The effects of liver and renal disease on stereoselective serum binding of flurbiprofen.

Stereoselectivity in the serum binding of flurbiprofen, a non-steroidal anti-inflammatory drug which is highly bound to albumin, was studied in patients with liver and renal disease. Subjects with renal disease or liver disease with ascites had significantly lower serum albumin concentrations than normals, resulting in higher free fractions of both the R(-) and S(+) enantiomers of flurbiprofen as determined by equilibrium dialysis. The ratio (+/- s.d.) of R/S-flurbiprofen free fractions was lower in the subjects with ascites (0.714 +/- 0.298) than in those without ascites (0.796 +/- 0.090) (P < 0.05).     

The effect of pregnenolone 16alpha-carbonitrile on the pharmacokinetics and metabolism of dapsone in rats.

The purpose of this study was to evaluate the effect of pregnenolone 16 alpha-carbonitrile (PCN) on the interconversion pharmacokinetics and metabolism of dapsone. To determine microsomal CYP3A activity and protein, eight rats (4 PCN, 4 corn oil) received a 1 mg kg(-1) intravenous bolus dose of dapsone, followed by blood and urine sampling. The formation clearance of dapsone hydroxylamine (CLf DDS-NOH) was calculated from the obtained samples. Interconversion pharmacokinetics estimates were obtained after 10 rats (5 PCN, 5 control) received 1 mg kg(-1) dapsone or 1.17 mg kg(-1) monoacetyldapsone, with a 24-h wash-out. Results from the interconversion analysis demonstrated that PCN significantly increased systemic clearance (CLs) of dapsone, but not its interconversion. The in-vivo/in-vitro correlation study demonstrated that PCN significantly increased CLs of dapsone (8.55 to 16.39mLmin(-1); P<0.01) and CLf DDS-NOH (0.13 to 0.18mLmin(-1); P<0.01). PCN treatment produced a 69% increase in CYP3A protein, and increased 6beta- and 2beta-hydroxytestosterone formation rates. Significant correlations were found between CLf DDS-NOH and either 6beta- (r2 = 0.925), 2beta-hydroxytestosterone (r2 = 0.92), or CYP3A1/2 protein (r2= 0.60). We conclude that PCN treatment produces significant increases in CLs (dapsone) and CLf (DDS-NOH) in rats. These changes were not due to changes in the reversible metabolism of dapsone. These results suggest that the formation clearance of dapsone hydroxylamine reflects alterations in CYP3A activity, despite the fact that it accounted for a small part of the systemic clearance of dapsone.     

Phenobarbital in the genetically obese Zucker rat. II. In vivo and in vitro assessments of microsomal enzyme induction

In vivo and in vitro alterations in drug metabolism and the extent of enzyme induction of the hepatic microsomal cytochrome P-450 system were evaluated in obese and lean Zucker and lean Sprague-Dawley rats. Phenobarbital enzyme-inducing regimens were administered p.o. to achieve similar steady-state phenobarbital plasma concentrations. Control rats received p.o. placebo solution. No significant intra- or inter-strain differences in antipyrine clearance (milliliters per hour) or apparent volume of distribution (liters) were observed between the placebo-treated lean Sprague-Dawley, lean Zucker and obese Zucker rats. Intra- and inter-strain differences in hepatic microsomal protein and cytochrome P-450 content were observed. Compared to placebo, antipyrine clearance (milliliters per hour) after chronic phenobarbital pretreatment was increased in the Sprague-Dawley (198%) and lean Zucker rats (131%), but not significantly altered in the obese Zucker rats. Similarly, increases in hepatic weight, whole liver microsomal protein and cytochrome P-450 content were also observed in the Sprague-Dawley (34, 124 and 352%, respectively) and the lean Zucker rats (24, 96 and 249%, respectively). However, no significant alterations in these parameters were observed in the obese Zucker rats after phenobarbital treatment. Results from these in vivo and in vitro studies implicate alterations in drug metabolism and genetic differences in cytochrome P-450 content in Zucker rats relative to the Sprague-Dawley strain. Obese Zucker rats failed to exhibit a significant induction response after phenobarbital pretreatment.     

Phenobarbital in the genetically obese Zucker rat. I. Pharmacokinetics after acute and chronic administration

We measured both pulmonary and plasma angiotensin converting enzyme (ACE) activity in conscious rabbits before and for 6 days after administration of captopril (2 mg/kg i.v.). Pulmonary ACE activity was measured by means of modified indicator-dilution techniques after bolus injection of [3H]benzoyl-phenyl-alanyl-alanyl-proline ( BPAP , synthetic substrate for ACE). Plasma ACE activity was also determined radiometrically with [3H] BPAP as substrate. In addition, we measured the systemic pressor response to i.v. bolus dose of angiotensin I both before and after captopril. Twenty-four hours after captopril, pulmonary metabolism of BPAP had decreased from control of 73 +/- 5 to 8 +/- 2%; even 6 days after drug treatment there was still evidence of inhibition. In contrast, plasma ACE activity was significantly (P less than .05) reduced within 15 min of treatment (to 20% of control levels) but recovered within 24 hr. The time course of changes in the pressor response to angiotensin I, after captopril, resembled that of plasma ACE activity. Mean systemic arterial blood pressure was 81 +/- 4 torr at control and reached its nadir at 24 hr (64 +/- 2 torr; P less than .05); thereafter a gradual recovery ensued, similar in time course to that of pulmonary ACE. These data suggest that inhibition of plasma ACE and also the pressor response to angiotensin I are unrelated temporally to the hypotensive effects of captopril.     

IGF1/insulin receptor kinase inhibition by BMS-536924 is better tolerated than alloxan-induced hypoinsulinemia and more effective than metformin in the treatment of experimental insulin-responsive breast cancer

Epidemiologic and experimental evidence suggest that a subset of breast cancer is insulin responsive, but it is unclear whether safe and effective therapies that target the insulin receptor (IR), which is homologous to oncogenes of the tyrosine kinase class, can be developed. We demonstrate that both pharmacologic inhibition of IR family tyrosine kinase activity and insulin deficiency have anti-neoplastic activity in a model of insulin-responsive breast cancer. Unexpectedly, in contrast to insulin deficiency, pharmacologic IR family inhibition does not lead to significant hyperglycemia and is well tolerated. We show that pharmacokinetic factors explain the tolerability of receptor inhibition relative to insulin deficiency, as the small molecule receptor kinase inhibitor BMS-536924 does not accumulate in muscle at levels sufficient to block insulin-stimulated glucose uptake. Metformin, which lowers insulin levels only in settings of hyperinsulinemia, had minimal activity in this normoinsulinemic model. These findings highlight the importance of tissue-specific drug accumulation as a determinant of efficacy and toxicity of tyrosine kinase inhibitors and suggest that therapeutic targeting of the IR family for cancer treatment is practical.     

Plasma Membrane Subdomain Compartmentalization Contributes to Distinct Mechanisms of Ceramide Action on Insulin Signaling

OBJECTIVE

Ceramide is now recognized as a negative regulator of insulin signaling by impairing protein kinase B (PKB)/Akt activation. In different cells, two distinct mechanisms have been proposed to mediate ceramide inhibition of PKB/Akt: one involving atypical protein kinase C zeta (PKCζ) and the other the protein phosphatase-2 (PP2A). We hypothesized that ceramide action through PKCζ or PP2A might depend on plasma membrane (PM) structural organization and especially on caveolin-enriched domain (CEM) abundance.

RESEARCH DESIGN AND METHODS

We have used different PKCζ mutant constructs or the PP2A inhibitor, okadaic acid (OKA), to selectively inhibit PKCζ- and PP2A-dependent pathways in cells expressing different caveolin-1 levels and evaluated the impact of insulin and ceramide on PKB/Akt activity in different PM subdomains.

RESULTS

Although the PKCζ-mediated negative effect of ceramide on insulin-stimulated PKB/Akt was dominant in adipocytes, a ceramide action through PP2A outside CEMs, prevented by OKA, was also unraveled. To test the importance of CEM to direct ceramide action through the PKCζ pathway, we treated 3T3-L1 preadipocytes devoid of CEMs with ceramide and we saw a shift of the lipid-negative action on PKB/Akt to a PP2A-mediated mechanism. In fibroblasts with low CEM abundance, the ceramide-activated PP2A pathway dominated, but could be shifted to a ceramide-activated PKCζ pathway after caveolin-1 overexpression.

CONCLUSIONS

Our results show that ceramide can switch from a PKCζ-dependent mechanism to a PP2A pathway, acting negatively on PKB/Akt, and hence revealing a critical role of CEMs of the PM in this process.Insulin is a hormone essential for tissue development, growth, energy storage, and maintenance of glucose homeostasis. Defects in insulin secretion and action are key factors in the development of metabolic diseases such as diabetes, obesity, hypertension, atherosclerosis, and cardiovascular diseases (1).The mechanism by which insulin resistance develops in peripheral tissue is not yet fully solved. Recent work has suggested that forcing cells to store fatty acids beyond their capacities could promote insulin resistance by inducing the accumulation of intracellular signaling molecules able to inhibit the action of insulin (2). Among these fatty acid–derived lipids, ceramides are the most active to negatively regulate intermediates of the insulin-signaling pathway and to inhibit insulin-dependent pathways such as the uptake of glucose into muscle and adipocytes (3,4).The process of insulin signal transduction is initiated by the activated insulin receptor kinase, which tyrosine phosphorylates intracellular target substrates, in particular the family of insulin receptor substrates (IRS 1–4 proteins) (5). Although numerous proteins can dock on activated IRS, it is generally accepted that phosphoinositide 3-kinase (PI 3-kinase) and signaling effectors that lie downstream from it, in particular protein kinase B (PKB, also known as Akt) and atypical protein kinase C ζ/λ (aPKCs), play crucial roles in glucose homeostasis (6). PI 3-kinase–generated membrane phosphatidylinositol-3,4,5-triphosphates (PIP₃s) recruit to the plasma membrane (PM) and activate both aPKCs and PKB/Akt (7,8). Once recruited, aPKCs are phosphorylated by a 3-phosphoinositide–dependent protein kinase-1 (PDK1) on their Thr410/403 site (9). On the other hand, binding of PIP₃s to the pleckstrin homology (PH) domain of PKB/Akt induces conformational changes in the kinase that expose two regulatory sites, Thr308 and Ser473 (for PKBα/Akt1). Phosphorylation of Thr308 is mediated by PDK1 and Ser473 phosphorylation by TORC2 (mammalian target of rapamycin)-rictor (rapamycin-insensitive companion of mTOR) complex (10). The importance of the activation of aPKCs and PKB/Akt by insulin in mediating glucose metabolism is now well documented in insulin-sensitive tissues. Mice lacking PKBβ (Akt2) become insulin resistant and develop severe diabetes (11), and recently, Farese et al. (12) have demonstrated the importance of PKCλ in skeletal muscle by selectively ablating this kinase in a mouse model. They showed that these mice developed insulin resistance, reduced glucose tolerance, and dyslipidemia, all common features of the metabolic syndrome.A consensus now exists that PKB/Akt is the primary target of ceramide. Indeed, defects in activation of this kinase induced by ceramide have been observed in cell types, such as white and brown adipocytes, skeletal and smooth muscles, mammary cells, and nerve cells (13). In some cells, ceramide acts on PKB/Akt through the direct activation of phosphatases such as the protein phosphatase-2A (PP2A) (14), a cytosolic serine/threonine phosphatase responsible for dephosphorylating PKB/Akt (15). Treatment of several cell types such as C2C12 muscle cells, PC12 nerve cells, and brown adipocytes with the PP2A inhibitor okadaic acid (OKA) (16) prevents the negative effects of ceramide on PKB/Akt (13). However, in L6 muscle cells and white adipose tissue, we and others have shown that ceramide inhibited insulin-stimulated glucose transport through a mechanism that does not involve a phosphatase (17,18). Ceramide activates PKCζ (19,20), which interacts and phosphorylates the PH domain of PKB/Akt on a Thr34 residue, preventing PKB/Akt to be recruited and activated at the PM in response to insulin (19).Thus, two mechanisms by which ceramide can inhibit PKB/Akt are described in different cell types. We hypothesized that a PKCζ- or PP2A-mediated action of ceramides might be dependent on the structure and compartmentalization of the PM that differs among cell types. It is now well recognized that the PM is not uniform but composed of subdomains with unique lipid compositions. In particular, specialized domains called caveolin-enriched domains (CEMs) have been shown to be important in mediating insulin action (21) and are enriched in ceramide (22,23). Moreover, we, as well as others, have shown that ceramide induced the recruitment of both PKCζ and PKB/Akt in CEMs (22,23). Thus, PKCζ- and PP2A-mediated mechanisms are likely to occur in different compartments. As a unifying hypothesis to explain why two different mechanisms by which ceramide inhibits PKB/Akt exist, we propose that the structure of the PM in different cell types, particularly the relative abundance of CEMs, might be a determining factor to direct the action of ceramide toward either the PKCζ or the PP2A mechanism. To test this hypothesis, we have used different PKCζ mutant constructs or OKA, to inhibit PKCζ- and PP2A-dependent pathways and evaluated the impact of both insulin and ceramide on PKB/Akt activity in CEMs and non-CEMs. Using cells with different levels of expression of caveolin, we demonstrated that ceramide switches from one mechanism to the other to inhibit the insulin activation of PKB/Akt.     

Fusion of visuo-ocular and vestibular signals in arm motor control

Keeping the finger pointing at an Earth-fixed object during body displacements can be achieved if compensatory arm movements counteract the effect of the rotation on the hand's position in space. Here we investigated the fusion of signals that originated from systems having different neurophysiological properties (i.e., the visuo-oculomotor and vestibular systems) in the production of such compensatory arm movements. To this end, we analyzed the subjects' performance in three conditions that differed according to the information they provided about relative target-body motion. This information originated either from the vestibular or visuo-oculomotor system, or from a combination of the two. To highlight the integration of visuo-oculomotor and vestibular signals, we compared the arm response to motion frequencies presumed to allow or not to allow optimal vestibular and oculomotor responses. When they could be used in isolation, the ocular signals allowed long-latency but precise kinematics control of the arm movement, whereas vestibular signals allowed accurate motor response early in the rotation but their contribution declined as body rotation developed. Optimal performance was obtained throughout the whole movement and for all rotation frequencies when the visuo-oculomotor and vestibular signals could be used together. This increase in hand-tracking performance could not be explained by a unimodal model or an additive model of vestibular and ocular cues, even when using weighted signals. Rather, the results supported a functional model in which vestibular and visuo-oculomotor signals have different influences on the temporal and spatial aspects of hand movement compensating for body motion.     

Loss of function of PTEN alters the relationship between glucose concentration and cell proliferation,increases glycolysis,and sensitizes cells to 2-deoxyglucose

PTEN loss of function enhances proliferation, but effects on cellular energy metabolism are less well characterized. We used an inducible PTEN expression vector in a PTEN-null glioma cell line to examine this issue. While proliferation of PTEN-positive cells was insensitive to increases in glucose concentration beyond 2.5 mM, PTEN-null cells significantly increased proliferation with increasing glucose concentration across the normal physiologic range to ∼10 mM, coinciding with a shift to glycolysis and “glucose addiction”. This demonstrates that the impact of loss of function of PTEN is modified by glucose concentration, and may be relevant to epidemiologic results linking hyperglycemia to cancer risk and cancer mortality.