Evaluation of selective competitive binding of basic drugs to alpha1-acid glycoprotein variants.

We examined the binding of various basic drugs to the F(1)S and A genetic variants of alpha(1)-acid glycoprotein (AGP), which were isolated from native human commercial AGP (total AGP) by chromatography on an immobilized copper(II) affinity adsorbent. The values of the dissociation constant (K(d)) of some basic drugs with the F(1)S variant in equilibrium dialysis differed characteristically from those with the A variant. The selective binding to these variants was evaluated by measuring the displacement ratio of dicumarol bound to the F(1)S variant or that of acridine orange bound to the A variant, using circular dichroism spectroscopy. There was reasonably good agreement between the K(d) values and displacement ratios. There was a characteristic difference between the values of inhibition constant (K(i)) of basic drugs towards dipyridamole binding to F(1)S and towards disopyramide binding to A in total AGP. We found that the K(i) values for dipyridamole binding were well correlated with the K(d) values for the F(1)S variant, whereas those for disopyramide binding were well correlated with the K(d) values for the A variant. In conclusion, the higher the affinity of basic drugs for AGP, the more they inhibit the binding of other basic drugs, and further, the inhibitory potency depends on the selectivity of binding to the AGP variants.

Bone-targeting of quinolones conjugated with an acidic oligopeptide.

PURPOSE: Osteomyelitis is a progressive infectious process resulting in inflammatory destruction and necrosis of bone. The long-term administration of high-dosage antibiotics is required to treat osteomyelitis, owing to the limited distribution of antibiotics within bone. Therefore, targeted delivery of antibiotics to bone promises to improve therapeutic effectiveness. METHODS: We synthesized quinolones such as levofloxacin and norfloxacin conjugated to an acidic oligopeptide, which works as a bone-targeting carrier after systemic administration. The therapeutic effectiveness of the conjugated quinolones in osteomyelitis was evaluated using a mouse model of osteomyelitis, created by inoculating Staphylococcus aureus into the tibia of mice. RESULTS: With intravenous injection, the conjugated quinolones selectively distributed to bone, reaching concentrations up to 100-fold those of non-conjugated quinolones. Single intravenous injection of levofloxacin as well as conjugated levofloxacin exhibited antibiotic effects in the osteomyelitis mouse model; conversely, neither conjugated nor non-conjugated norfloxacin was effective. The antibiotic effect of conjugated levofloxacin persisted to at least 6 days after injection, whereas the effect of non-conjugated levofloxacin was temporary. CONCLUSION: The selective bone delivery of quinolones conjugated with an acidic oligopeptide may be effective in treating osteomyelitis, although the resulting concentration of antibiotic may be insufficient to completely kill S. aureus.

Inhibitory mechanisms of flavonoids on insulin-stimulated glucose uptake in MC3T3-G2/PA6 adipose cells.

We assessed the effects of different classes of flavonoids on insulin-stimulated 2-deoxy-D-[1-(3)H]glucose uptake by mouse MC3T3-G2/PA6 cells differentiated into mature adipose cells. Among the flavonoids examined, the flavones, apigenin and luteolin, the flavonols, kaempferol, quercetin and fisetin, an isoflavone, genistein, a flavanonol, silybin, and the flavanols, (-)-epigallocatechin gallate (EGCG) and theaflavins, significantly inhibited insulin-stimulated glucose uptake. Key structural features of flavonoids for inhibition of insulin-stimulated glucose uptake are the B-ring 4'- or 3',4'-OH group and the C-ring C2-C3 double bond of the flavones and flavonols, the A-ring 5-OH of isoflavones, and the galloyl group of EGCG and theaflavins. Luteolin significantly inhibits insulin-stimulated phosphorylation of insulin receptor-beta subunit (IR-beta), and apigenin, kaempferol, quercetin and fisetin, also tended to inhibit the IR-beta phosphorylation. On the other hand, isoflavones, flavanols or flavanonols did not affect insulin-stimulated IR-beta phosphorylation. Apigenin, luteolin, kaempferol, quercetin and fisetin also appeared to inhibit insulin-stimulated activation of Akt, a pivotal downstream effector of phosphatidylinositol 3-kinase (PI3K), and suppressed insulin-dependent translocation of a glucose transporter, (GLUT)4, into the plasma membrane. Although genistein, silybin, EGCG and theaflavins had no effect on the insulin-stimulated activation of Akt, they blocked insulin-dependent GLUT4 translocation. These results provide novel insights into the modulation by flavonoids of insulin's actions, including glucose uptake in adipocytes.

Caffeine dose-dependently potentiates the antitumor effect of cisplatin on osteosarcomas.

BACKGROUND: Caffeine-assisted chemotherapy has been used in the treatment of osteosarcomas. However, there is little in vivo evidence for this treatment, and thus we sought to verify the dose and effect of caffeine in combination with cisplatin in osteosarcoma-bearing rats. MATERIALS AND METHODS: Seven-week-old male Fischer rats were transplanted with chemical carcinogen-induced osteosarcoma, selected lung metastatic lesions tumor block. The osteosarcoma-bearing rats were treated with saline, caffeine, cisplatin or cisplatin and caffeine. RESULTS: The most growth inhibition was observed in the co-administration group. When three different dosing schedules of caffeine were given, the extent of tumor inhibition was closely correlated with the average plasma concentration of caffeine. The cisplatin concentration in the tumor was significantly increased when caffeine was co-administered. CONCLUSION: This study confirms that a high concentration of caffeine (about 0.4 mM) is effective in enhancing the antitumor effects of cisplatin.

Targeted drug delivery to bone: pharmacokinetic and pharmacological properties of acidic oligopeptide-tagged drugs.

Site-specific drug delivery to bone is considered to be achievable by utilizing acidic amino acid homopeptides. We found that fluorescence-labeled acidic amino acid (L-Asp or L-Glu) homopeptides containing six or more residues bound strongly to hydroxyapatite, which is a major component of bone, and were selectively delivered to and retained in bone after systemic administration. We explored the applicability of this result for drug delivery by conjugation of estradiol and levofloxacin with an L-Asp hexapeptide. We also similarly tagged an enzyme, tissue-nonspecific alkaline phosphatase, to see whether this would improve the efficacy of enzyme replacement therapy. The L-Asp hexapeptide-tagged drugs, including the enzyme, were selectively delivered to bone in comparison with the untagged drugs. It was expected that the ester linkage to the hexapeptide would be susceptible to hydrolysis in situ, releasing the drug or enzyme from the acidic oligopeptide. An in vivo experiment confirmed the efficacy of L-Asp hexapeptide-tagged estradiol and levofloxacin, although there was some loss of bioactivity of estradiol and levofloxacin in vitro, suggesting that the acidic hexapeptide was partly removed by hydrolysis in the body after delivery to bone. The adverse effect of estradiol on the uterus was greatly reduced by conjugation to the hexapeptide. These results support the usefulness of acidic oligopeptides as bone-targeting carriers for therapeutic agents. We present some pharmacokinetic and pharmacological properties of the L-Asp hexapeptide-tagged drugs and enzyme.

Soybean fat supplementation controls insulin resistance caused by fat-free total parenteral nutrition.

Conscious young adult male rats were given total parenteral nutrition (TPN) with or without soybean fat for 4 days. Those given fat-free TPN developed severe fatty liver, with hyperglycaemia, hyperinsulinaemia, and hypotriglyceridaemia. These disorders were clearly improved by supplementing TPN with soybean fat, in an amount equivalent to 20% of total calories, and correspondingly reducing glucose. Insulin resistance also developed over a 4-day infusion of fat-free TPN in mature rats. Even after over-night fasting after stopping the TPN infusion, the levels of serum glucose and insulin were higher in the fat-free TPN group than in the control group, and intravenous glucose tolerance test results indicated insulin resistance in the fat-free TPN group. The HOMA-IR index of insulin resistance was significantly improved by supplementation with soybean fat. In conclusion, fat-free TPN infusion induced hyperglycaemia and hyperinsulinaemia, leading to fatty liver and insulin resistance. TPN with glucose should be supplemented with soybean fat emulsion as replacement for part of the glucose calories.

Mechanism of decrease of oral bioavailability of cyclosporin A during immunotherapy upon coadministration of amphotericin B.

The trough level of blood concentration of cyclosporin A (CyA) in a patient receiving immunotherapy was observed to decrease following coadministration of amphotericin B (AMB). This clinical observation was confirmed experimentally in Wistar rats intravenously given AMB (1.5 or 3.0 mg/kg) or saline (control) for 4 days, followed by CyA (10 mg/kg). The blood concentration of CyA after i.v. or p.o. administration in both AMB groups was significantly decreased compared with the control. The oral bioavailability of CyA after 1.5 or 3.0 mg/kg AMB treatment was decreased to 67% or 46%, respectively, of that of the control group. AMB treatment increased the expression levels of mdr1a and mdr1b mRNAs in the duodenum to about three times the control, and expression of CYP3A2 mRNA in the liver was increased to about twice the control. The P-gp and CYP3A2 proteins were increased significantly. These findings suggest that the oral bioavailability of CyA is reduced as a result of both increased efflux transport via P-glycoprotein in the duodenum and an increased first-pass effect of CYP3A2-mediated hepatic metabolic activity, induced by AMB. It is suggested that careful monitoring of CyA levels is necessary in the event of AMB administration to patients receiving immunotherapy with CyA.

Influence of chronic hepatic failure on disposition kinetics of valproate excretion through a phase II reaction in rats treated with carbon tetrachloride.

The influence of chronic hepatic failure on the disposition kinetics of valproate (VPA) excretion via a phase II reaction was examined in rats treated with carbon tetrachloride (1.0 mg/kg, s.c., 3 times a week) for 2 or 3 months. There was no significant difference in the plasma concentration-time courses of VPA among the control and two treated groups up to 120 min after i.v. administration of VPA (75 mg/kg), but subsequently the plasma concentrations of the treated groups declined significantly below the control levels. Expression of Mrp2 mRNA in the liver of the treated groups was significantly lower than in the control group; conversely that in the kidney was significantly higher. The enzyme activity of UGTs in the liver of the treated groups decreased significantly, but UGT1A8 mRNA expression in the duodenum was increased about 3-fold. Cumulative excretion of VPA glucuronide (VPA-G) in bile of the treated groups was reduced significantly, while that in urine was markedly increased. In conclusion, the area under the VPA plasma concentration-time curve was decreased significantly in rats with chronic hepatic failure owing to increased excretion of VPA-G via the kidney as a result of induction of Mrp2, and inhibition of enterohepatic circulation of VPA-G.

Circadian rhythm of cytochrome P4502E1 and its effect on disposition kinetics of chlorzoxazone in rats.

The aim of this report is to study the circadian rhythm of cytochrome P4502E1 (CYP2E1) and its effect on the disposition kinetics of chlorzoxazone in male Wistar rats. The rats were housed under a 12-h light/dark cycle (lights from 9:00 to 21:00) with food and water ad libitum for 3 months. It was found that the expression of microsomal CYP2E1 mRNA in the liver during the dark phase was significantly lower than during the light phase, whereas the content of CYP2E1 protein and its hydroxylation activity were significantly higher. Therefore, chlorzoxazone 20 mg/kg was intravenously administered at 12:00 (light phase group) or 24:00 (dark phase group) to determine the effect on the disposition kinetics. The value of the area under the plasma concentration-time curve from 0 to 8 h (AUC(0-8 h)) of chlorzoxazone showed no significant difference between the two groups. However, the value of chlorzoxazone half-life in plasma of the light phase group was significant longer than the dark phase group. The AUC(0-8 h) of 6-hydroxychlorzoxazone, a metabolite formed from chlorzoxazone mainly by CYP2E1, was significantly higher in the dark phase than in the light phase. In conclusion, microsomal CYP2E1 shows a substantial circadian variation in rats, and this was associated with a decrease of chlorzoxazone half life, and an increase of 6-hydroxychlorzoxazone production. Therefore, the temporal variations of therapeutic response and toxicological effects may have to be taken into consideration for other xenobiotics that are predominantly metabolized by CYP2E1, particularly those with a short half-life.

Involvement of alpha1-acid glycoprotein in inter-individual variation of disposition kinetics of ropivacaine following epidural infusion in off-pump coronary artery bypass grafting.

The influence of drug interaction and protein variants on the binding disposition of ropivacaine to alpha1-acid glycoprotein (AGP) was examined. The subjects were five patients who received epidural infusion of ropivacaine for 24-54 h in off-pump coronary artery bypass grafting followed by drug combination therapy, and 10 healthy volunteers. The post-operation plasma albumin concentration showed little overall change, while the AGP concentration in the five patients decreased for 6 h, then increased gradually to about 3-times the initial value by 54 h. The unbound fraction in plasma (fu) of ropivacaine gradually decreased as the AGP concentration increased, but there was large inter-individual variation among the five patients. In contrast, there was a good correlation between the fu value and AGP concentration when ropivacaine was added to blood samples from the 10 healthy volunteers. Among the volunteers, eight showed F1S variants and two showed F1 variant without S variant of AGP. The fu value of ropivacaine did not differ between these two groups. However, when ropivacaine was added in combination with dipyridamole, the fu values of ropivacaine in blood from volunteers with F1S variants were greater than those in blood from volunteers without S variant. In the case of co-administration of disopyramide or lidocaine, there was no such difference. Among the patients, one showed F1S variants and four showed F1 variant without S variant. The results indicate that variability in the side-effects of therapy with ropivacaine alone is caused by the change of the unbound concentration upon changes in the AGP concentration. However, in combination therapy, it is also important to consider the AGP variant-dependence of the inhibitory effect of concomitantly administered drugs.

Structure-activity relationship of flavonoids for inhibition of epidermal growth factor-induced transformation of JB6 Cl 41 cells.

We found that quercetin, myricetin, quercetagetin, fisetin, (-)-epigallocatechin gallate (EGCG), and theaflavins, among 24 flavonoids examined, markedly inhibited epidermal growth factor (EGF)-induced cell transformation of mouse epidermal JB6 Cl 41 cells. The six flavonoids suppressed the EGF-induced activation of activator protein 1 (AP-1). In addition, myricetin, quercetagetin, EGCG, and theaflavins directly inhibited EGF-induced phosphatidylinositol 3-kinase (PI3K) activation. The important structural features of flavonoids for cell transformation-inhibitory activity are 3'- and 4'-OH on the B-ring, 3-OH on the C-ring, C2=C3 double bond in the C-ring, and the phenylchromone (C6-C5-C6) skeleton in the flavonols, and the galloyl group in EGCG and theaflavins. Our results provide new insight into possible mechanisms of the anti-carcinogenic effects of flavonoids, and could help to provide a basis for the design of novel cancer chemopreventive agents.

Obesity-induced increase of CYP2E1 activity and its effect on disposition kinetics of chlorzoxazone in Zucker rats.

This study was designed to investigate the induction of CYP2E1 in obese Zucker rats and its effect on the disposition kinetics of chlorzoxazone (CZX). CZX 20mg/kg was administered to three groups of rats: normal Zucker rats fed a normal diet (ND), normal Zucker rats fed a high-fat diet (HF), and genetically obese Zucker rats fed a normal diet (OB). The values of the area under the plasma concentration-time curve from 0 to infinity (AUC(infinity)) of CZX were in the order of ND>HF>OB rats. The AUC(infinity) values of total 6-hydroxychlorzoxazone (6OHCZX-T), which is considered to be a CYP2E1 metabolic marker, were in the opposite order. The values of the AUC(infinity) ratio (6OHCZX-T/CZX) in ND, HF and OB rats were approximately 0.2, 0.3 and 0.4, respectively. The CZX concentration in fat was much higher than the concentrations in plasma, liver and kidney in all groups. Induction of CYP2E1 protein was greater in both liver and fat of OB rats than in those of HF rats. Microsomal activity of CYP2E1 in liver and fat was also in the order of OB>HF>NM rats. These results suggest that CYP2E1 may be induced in liver and fat of obese patients, thereby potentially altering the disposition kinetics of not only CZX, but also other lipophilic drugs metabolized by CYP2E1.

Development of dosage design of hepatic metabolizing drugs using serum albumin level in chronic hepatic failure.

We have previously reported good correlations among serum aminotransferase (AST) activity, metabolic enzyme activity of CYPs, and total clearance (CL(tot)) of probe drugs in rats with acute hepatic failure induced by CCl4. In this study, we searched for new biochemical indicators that correlate with hepatic function and tried to simulate appropriate drug dosage in chronic hepatic failure. Model rats were prepared by administration of CCl4 (1 ml/kg, s.c., 3 times/week) and used at 48 h after the last administration. Serum albumin concentration was time-dependently decreased and correlated well with 3 major biologic determinants of drug clearance, hepatic blood flow (HBF), intrinsic clearance (CL(int)), and the unbound fraction of drugs in plasma (fp) after intravenous administration of cyclophosphamide, tolbutamide, zonisamide, and chlorzoxazone (as probe drugs for low hepatic extraction) and propranolol and lidocaine (as high-hepatic extraction drugs). By calculating these parameters based on prediction equations by the level of albumin, CL(tot) was obtained. As a result of having evaluated this model using administration of cyclosporin, there was a statistically significant relationship between predicted CL(tot) and observed CL(tot). In conclusion, the value of serum albumin level is a useful parameter that correlates well with chronic hepatic function. We have shown that this quantitative administering design using serum albumin level can predict appropriate dosages of hepatic metabolizing drugs in chronic hepatic failure.

Site-dependent contributions of P-glycoprotein and CYP3A to cyclosporin A absorption, and effect of dexamethasone in small intestine of mice.

We examined whether the oral bioavailability of cyclosporin A is controlled primarily by P-glycoprotein (P-gp) or CYP3A in the small intestine. In situ loop method was used to evaluate the uptake of cyclosporin A (40nmol) at the upper and lower intestine of wild-type and mdr1a/1b knockout mice treated or not treated with dexamethasone (75mg/kg/day, 7 days, i.p.). Expression of CYP3A mRNA in the control group was higher in the upper than the lower intestine, while that of the multidrug resistance-1a (mdr1a) mRNA was in the opposite order. Dexamethasone administration potently induced CYP3A and mdr1a mRNAs in the lower and upper intestine, respectively. At 45min after cyclosporin A administration into an upper intestinal loop of the control group of wild-type mice, the ratio of residual cyclosporin A to dose did not differ significantly from that of mdr1a/1b knockout mice, whereas in dexamethasone-treated wild-type mice, the residual ratio was increased significantly. The ratio of the cyclosporin A metabolite M17 to cyclosporin A in portal venous blood at an upper intestinal loop of mdr1a/1b knockout mice was much higher than that a lower intestinal loop. The M17/cyclosporin A ratio of portal venous blood at a lower intestinal loop in mdr1a/1b knockout mice was increased significantly by dexamethasone treatment. These results suggest that, under physiological conditions, the oral bioavailability of cyclosporin A is mainly controlled by CYP3A in the upper intestine, rather than liver, but when P-gp is induced by steroid, the intestinal absorption of cyclosporin A may be inhibited.

Phosphodiesterase 4 inhibitor rolipram potentiates the inhibitory effect of calcitonin on osteoclastogenesis.

To assess the combination effect of calcitonin and the phosphodiesterase 4 inhibitor rolipram on osteoclastogenesis, adherent cell-depleted bone marrow cells from mouse tibia and femur (ACD-BMCs), which were cultured in the presence of 25 ng/ml colony-stimulating factor 1 (CSF-1) and 100 ng/ml soluble receptor activator of NF-kappaB ligand (sRANKL), were utilized. Calcitonin inhibited formation of tartrate-resistant acid phosphatase-positive multinucleated cells, as mature osteoclasts, by 70% even at 20 pM, whereas rolipram (10 microM) scarcely affected osteoclast formation; in contrast, the combination of both agents led to significant inhibition of multinucleation and pit formation ability of osteoclasts. The combined administration of calcitonin and rolipram attenuated calcitonin receptor mRNA expression in comparison to treatment with either agent alone, whereas expression of RANK and CSF-1 receptor mRNAs was unchanged. Alone, these agents scarcely elevated intracellular cyclic AMP (cAMP) concentration; however, combination treatment with both agents significantly increased cAMP concentration in osteoclast progenitors and osteoclasts. The combination effect was abolished by H-89, an inhibitor of protein kinase A. It appears that rolipram inhibited hydrolysis of cAMP formed by calcitonin in cells and potentiated the inhibitory effect of calcitonin on osteoclastogenesis. The escape phenomenon following calcitonin treatment may also be prevented by concomitant treatment with the phosphodiesterase 4 inhibitor.

Pharmacokinetic advantage of an intranasal preparation of a novel anti-osteoporosis drug, L-Asp-hexapeptide-conjugated estradiol.

We examined the usefulness of intranasal (i.n.) administration of a novel osteotropic prodrug of estradiol, estradiol-17beta-succinate-(L-aspartate)6 (E2.17D6), for selective drug delivery to bone. E2.17D6 alone or with 5% 2,6-di-O-methyl-beta-cyclodextrin (DMbetaCD), 5% beta-cyclodextrin (betaCD), or 10% hydroxypropyl cellulose (HPC) as an absorption enhancer was administered to ovariectomized (OVX) mice via the i.n. route. The oral and nasal bioavailability after p.o. or i.n. administration of E2.17D6 (3.7 micromol/kg) in mice amounted to 9.9 and 23.0% of the dose, respectively. The values of nasal bioavailability of E2.17D6 administered with DMbetaCD, betaCD, and HPC were 74.9, 55.8, and 49.1%, respectively. The plasma concentration of E2.17D6 after i.n. administration of E2.17D6-DMbetaCD decreased rapidly to the endogenous level by 6 h, but the concentration in the bone was about 200 times higher than that in plasma, and decreased slowly over a period of about a week. When E2 (total dose 4.4 micromol/kg, i.n., every 3rd day) was administered to OVX mice for 35 d, bone mineral density (BMD), liver weight, and uterus weight increased, whereas E2.17D6-DMbetaCD (total dose 0.44 to 8.8 micromol/kg, i.n., every 7th day) increased only BMD in a dose-dependent manner. In conclusion, intranasally administered E2.17D6-DMbetaCD has a potent antiosteoporotic effect without side effects, and has potential to provide an improved quality of life for patients with osteoporosis.

Serum aminotransferase activity as a predictor for estimation of total clearance of hepatically metabolized drugs in rats with acute hepatic failure.

The levels of serum aminotransferase activity, including aspartate aminotransferase (AST), in rats with acute hepatic failure at 24 h after an oral administration of CCl4 (0.01-0.5 ml/kg) were about 15-50 times higher (up to nearly 5000 IU/l) than those of vehicle control rats (about 85 IU/l). The values of total clearance (CL(tot)) of cyclosporin A, doxorubicin, tacrolimus and zonisamide in the CCl4-treated rats were decreased to about 1/2-1/3 of those in control rats. There were good correlations between AST activity and hepatic intrinsic clearance (CL(int)) (r=0.733-0.949) for the above drugs, as well as for chlorzoxazone, caffeine, lidocaine and tolbutamide after the intravenous administration of each drug in rats with acute hepatic failure. However, the slope of the linear regression equation, i.e., the ratio of decrease of CL(int) against increase of AST activity, differed markedly among these drugs. We found that there is a good correlation (r=0.953) between the values of the slope and the CL(int) of normal rats for these drugs, except for caffeine. In summary, the linear regression equation enables us to predict the decrease of CL(tot) in rats with acute hepatic failure to be predicted from the increase in serum AST activity. This approach may be useful as a guide for the dose modification of drugs for patients with acute hepatic failure.

Contributions of intestinal P-glycoprotein and CYP3A to oral bioavailability of cyclosporin A in mice treated with or without dexamethasone.

The contributions of P-glycoprotein (P-gp) and CYP3A to the oral bioavailability (BA) of cyclosporin A (CyA) were separately evaluated by using wild-type and mdr1a/1b knockout mice treated with dexamethasone (DEX). Mice were treated with DEX (1 or 75 mg/kg/day, i.p.) daily for 7 days, and the blood concentrations of CyA were measured after an i.v. or p.o. dose of CyA (10mg/kg) at 1.5h after the last DEX treatment. The BA values of CyA in wild-type and mdr1a/1b knockout mice were similar, 0.25 and 0.287, respectively. As regards expression of mdr1a and CYP3A mRNAs, expression of mdr1a mRNA was weakest in the duodenum, the main absorption site of CyA, along the whole intestine of wild-type mice, while expression of CYP3A was strongest in the duodenum of both types of mice. After treatment with 1 and 75 mg/kg DEX, the BA values decreased to 43 and 25% of the control in wild-type mice, respectively, and to 89 and 73% of the control in mdr1a/1b knockout mice, respectively. Expression of mdr1a mRNA in duodenum of wild-type mice was potently induced by DEX treatment. The expression of CYP3A mRNA in liver and duodenum of both strains was enhanced only by high-DEX treatment. These results suggest that P-glycoprotein plays only a small role in the absorption of CyA under physiological conditions, but the protein is readily induced by DEX and then functions as a more substantial absorption barrier to CyA than does CYP3A in the intestine.

Insulin signaling in adipocytes differentiated from mouse stromal MC3T3-G2/PA6 cells.

The stromal MC3T3-G2/PA6 (PA6) cells from mouse clavaria did not require insulin for differentiation into mature adipose cells, although insulin is well known to play a key role in adipocyte differentiation. Large lipid droplets were observed in the cytoplasm of PA6 cells, and mRNA expression of the adipose specific proteins (aP2, PPARgamma, C/EBPalpha, FAS, GLUT4, leptin, and adiponectin) as differentiation markers appeared or increased clearly in the cells at 8 d after stimulation without insulin. In addition, the glycerol released from the cells (lipolysis) was increased in a concentration-dependent manner by isoproterenol. However, the isoproterenol-induced lipolysis in the cells was not influenced by treatment with insulin, although that was observed in extramedullary adipocytes, 3T3-L1 cells. On the other hand, the 2-deoxy-D-[1-3H]glucose uptake in differentiated PA6 cells also increased by insulin, as shown in other adipose cells. In the cells, insulin induced the phosphorylation of extracellular signal-regulated kinases (Erks), Akt at Ser 473 and ribosomal p70 S6 protein kinase (p70 S6K) at Thr 389, and the insulin-induced 2-deoxy-D-[1-3H]glucose uptake was inhibited by pre-treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or ML-9, an Akt inhibitor. These results suggest that the insulin signal for adipogenesis (lipogenesis) and lipolysis in bone marrow stroma PA6 cells differs from extramedullary adipocytes, such as 3T3-L1 cells.