Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Endothelial cell CD36 regulates membrane ceramide formation, exosome fatty acid transfer and circulating fatty acid levels.

Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we examine how ECs transfer FAs. FA interaction with apical membrane CD36 induces Src phosphorylation of caveolin-1 tyrosine-14 (Cav-1Y14) and ceramide generation in caveolae. Ensuing fission of caveolae yields vesicles containing FAs, CD36 and ceramide that are secreted basolaterally as small (80-100 nm) exosome-like extracellular vesicles (sEVs). We visualize in transwells EC transfer of FAs in sEVs to underlying myotubes. In mice with EC-expression of the exosome marker emeraldGFP-CD63, muscle fibers accumulate circulating FAs in emGFP-labeled puncta. The FA-sEV pathway is mapped through its suppression by CD36 depletion, blocking actin-remodeling, Src inhibition, Cav-1Y14 mutation, and neutral sphingomyelinase 2 inhibition. Suppression of sEV formation in mice reduces muscle FA uptake, raises circulating FAs, which remain in blood vessels, and lowers glucose, mimicking prominent Cd36-/- mice phenotypes. The findings show that FA uptake influences membrane ceramide, endocytosis, and EC communication with parenchymal cells.

Temperature dependence of glucose transport in erythrocytes from normal and alloxan-diabetic rats.

Alloxan diabetes increased 3-O-methylglucose transport rates in rat red blood cells (RBC) at temperatures below 30 degrees C and decreased them above 30 degrees C. Preincubation of RBC from control rats with 20 mM glucose, 3-O-methylglucose, 2-deoxyglucose or xylose greatly elevated transport at 14 degrees C by increasing Vmax. The effect was slight at 40 degrees C. Preincubation with glucose or deoxyglucose alone caused a 50% depression of transport rates at 40 degrees C as a result of a rise in the Km, which is similar to findings in cells from alloxan-diabetic rats. Measurement of intracellular glucose metabolites suggested inhibition of glycolysis in cells from diabetic rats and a positive correlation between the level of intracellular hexose monophosphates and transport inhibition. Membrane fatty-acid and cholesterol composition and membrane lipid-ordering as monitored by electron paramagnetic resonance were not altered by alloxan diabetes. It is concluded that intracellular sugar and sugar metabolism alter the temperature dependence of glucose transport kinetics. Glucose metabolism can feed back to inhibit transport by increasing the transport Km at physiological temperatures only.

Evidence that downregulation of hexose transport limits intracellular glucose in 3T3-L1 fibroblasts.

Measurements of initial glucose entry rate and intracellular glucose concentration in cultured cells are difficult because of rapid transport relative to intracellular volume and a substantial extracellular space from which glucose cannot be completely removed by quick exchanges of medium. In 3T3-L1 cells, we obtained good estimates of initial entry of [14C]methylglucose and D-[14C]glucose with 1) L-[3H]glucose as an extracellular marker together with the [14C]glucose or [14C]methylglucose in the substrate mixture, 2) sampling times as short as 2 s, 3) ice-cold phloretin-containing medium to stop uptake and rinse away the extracellular label, and 4) nonlinear regression of time courses. Methylglucose equilibrated in two phases--the first with a half-time of 1.7 s and the second with a half-time of 23 s; it eventually equilibrated in an intracellular space of 8 microliters/mg protein. Entry of glucose remained almost linear for 10 s, making its transport kinetics easier to study (Km = 5.7 mM, Vmax = 590 nmol.s-1.ml-1 cell water). Steady-state intracellular glucose concentration was 75-90% of extracellular glucose concentration. Cells grown in a high-glucose medium (24 mM) exhibited a 67% reduction of glucose-transport activity and a 50% reduction of steady-state ratio of intracellular glucose to extracellular glucose.

Two antiatherogenic effects of progesterone on human macrophages; inhibition of cholesteryl ester synthesis and block of its enhancement by glucocorticoids.

The effects of progesterone and estradiol on cholesteryl ester (CE) formation by monocyte-derived human macrophages were examined. Formation was assessed from incorporation of 14C-cholesterol during a 20-h incubation with hormone and from that of 3H-oleate (3 h) after hormone removal. Progesterone inhibited cholesterol into CE and decreased CE cellular levels. Inhibition: 1) was reversed by progesterone removal; 2) was independent of the progesterone receptor (not blocked by the receptor antagonist RU40555); and 3) exhibited specific structural requirements; 11alpha-OH-progesterone was inhibitory, whereas its stereoisomer 11beta-OH-progesterone was not. In contrast to progesterone, estradiol was ineffective. We had reported that dexamethasone enhanced CE accumulation by human macrophages (1). In this study, we describe similar effects of the endogenous steroid, cortisol, and of the most widely prescribed glucocorticoid, prednisolone. Both steroids increased CE formation from two folds, in the presence of cholesterol-liposomes, to five folds, in the presence of modified low-density lipoprotein. Progesterone (0.1-1 micromol/L), added during glucocorticoid treatment, blocked this increase. The progesterone block: 1) was duplicated by the steroid receptor inhibitor RU40555; 2) was not reversed by hormone removal; and 3) reflected inhibition of glucocorticoid-induced increases in messenger RNA for acyl-CoA-cholesterol:acyl transferase. Thus, progesterone exerted two effects on macrophages: it acutely inhibited CE formation, and it prevented glucocorticoid-induced increases in acyl-CoA-cholesterol-acyl transferase gene expression and CE synthesis.

Role of CD36 in membrane transport and utilization of long-chain fatty acids by different tissues.

The transmembrane glycoprotein CD36 has been identified in isolated cell studies as a putative transporter of long-chain fatty acids. To examine the physiological role of CD36, we studied FA uptake and metabolism by tissues of CD36 null mice after injection with two fatty acid analogs. Compared to controls, uptake was substantially reduced (50-80%) in heart, skeletal muscle, and adipose tissues of null mice. The reduction in uptake was associated with a large decrease in fatty acid incorporation into triglycerides, which could be accounted for by an accumulation of diacylglycerides. Thus CD36 facilitates a major fraction of fatty acid uptake by myocardial, skeletal muscle, and adipose tissues, where it is highly expressed. Its role in other tissues where its expression is low and cell-specific could not be determined in these studies.

CD36 gene deletion reduces fat preference and intake but not post-oral fat conditioning in mice.

Several findings suggest the existence of a "fatty" taste, and the CD36 fatty acid translocase is a candidate taste receptor. The present study compared fat preference and acceptance in CD36 knockout (KO) and wild-type (WT) mice using nutritive (triglyceride and fatty acid) and nonnutritive (Sefa Soyate oil) emulsions. In two-bottle tests (24 h/day) naive KO mice, unlike WT mice, displayed little or no preference for dilute soybean oil, linoleic acid, or Sefa Soyate emulsions. At high concentrations (2.5-20%), KO mice developed significant soybean oil preferences, although they consumed less oil than WT mice. The postoral actions of fat likely conditioned these preferences. KO mice, like WT mice, learned to prefer a flavored solution paired with intragastric soybean oil infusions. These findings support CD36 mediation of a gustatory component to fat preference but demonstrate that it is not essential for fat-conditioned flavor preferences. The finding that oil-naive KO mice failed to prefer a nonnutritive oil, assumed to provide texture rather than taste cues, requires explanation. Finally, CD36 deletion decreased fat consumption and enhanced the ability of the mice to compensate for the calories provided by their optional fat intake.

CD36-dependent fatty acid uptake regulates expression of peroxisome proliferator activated receptors.

CD36 is an important regulator of lipid metabolism in vivo due to its role in the facilitated uptake of long-chain FAs (fatty acids). CD36-deficient mice display reduced TAG (triacylglycerol) in muscle, but elevated hepatic TAG. Also, insulin sensitivity is enhanced peripherally, while it appears impaired in the liver. Tissues such as muscle, which normally express high levels of CD36, shift to high glucose utilization in CD36 deficiency, so we hypothesized that this shift must involve adaptive changes in the PPAR (peroxisome-proliferator-activated receptor) transcription factors which regulate FA metabolism. To test this, we examined mRNA levels for the three PPAR isoforms in tissues of WT (wild-type) and CD36-deficient mice following the administration of saline, glucose or olive oil by intragastric gavage. Compared with WT mice, CD36-null mice had 5-10-fold increased PPAR mRNA in adipose tissue in the basal state, and did not exhibit diet-induced changes. Correlations between adipose PPAR mRNA abundance and plasma lipids were observed in WT mice, but not in CD36-null mice. The opposite was true for hepatic PPAR mRNA levels, which correlated with plasma FA, TAG and/or glucose only in CD36-null mice. No significant differences were observed in PPAR mRNA levels in the intestine, where CD36 does not impact on FA uptake. The data suggest that CD36 and the PPARs are components of the FA-sensing machinery to respond to changes in FA flux in a tissue-specific manner.

The gene-nutrient-gene loop.

It is increasingly apparent that the genetic influence on the development and severity of a particular phenotype (e.g. diabetes, hyperlipidemia, hypertension, and coronary heart disease) can be strongly modulated by diet. In turn, the response of the phenotype to dietary intervention is determined by the individual genotype. The reviews in this issue provide striking examples of recent progress related to the molecular basis of nutrient-gene interactions. As our understanding of these interactions improves, we should be better equipped to identify individuals at risk of specific pathologies and make a better assessment of the risk involved. Nutritional support could then be tailored to the individual genotype to favour beneficial phenotypic expression or to suppress that leading to pathology and disease.

Expression of the adipocyte fatty acid-binding protein in streptozotocin-diabetes: effects of insulin deficiency and supplementation.

The adipocyte fatty acid-binding protein, aP2 or ALBP, is an abundant cytosolic protein postulated to function in binding and intracellular transport of long-chain fatty acids. In this report, we investigated levels of aP2 mRNA and protein and transcriptional activity of the aP2 gene in tissues from streptozotocin-diabetic rats at different time periods following the induction of diabetes. An average 75% decrease in mRNA for aP2 (relative to mRNA for beta-actin) was observed in all diabetic rats at 7 days post-STZ injection. Insulin supplementation rapidly (2 h) restored aP2 mRNA and the insulin effect was cycloheximide-sensitive. Nuclear transcription assays measured a 60% decrease in transcription of the aP2 gene in diabetic rats that was reversed by insulin administration. Levels of aP2 protein were still high, in some cases, 1 day after the decrease in mRNA levels consistent with a long half-life of the protein. Decreases in aP2 protein were rapidly reversed by insulin administration. There were no changes in aP2 protein in the absence of changes in aP2 mRNA supporting a pretranslational mechanism of regulation. The decrease in aP2 mRNA was delayed in onset when compared with the rapid decline (at day 2 of diabetes) of mRNA for the lipogenic enzyme, fatty acid synthase, and with the accelerated depletion of adipose tissue lipid. Adipose tissue weight and lipid content had decreased by more than 80% 3 days before any significant changes in aP2 expression were observed. Changes in aP2 could not be related to changes in the levels of circulating fatty acids that regulate aP2 expression in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of basal glucose transporter Km in the adipocyte by insulin and other factors.

We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent Km of approximately 35 mM. Under those conditions insulin stimulated transport predominantly by decreasing the transport Km (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899). Our findings were in contrast with earlier reports that the Km of basal glucose transport was low (3-5 mM) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37 degrees C, cell agitation for 10 min prior to the transport assay decreased the basal Km from 35 to 12 mM. Deprivation of metabolic substrate produced a further reduction down to 2 mM. Refeeding starved cells with 1 mM glucose returned the Km back up to 12 mM in agitated cells and to 40 mM in stabilized cells. The effects of agitation to lower and of glucose to raise the basal Km were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the Vmax of basal transport and were without effect on both Km and Vmax in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23 degrees C caused both basal Km and Vmax to drop proportionately from 25 to 5 mM, and 13 to 3.6 nmol/(microliter X min), respectively. In insulin-stimulated cells, only the Vmax was decreased (Km went from 3.5 to 3 mM, Vmax from 45 to 17 nmol/(microliter X min]. The results support the concept that experimental conditions can produce large changes in the Km of basal glucose transporters. Furthermore they explain why, under certain assay conditions (with temperatures around 23 degrees C or with deprivation of metabolic substrate), the effect of insulin on transport Km is not observed. Our data also suggest that basal transport characteristics do not persist in insulin-treated cells. We would propose that one of the actions of insulin (in addition to raising Vmax) is to change the characteristics of basal transporters by overriding metabolic factors which keep the Km high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores.

Binding of sulfosuccinimidyl fatty acids to adipocyte membrane proteins: isolation and amino-terminal sequence of an 88-kD protein implicated in transport of long-chain fatty acids.

We recently reported (Harmon et al., J. Membrane Biol. 124:261-268, 1991) that sulfo-N-succinimidyl derivatives of long-chain fatty acids (SS-FA) specifically inhibited transport of oleate by rat adipocytes. These compounds bound to an 85-90 kD membrane protein which was also labeled by another inhibitor of FA transport [3H]DIDS (4,4'-diisothiocyanostilbene-2-2'-sulfonate). These results indicated that the protein was a strong candidate as the transporter for long-chain fatty acids. In this report we determined that the apparent size of the protein is 88 kD and its isoelectric point is 6.9. We used [3H]SS-oleate (SSO), which specifically labels the 88-kD protein, to isolate it from rat adipocyte plasma membranes. Identification of 15 amino acids at the N-terminus region revealed strong sequence homology with two previously described membrane glycoproteins: CD36, a ubiquitous protein originally identified in platelets and PAS IV, a protein that is enriched in the apical membranes of lipid-secreting mammary cells during lactation. Antibody against PAS IV cross-reacted with the adipocyte protein. This, together with the N-terminal sequence homology, suggested that the adipocyte protein belongs to a family of related intrinsic membrane proteins which include CD36 and PAS IV.

Glycerolipid synthesis in isolated adipocytes: substrate dependence and influence of norepinephrine.

Studies of fatty acid (FA) esterification by adipocytes have led to conflicting views with respect to how the process is regulated by norepinephrine (NE). It remains unclear whether NE directly modulates the pathway or whether its effects are indirect and reflect its well-known action to activate lipolysis. Changes in lipolysis can complicate estimation of esterification rates by altering both medium FA and the hydrolysis of newly formed FA esters. In this report, we describe an experimental approach that determined the effect of NE on FA esterification, amidst the complications introduced by activation of lipolysis. Esterification rates were estimated from the simultaneous incorporations (0.1-60 min) of [14C]glucose and [3H]oleate into diglyceride (DG), phospholipid (PL), and triglyceride (TG). Saturation kinetics of incorporation rates, with respect to FA, and more specifically to unbound or albumin-free FA (ubFA), were determined in both basal and NE-treated cells. To obtain true estimates of ester synthesis, incorporation rates were adjusted for label loss from breakdown of labeled esters. Our findings were: 1) In basal versus NE-treated cells, [3H]oleate, on its pathway to esterification, was diluted, respectively, by 2 and 50% of measured cell FA, and the diluting FA appeared derived from lipolysis. 2) Syntheses of PL, DG, and TG, estimated from incorporation of [14C]glucose, saturated at low ubFA. The Km for TG synthesis (0.06 microM) was within the physiological range of ubFA which meant that changes in plasma FA will modulate TG synthesis. PL synthesis, on the other hand (Km less than 0.01 microM), would be largely saturated under physiological conditions. 3) NE treatment increased the molar ratio of FA to albumin in the medium an average 8-fold and ubFA about 87-fold. In addition, NE accelerated hydrolysis of labeled PL and DG. Adjusting incorporation rates for these changes indicated that NE does not directly regulate glyceride synthesis. The assays described should allow estimation of glycerolipid synthesis under various metabolic or disease states and will distinguish direct effects from those reflecting changes in FA concentration or in hydrolysis of labeled FA esters.

Increased affinity predominates in insulin stimulation of glucose transport in the adipocyte.

The kinetics for transport of glucose and 3-O-methylglucose (MeGlc) were determined in preparations of rat adipocytes characterized by low basal rates which could be increased consistently more than 30-fold by addition of insulin (10 nM). In basal cells, the Km of [14C]glucose uptake was about 75 mM. The Ki for glucose inhibition of [14C]MeGlc uptake was similarly high (105 mM). These results were further confirmed by studies of basal glucose consumption which remained linear up to the highest glucose concentration used (30 mM) and by measurements of MeGlc transport kinetics (net and equilibrium exchange Km were both about 35 mM). Basal glucose uptake was determined to be stereospecific and cytochalasin sensitive (greater than 90%) and thus could not be dismissed as nonmediated diffusion. Insulin treatment decreased the transport Km for glucose and MeGlc to one-tenth the values measured in basal cells. The Vmax for glucose was doubled and that for MeGlc quadrupled. We conclude that insulin brings about a great reduction in the Km of hexose transport in adipocytes. This can only be perceived if activation of basal cells by experimental manipulations is avoided. The decrease in Km is the major kinetic factor involved in the stimulation of glucose transport by insulin.

Insulin antagonizes epinephrine activation of the membrane transport of fatty acids. Potential site for hormonal suppression of lipid mobilization.

Membrane transport of long chain fatty acids in the isolated rat adipocyte can be strongly stimulated by epinephrine (Abumrad, N. A., Perry, P. R., and Whitesell, R. R. (1985) J. Biol. Chem. 260, 9969-9971). We now report that insulin at physiological concentrations can completely block or reverse the epinephrine effect. Insulin was optimally effective at a concentration of about 0.1 nM in inhibiting transport activation by 0.3 and 3 microM epinephrine (0.1 and 1.0 microgram/ml). High concentrations of insulin (above 1 nM) were generally less effective and this was particularly true at the highest dose of epinephrine (1.0 microgram/ml). The insulin effect was shown to be on the transport process since insulin inhibited epinephrine activation of transport in both directions (influx and efflux). No effect of insulin on basal transport was observed over a wide range of concentrations (0.01-10 nM). Insulin's antagonism of transport activation by epinephrine appeared dependent on ATP metabolism since it was abolished by preincubating the cells with dinitrophenol (1 mM). Dinitrophenol, however, could not reverse the insulin effect when exposure to the hormone preceded that to dinitrophenol, consistent with an action of insulin at the transport step. The data indicate that regulation of the membrane transport of fatty acids is a potential site for insulin's action to suppress lipid mobilization.

Evidence for functionally distinct glucose transporters in basal and insulin-stimulated adipocytes.

The activity and Km of glucose transport of rat adipocytes are quite variable in the basal state. This could be due to differing levels of highly saturable transport against a background of less saturable transport. Such heterogeneity could lead to differing conclusions as to the Km of basal cells compared to insulin-stimulated cells depending on the choice of substrate, the range of concentrations tested, and the rigor of data analysis. In the present work, we used a cell preparation which was stable and partially activated by constant agitation. We used a two-component model to fit the concentration dependence of D-glucose uptake. We defined two parallel pathways of glucose entry, a high-affinity/low-capacity pathway and a low-affinity/high-capacity pathway. Both pathways were stereospecific and were inhibited by cytochalasin B. The low-affinity pathway in basal cells had 97% of the total capacity (Vmax) with a high Km (greater than 50 mM). A second pathway had a very low Km (less than 1 mM) and only 3% of the total capacity, but contributed to 30-60% of glucose uptake at 8 mM glucose. In insulin-stimulated cells, a pathway with a Km of 4-5 mM dominated and contributed 85% of glucose transport. The low-affinity but not the very high affinity pathway persisted in stimulated cells, but its contribution was only 10-15% of transport at 8 mM glucose. These results suggest the presence of at least two functionally distinct transporters whose respective contributions can be characterized by nonlinear regression of data over a wide range of glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of endogenous triglyceride breakdown in the mouse diaphragm.

The control of endogenous triglyceride breakdown was studied in vitro, in the incubated intact mouse diaphragm. Isoproterenol (2 microgram/ml) produced parallel increases in glycerol and free fatty acid release, and in tissue cyclic AMP levels, suggesting that cyclic AMP mediates the action of the catecholamine on triglyceride mobilization. In addition to cyclic AMP, calcium seems to be involved in the action of isoproterenol because preincubation of hemidiaphragms in the presence of the calcium ionophore A23187 decreased the lipolytic effect of the drug. Insulin (12.5 mU/ml) antagonized the action of isoproterenol on triglyceride breakdown (it decreased glycerol and free fatty acid release) without altering its stimulatory effect on cyclic AMP levels. On the other hand, no detectable effect on lipolysis was observed with carbachol in control and denervated hemidiaphragms, although the latter possess acetylcholine receptors over the entire surface area of the muscle. It was concluded that catecholamines control triglyceride breakdown in muscle while the cholinergic system does not seem to be involved. Cyclic AMP, calcium, and insulin all affect lipolysis in muscle and the interrelationships remain to be elucidated.

Lipolysis and cyclic AMP response to isoproterenol in diaphragms from control and dystrophic mice.

A comparison was made of the sensitivity of lipolysis (glycerol and free fatty acid release) and of cyclic AMP production to the action of isoproterenol in diaphragms from control and dystrophic Bar Harbor mice at 7 weeks of age. An increased lipolytic response was observed in diaphragms from dystrophic mice that was more apparent in the males, and was demonstrable when cyclic AMP was used instead of isoproterenol. The increased glycerol and free fatty acid release in response to isoproterenol and cyclic AMP cannot be explained by a higher triglyceride content of diaphragms from dystrophic mice, because it was found to be similar to that of controls when it was estimated by biochemical and light microscopic techniques. The increased lipolytic response was not paralleled by changes in cyclic AMP levels, which were found to be similar in diaphragms from control and dystrophic mice, whether in the basal or the stimulated state. It was concluded that the lipolytic apparatus in muscles from dystrophic mice shows an increased sensitivity to isoproterenol that seems to be related to events more intracellular than the cAMP production step.