

Hyperuricemia is recognized as a lifestyle-related disease and the prevalence is increasing in many parts of the world. Excessive intake of purines increases serum uric acid concentration and is thought to be one factor causative of hyperuricemia. Therefore, it is thought that prevention of hyperuricemia by dietary control may be very important. This is an article of our basic research regarding the role of dietary fiber in the suppression of hyperuricemia induced by dietary purines in rats; in addition, clinical research suggesting the possibility that dietary fiber intake has a beneficial effect on the prevention or suppression of hyperuricemia in humans is discussed. Our own studies reveal that the test dietary fiber significantly suppresses the elevation of serum uric acid concentration induced by dietary RNA (12.3-46.2%), adenosine-5'-monophosphate (9.5-23.2%), adenosine (10.7-20.4%), or adenine (16.3-38.9%) and suppresses experimental hyperuricemia in rats. The mechanism is presumed to be mediated by suppression of the digestion and/or absorption of dietary purines by dietary fiber. In clinical studies, intake of dietary fiber decreases serum uric acid concentrations. Reports of recent epidemiological studies indicate that higher levels of hyperuricemia increase the prevalence of its comorbidities (e.g., chronic kidney disease, obesity, hypertension, diabetes, and cardiovascular disease) and dietary fiber intake is associated with significantly lower risk for hyperuricemia and its comorbidities. We wish to emphasize the importance of recognizing the validity of increased dietary fiber intake as a preventive or suppressive method for hyperuricemia and its comorbidities.

Metabolic abnormalities induced by mitochondrial dysfunction in skeletal muscle of the renal carcinoma Eker (TSC2+/-) rat model.

Tuberous sclerosis complex 2 (TSC2) is a mediator of insulin signal transduction, and a loss of function in TSC2 induces hyperactivation of mTORC1 pathway, which leads to tumorigenesis. We have previously demonstrated that Eker rat model, which is heterozygous for a TSC2 mutation, exhibits hyperglycemia and hyperketonemia. The present study was to investigate whether these changes also can affect metabolism in skeletal muscle of the Eker rat. Wild-type (TSC2+/+) and Eker (TSC2+/-) rats underwent an oral glucose tolerance test, and the latter showed decrease in whole-body glucose utilization. Additionally, reductions in the expression of glycolysis-, lipolysis-, and ketone body-related genes in skeletal muscle were observed in Eker rats. Furthermore, ATP content and mitochondrial DNA copy number were lower in skeletal muscle of Eker rats. These data demonstrate that heterozygous to mutation TSC2 not only affects the liver metabolism, but also skeletal muscle metabolism, via mitochondrial dysfunction.

High dietary fat-induced obesity in Wistar rats and type 2 diabetes in nonobese Goto-Kakizaki rats differentially affect retinol binding protein 4 expression and vitamin A metabolism.

Obesity is a major risk factor for type 2 diabetes, which is caused mainly by insulin resistance. Retinol binding protein 4 (RBP4) is the only specific transport protein for retinol in the serum. RBP4 level is increased in the diabetic state and high-fat condition, indicating that retinol metabolism may be affected under these conditions. However, the precise effect of diabetes and high fat-induced obesity on retinol metabolism is unknown. In this study, we examined differences in retinol metabolite levels in rat models of diet-induced obesity and type 2 diabetes (Goto-Kakizaki [GK] rat). Four-week-old male Wistar and GK rats were given either a control diet (AIN-93G) or a high-fat diet (HFD, 40% fat kJ). After 15 weeks of feeding, the RBP4 levels increased by 2-fold in the serum of GK rats but not HFD-fed rats. The hepatic retinol concentration of HFD-fed rats was approximately 50% that of the controls (P < .01). In contrast, the renal retinol concentrations of GK rats increased by 70% (P < .01). However, expression of RARß in the kidney, which was induced in a retinoic acid-dependent manner, was downregulated by 90% (P < .01) in GK rats. In conclusion, diabetes and obesity affected retinol metabolism differently, and the effects were different in different peripheral tissues. The impact of HFD may be limited to the storage of hepatic vitamin A as retinyl palmitate. In particular, our data indicate that renal retinoic acid production might represent an important target for the treatment of type 2 diabetes mellitus.

The tuberous sclerosis complex model Eker (TSC2+/-) rat exhibits hyperglycemia and hyperketonemia due to decreased glycolysis in the liver.

Tuberous sclerosis complex (TSC) presents as benign tumors that affect the brain, kidneys, lungs and skin. The inactivation of TSC2 gene, through loss of heterozygosity is responsible for tumor development in TSC. Since TSC patients are carriers of heterozygous a TSC2; mutation, to reveal the risk factors which these patients carry prior to tumor development is important. In this experiment, Eker rat which carry a mutation in this TSC2 gene were analyzed for their metabolic changes. Wild-type (TSC2+/+) and heterozygous mutant TSC2 (TSC2+/-) Eker rats were raised for 100 days. As a result, the Eker rats were found to exhibit hyperglycemia and hyperketonemia. However the high ketone body production in the liver was observed without accompanying increased levels of plasma free fatty acids or insulin. Further, production of the ketone body ß-hydroxybutyrate was inhibited due to the low NADH/NAD(+) ratio resulting from the restraint on glycolysis, which was followed by inhibition of the malate-aspartate shuttle and TCA cycle. Therefore, we conclude that glycolysis is restrained in the livers of TSC2 heterozygous mutant rats, and these defects lead to abnormal production of acetoacetate.

Vitamin B12 deficiency-induced increase of osteoclastic bone resorption caused by abnormal renal resorption of inorganic phosphorus via Napi2a.

Vitamin B12 deficiency is a risk factor for bone disorders via mechanisms not fully understood. In this study, an increase in serum inorganic phosphorus (Pi) concentrations was associated with a vitamin B12 deficiency. Napi2a, a renal cotransporter for Pi reabsorption, accumulated on plasma membranes in a vitamin B12 deficiency suggests that vitamin B12 plays an important role in Pi homeostasis.

Elevation of urinary methylmolonic acid induces the suppression of megalin-mediated endocytotic cycles during vitamin B12 deficiency.

Megalin is a scavenger receptor that serves in the endocytosis of a highly diverse group of ligands that includes Vitamin B12. We found an accumulation of megalin closed to apical region in renal proximal tubule cells of Vitamin B12-deficient rats. Interestingly, Vitamin B12 levels also controlled resorption of renal retinol binding protein. Using L2 yolk sac cells, megalin localized to the submembrane compartment by methylmalonic acid (MMA), which accumulates during vitamin B12 deficiency. In addition, MMA inhibited megalin-mediated endocytosis via YWTD repeats motif in an ectodomain of megalin. Therefore, megalin endocytosis may be regulated by MMA.

Influences of maternal B12 and methionine intake during gestation and lactation on testicular development of offspring in rats.

The influence of maternal vitamin B12 malnutrition on testicular development of offspring was examined using soy protein-based B12-deficient diets with or without 0.5% DL-methionine supplementation. Dams were fed the B12-deficient diet throughout gestation and lactation, whereas dams in a control group were fed a control diet which contained cyanocobalamin in the B12-deficient diet without methionine. Offspring born to dams fed the B12-deficient diet without methionine showed poor testicular development, e.g. decreased numbers of seminiferous tubules containing healthy spermatocytes and a high ratio of apoptotic cells per all germ cells. The abnormality was rarely observed in the group fed the B12-deficient diet with methionine. It was likely that the testicular abnormality of offspring was caused by B12-deficiency post partum and was prevented by the methionine supplementation. These observations suggested that maternal B12 nutritional status during the pre-weaning period is quite important for spermatogenesis of male offspring and that the requirement of B12 for testicular development is to produce active B12-dependent methionine synthase.

Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells.

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.

Identification of glucose transporter 4 knockdown-dependent transcriptional activation element on the retinol binding protein 4 gene promoter and requirement of the 20 S proteasome subunit for transcriptional activity.

Retinol binding protein 4 (RBP4) is the transport protein that carries retinol in blood. RBP4 was described recently as a new adipokine that reduced insulin sensitivity. Mice lacking glucose transporter 4 (GLUT4) in adipocytes have enhanced Rbp4 gene expression; however, the molecular mechanism is unknown. We found a G4KA (GLUT4 knockdown-dependent transcriptional activation) element located approximately 1.3 kb upstream of the Rbp4 promoter. Mutations within the G4KA sequence significantly reduced expression of the Rbp4 promoter-reporter construct in G4KD-L1 (GLUT4 knockdown 3T3-L1) adipocyte cells. In a yeast one-hybrid screen of a G4KD-L1 cell cDNA library, using the G4KA element as bait, we identified subunits of the 20 S proteasome, PSMB1 and PSMA4, as binding partners. In chromatin immunoprecipitation assays, both subunits bound to the G4KA element; however, only PSMB1 was tightly bound in the GLUT4 knockdown model. PSMB1 RNA interference, but not PSMA4, significantly inhibited Rbp4 transcription. Nuclear transportation of PSMB1 was increased in G4KD-L1 cells. These results provide evidence for an exclusive proteasome subunit-related mechanism for transcriptional activation of RBP4 within a GLUT4 knockdown model.

N-Formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam, KNK437 induces caspase-3 activation through inhibition of mTORC1 activity in Cos-1 cells.

The mammalian target of rapamycin complex 1 (mTORC1: mTOR-raptor interaction) and heat shock protein 70 (Hsp70) regulate various cellular processes and are crucial for the progression of many cancers and metabolic diseases. In the recent study, we reported that interaction of Hsp70 with tuberous sclerosis complex 1 (TSC1) regulated apoptosis. This study was designed to elucidate the underlying mechanism in Cos-1 cells. Here, we show that N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam (KNK437), which inhibits the expression level of Hsp70, abrogated phosphorylation of mTOR and S6K in response to insulin, and inhibited mTORC1 activity via disruption of an interaction between mTOR and raptor. In addition, KNK437 did not alter TSC1/2 complex formation. Furthermore, KNK437 inhibited the mTOR-raptor interaction on the outer membrane of the mitochondria and triggered caspase-3 activation. A reduction in the level of Hsp70 could result in the inhibition of the mTORC1 signaling pathway, thereby inducing apoptosis.

Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis.

Western blots are widely used for analysis of the expression levels of specific proteins. Blotting is conducted after sodium dodecyl sulfate or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels (one of which is stained) usually must be prepared, leading to the consumption of precious sample. Thus, we have developed a convenient and efficient Western blot method using a stained gel. This simple modification should be beneficial for the analysis of samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.

Phosphorylated hamartin-Hsp70 complex regulates apoptosis via mitochondrial localization.

The products of the tuberous sclerosis complex (TSC) genes, hamartin and tuberin, form a heterodimer. Recently we reported that hamartin directly interacted with Hsp70. However, the physiological implications of this interaction have not yet been clearly defined. Here we show that hamartin localized to the outer membrane of the mitochondria in an Hsp70-dependent manner. Moreover, phosphorylation of the T417 residue of hamartin was required for its localization to the mitochondria as well as its interaction with Hsp70. A non-phosphorylatable hamartin mutant at residue T417 was unable to localize to the mitochondria and suppress apoptosis, whereas non-phosphorylatable hamartin mutants T357A and T390A localized to the mitochondria and suppressed apoptosis. Importantly, non-phosphorylatable mutants (T357A, T390A and T417A) promoted apoptosis after treatment with Hsp 70-inhibitor KNK437. We conclude that hamartin inhibited apoptosis by localizing to the mitochondria and that its phosphorylation and binding to Hsp70 was required for facilitation of this process.

Hamartin-Hsp70 interaction is necessary for Akt-dependent tuberin phosphorylation during heat shock.

Hamartin and tuberin interact directly to regulate cell growth negatively. In this study, far-western blotting revealed that hamartin binds directly Heat shock protein 70 (Hsp70), even in the absence of tuberin. While the hamartin-tuberin complex acts as a sensor for a variety of types of stress, it is unclear how the complex is regulated under stress conditions. We found that the hamartin-Hsp70 interaction is stabilized during heat shock. On the other hand, tuberin underwent degradation through phosphorylation in an Akt-dependent manner. Furthermore, we found that when Hsp70 expression was inhibited by N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolactam (KNK437), Akt phosphorylation on site Ser308 diminished and tuberin was not phosphorylated at Thr1462 during heat shock. We conclude that both hamartin and Hsp70 increase in response to heat shock, whereas tuberin is phosphorylated and thereafter degraded via the PI3K/Akt pathway. Through this pathway, hamartin-Hsp70 plays a crucial role as a scaffolding protein that transfers the Akt signal to tuberin.

Iron deficiency down-regulates the Akt/TSC1-TSC2/mammalian Target of Rapamycin signaling pathway in rats and in COS-1 cells.

Iron deficiency (ID) is one of the most commonly known forms of nutritional deficiencies. Low body iron is thought to induce neurologic defects but may also play a protective role against cancer development by cell growth arrest. Thus, ID may affect cellular pathways controlling cell growth and proliferation, the mechanism of which is still not fully understood. The serine/threonine protein kinase Akt and its downstream target, the mammalian Target of Rapamycin (mTOR), is known to play a crucial role in the regulation of cell growth and survival. Therefore, we hypothesized that Akt/mTOR pathway could be influenced by ID. Three-week-old male Wistar-strain rats were divided into 3 groups and the 2 groups had free access to a control diet (C group) or an iron-deficient diet (D group). The third group (PF group) were pair-fed the control diet to the mean intake of the D group. After 4 weeks, rats were killed and their brains were sampled. In separate experiments, COS-1 cells were cultured with or without the iron chelator deferoxamine. Western blots of brain samples and COS-1 lysates were used to analyze the expression and phosphorylation state of Akt, TSC2, mTOR, and S6 kinase proteins implicated in the Akt/mTOR pathway. Using 2 different ID models, we show for the first time that iron deficiency depresses Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity.

Change of epigenetic control of cystathionine beta-synthase gene expression through dietary vitamin B12 is not recovered by methionine supplementation.

BACKGROUND/AIMS: Vitamin B12 (B-12) is an essential cofactor for methionine synthase, and methionine is critical for the methylation of various biological molecules including DNA. Whether changes in B-12 levels can alter specific gene expression through DNA methylation and whether dietary methionine has any effect on general DNA methylation status still remains controversial. METHODS: We raised severely B-12-deficient rats as well severely-B-12 deficient rats but supplemented with 5% methionine. mRNA levels of methionine cycle-related enzymes were analyzed. RESULTS: Gene expression patterns changed under B-12-deficient conditions but were recovered by dietary methionine supplementation to B-12-deficient rats. However, cystathionine beta-synthase mRNA levels, which had decreased under B-12-deficient conditions, did not recover with supplementary dietary methionine. The CpG island of the cystathionine beta-synthase promoter was hypomethylated in B-12-deficient rats, and showed no recovery after methionine addition. CONCLUSIONS: Dietary B-12 can affect epigenetic machinery by regulating DNA methylation status and dietary methionine may have small effects on DNA methylation.

Rab5 affinity chromatography without nonhydrolyzable GTP analogues.

Rab5 is an important small GTPase involved in endocytosis and membrane trafficking. Rab5-binding proteins can be identified using Rab5 affinity chromatography with nonhydrolyzable GTP analogues such as GTPgammaS or GppNHp. However, this method requires significant quantities of the GTP analogue and is thus time-consuming and expensive. In the present report we show a faster and more cost-effective method that does not use a GTP analogue but uses constitutively the active Rab5 mutant (Rab5Q79L) as a ligand. To validate this method, the binding of EEA-1 was confirmed and several novel Rab5-binding proteins were also identified by 2-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).

Lipid-soluble components of honeybee-collected pollen exert antiallergic effect by inhibiting IgE-mediated mast cell activation in vivo.

It was shown previously that bee-collected pollen (bee pollen, BP), inhibited in vitro murine mast cell activation. This study further analysed the antiallergic effect of BP in vivo by measuring cutaneous mast cell activation using a passive cutaneous anaphylaxis reaction. Daily oral administration of BP to mice, dose-dependently reduced the cutaneous mast cell activation elicited by IgE and specific antigens. Administration of BP also reduced the plasma concentration of malondialdehyde (MDA), an indicator of lipid peroxidation. The inhibitory effect of BP was mostly in a lipid- but not in water-soluble fraction. The HPLC analysis of isoflavones in BP revealed that genistein was a major isoflavone. However, administration of genistein alone at the concentration found in BP, did not show an inhibitory effect as observed in whole BP, suggesting that component(s) other than genistein would be responsible for the inhibitory effect of BP. These results first reveal that lipid-soluble components of BP exert an antiallergic action by inhibiting the FcåRI-mediated cutaneous mast cell activation.

Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis.

Western blot analysis has been a useful method for analysis of expression levels of specific proteins and is conducted after sodium dodecyl sulfate (SDS) or native polyacrylamide gel electrophoresis without staining the gel. However, when it is necessary to analyze the gel, duplicate polyacrylamide gels usually must be prepared, one of which is stained, leading to the consumption of precious sample. Thus, we developed a convenient and efficient Western blotting method using a stained gel. This simple modification should be beneficial for analyzing samples that are limited in quantity and/or samples for which the stained gel serves as the loading control.

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction.

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.