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Biochemical Pharmacology (v.72, #12)

Editorial Advisory Board (pp. iii).

Recognition forces in ligand–protein complexes: Blending information from different sources by Giuseppe Ermondi; Giulia Caron (pp. 1633-1645).

A variety of ligands interact with proteins in many biological processes; shape complementarity, electrostatic forces and hydrophobicity are the main factors governing these interactions. Although this is accepted by the scientific community, confusion about the significance of certain terms (e.g. hydrophobicity, salt bridge) and the difficulty of discussing the balance of acting forces rather than their single contributions, are two of the main problems encountered by researchers working in the field. These difficulties are sometimes enhanced by the unskilled use of informatics tools, which give great help in understanding the topic (especially from the visual standpoint), but only if used critically. After explaining some general chemical concepts, the commentary discusses the main forces governing ligand–protein interactions, focusing on those generating confusion among scientists with different backgrounds. Three examples of ligand–protein interactions are then discussed to illustrate the advantages and drawbacks of some in silico tools, highlighting the main interactions responsible for complex formation. The same examples are used to point out the limits in separating forces that are mandatory for occurrence of a given interaction and additional forces.

Keywords: Abbreviations; α; mean polarizability; aq; aqueous state; COX-1; cyclooxygenase isoenzyme; CSD; Cambridge Structural Database; E; internal energy; E; ′; electric field; F; (; r; ); force; G; Gibbs free energy; g; gas state (or vacuum); H; enthalpy; Ĥ; Hamiltonian operator; hERG; human ether á go-go related gene; MD; molecular dynamics; MIF; molecular interaction field; r; distance between particles; P; pressure; PDB; Protein Data Bank; p; permanent dipole moment; p; _ind; induced dipole moment; q; charge of a particle; S; entropy; U; potential function; V; volume of the systemBinding; Intermolecular forces; Hydrophobic effect; Solvated systems; Crystallography; Contacts

Human equilibrative nucleoside transporter-1 (hENT1) is required for the transcriptomic response of the nucleoside-derived drug 5′-DFUR in breast cancer MCF7 cells by Míriam Molina-Arcas; Gema Moreno-Bueno; Pedro Cano-Soldado; Héctor Hernández-Vargas; F. Javier Casado; José Palacios; Marçal Pastor-Anglada (pp. 1646-1656).

Nucleoside analogues are broadly used in cancer treatment. Although nucleoside metabolism is a necessary step in the development of their cytotoxicity, mediated transport across the plasma membrane might be needed for nucleoside-derived drugs to exert their pharmacological action. In this study, we have addressed the question of whether particular plasma membrane transporters contribute to the transcriptomic response associated with nucleoside-derived drug therapy. Firstly, we have characterized the nucleoside transporters responsible for 5′-DFUR uptake into the breast cancer cell line MCF7. 5′-DFUR is the immediate precursor of 5-FU and a metabolite of the orally administered pro-drug capecitabine, currently used in the treatment of breast cancer and other solid tumors. Although 5′-DFUR is a substrate for both plasma membrane equilibrative nucleoside carriers, hENT1 shows higher affinity for this molecule than hENT2. Inhibition of hENT1 function partially protected MCF7 cells from 5′-DFUR-induced cytotoxicity. Secondly, we have used a pharmacogenomic approach to determine how inhibition of hENT1 function contributes to the transcriptomic response associated to 5′-DFUR treatment. Under hENT1 inhibition most of the transcriptional targets of 5′-DFUR action, which were genes associated with apoptosis and cell cycle progression were blocked. This study demonstrates that although 5′-DFUR is substrate for both equilibrative nucleoside carriers, hENT1 function is essential for the full transcriptional response to 5′-DFUR treatment.

Keywords: hENT1; 5′-DFUR; Nucleoside transporter; cDNA microarray; Breast cancer

Mitochondrial permeability transition induced by novel pyridothiopyranopyrimidine derivatives: Potential new antimitochondrial antitumour agents by Lisa Dalla Via; Anna Maria Marini; Silvia Salerno; Antonio Toninello (pp. 1657-1667).

New pyridothiopyranopyrimidine derivatives (PTP1 and PTP2) were synthesised. Evaluation of the antiproliferative activity showed a significant capacity of the two compounds to inhibit cell growth. Investigation of the mechanism of action reveals that PTP1 interferes with the mitochondrial functions by inducing both swelling of the mitochondrial matrix and collapse of the electrical potential. These phenomena are fully prevented by typical inhibitors of the mitochondrial permeability transition, and are accompanied by the release of cytochrome c in the cytosol. The estimation of the redox state of thiol groups and glutathione suggests that the induction of permeability transition mediated by PTP1 is the result of an oxidative stress. The ability of cyclosporin A to prevent the antiproliferative effect of PTP1 indicates the induction of mitochondrial permeability transition as the molecular event responsible for the inhibition of cell growth. PTP1 also induces DNA fragmentation in intact cells.As regards PTP2, the presence of the p-toluensulphonamido group makes the lead chromophore unable to induce any effect on mitochondria.

Keywords: Pyridothiopyranopyrimidine derivatives; Antiproliferative activity; Mitochondria; Reactive oxygen species; Permeability transition

Induction of G2/M phase arrest and apoptosis of human leukemia cells by potent antitumor triazoloacridinone C-1305 by Ewa Augustin; Anna Moś-Rompa; Anna Skwarska; Jacek M. Witkowski; Jerzy Konopa (pp. 1668-1679).

In this study, we show that triazoloacridinone derivative C-1305, a potent antitumor compound, in human lymphoblastic (MOLT4) and promyelocytic (HL60) leukemia cells induces G2/M arrest followed by apoptosis. In both type of cells, C-1305 at biological relevant concentrations corresponding to EC₉₀ value, induced a significant increase in the fraction of G2/M cells. The cell cycle perturbations were accompanied by the appearance of sub-G1 fraction, which can be considered as the apoptotic cells population. In both human leukemia cells apoptosis was additionally proved by appearance of DNA fragmentation, activation of caspase-3, PARP cleavage, externalization of phosphatydilserine as well as decrease of the mitochondrial transmembrane potential Δ Ψ_m and ATP depletion. Treatment of lymphoblastic MOLT4 cells with the C-1305 at EC₉₀ concentration, caused massive death by apoptosis. Compared to MOLT4 cells, the capacity of HL60 cells to execute apoptosis after C-1305 treatment at equitoxic dose was significantly weaker, but very effective at high concentration (4× EC₉₀). These differences could originate from different sensitivity of both cell types to cytotoxic action of C-1305 (EC₅₀ value for MOLT4 cells was 8 times lower than for HL60 cells and the EC₉₀ value was 14 times lower, respectively). Collectively, these results show that C-1305 is a novel and potent compound which induces G2/M arrest and subsequent apoptosis of human leukemia cells. This strong ability to induce apoptosis of tumor cells support the view that C-1305 could be consider as a new potent and promising antitumor agent.

Keywords: Triazoloacridinones; C-1305; G2/M arrest; Apoptosis; Caspases; Mitochondria

Cryptotanshinone from Salvia miltiorrhiza BUNGE has an inhibitory effect on TNF-α-induced matrix metalloproteinase-9 production and HASMC migration via down-regulated NF-κB and AP-1 by Seok-Jong Suh; Un-Ho Jin; Hee-Jung Choi; Hyen Wook Chang; Jong-Keun Son; Seung Ho Lee; Su-Jin Jeon; Kun-Ho Son; Young-Chae Chang; Young-Choon Lee; Cheorl-Ho Kim (pp. 1680-1689).

Matrix metalloproteinases (MMP-9 and MMP-2) production and smooth muscle cell (SMC) migration may play key roles in the phathogenesis of neointima formation and atherosclerosis. Especially inducible MMP-9 expression was directly involved in the cancer cell invasion and SMC migration through vascular wall. In this study, we reveal that cryptotanshinone (CT) purified from Salvia miltiorrhiza BUNGE had an inhibitory effect on MMP-9 production and migration of human aortic smooth muscle cells treated with TNF-α in a dose-dependent manner. The down regulation of transcription of MMP-9 mRNA was evidenced by RT-PCR and MMP-9 promoter assay using luciferase reporter gene. Eletrophoretic mobility shift assay showed NF-κB and AP-1 nuclear translocations were suppressed. In addition, Western blot analysis indicated that extracellular signal regulated kinase 1 and 2, p38 and JNK MAP kinase signaling pathways were inhibited. From the results, it is suggested that CT has anti-atherosclerosis and anti-neointimal formation activity.

Keywords: Cryptotanshinone; Salviae miltiorrahizae (; Salvia miltiorrhiza; Bunge); Matrix metalloproteinase-9; Human aortic smooth muscle cells; TNF-α; Migration

Synergistic effects of the anti-cholinergic R, R-glycopyrrolate with anti-inflammatory drugs by Andreas Pahl; Artur Bauhofer; Ursula Petzold; Peter J. Cnota; Joachim Maus; Kay Brune; Stefan Szelenyi (pp. 1690-1696).

Currently, much effort is geared towards developing therapies that impact on the inflammation in respiratory diseases such as asthma and COPD, assuming that this will improve disease pathology. R, R-Glycopyrrolate, a quaternary ammonium compound, is a muscarinic receptor antagonist with the potential to be used as a long-acting bronchodilator in patients with asthma and COPD. In this study we evaluated whether the combination of R, R-glycopyrrolate with known anti-inflammatory drugs results in synergistic effects. Human primary monocytes were used as an in vitro model system. M3, M4, M1 and M2 receptors were expressed in these cells in descending order. The combinatory effects of the drugs on the release of TNF-α after lipopolysaccharide stimulation were analyzed. R, R-Glycopyrrolate alone did not affect LPS induced TNF-α release. The PDE4 inhibitor rolipram dose dependently inhibited the TNF-α release. Maximum inhibition was around 70%. The IC₃₅ for rolipram was 68.9±15.2nM. The simultaneous administration of 10μM R, R-glycopyrrolate reduced the IC₃₅ to 1.70±1.18nM. The anti-histamine azelastine inhibited TNF-α release dose dependently. The simultaneous administration of R, R-glycopyrrolate did not influence the action of azelastine. The corticosteroid budesonide inhibited the TNF-α release dose dependently with an IC₅₀ of 0.55±0.13nM. The simultaneous administration of 10μM R, R-glycopyrrolate reduced the IC₅₀ to 0.13±0.03nM. Finally, R, R-glycopyrrolate was most effective in the triple combination with budesonide and rolipram in the reduction of TNF-α release. In conclusion, R, R-glycopyrrolate acts synergistically with the PDE4 inhibitor rolipram and the steroid budesonide in inhibiting inflammatory mediators.

Keywords: Asthma; COPD; Inflammation; Anti-cholinergic; R; ,; R; -Glycopyrrolate

R-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone (WIN-2) ameliorates experimental autoimmune encephalomyelitis and induces encephalitogenic T cell apoptosis: Partial involvement of the CB₂ receptor by Antonio J. Sánchez; Paz González-Pérez; Ismael Galve-Roperh; Antonio García-Merino (pp. 1697-1706).

Many reports have shown that cannabinoids might be beneficial in the symptomatic treatment of multiple sclerosis (MS). We have investigated the therapeutic properties of the non-selective cannabinoid receptor agonist WIN-2 as a suppressive drug in the experimental autoimmune encephalomyelitis (EAE) model of MS. In the passive variety of EAE, induced in Lewis rats by adoptive transfer of myelin-reactive T cells, WIN-2 ameliorates the clinical signs and diminishes the cell infiltration of the spinal cord. Due to the involvement of cannabinoids in the regulation of cell death and survival, we investigated the effects of WIN-2 on the encephalitogenic T cell population. WIN-2 induced a profound increase of apoptosis in a dose- and time-dependent manner. The potential involvement of cannabinoid receptors (CB) was investigated by encephalitogenic T cell stimulation in the presence of the CB₁ (SR141716A) and CB₂ (SR144528) antagonists, pertussis toxin (PTX) and the inactive enantiomer WIN-3. WIN-2-induced apoptosis was partially blocked by SR144528 and PTX, whereas, WIN-3 only exerted a mild effect on cell viability. These results point to the partial involvement of CB₂ receptor together with other receptor-independent mechanism or by yet unknown cannabinoid receptors. Moreover, WIN-2 induced the extrinsic pathway of apoptosis, as shown by caspase-10 and -3 activation. These results suggest that cannabinoid-induced apoptosis of encephalitogenic T cells may cooperate in their anti-inflammatory action in EAE models. The partial involvement of CB₂ receptors in WIN-2 action may open new therapeutic doors in the management of MS by non-psychoactive selective cannabinoid agonists.

Keywords: Abbreviations; EAE; experimental autoimmune encephalomyelitis; MS; multiple sclerosis; TMEV; Theiler's murine encephalomyelitis virus; CNS; central nervous system; CREAE; chronic/relapsing EAE; CB; cannabinoid receptors; WIN-2; R; -(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone; WIN-3; S; -(−)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone; gp-MBP; guinea pig myelin basic protein; LNCs; lymph node cells; TRPV1; transient receptor potential vanilloid 1; SR1 (SR141716A); N; -(piperidine-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; SR2 (SR144528); N; -[(1; S; )-endo-1,3,3-trimethyl bicyclo [2,2,1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI; 4,6-diamidino-2-phenylindole; PTX; pertussis toxin; JWH-015; (2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone; Δ; ⁹; -THC; delta(9)-tetrahydrocannabinol; Δ; ⁸; -THC; delta(8)-tetrahydrocannabinol; ACEA; arachidonoyl-2-chloroethylamide; JWH-133; 3-(1′-dimethylbutyl)-1-deoxy-Δ; ⁸; -THC; HU-211; (+)-(3; S; ,4; S; )-7-hydroxy-delta(6)-tetrahydrocannabinol 1,1-dimethylheptyl; DMSO; dimethylsulfoxide; PBS; phosphate buffered saline; FCS; fetal calf serum; SDS; sodium dodecyl sulfate; DMF; dimethylformamide; PMSF; phenylmethylsulfonyl fluoride; DMEM; Dulbecco's modified Eagle mediumApoptosis; Cannabinoids; Experimental autoimmune encephalomyelitis; Inflammation; Multiple sclerosis; T cells

Anti-allodynic property of flavonoid myricitrin in models of persistent inflammatory and neuropathic pain in mice by Flavia Carla Meotti; Fabiana Cristina Missau; Juliano Ferreira; Moacir Geraldo Pizzolatti; Cristina Mizuzaki; Cristina Wayne Nogueira; Adair R.S. Santos (pp. 1707-1713).

The aim of the present study was to investigate the effects of myricitrin, a flavonoid with anti-inflammatory and antinociceptive action, upon persistent neuropathic and inflammatory pain. The neuropathic pain was caused by a partial ligation (2/3) of the sciatic nerve and the inflammatory pain was induced by an intraplantar (i.pl.) injection of 20μL of complete Freund's adjuvant (CFA) in adult Swiss mice (25–35g). Seven days after sciatic nerve constriction and 24h after CFA i.pl. injection, mouse pain threshold was evaluated through tactile allodynia, using Von Frey Hair (VFH) filaments. Further analyses performed in CFA-injected mice were paw edema measurement, leukocytes infiltration, morphological changes and myeloperoxidase (MPO) enzyme activity. The intraperitoneal (i.p.) treatment with myricitrin (30mg/kg) significantly decreased the paw withdrawal response in persistent neuropathic and inflammatory pain and decreased mouse paw edema. CFA injection increased 4-fold MPO activity and 27-fold the number of neutrophils in the mouse paw after 24h. Myricitrin strongly reduced MPO activity, returning to basal levels; however, it did not reduce neutrophils migration. In addition, myricitrin treatment decreased morphological alterations to the epidermis and dermis papilar of mouse paw. Together these results indicate that myricitrin produces pronounced anti-allodynic and anti-edematogenic effects in two models of chronic pain in mice. Considering that few drugs are currently available for the treatment of chronic pain, the present results indicate that myricitrin might be potentially interesting in the development of new clinically relevant drugs for the management of this disorder.

Keywords: Myricitrin; Allodynia; Neuropathy; Inflammation; Antioxidant

Indole-3-carbinol mediated cell cycle arrest of LNCaP human prostate cancer cells requires the induced production of activated p53 tumor suppressor protein by Jocelyn C. Hsu; Anurupa Dev; Aimee Wing; Christine T. Brew; Leonard F. Bjeldanes; Gary L. Firestone (pp. 1714-1723).

Indole-3-carbinol (I3C), a dietary compound found naturally in cruciferous vegetables of the Brassica genus such as broccoli and brussels sprouts, induces a G1 growth arrest of human reproductive cancer cells. We previously reported that in LNCaP prostate cancer cells, I3C down-regulated cyclin-dependent kinase (CDK) 2 activity. In our current study, Western blotting and quantitative RT-PCR demonstrated that I3C treatment increased both the transcripts and protein levels of the CDK2 inhibitor p21^waf1/cip1 (p21). Transfection of luciferase reporter plasmids containing wild-type and mutated p21 promoter fragments revealed that I3C induced p21 gene transcription through a p53 DNA binding element. Oligonucleotide precipitation showed that I3C increased the level of activated p53 nuclear protein that is competent to bind its DNA target site on the p21 promoter. Ablation of p53 production using short interfering RNA (siRNA) prevented that the I3C induced G1 arrest and up-regulation of p21 expression. Western blots using p53 phospho-specific antibodies revealed that I3C treatment increased the levels of three phosphorylated forms of p53 (Ser15, Ser37, Ser392) that are known to contribute to p53 protein stability and greater transactivation potential. Taken together, our results establish that the I3C induced G1 arrest of human prostate cancer cells requires the induced production of the activated phosphorylated forms of p53, which stimulate transcription of the CDK2 inhibitor p21.

Keywords: Abbreviations; I3C; indole-3-carbinol; DIM; 3,3′-diindolylmethane; TRYP; tryptophol; LNCaP; Lymph node carcinoma of the prostate; CDK; cyclin-dependent kinase; CKI; cyclin-dependent kinase inhibitor; ser; serine; siRNA; short interfering RNA; RNAi; RNA interferenceIndole-3-carbinol; Prostate cancer; p21; ^waf1/cip1; p53; LNCaP

Tyramine in the assessment of regional adrenergic function by F. Adams; M. Boschmann; K. Schaller; G. Franke; K. Gorzelniak; J. Janke; S. Klaus; F.C. Luft; M. Heer; J. Jordan (pp. 1724-1729).

Regional adrenergic function is difficult to assess in humans. Tyramine given through a microdialysis probe may be a useful tool in this regard. However, tyramine data is hard to interpret given the drug's complex mode of action. We characterized the response to tyramine, isoproterenol, and dopamine in adipose tissue with microdialysis probes in normal subjects. We measured glycerol concentrations to follow changes in lipolysis and monitored tissue perfusion with ethanol dilution. During perfusion with tyramine, dialysate glycerol concentration increased dose-dependently from 83±8μM at baseline to 181±18μM at 3.5mM tyramine ( p<0.001) followed by a fall down to 121±9μM at 35mM tyramine ( p<0.001). Propranolol almost completely blocked this response. A similar lipolytic response was not observed in isolated human adipocytes. Dopamine <35μM did not replicate the tyramine-induced lipolysis; however, dopamine >35μM potently inhibited lipolysis. We conclude that tyramine-induced lipolysis is explained by a pre-synaptic mechanism. Tyramine applied through a microdialysis probe in concentrations up to 3.5mM can be used to assess pre- and post-synaptic mechanisms regulating lipid mobilization.

Keywords: Norepinephrine; Dopamine; Adrenoreceptors; Autonomic nervous system; Lipolysis; Glycerol

Identification of glyburide metabolites formed by hepatic and placental microsomes of humans and baboons by Selvan Ravindran; Olga L. Zharikova; Ronald A. Hill; Tatiana N. Nanovskaya; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 1730-1737).

Glyburide (glibenclamide) is a second-generation sulfonylurea used for treatment of type-2 and gestational diabetes mellitus. To date, two glyburide metabolites have been identified in maternal urine: namely, 4- trans-hydroxycyclohexyl glyburide and 3- cis-hydroxycyclohexyl glyburide. The use of glyburide to treat gestational diabetes prompted us to investigate its metabolism by the placenta. The metabolism of glyburide by microsomal preparations from human and baboon placenta was compared with metabolism by their livers. The metabolites formed by the microsomes of the four tissues were identified by high-performance liquid chromatography–mass spectrometry using retention times, ion current (extracted at m/ z 510), and selected-ion monitoring. The data obtained revealed the formation of six distinct hydroxylated derivatives of glyburide by each of the four microsomal preparations. However, the amounts of the six metabolites formed by the placentas were a fraction of that formed by the livers. Moreover, the relative quantities of each metabolite formed differed between species as well as between the two tissues. Also, the structure of the unidentified metabolites was determined by comparison with synthesized standards. These metabolites were identified as the 4- cis-hydroxycyclohexyl glyburide, 3- trans-hydroxycyclohexyl glyburide, and 2- trans-hydroxycyclohexyl glyburide. Therefore, one glyburide metabolite remains to be identified, but the data we obtained allowed us to suggest its structure.

Keywords: Gestational diabetes; Human placenta; Baboon placenta; Glyburide; Metabolism; Mass spectrometry; Pregnancy

Cloning of a cDNA encoding a novel marmoset CYP2C enzyme, expression in yeast cells and characterization of its enzymatic functions by Shizuo Narimatsu; Fumihiro Torigoe; Yumi Tsuneto; Keita Saito; Nobumitsu Hanioka; Kazufumi Masuda; Takashi Katsu; Shigeo Yamamoto; Shigeru Yamano; Takahiko Baba; Atsuro Miyata (pp. 1738-1748).

We cloned a cDNA encoding a novel CYP2C enzyme, called P450 M-2C, from a marmoset liver. The deduced amino acid sequence showed high identities to those of human CYP2C8 (87%), CYP2C9 (78%) and CYP2C19 (77%). The P450 M-2C enzyme expressed in yeast cells catalyzed p-methylhydroxylation of only tolbutamide among four substrates tested, paclitaxel as a CYP2C8 substrate, diclofenac and tolbutamide as CYP2C9 substrates and S-mephenytoin as a CYP2C19 substrate. p-Methylhydroxylation of tolbutamide by marmoset liver microsomes showed monophasic kinetics, and the apparent K_m value (1.2mM) for the substrate was similar to that of the recombinant P450 M-2C (1.8mM). Although all of the recombinant human CYP2C8, CYP2C9 and CYP2C19 expressed in yeast cells catalyzed tolbutamide p-methylhydroxylation, the kinetic profile of CYP2C8 was most similar to that of P450 M-2C. Tolbutamide oxidation by the marmoset liver microsomes and the recombinant P450 M-2C was inhibited most effectively by quercetin, a CYP2C8 inhibitor, followed by omeprazole, a CYP2C19 inhibitor, whereas sulfaphenazole, a CYP2C9 inhibitor, was less potent under the conditions used. These results indicate that P450 M-2C is the major tolbutamide p-methylhydroxylase in the marmoset liver.

Keywords: Abbreviations; CYP; cytochrome P450; TB; tolbutamide; PT; paclitaxel; DF; diclofenac; S; -MP; S; -mephenytoin; G-6-P; glucose 6-phosphateMarmoset; CYP2C8; Yeast cell; Tolbutamide; Quercetin

Elevated K-ras activity with cholestyramine and lovastatin, but not konjac mannan or niacin in lung—–Importance of mouse strain by Richard J. Calvert; Shirley Tepper; Wafa Kammouni; Lucy M. Anderson; David Kritchevsky (pp. 1749-1755).

Our previous work established that hypocholesterolemic agents altered K-ras intracellular localization in lung. Here, we examined K-ras activity to define further its potential importance in lung carcinogenesis. K-ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeks, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% konjac mannan (KM), or were injected with lovastatin 25mg/kg three or five times weekly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeks, serum cholesterol was assayed with a commercial kit. Activated K-ras protein from lung was affinity precipitated with a Raf-1 ras binding domain-glutathione- S-transferase fusion protein bound to glutathione-agarose beads, followed by Western blotting, K-ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-ras activity increased 1.8-fold versus control ( p=0.009). In normal lung with wild-type K-ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-ras activity increased 2.1-fold ( p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-ras activity. These data are consistent with the concept that K-ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.

Keywords: Lung; Mice; Cholestyramine; Lovastatin; Konjac mannan; Niacin; K-ras activity; Cholesterol

Erratum to “Glucose utilization is suppressed in the gut of insulin-resistant high fat-fed rats and is restored by metformin�? [Biochem. Pharmacol. 72 (2) (2006) 198–203] by Gilles Mithieux; Fabienne Rajas; Carine Zitoun (pp. 1756-1756).

Erratum to: “Glucose utilization is suppressed in the gut of insulin-resistant high fat-fed rats and is restored by metformin�? [Biochem. Pharmacol. 72 (2) (2006) 198–203] by Gilles Mithieux; Fabienne Rajas; Carine Zitoun (pp. 1757-1762).

It has been recently suggested that the small intestine (SI) has the capacity to contribute to endogenous glucose production (EGP), in addition to the liver and kidney. The aim of this work was: (1) to estimate the role of SI glucose fluxes in glucose homeostasis in insulin resistance states (induced by high-fat (HF) feeding); (2) to assess the effect of metformin, an anti-diabetic molecule, on these fluxes. Rats were fed for 6 weeks on a HF-diet, supplemented or not with metformin (HF-Met) at a daily dosage of 50mg/kg during the last week. We combined arterio-venous glucose balance measurements and isotopic dilution techniques to separate basal intestinal glucose uptake (IGU) and release (IGR). Contrary to what was observed in control starch-fed rats, IGU was negligible in HF-fed rats: 0.6±2.4μmol/kgmin (mean±S.E.M., n=9). It was restored to a level close to that of control rats in the HF-Met group: 13.0±6.7μmol/kgmin (mean±S.E.M., n=9, p<0.05 compared to the non-treated group). Similarly, IGR was close to zero in HF-fed rats (−3.8±2.6μmol/kgmin), but was significant in HF-Met rats (7.4±4.4μmol/kgmin, p<0.05 compared to non-treated rats). These data strongly suggest that the impairment of glucose uptake in the SI might be a crucial feature of insulin resistance states and that a key beneficial effect of metformin in these situations might be to restore a normal glucose metabolism in this tissue.

Keywords: Metformin; Glucose metabolism; Small intestine; Glucose tracers; Endogenous Glucose production; Glucose-6 phosphatase

Author index Volume 72 (pp. 1763-1767).

Subject index Volume 72 (pp. 1768-1792).

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Biochemical Pharmacology (v.72, #12)