Does the gastroprotective action of a medicinal plant ensure healing effects? An integrative study of the biological effects of Serjania marginata Casar. (Sapindaceae) in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The chemical composition and pharmacological activities of geopropolis produced by Melipona fasciculata Smith in northeast Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reducing Myofibroblast Arrhythmogeneicity by Pharmacological Targeting of Their Cytoskeleton

Introduction: Slow conduction and ectopic activity are key elements of cardiac arrhythmogenesis. Both anomalies can be caused by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. Because MFBs are characterized by the expression of {alpha}-smooth muscle actin ({alpha}-SMA) containing stress fibers, we investigated whether pharmacological interference with stress fiber formation might affect myofibroblast arrhythmogenicity. Methods: Experiments were done with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse propagation characteristics were measured optically using voltage sensitive dyes. Electrophysiological characteristics of single MFBs were assessed using patch clamp techniques. Actin polymerization was inhibited by latrunculin B (LtB). Data are given as mean±S.D. (n=5 to 22). Results: As assessed by immunocytochemistry, exposure of MFBs to LtB (0.3–10 µmol/L) profoundly disrupted stress fiber formation. This led, within minutes, to a dramatic change in cell morphology with MFBs assuming an astrocyte-like shape. In pure cardiomyocyte preparations, LtB had negligible effects on impulse conduction velocity ({theta}) and maximal action potential upstroke velocities (dV/dtmax). In contrast, LtB applied to MFB coated cardiomyocyte strands substantially increased {theta} from 247±32 to 371±26 mm/s and dV/dtmax from 40±7 to 81±1 %APA/ms, i.e., to values similar to those of pure cardiomyocyte strands (342±13 mm/s; 82±1 %APA/ms). Moreover, LtB at 1 µmol/L completely abolished MFB induced ectopic activity. LtB induced normalization of electrophysiologic parameters can be explained by the finding that LtB hyperpolarized MFBs from –25 mV to –50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause slow conduction and ectopic activity. Conclusions: Pharmacological interference with the cytoskeleton of cardiac MFBs alters their electrophysiological phenotype to such an extent that detrimental effects on cardiomyocyte electrophysiology are completely abolished. This observation might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs.

Design, synthesis and pharmacological characterization of analogs of 2-aminoethyl diphenylborinate (2-APB), a known store-operated calcium channel blocker, for inhibition of TRPV6-mediated calcium transport

2-Aminoethyl diphenylborinate (2-APB) is a known modulator of the IP3 receptor, the calcium ATPase SERCA, the calcium release-activated calcium channel Orai and TRP channels. More recently, it was shown that 2-APB is an efficient inhibitor of the epithelial calcium channel TRPV6 which is overexpressed in prostate cancer. We have conducted a structure-activity relationship study of 2-APB congeners to understand their inhibitory mode of action on TRPV6. Whereas modifying the aminoethyl moiety did not significantly change TRPV6 inhibition, substitution of the phenyl rings of 2-APB did. Our data show that the diaryl borinate moiety is required for biological activity and that the substitution pattern of the aryl rings can influence TRPV6 versus SOCE inhibition. We have also discovered that 2-APB is hydrolyzed and transesterified within minutes in solution.

Illuminating somatostatin analog action at neuroendocrine tumor receptors

Somatostatin analogs for the diagnosis and therapy of neuroendocrine tumors (NETs) have been used in clinical applications for more than two decades. Five somatostatin receptor subtypes have been identified and molecular mechanisms of somatostatin receptor signaling and regulation have been elucidated. These advances increased understanding of the biological role of each somatostatin receptor subtype, their distribution in NETs, as well as agonist-specific regulation of receptor signaling, internalization, and phosphorylation, particularly for the sst2 receptor subtype, which is the primary target of current somatostatin analog therapy for NETs. Various hypotheses exist to explain differences in patient responsiveness to somatostatin analog inhibition of tumor secretion and growth as well as differences in the development of tumor resistance to therapy. In addition, we now have a better understanding of the action of both first generation (octreotide, lanreotide, Octreoscan) and second generation (pasireotide) FDA-approved somatostatin analogs, including the biased agonistic character of some agonists. The increased understanding of somatostatin receptor pharmacology provides new opportunities to design more sophisticated assays to aid the future development of somatostatin analogs with increased efficacy.

Type I hypersensitivity and its pharmacological modulation in {\it Trichinella\/}-induced intestinal fluid secretion and rapid rejection of the parasite in immunized rats

Studies were performed to test the hypothesis that type I hypersensitivity underlies worm induced intestinal fluid secretion and the rapid rejection of Trichinella spiralis from immunized rats, and the two events may be related in a cause-effect manner.^ Two approaches were taken. One was to determine whether inhibition of anaphylaxis-mediated Cl$\sp{-}$ and fluid secretion accompanying a secondary infection impedes worm rejection from immune hosts. The other was to determine whether induction of intestinal fluid secretion in nonimmune hosts interfered with worm establishment. In both studies, fluid secretion was measured volumetrically 30 min after a challenge infection and worms were counted.^ In immunized rats indomethacin did not affect the worm-induced fluid secretion when used alone, despite inhibiting mucosal prostaglandin synthesis. Fluid secretion was reduced by treatment with diphenhydramine and further reduced by the combination of diphenhydramine and indomethacin. The paradoxical effects of indomethacin when used alone compared with its coadministration with diphenhydramine is explained by the enhancing effect of indomethacin on histamine release. Abolishing net fluid secretion in these studies had no effect on rapid worm rejection in immune hosts.^ Worm establishment was reduced in recipients of immune serum containing IgE antibodies. Net intestinal fluid secretion induced in normal rats by PGE$\sb2$, cholera toxin, or hypertonic mannitol solution had no effect on worm establishment compared with untreated controls.^ In a related experiment, worm-induced intestinal fluid secretion and worm rejection in immune rats were partially blocked by concurrent injection with 5-HT$\sb2$ and 5-HT$\sb3$ blockers (Ketanserin and MDL-72222), suggesting that 5-HT is involved. This possible involvement was supported in that treatment of nonimmune rats with 5-HT significantly inhibited worm establishment in the intestine.^ Results indicate that anaphylaxis is the basis for both worm-induced intestinal fluid secretion and rapid rejection of T. spiralis in immune rats, but these events are independent of one another. 5-HT is a possible mediator of worm rejection, however, its mechanism of action is related to something other than fluid secretion. ^

A PHARMACOLOGICAL EVALUATION OF THE MECHANISM OF CYTOTOXICITY OF 9-BETA-D- XYLOFURANOSYLADENINE IN CHINESE HAMSTER OVARY CELLS

The biochemical determinants of cytotoxicity of the purine nucleoside analog, 9-(beta)-D-xylofuranosyladenine (xyl-A) were studied in wild-type Chinese hamster ovary cells and in nucleoside kinase deficient mutants. It was found that {('3)H}xyl-A was readily phosphorylated to the triphosphate level in both the wild-type and deoxycytidine kinase deficient mutant, but not by the adenosine kinase deficient cells. Values for the apparent Km and Vmax of this uptake process were 43.9 (mu)M and 118.7 nmol/min/10('9) cells, respectively. Cloning procedures indicated that the viability of CHO cells was decreased 90 per cent by a 5-hr incubation with 10 (mu)M xyl-A. However, the toxicity of xyl-A was increased 100-fold by the addition of a nontoxic concentration (10 (mu)M) of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to the medium. High-pressure liquid chromatographic analysis indicated that after 5 hr, the concentration of 9-(beta)-D-xylofuranosyladenine 5'-triphosphate (xyl-ATP) in cells incubated with xyl-A plus EHNA was 2.0 mM, four times greater than in those cells incubated with xyl-A alone. Incubation with xyl-A plus EHNA had no significant effect on the cellular concentrations of 5-phosphoribosyl-1-pyrophosphate after 1 hr whereas, treatment with 3'-dexoyadenosine (cordycepin) decreased the concentration of this metabolite. Determinations of the cellular nucleoside triphosphates indicated that under conditions that resulted in an intracellular accumulation of 500 (mu)M xyl-ATP, the endogenous concentrations of neither the ribonucleoside triphosphates nor deoxyribonucleoside triphosphates were significantly different from those of control cells. The ID(,50) for {('3)H}thymidine incorporation into DNA, 105 (mu)M xyl-ATP, was four-fold less than the ID(,50) for {('3)H}uridine incorporation into RNA suggesting that the process of DNA synthesis is more sensitive to the presence of xyl-ATP. When removed from exogenous xyl-A, CHO cells failed to recover their ability to synthesize RNA and DNA, although the intracellular xyl-ATP concentration decreased to less than 35 (mu)M. The selective inhibition of RNA synthesis by 6-azauridine did not prevent the expression of toxicity by xyl-ATP. However, the selective inhibition of DNA synthesis by ara-C significantly spared toxicity in cells that had accumulated an otherwise lethal concentration of xyl-ATP. It is shown that in cells which had accumulated 1.27 mM {('3)H}xyl-ATP, {('3)H}xyl-A was found to terminate cellular RNA chains at a frequency of 1.42 (mu)mol of {('3)H}xyl-A 3' termini per mol of mononucleotide. These results indicate that a general mechanism for the toxicity of xyl-A to CHO cells includes the cellular accumulation of xyl-ATP, which serves as a substrate for RNA synthesizing enzymes and subsequently is incorporated into nascent RNA transcripts as a chain terminator. A specific mechanism involving the premature termination of RNA primers required for the initiation of DNA synthesis is proposed to account for the inhibitory action of xyl-ATP on DNA synthesis. ^

The mechanism of the pharmacological actions of dexrazoxane on different tissue types

Approximately 6,600 people die from acute myelogenous leukemia (AML) on an annual basis. During the past 10 to 15 years, there has been gradual overall improvements in the therapy of this disease, yet the majority of patients with AML succumb to this disease. In an attempt to improve current therapeutic strategies for AML, we became interested in a commercially available drug, dexrazoxane, which protects against anthracycline-induced cardiotoxicity. We have investigated dexrazoxane's (DEX) effects on different tissue types in an effort to determine its unique mechanism of action. Colony forming assays were used to evaluate stem-cell renewal of myeloid cells in vitro and median effect analysis was used to evaluate antagonism, synergism, or additivity. The anthracyclines, doxorubicin, daunorubicin, and idarubicin were individually combined with DEX in leukemic myeloid models to determine if the combination of the two drugs resulted in a synergistic, additive or antagonistic effect. Etoposide and cytosine arabinoside were also evaluated in combination with DEX using the same in vitro model and evaluated similarly. ^ Dexrazoxane in combination with any of the anthracyclines was schedule dependent. The combination of DEX and anthracycline resulted in a greater antitumor effect than anthracycline alone except for DEX administered 24 hours before doxorubicin or daunorubicin. These data were corroborated through median effect analysis. Etoposide in combination with dexrazoxane was synergistic for all combinations, and the combination of cytosine arabinoside and DEX was schedule dependent. In contrast, using an in vivo gastrointestinal model, DEX in combination with doxorubicin was antagonistic for almost all of the ratios used, except for the highest. A Withers' assay was used to evaluate toxicity on jejunal crypt cells. No effect was apparent for the combination of idarubicin and DEX, however, as seen with RZ, DEX in addition to radiation greatly potentiated the cytotoxic effects of radiation on crypts. These paradoxical effects of dexrazoxane were initially enigmatic, but after additional investigation, we propose a model that explains our findings. We conclude that DEX in combination with anthracyclines produces an additive to synergistic antileukemic response and may have therapeutic potential clinically. Additionally, DEX protects the gastrointestinal tract from doxorubicin toxicity, which could have clinical implications for the administration of greater doses of doxorubicin. ^

A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans

The molecular mechanisms underlying general anesthesia are unknown. For volatile general anesthetics (VAs), indirect evidence for both lipid and protein targets has been found. However, no in vivo data have implicated clearly any particular lipid or protein in the control of sensitivity to clinical concentrations of VAs. Genetics provides one approach toward identifying these mechanisms, but genes strongly regulating sensitivity to clinical concentrations of VAs have not been identified. By screening existing mutants of the nematode Caenorhabditis elegans, we found that a mutation in the neuronal syntaxin gene dominantly conferred resistance to the VAs isoflurane and halothane. By contrast, other mutations in syntaxin and in the syntaxin-binding proteins synaptobrevin and SNAP-25 produced VA hypersensitivity. The syntaxin allelic variation was striking, particularly for isoflurane, where a 33-fold range of sensitivities was seen. Both the resistant and hypersensitive mutations decrease synaptic transmission; thus, the indirect effect of reducing neurotransmission does not explain the VA resistance. As assessed by pharmacological criteria, halothane and isoflurane themselves reduced cholinergic transmission, and the presynaptic anesthetic effect was blocked by the resistant syntaxin mutation. A single gene mutation conferring high-level resistance to VAs is inconsistent with nonspecific membrane-perturbation theories of anesthesia. The genetic and pharmacological data suggest that the resistant syntaxin mutant directly blocks VA binding to or efficacy against presynaptic targets that mediate anesthetic behavioral effects. Syntaxin and syntaxin-binding proteins are candidate anesthetic targets.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

The mode of action of aspirin-like drugs: effect on inducible nitric oxide synthase.

Nitric oxide synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases. We report that exposure of lipopolysaccharide-stimulated murine macrophages to therapeutic concentrations of aspirin (IC50 = 3 mM) and hydrocortisone (IC50 = 5 microM) inhibited the expression of iNOS and production of nitrite. In contrast, sodium salicylate (1-3 mM), indomethacin (5-20 microM), and acetaminophen (60-120 microM) had no significant effect on the production of nitrite at pharmacological concentrations. At suprapharmacological concentrations, sodium salicylate (IC50 = 20 mM) significantly inhibited nitrite production. Immunoblot analysis of iNOS expression in the presence of aspirin showed inhibition of iNOS expression (IC50 = 3 mM). Sodium salicylate variably inhibited iNOS expression (0-35%), whereas indomethacin had no effect. Furthermore, there was no significant effect of these nonsteroidal anti-inflammatory drugs on iNOS mRNA expression at pharmacological concentrations. The effect of aspirin was not due to inhibition of cyclooxygenase 2 because both aspirin and indomethacin inhibited prostaglandin E2 synthesis by > 75%. Aspirin and N-acetylimidazole (an effective acetylating agent), but not sodium salicylate or indomethacin, also directly interfered with the catalytic activity of iNOS in cell-free extracts. These studies indicate that the inhibition of iNOS expression and function represents another mechanism of action for aspirin, if not for all aspirin-like drugs. The effects are exerted at the level of translational/posttranslational modification and directly on the catalytic activity of iNOS.

Pharmacological discrimination of calcitonin receptor:receptor activity-modifying protein complexes

Calcitonin (CT) receptors dimerize with receptor activity-modifying proteins (RAMPs) to create high-affinity amylin (AMY) receptors, but there is no reliable means of pharmacologically distinguishing these receptors. We used agonists and antagonists to define their pharmacology, expressing the CT (a) receptor alone or with RAMPs in COS-7 cells and measuring cAMP accumulation. Intermedin short, otherwise known as adrenomedullin 2, mirrored the action of αCGRP, being a weak agonist at CT(a), AMY 2(a), and AMY3(a) receptors but considerably more potent at AMY1(a) receptors. Likewise, the linear calcitonin gene-related peptide (CGRP) analogs (Cys(ACM)2,7)hαCGRP and (Cys(Et) 2,7)haCGRP were only effective at AMY1(a) receptors, but they were partial agonists. As previously observed in COS-7 cells, there was little induction of the AMY2(a) receptor phenotype; thus, AMY 2(a) was not examined further in this study. The antagonist peptide salmon calcitonin8-32 (sCT8-32) did not discriminate strongly between CT and AMY receptors; however, AC187 was a more effective antagonist of AMY responses at AMY receptors, and AC413 additionally showed modest selectivity for AMY1(a) over AMY3(a) receptors. CGRP8-37 also demonstrated receptor-dependent effects. CGRP 8-37 more effectively antagonized AMY at AMY1(a) than AMY3(a) receptors, although it was only a weak antagonist of both, but it did not inhibit responses at the CT(a) receptor. Low CGRP 8-37 affinity and agonism by linear CGRP analogs at AMY 1(a) are the classic signature of a CGRP2 receptor. Our data indicate that careful use of combinations of agonists and antagonists may allow pharmacological discrimination of CT(a), AMY1(a), and AMY3(a) receptors, providing a means to delineate the physiological significance of these receptors. Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics.

A review of non-pharmacological and pharmacological management of seasonal and perennial allergic conjunctivitis

Allergic eye disease encompasses a group of hypersensitivity disorders which primarily affect the conjunctiva and its prevalence is increasing. It is estimated to affect 8% of patients attending optometric practice but is poorly managed and rarely involves ophthalmic assessment. Seasonal allergic conjunctivitis (SAC) is the most common form of allergic eye disease (90%), followed by perennial allergic conjunctivitis (PAC; 5%). Both are type 1 IgE mediated hypersensitivity reactions where mast cells play an important role in pathophysiology. The signs and symptoms are similar but SAC occurs periodically whereas PAC occurs year round. Despite being a relatively mild condition, the effects on the quality of life can be profound and therefore they demand attention. Primary management of SAC and PAC involves avoidance strategies depending on the responsible allergen(s) to prevent the hypersensitivity reaction. Cooled tear supplements and cold compresses may help bring relief. Pharmacological agents may become necessary as it is not possible to completely avoid the allergen(s). There are a wide range of anti-allergic medications available, such as mast cell stabilisers, antihistamines and dual-action agents. Severe cases refractory to conventional treatment require anti-inflammatories, immunomodulators or immunotherapy. Additional qualifications are required to gain access to these medications, but entry-level optometrists must offer advice and supportive therapy. Based on current evidence, the efficacy of anti-allergic medications appears equivocal so prescribing should relate to patient preference, dosing and cost. More studies with standardised methodologies are necessary elicit the most effective anti-allergic medications but those with dual-actions are likely to be first line agents.

Investigation into the biochemical changes in Tourette Syndrome with a potential for pharmacological manipulation

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, which accounts for over 95% of tryptophan metabolism. Two previous studies by this research group reported elevated plasma KYN in Tourette syndrome (TS) patients when compared with age and sex matched controls and another study showed that KYN potentiated 5-HT2A-mediated head-shakes (HS) in rodents. These movements have been suggested to model tics in TS. This raised the questions how KYN acts in eliciting this response and whether it is an action of its own or of a further metabolite along the kynurenine pathway. In the liver, where most of the kynurenine pathway metabolism takes place under physiological conditions, the first and the rate limiting enzyme is tryptophan-dioxygenase (TDO) which can be induced by cortisol. In extrahepatic tissues the same step of the pathway is catalyzed by indoleamine-dioxygenase (IDO), which is induced by cytokines, predominantly interferon-y (INF-y). Plasma neopterin, which shows parallel increase with KYN following immune stimulation, was also found elevated in one of these studies positively correlating with KYN. In the present work animal studies suggested that KYN potentiates and quinolinic acid (QUINA) dose dependently inhibits the 5-HT2A-mediated HS response in mice. The potentiating effect seen with KYN was suggested to be an effect of KYN itself. Radioligand binding and phosphoinositide (PI) hydrolysis studies were done to explore the mechanisms by which kynurenine pathway metabolites could alter a 5-HT2A-receptor mediated response. None of the kynurenine pathway metabolites tested showed direct binding to 5-HT2A-receptors. PI hydrolysis studies with KYN and QUINA showed that KYN did not have any effect while QUINA inhibited 5-HT2A-mediated PI hydrolysis. Plasma cortisol determination in TS patients with elevated plasma KYN did not show elevated plasma cortisol levels, suggesting that the increase of plasma KYN in these TS patients is unlikely to be due to an increased TDO activity induced by increased cortisol. Attention deficit hyperactivity disorder (ADHD) is commonly associated with TS. Salivary cortisol detected in a group of children primarily affected with ADHD showed significantly lower salivary cortisol levels when compared with age and sex matched controls. Plasma tryptophan, KYN, neopterin, INF-y and KYN/tryptophan ratio and night-time urinary 6-sulphatoxymelatonin (aMT6s) excretion measured in a group of TS patients did not show any difference in their levels when compared with age and sex matched controls, but TS patients failed to show the expected positive correlation seen between plasma INF-y, neopterin and KYN and the negative correlation seen between plasma KYN and night-time urinary aMT6s excretion seen in healthy controls. The relevance of the kynurenine pathway, melatonin secretion and cortisol to Tourette Syndrome and associated conditions and the mechanism by which KYN and QUINA alter the 5-HT2A-receptor mediated HS response are discussed.

Investigation of potential intervention targets to improve insulin action: a therapeutic approach

Impaired insulin action (insulin resistance) is a key factor in the pathogenesis of diabetes mellitus. To investigate therapeutic targets against insulin resistance, this thesis explores the mechanism of action of pharmacological agents and exogenous peptides known or suspected to modify insulin action. These included leptin, a hormone primarily involved in the regulation of body weight; sibutramine, an antiobesity agent; plant-derived compounds (pinitol and chamaemeloside) and agents known to affect insulin sensitivity, e.g. metformin, tolbutamide, thiazolidinediones, vanadyl sulphate and thioctic acid. Models used for investigation included the L6 skeletal muscle cell line and isolated skeletal muscles. In vivo studies were undertaken to investigate glycaemia, insulinaemia, satiety and body weight in streptozotocin-induced diabetic mice and obese (ob/ob) mice. Leptin acutely altered insulin action in skeletal muscle cells via the short form of the leptin receptor. This direct action of leptin was mediated via a pathway involving PI 3-kinase but not Jak2. The active metabolites of sibutramine had antidiabetic properties in vivo and directly improved insulin sensitivity in vitro. This effect appeared to be conducted via a non-PI 3-kinase-mediated increase in protein synthesis with facilitated glucose transport, and was independent of the serotonin and noradrenaline reuptake inhibition produced by sibutramine. Pinitol (a methyl inositol) had an insulin mimetic effect and was an effective glucose-lowering agent in insulinopenic states, acting directly on skeletal muscle. Conversely chamaemeloside appeared to improve glucose tolerance without directly altering glucose transport. Metformin directly increased basal glucose uptake independently of PI 3-kinase, possibly via an increase in the intrinsic activity of glucose transporters. Neither tolbutamide nor thiazolidinediones directly altered insulin sensitivity in L6 skeletal muscle cells: however vanadyl sulphate and thioctic acid increased glucose transport but appeared to exert toxic effects at therapeutic concentrations. Examination of glucose transport in skeletal muscle in this thesis has identified various components of post-receptor insulin signalling pathways which may be targeted to ameliorate insulin resistance. Type 2 Diabetes Mellitus Obesity L6 Skeletal Muscle Cells Glucose Transport Insulin Signalling 2