Mouse Liver Plasma Membranes Research Articles

Thematic Review Series: Proteomics. Proteomic analysis of lipid-protein complexes

There is intense interest in comprehensive proteomic approaches for analyzing integral membrane proteins and lipoproteins. Key features of mass spectrometric analysis center on enriching biological material for proteins of interest, efficiently digesting them, extracting the resulting peptides, and using fractionation methods to comprehensively sample proteins or peptides by tandem mass spectrometry. However, lipid-associated proteins are generally rich in hydrophobic domains and are often low in abundance. These features, together with the associated lipid, make their mass spectrometric analysis technically challenging. In this article, we review analytical strategies for successful proteomic analysis of lipid-associated proteins.

Activity of the Bile Salt Export Pump (ABCB11) Is Critically Dependent on Canalicular Membrane Cholesterol Content

Mutations in ATP8B1 cause severe inherited liver disease. The disease is characterized by impaired biliary bile salt excretion (cholestasis), but the mechanism whereby impaired ATP8B1 function results in cholestasis is poorly understood. ATP8B1 is a type 4 P-type ATPase and is a flippase for phosphatidylserine. Atp8b1-deficient mice display a dramatic increase in the biliary extraction of cholesterol from the canalicular (apical) membrane of the hepatocyte. Here we studied the hypothesis that disproportionate cholesterol extraction from the canalicular membrane impairs the activity of the bile salt transporter, ABCB11, and as a consequence causes cholestasis. Using single pass liver perfusions, we show that not only ABCB11-mediated transport but also Abcc2-mediated transport were reduced at least 4-fold in Atp8b1 deficiency. We show that canalicular membranes of cholestatic Atp8b1-deficient mice have a dramatically reduced cholesterol to phospholipid ratio, i.e. 0.75 +/- 0.24 versus 2.03 +/- 0.71 for wild type. In vitro depletion of cholesterol from mouse liver plasma membranes using methyl-beta-cyclodextrin demonstrated a near linear relation between cholesterol content of the membranes and ATP-dependent taurocholate transport. Abcc2-mediated transport activity was not affected up to 30% of membrane cholesterol depletion but declined to negligible levels at 70% of membrane cholesterol depletion. These effects were reversible as cholesterol repletion of the liver membranes completely restored Abcb11- and Abcc2-mediated transport. Our data demonstrate that membrane cholesterol content is a critical determinant of ABCB11/ABCC2 transport activity, provide an explanation for the etiology of ATP8B1 disease, and suggest a novel mechanism protecting the canalicular membrane against luminal bile salt overload.

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

Plasma membrane (PM) proteome is one of the major subproteomes present in the cell, and is very important in liver function. In the present work, C57 mouse liver PM was purified by density-gradient centrifugation. The purified PM was verified by electron microscope analysis and Western blotting. The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction. Proteins were separated by 2DE and 1DE, trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry. In all, 547 non-redundant mouse liver PM proteins were identified, of which 34% contributed to plasma membrane or plasma membrane-related proteins. This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.

Proteomic analysis of mouse liver plasma membrane: Use of differential extraction to enrich hydrophobic membrane proteins

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.

Mouse liver plasma membrane redox system activity is altered by aging and modulated by calorie restriction

Caloric restriction (CR) is known as the only non-genetic method proven to slow the rate of aging and extend lifespan in animals. Free radicals production emerges from normal metabolic activity and generates the accumulation of oxidized macromolecules, one of the main characteristics of aging. Due to its central role in cell bioenergetics, a great interest has been paid to CR-induced modifications in mitochondria, where CR has been suggested to decrease reactive oxygen species production. The plasma membrane contains a trans-membrane redox system (PMRS) that provides electrons to recycle lipophilic antioxidants, such as α-tocopherol and coenzyme Q (CoQ), and to modulate cytosolic redox homeostasis. In the present study, we have investigated age differences in the PMRS in mouse liver and their modulation by CR. Aging induced a decrease in the ratio of CoQ10/CoQ9 and α-tocopherol in liver PM from AL-fed mice that was attenuated by CR. CoQ-dependent NAD(P)H dehydrogenases highly increased in CR old mice liver PMs. On the other hand, the CoQ-independent NADH-FCN reductase activity increased in AL-fed animals; whereas, in mice under CR this activity did not change during aging. Our results suggest that liver PMRS activity changes during aging and that CR modulates these changes. By this mechanism CR maintains a higher antioxidant capacity in liver PM of old animals by increasing the activity of CoQ-dependent reductases. Also, the putative role of PMRS in the modulation of redox homeostasis of cytosol is implicated.

Apolipoprotein C-III can specifically bind to hepatic plasma membranes.

Apo C-III plays an important role in the metabolism of plasma triglyceride, which can delay the catabolism of triglyceride-rich lipoproteins by interfering with apo E-mediated receptor clearance of remnant particles from plasma. The mechanism of the interference has not yet been defined. To further explore the role of apo C-III, we first injected mice with 125I-apo C-III. The measurement of radioactivity showed that liver took up 3.3-10 fold as much radioactivity as other organs such as heart, spleen, lung, kidney, stomach, large intestine, small intestine, and muscle. This was confirmed by incubating the tissue homogenates of the organs with 125I-apo C-III that the radiolabeled apo C-III specifically bound to only hepatic homogenate. To investigate which subcellular part or parts of hepatic cells play the role of binding to apo C-III, hepatic cell components of nucleus, mitochondria, microsomes and plasma membranes were then incubated with 125I-apo C-III. The radiolabeled apo C-III could specifically bind to only hepatic plasma membranes. Finally hepatic plasma membranes were purified to study the characteristics of the specific binding with apo C-III. Addition of increasing concentration of 125I-apo C-III to human hepatic plasma membranes revealed saturable binding to membranes with a Kd of 0.31 +/- 0.07 micromol/l. The maximum specific binding capacity was 1.74 +/- 0.45 microg apo C-III/mg membrane protein. In competition studies using unlabeled apo C-III and isolated lipoproteins HDL, LDL and VLDL, only apo C-III and VLDL effectively competed with 125I-apo C-III for membrane binding. The binding of 125I-apo C-III to human liver plasma membranes was Ca2+-independent, and was abolished when plasma membranes were treated with trypsin. The characteristics of 125I-apo C-III binding to mouse liver plasma membranes were similar to those of human liver plasma membranes with the exception of a binding maximum of 1.52 +/- 0.39 microg apo C-III/mg membrane protein. We conclude that apo C-III exhibits high-affinity binding to hepatic plasma membranes, which is saturable, reverse and specific.

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences

Radioligand binding studies with [ 3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V 1A subtype while kidney binding sites belong to the V 2 receptor subtype. The affinity of each ligand for mouse V 1A receptors was very similar to that for rat V 1A receptors, showing differences in K i values of less than 3-fold. In contrast, several peptide (d(CH 2) 5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V 2 receptors, with differences in K i values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V 2 receptors show significant pharmacologic differences.

Connexins and the vacuolar proteolipid-like 16-kDa protein are not directly associated with each other but may be components of similar or the same gap junctional complexes

Gap junction preparations made from mouse liver plasma membranes by alkali extraction contain variable proportions of connexins (Cx32 and Cx26) and the 16-kDa protein which is closely related or may be identical to the 16-kDa proteolipid (subunit c) of the vacuolar H +-ATPase and the mediatophore complex. The absence of a stoichiometric relationship suggests that connexins and the 16-kDa protein are not subunits of the same channel complex, but analysis of alkali preparations by isopycnic centrifugation shows both types of protein are in membrane structures of the same buoyant density. Electron microscopic analysis of alkali preparations shows a homogeneous population of gap junctions of uniform morphology and width, suggesting the proteins are in the same or similar structures. The structures containing connexins and the 16-kDa protein can be separated by treatment of the plasma membranes with Triton X-100. After such treatment, the connexins remain associated with dense cellular or extracellular material and the gap junctional structures, after further extraction with N-lauroyl sarcosine and urea, contain only the 16-kDa protein. These detergent-extracted gap junctions are thinner (14.1 nm) than those in alkali preparations (18.4 nm).

Insulin receptors prepared with iodoacetamide show enhanced autophosphorylation and receptor kinase activity.

In this study, we found that adding iodoacetamide to the homogenization buffer used in the preparation of mouse or rat liver plasma membranes resulted in an increase of insulin receptor autophosphorylation by 4-5-fold and receptor kinase activity by about 2-fold. Similar effects were obtained with iodoacetate and p-chloromercuriphenyl sulfonate. The effect of iodoacetamide was minimal when it was added to membranes prepared without the thiol reagent. The enhancing effect of iodoacetamide on insulin receptor autophosphorylation was the result of a more than 2-fold decrease in the Km and a more than 3-fold increase in Vmax for ATP. The presence of iodoacetamide in the preparation of plasma membranes also greatly increased the solubilization of the insulin receptor from the plasma membrane by Triton X-100. We propose that iodoacetamide acts to alkylate some unknown thiols released during tissue homogenization and that in its absence these thiols formed mixed disulfides with the insulin receptor, thus adversely affecting the process of receptor activation by insulin.

Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver

The specific binding of [3H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (KD = 8.8 nM and Bmax = 1477 fmol/mg protein) and the other one has low affinity and high binding capacity (KD = 91 nM and Bmax = 9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (KD = 11.2 nM and Bmax = 1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na+,K(+)-ATPase digitalis receptor. Probably it is of the same nature that the one determinate of [3H]cortisol and [3H]corticosterone in mouse liver plasma membrane. Beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities.

Effects of chronic ethanol administration on plasma-membrane- bound glycosyltransferase activities

Physiological and membranous modifications induced by a 4-week ethanol administration in mouse liver plasma membrane were studied. Galactosyl- and glucosyltransferase activities were stimulated in the presence of dolichylphosphate alone or with phosphatidyl-choline. The galactosyltransferase activity was inhibited by chronic ethanol administration. These enzymes were modulated by different phospholipids. The phosphatidic acid was the most efficient activator. Ethanol provoked an inhibition of the galactosyltransferase activity whatever the phospholipid used, as well as an inhibition of the glucosyltransferase activity, chiefly in presence of phosphatidyl-inositol. The preincubation of control or treated mouse liver plasma membranes with liposomes loaded by dolichylphosphate and cholesterol greatly enhanced the enzymatic activities without removing the inhibition by ethanol treatment.

Evidence for the presence of specific binding sites for corticoids in mouse liver plasma membranes.

The specific binding of [3H]cortisol to plasma membranes purified from mouse liver, studied by the ultrafiltration method, shows the existence of specific binding sites for cortisol. The kinetic parameters of this binding are KD = 4.4 nM and Bmax = 685 fmol/mg protein in presence of 1 microM of corticosterone. With respect to the binding of 4 nM [3H]cortisol to the membrane, the affinities of the steroids decreased in the following order: deoxycorticosterone greater than corticosterone greater than progesterone greater than cortisol greater than prednisolone greater than testosterone greater than 20 beta-hydroxyprogesterone greater than cortisone. Estradiol, dexamethasone, ouabain and triamcinolone acetonide do not have affinity for this binding site. Neither Ca2+ nor Mg2+ affected the binding of [3H]cortisol to the plasma membranes. Likewise, the presence of agonists and antagonists of alpha and beta-adrenergic receptors did not modify the binding of [3H]cortisol. The results suggest that the plasma membrane binding site characterized is more specific for corticoids and is different from nuclear glucocorticoid and progesterone receptors.

Characterization of Specific Binding Sites for Corticosterone in Mouse Liver Plasma Membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.

Effect of dietary N-6 and N-3 polyunsaturated fatty acids on composition and enzyme activities in liver plasma membrane of mice

The effects of increasing amounts of dietary linoleic acid (18:2n-6) and 5 and 10 wt. % menhaden oil on the fatty acid composition and enzyme activities of mouse liver plasma membrane was studied. Groups of male mice were fed a diet containing 20 wt.% fat for 2 weeks and 15%, 35% and 47% of the total fat was composed of 18:2n-6. The increased amount of 18:2n-6 in the diet did not alter 18:2n-6, arachidonic acid (20:4n-6_ nor total polyunsaturated fatty acid (PUFA) composition of mouse liver plasma membranes. This did not affect membrane bound 5′-nucleotidase activity but moderately decreased Na +-K + ATPase activity in plasma membranes when high levels of 18:2n-6 (47%) was used in the diet. The inclusion of 5 or 10 wt% of menhaden oil in the diets containing (13%) 18:2n-6 resulted in the incorporation of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) which replaced 18:2n-6 (20–40%) and 20:4n-6 (65–80%) in the membrane phospholipids. The 5′-nucleotidase and Na +-K + ATPase activity of plasma membrane were not significantly affected by these changes in membrane fatty acid.

Growth-stimulatory action of plasma membrane-associated growth factor: A synergistic effect with platelet-poor plasma

Growth factor activity was partially purified from mouse liver plasma membranes and its growth-stimulatory action on cultured mouse fibroblasts was studied. The plasma membrane-associated growth factor (PMGF) was unable to support the proliferation of mouse fibroblasts in monolayer when added as the sole source of growth factor. However, it stimulated the growth of fibroblasts in the presence of CM-Sephadex-treated human platelet-poor plasma (h-CMP) which by itself is not growth-stimulatory. The stimulation of DNA synthesis in quiescent fibroblasts was also observed upon the addition of PMGF and h-CMP. Under the same conditions, both platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) showed the same effect as did PMGF. The synergistic action of h-CMP with PMGF on quiescent cells was partially reproduced by insulin at microgram quantities or by insulin-like growth factor I(IGF-I) at nanogram quantities. Thus, the data presented here indicates that the action of PMGF is similar to that of the family of growth factors termed ‘competence factor’, and distinct from that of plasma growth factors termed ‘progression factor’.

Hepatic adrenergic receptors in the genetically diabetic C57 BL/KsJ (db/db) mouse

1. 1. At 30 weeks of age, homozygote diabetic C57 BL KsJ (db/db) mice were grossly obese, lethargic and displayed moderate hair loss relative to heterozygote control C 57 BL KsJ (db/+) mice. 2. 2. In diabetic mice, compared to control, the total body weights, liver weight: body weight ratios, and blood glucose levels were increased 2.3 fold, 20% and 3.1 fold, respectively. 3. 3. Analysis of plasma membranes isolated from control and diabetic mouse liver established that comparable purity levels were achieved since relative specific activities of the plasma membrane markers 5′-nucleotidase and γ-glutamyltranspeptidase were similar: 10.2 and 11.4 fold with respect to 5′-nucleotidase in control and diabetic states respectively; and 8.0 and 8.3 fold with respect to γ-glutamyltranspeptidase in control and diabetic states respectively. 4. 4. A select effect of diabetes on γ-glutamyltranspepetidase, however, was observed. The activity of this enzyme was found to be reduced 16% in diabetic liver compared to control liver. 5. 5. Assessment of [ 3H]prazosin and [ 3H]dihydrolalprenolol binding to mouse liver plasma membranes indicated that although there was no difference in β-adrenergic receptor binding in control and diabetic states, α 1-adrenergic receptor binding was found to be reduced 43% in diabetic mouse liver plasma membranes. 6. 6. Scatchard analyses of kinetic studies indicate that the reduction is a reflection of decreases in α 1-adrenergic receptor numbers with no change in α 1 receptor affinity in the diabetic state: since for diabetic and control liver plasma membranes, K d values were 3.40 ± 0.02 nM and 3.40 ± 0.01 nM respectively; and B max were 650.12 ± 16.44 fmol mg −1 and 380.76 ± 12.92 fmol mg −1, respectively.

Antibody to liver membrane antigens in chronic active hepatitis. IV. Exclusion of specific reactivity to polypeptides and glycolipids by immunoblotting.

The reactivity of sera was examined in patients with autoimmune chronic active hepatitis and other liver diseases by immunoblotting. Polypeptides and glycolipids of liver plasma membrane, liver-specific lipoprotein and kidney membrane were separated and probed with sera from patients and from a rabbit immunized with mouse liver plasma membrane. Chronic active hepatitis sera reacted with a number of polypeptides in the liver plasma membrane preparations; similar but weaker reactivity was observed with sera from patients with other diseases and in some healthy subjects. Chronic active hepatitis sera did not react with glycolipids from liver plasma membrane. The immune rabbit serum reacted with two polypeptides of 180 kd present in liver plasma membrane but absent from kidney membrane, with two polypeptides of 50 kd which were nonliver-specific but species-specific, and with three major glycolipid components of liver plasma membrane: this reactivity thus differed markedly from that of the chronic active hepatitis sera. In studies using dot-blotting, it was found that solubilization of liver plasma membrane in detergents resulted in a marked reduction of the reactivity to liver plasma membrane of chronic active hepatitis sera, but little change in the reactivity of the chronic active hepatitis and other sera with liver-specific lipoprotein by immunoblotting indicated that liver-specific lipoprotein consisted of constituents of liver plasma membrane together with intracellular proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.

A tight junction-enriched membrane fraction has been used as immunogen to generate a monoclonal antiserum specific for this intercellular junction. Hybridomas were screened for their ability to both react on an immunoblot and localize to the junctional complex region on frozen sections of unfixed mouse liver. A stable hybridoma line has been isolated that secretes an antibody (R26.4C) that localizes in thin section images of isolated mouse liver plasma membranes to the points of membrane contact at the tight junction. This antibody recognizes a polypeptide of approximately 225,000 D, detectable in whole liver homogenates as well as in the tight junction-enriched membrane fraction. R26.4C localizes to the junctional complex region of a number of other epithelia, including colon, kidney, and testis, and to arterial endothelium, as assayed by immunofluorescent staining of cryostat sections of whole tissue. This antibody also stains the junctional complex region in confluent monolayers of the Madin-Darby canine kidney epithelial cell line. Immunoblot analysis of Madin-Darby canine kidney cells demonstrates the presence of a polypeptide similar in molecular weight to that detected in liver, suggesting that this protein is potentially a ubiquitous component of all mammalian tight junctions. The 225-kD tight junction-associated polypeptide is termed "ZO-1."

Inhibition of lymphocyte proliferation by detergent-solubilized mouse liver membranes.

The role of phenomena analogous to fibroblast contact inhibition in lymphocyte growth regulation is controversial, although it is clear that direct cell-cell contact is vital to immunoregulation and accessory cell function. An extract of mouse liver plasma membrane proteins, referred to as suppressive liver extract (SLE), that suppresses the growth of 3T3 fibroblasts also inhibited the mitogen-induced proliferation of murine lymphocytes. A dose of 20 micrograms/ml SLE was less than 95% suppressive of proliferation in both mouse T and mouse B cells treated with a variety of mitogens. B cell growth factor, while increasing DNA synthesis overall in mitogen-stimulated B cells, did not change the extent of SLE suppression, which suggests that the SLE does not interfere with lymphocyte-growth factor interactions. In exploring a sequence of B cell activation events, we discovered that SLE had no effect on the early activation event of increased phosphatidylinositol turnover. Blastogenesis, however, was inhibited in mitogen-stimulated, SLE-treated B cells. The maximum suppressive effect was observed if the SLE was added within 8-12 h of the mitogenic stimulus. SLE did not affect the viability of cells in culture. These results point to a possible unity of regulatory mechanisms between contact inhibition in fibroblasts and the processes of immunoregulation.

Effects of insulin on the lipid structure of liver plasma membrane measured with fluorescence and ESR spectroscopic methods

Insulin increased the lipid order of rat and mouse liver plasma membrane domains sampled by the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in a concentration-dependent saturable manner. The ordering is half maximal at 5.1 · 10 −11 M and fully saturated at 1.7 · 10 −10 M insulin. Membranes prepared from obese hyperglycemic (ob / ob) mice demonstrated a right-shift in the dose-dependent ordering induced by insulin, such that ordering was half maximal at 1.2 · 10 −10 M and fully saturated at 2.0 · 10 −10 M . Insulin also increased the order of rat liver plasma membranes labeled with the cis- and trans-parinaric acid methyl esters. The ordering caused by insulin as detected with cis methyl parinarate was complete within approx. 15 min. after hormone addition at 37°C, and the ordering was approximately double that observed with the trans isomer. Additional ESR experiments demonstrated that the addition of insulin increased the outer hyperfine splittings of spectra recorded from membranes labeled with the steroid-like spin labels, nitroxide cholestane and nitroxide androstane, but not the fatty acid spin probe, 5-nitroxide stearate. Studies utilizing model membrane systems strongly suggest that the 5-nitroxide stearate samples a cholesterol-poor domain of the membrane, while the steroid-like probes preferentially sample cholesterol-rich regions of the membrane. Finally, insulin-induced membrane ordering was dose-dependently inhibited by cytochalasin B in the range 1–50 μM. From these results, we conclude that (1) the ordering effect of insulin addition to isolated liver plasma membrane fractions occurs within the physiological range of hormone concentration, and the dose-response is right-shifted in membranes from ‘insulin resistant’ animals; (2) the relative responses of the fluorescent and spin probes suggest that the effects of insulin are confined to specific domains within the membrane matrix; and (3) the direct effects of insulin on the membranes may involve protein components having cytochalasin B binding sites.