Artemisia dracunculus L. extract ameliorates insulin sensitivity by attenuating inflammatory signalling in human skeletal muscle culture.

AIMS: Bioactives of Artemisia dracunculus L. (termed PMI 5011) have been shown to improve insulin action by increasing insulin signalling in skeletal muscle. However, it was not known if PMI 5011's effects are retained during an inflammatory condition. We examined the attenuation of insulin action and whether PMI 5011 enhances insulin signalling in the inflammatory environment with elevated cytokines. METHODS: Muscle cell cultures derived from lean, overweight and diabetic-obese subjects were used. Expression of pro-inflammatory genes and inflammatory response of human myotubes were evaluated by real-time polymerase chain reaction (RT-PCR). Insulin signalling and activation of inflammatory pathways in human myotubes were evaluated by multiplex protein assays. RESULTS: We found increased gene expression of monocyte chemoattractant protein 1 (MCP1) and TNFα (tumour necrosis factor alpha), and basal activity of the NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway in myotubes derived from diabetic-obese subjects as compared with myotubes derived from normal-lean subjects. In line with this, basal Akt phosphorylation (Ser473) was significantly higher, while insulin-stimulated phosphorylation of Akt (Ser473) was lower in myotubes from normal-overweight and diabetic-obese subjects compared with normal-lean subjects. PMI 5011 treatment reduced basal phosphorylation of Akt and enhanced insulin-stimulated phosphorylation of Akt in the presence of cytokines in human myotubes. PMI 5011 treatment led to an inhibition of cytokine-induced activation of inflammatory signalling pathways such as Erk1/2 and IkBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha)-NFkB and moreover, NFkB target gene expression, possibly by preventing further propagation of the inflammatory response within muscle tissue. CONCLUSIONS: PMI 5011 improved insulin sensitivity in diabetic-obese myotubes to the level of normal-lean myotubes despite the presence of pro-inflammatory cytokines.

Inhibition of carnitine palymitoyltransferase1b induces cardiac hypertrophy and mortality in mice.

Recent reports suggest that short-term pharmacological carnitine palmitoyltransferase 1 (Cpt1) inhibition improves skeletal muscle glucose tolerance and insulin sensitivity. Although this appears promising for the treatment of diabetes, these Cpt1 inhibitors are not specific to skeletal muscle and target multiple Cpt1 isoforms. To assess the effects of inhibiting the Cpt1b isoform we generated mice with a heart- and skeletal muscle-specific deletion of the Cpt1b, Cpt1b(HM-/-). These mice seem to develop normally with similar bodyweights as control mice. However, premature mortality was observed by 15 weeks of age in the Cpt1b(HM-/-) mice. The hearts of Cpt1b(HM-/-) mice were four times the size of controls. Cpt1b(HM-/-) mice were also subject to stress-induced seizures that accompanied an increased risk for premature mortality. Our data suggests that prolonged Cpt1b inhibition poses severe cardiac risk and emphasizes that attempts to improve insulin sensitivity by targeting Cpt1 with current inhibitors is not viable.

Carnitine revisited: potential use as adjunctive treatment in diabetes.

AIMS/HYPOTHESIS: This study examined the efficacy of supplemental L: -carnitine as an adjunctive diabetes therapy in mouse models of metabolic disease. We hypothesised that carnitine would facilitate fatty acid export from tissues in the form of acyl-carnitines, thereby alleviating lipid-induced insulin resistance. MATERIALS AND METHODS: Obese mice with genetic or diet-induced forms of insulin resistance were fed rodent chow +/- 0.5% L: -carnitine for a period of 1-8 weeks. Metabolic outcomes included insulin tolerance tests, indirect calorimetry and mass spectrometry-based profiling of acyl-carnitine esters in tissues and plasma. RESULTS: Carnitine supplementation improved insulin-stimulated glucose disposal in genetically diabetic mice and wild-type mice fed a high-fat diet, without altering body weight or food intake. In severely diabetic mice, carnitine supplementation increased average daily respiratory exchange ratio from 0.886 +/- 0.01 to 0.914 +/- 0.01 (p < 0.01), reflecting a marked increase in systemic carbohydrate oxidation. Similarly, under insulin-stimulated conditions, carbohydrate oxidation was higher and total energy expenditure increased from 172 +/- 10 to 210 +/- 9 kJ kg fat-free mass(-1) h(-1) in the carnitine-supplemented compared with control animals. These metabolic improvements corresponded with a 2.3-fold rise in circulating levels of acetyl-carnitine, which accounts for 86 and 88% of the total acyl-carnitine pool in plasma and skeletal muscle, respectively. Carnitine supplementation also increased several medium- and long-chain acyl-carnitine species in both plasma and tissues. CONCLUSIONS/INTERPRETATION: These findings suggest that carnitine supplementation relieves lipid overload and glucose intolerance in obese rodents by enhancing mitochondrial efflux of excess acyl groups from insulin-responsive tissues. Carefully controlled clinical trials should be considered.

Secreted proteins and genes in fetal and neonatal pig adipose tissue and stromal-vascular cells.

Although microarray and proteomic studies have indicated the expression of unique and unexpected genes and their products in human and rodent adipose tissue, similar studies of meat animal adipose tissue have not been reported. Thus, total RNA was isolated from stromal-vascular (S-V) cell cultures (n = 4; 2 arrays; 2 cultures/array) from 90-d (79% of gestation) fetuses and adipose tissue from 105-d (92% of gestation) fetuses (n = 2) and neonatal (5-d-old) pigs (n = 2). Duplicate adipose tissue microarrays (n = 4) represented RNA samples from a pig and a fetus. Dye-labeled cDNA probes were hybridized to custom microarrays (70-mer oligonucleotides) representing more than 600 pig genes involved in growth and reproduction. Microarray studies showed significant expression of 40 genes encoding for known adipose tissue secreted proteins in fetal S-V cell cultures and adipose tissue. Expression of 10 genes encoding secreted proteins not known to be expressed by adipose tissue was also observed in neonatal adipose tissue and fetal S-V cell cultures. Additionally, the agouti gene was detected by reverse transcription-PCR in pig S-V cultures and adipose tissue. Proteomic analysis of adipose tissue and fetal and young pig S-V cell culture-conditioned media identified multiple secreted proteins including heparin-like epidermal growth factor-like growth factor and several apolipoproteins. Another adipose tissue secreted protein, plasminogen activator inhibitor-1, was identified by ELISA in S-V cell culture media. A group of 20 adipose tissue secreted proteins were detected or identified using the gene microarray and the proteomic and protein assay approaches including apolipoprotein-A1, apolipoprotein-E, relaxin, brain-derived neurotrophic factor, and IGF binding protein-5. These studies demonstrate, for the first time, the expression of several major secreted proteins in pig adipose tissue that may influence local and central metabolism and growth.

Overexpression of agouti protein and stress responsiveness in mice.

Ectopic overexpression of agouti protein, an endogenous antagonist of melanocortin receptors' linked to the beta-actin promoter (BAPa) in mice, produces a phenotype of yellow coat color, Type II diabetes, obesity and increased somatic growth. Spontaneous overexpression of agouti increases stress-induced weight loss. In these experiments, other aspects of stress responsiveness were tested in 12-week-old male wild-type mice and BAPa mice. Two hours of restraint on three consecutive days produced greater increases in corticosterone and post-stress weight loss in BAPa than wild-type mice. In Experiment 2, anxiety-type behavior was measured immediately after 12 min of restraint. This mild stress did not produce many changes indicative of anxiety, but BAPa mice spent more time in the dark side of a light-dark box and less time in the open arms of an elevated plus maze than restrained wild-type mice. In a defensive withdrawal test, grooming was increased by restraint in all mice, but the duration of each event was substantially shorter in BAPa mice, possibly due to direct antagonism of the MC4-R by agouti protein. Thus, BAPa mice showed exaggerated endocrine and energetic responses to restraint stress with small differences in anxiety-type behavior compared with wild-type mice. These results are consistent with observations in other transgenic mice in which the melanocortin system is disrupted, but contrast with reports that acute blockade of central melanocortin receptors inhibits stress-induced hypophagia. Thus, the increased stress responsiveness in BAPa mice may be a developmental compensation for chronic inhibition of melanocortin receptors.

Agouti regulates adipocyte transcription factors.

Agouti is a secreted paracrine factor that regulates pigmentation in hair follicle melanocytes. Several dominant mutations cause ectopic expression of agouti, resulting in a phenotype characterized by yellow fur, adult-onset obesity and diabetes, increased linear growth and skeletal mass, and increased susceptibility to tumors. Humans also produce agouti protein, but the highest levels of agouti in humans are found in adipose tissue. To mimic the human agouti expression pattern in mice, transgenic mice (aP2-agouti) that express agouti in adipose tissue were generated. The transgenic mice develop a mild form of obesity, and they are sensitized to the action of insulin. We correlated the levels of specific regulators of insulin signaling and adipocyte differentiation with these phenotypic changes in adipose tissue. Signal transducers and activators of transcription (STAT)1, STAT3, and peroxisome proliferator-activated receptor (PPAR)-gamma protein levels were elevated in the transgenic mice. Treatment of mature 3T3-L1 adipocytes recapitulated these effects. These data demonstrate that agouti has potent effects on adipose tissue. We hypothesize that agouti increases adiposity and promotes insulin sensitivity by acting directly on adipocytes via PPAR-gamma.

Regulation of leptin by agouti.

Dominant mutations at the mouse Agouti locus lead to ectopic expression of the Agouti gene and exhibit diabetes, obesity, and yellow coat color. Obese yellow mice are hyperinsulinemic and hyperleptinemic, and we hypothesized that Agouti directly induces leptin secretion. Accordingly, we used transgenic mice expressing agouti in adipocytes (under the control of aP2 promoter, aP212) to examine changes in leptin levels. Agouti expression in adipose tissue did not significantly alter food intake, weight gain, fat pad weight, or insulinemia; however, the transgenic mice were hyperglycemic. We demonstrated that plasma leptin levels are approximately twofold higher in aP212 transgenic mice compared with their respective controls, whereas ubiquitous expression of agouti (under the control of beta-actin promoter, BAP20) led to a sixfold increase in leptin. Insulin treatment of aP212 mice increased adipocyte leptin content without affecting plasma leptin levels. These findings were further confirmed in vitro in 3T3-L1 adipocytes treated with recombinant Agouti protein and/or insulin. Agouti but not insulin significantly increased leptin secretion, indicating that insulin enhances leptin synthesis but not secretion while Agouti increases both leptin synthesis and secretion. This increased leptin synthesis and secretion was due to increased leptin mRNA levels by Agouti. Interestingly, agouti regulation of leptin was not mediated by melanocortin receptor 4, previously implicated in agouti regulation of food intake. These results suggest that increased leptin secretion by agouti may serve to limit agouti-induced obesity, independent of melanocortin receptor antagonism, and indicate that interaction between obesity genes may play a key role in obesity.

Macronutrient diet intake of the lethal yellow agouti (Ay/a) mouse.

To examine the effect of chronic endogenous melanocortin receptor (MC-R) antagonism on macronutrient diet selection, Ay/a mice that ectopically overexpress the MC-R antagonist, agouti, were fed a three-choice macronutrient diet of pure fat, carbohydrate, and protein. Ay/a mice gained more weight and consumed a greater proportion of their daily intake from fat and less from carbohydrate than wild-type littermates did. The increased fat preference was present immediately, and persisted throughout the 7-week long experiment. Protein intake was greater for Ay/a mice; however, the proportion of protein intake to total intake was similar between mouse types. Ovarian fat pads of Ay/a mice comprised a greater percentage of total body weight that that from wild-type littermates. These results suggest that endogenous inhibition of MC-Rs mediate the increased fat intake in growing mice.

An agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice.

Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agoutiDeltabasic) was expressed in transgenic mice under the control of the beta-actin promoter (BAPaDeltabasic). Three independent lines of BAPaDeltabasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agoutiDeltabasic protein was functional in vivo. However, none of the BAPaDeltabasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agoutiDeltabasic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agoutiDeltabasic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPaDeltabasic transgenic mice. These findings implicate a functional role for the agouti basic domain in vivo, possibly influencing the biogenesis of secreted agouti protein or modulating protein-protein interactions that contribute to effective antagonism of melanocortin receptors.

The agouti gene product stimulates pancreatic [beta]-cell Ca2+ signaling and insulin release.

Ubiquitous expression of the mouse agouti gene results in obesity and hyperinsulinemia. Human agouti is expressed in adipose tissue, and we found recombinant agouti protein to stimulate lipogenesis in adipocytes in a Ca(2+)-dependent fashion. However, adipocyte-specific agouti transgenic mice only became obese in the presence of hyperinsulinemia. Because intracellular Ca(2+) concentration ([Ca(2+)](i)) is a primary signal for insulin release, and we have shown agouti protein to increase [Ca(2+)](i) in several cell types, we examined the effects of agouti on [Ca(2+)](i) and insulin release. We demonstrated the expression of agouti in human pancreas and generated recombinant agouti to study its effects on Ca(2+) signaling and insulin release. Agouti (100 nM) stimulated Ca(2+) influx, [Ca(2+)](i) increase, and a marked stimulation of insulin release in two beta-cell lines (RIN-5F and HIT-T15; P < 0. 05). Agouti exerted comparable effects in isolated human pancreatic islets and beta-cells, with a 5-fold increase in Ca(2+) influx (P < 0.001) and a 2.2-fold increase in insulin release (P < 0.01). These data suggest a potential role for agouti in the development of hyperinsulinemia in humans.

IL-17 stimulates granulopoiesis in mice: use of an alternate, novel gene therapy-derived method for in vivo evaluation of cytokines.

IL-17 is a novel cytokine secreted principally by CD4+ T cells. It has been shown to support the growth of hemopoietic progenitors in vitro; however, its in vivo effects are presently unknown. Adenovirus-mediated gene transfer of the murine IL-17 cDNA targeted to the liver (5 x 10(9) plaque-forming units (PFU) intravenous) resulted in a transiently transgenic phenotype, with dramatic effects on in vivo granulopoiesis. Initially, there was a significant increase (fivefold) in the peripheral white blood count (WBC), including a 10-fold rise in the absolute neutrophil count. This was associated with a doubling in the spleen size over 7-14 days after gene transfer, which returned to near baseline by day 21, although the white blood cell count remained elevated. There was a profound stimulation of splenic hemopoiesis as demonstrated by an increase in total cellularity by 50% 7 days after gene transfer and an increase in hemopoietic colony formation. A maximal increase in frequency of high proliferative potential colonies (HPPC) (11-fold) and CFU-granulocyte-macrophage (GM) and CFU-granulocyte-erythrocyte-megakaryocyte-monocyte (GEMM) (CFU) (6-fold) was seen on day 3 after IL-17 gene transfer. Both CFU and HPPC remained significantly elevated in the spleen throughout day 21, but at reduced levels compared with day 3. Bone marrow CFU and HPPC were elevated on day 3 only by 75% and 25%, respectively, without changes in total cellularity. Thus, murine IL-17 is a cytokine that can stimulate granulopoiesis in vivo. Since IL-17 is principally produced by CD4+ T cells, this cytokine could have therapeutic implications in AIDS-related bone marrow failure and opportunistic infections.

The role of the agouti gene in the yellow obese syndrome.

The yellow obese syndrome in mice encompasses many pleiotropic effects including yellow fur, maturity-onset obesity, hyperinsulinemia, insulin resistance, hyperglycemia, increased skeletal length and lean body mass, and increased susceptibility to neoplasia. The molecular basis of this syndrome is beginning to be unraveled and may have implications for human obesity and diabetes. Normally, the agouti gene is expressed during the hair-growth cycle in the neonatal skin where it functions as a paracrine regulator of pigmentation. The secreted agouti protein antagonizes the binding of the alpha-melanocyte-stimulating hormone to its receptor (melanocortin 1 receptor) on the surface of hair bulb melanocytes, causing alterations in intracellular cAMP levels. Widespread, ectopic expression of the mouse agouti gene is central to the yellow obese phenotype, as demonstrated by the molecular cloning of several dominant agouti mutations and the ubiquitous expression of the wild-type agouti gene in transgenic mice. Recent experiments have revealed that the hypothalamus and adipose tissue are biologically active target sites for agouti in the yellow obese mutant lines.

Combined effects of insulin treatment and adipose tissue-specific agouti expression on the development of obesity.

The agouti gene product is a secreted protein that acts in a paracrine manner to regulate coat color in mammals. Several dominant mutations at the agouti locus in mice cause the ectopic, ubiquitous expression of agouti, resulting in a condition similar to adult-onset obesity and non-insulin-dependent diabetes mellitus. The human agouti protein is 85% homologous to mouse agouti; however, unlike the mouse agouti gene, human agouti is normally expressed in adipose tissue. To address whether expression of agouti in human adipose tissue is physiologically relevant, transgenic mice were generated that express agouti in adipose tissue. Similar to most humans, these mice do not become obese or diabetic. However, we found that daily insulin injections significantly increased weight gain in the transgenic lines expressing agouti in adipose tissue, but not in nontransgenic mice. These results suggest that insulin triggers the onset of obesity and that agouti expression in adipose tissue potentiates this effect. Accordingly, the investigation of agouti's role in obesity and non-insulin-dependent diabetes mellitus in mice holds significant promise for understanding the pathophysiology of human obesity.

The effects of calcium channel blockade on agouti-induced obesity.

We have previously observed that obese viable yellow (Avy/a) mice exhibit increased intracellular Ca2+ ([Ca2+]i) and fatty acid synthase (FAS) gene expression; further, recombinant agouti protein increases in cultured adipocytes and these effects are inhibited by Ca2+ channel blockade. Accordingly, we determined the effect of Ca2+ channel blockade (nifedipine for 4 wk) on FAS and obesity in transgenic mice expressing the agouti gene in a ubiquitous manner. The transgenic mice initially were significantly heavier (30.5+/-0.6 vs. 27.3+/-0.3 g; P<0.001) and exhibited a 0.81 degrees C lower initial core temperature (P<0.0005), an approximately twofold increase in fat pad weights (P=0.002), a sevenfold increase in adipose FAS activity (P=0.009), and a twofold increase in plasma insulin level (P<0.05) compared to control mice. Nifedipine treatment resulted in an 18% decrease in fat pad weights (P<0.007) and a 74% decrease in adipose FAS activity (P=0.03), normalized circulating insulin levels and insulin sensitivity (P<0.05), and transiently elevated core temperature in the transgenic mice, but was without effect in the control mice. These data suggest that agouti regulates FAS, fat storage, and possibly thermogenesis, at least partially, via a [Ca2+]i-dependent mechanism, and that Ca2+ channel blockade may partially attenuate agouti-induced obesity.

Insulin regulates enzyme activity, malonyl-CoA sensitivity and mRNA abundance of hepatic carnitine palmitoyltransferase-I.

The regulation of hepatic mitochondrial carnitine palmitoyltransferase-I (CPT-I) was studied in rats during starvation and insulin-dependent diabetes and in rat H4IIE cells. The Vmax. for CPT-I in hepatic mitochondrial outer membranes isolated from starved and diabetic rats increased 2- and 3-fold respectively over fed control values with no change in Km values for substrates. Regulation of malonyl-CoA sensitivity of CPT-I in isolated mitochondrial outer membranes was indicated by an 8-fold increase in Ki during starvation and by a 50-fold increase in Ki in the diabetic state. Peroxisomal and microsomal CPT also had decreased sensitivity to inhibition by malonyl-CoA during starvation. CPT-I mRNA abundance was 7.5 times greater in livers of 48-h-starved rats and 14.6 times greater in livers of insulin-dependent diabetic rats compared with livers of fed rats. In H4IIE cells, insulin increased CPT-I sensitivity to inhibition by malonyl-CoA in 4 h, and sensitivity continued to increase up to 24 h after insulin addition. CPT-I mRNA levels in H4IIE cells were decreased by insulin after 4 h and continued to decrease so that at 24 h there was a 10-fold difference. The half-life of CPT-I mRNA was 4 h in the presence of actinomycin D or with actinomycin D plus insulin. These results suggest that insulin regulates CPT-I by inhibiting transcription of the CPT-I gene.

Changes in carnitine palmitoyltransferase-I mRNA abundance produced by hyperthyroidism and hypothyroidism parallel changes in activity.

To study the regulation of carnitine palmitoyltransferase-I by thyroid hormone, a cDNA was obtained by PCR amplification of DNA obtained by reverse transcription of rat liver RNA. CPT-I mRNA abundance was measured in livers of hyperthyroid, euthyroid and hypothyroid rats. In hypothyroid rats, the CPT-I mRNA levels decreased 40-fold relative to that of the hyperthyroid animals. These changes paralleled alterations in enzyme activity. These data suggest that CPT-I is regulated at the transcriptional level by thyroid hormone.

Hepatic carnitine palmitoyltransferase-I has two independent inhibitory binding sites for regulation of fatty acid oxidation.

Partial proteolysis of carnitine palmitoyltransferase (CPT-I) in intact mitochondria results in greatly diminished sensitivity to inhibition by its physiological inhibitor, malonyl-CoA, but inhibition by succinyl-CoA and methylmalonyl-CoA was affected to a lesser extent, whereas inhibition by coenzyme A, acetyl-CoA, and propionyl-CoA was not affected at all by proteinase treatment. These data suggested that inhibitors that are coenzyme A esters of short-chain dicarboxylic acids bind to a regulatory malonyl-CoA binding site located on the cytoplasmic face of the mitochondrial outer membrane while coenzyme A esters of monocarboxylic acids and free coenzyme A act at the active site in the mitochondrial intermembrane space. All inhibitors whose potency was altered by proteinase action provided protection against proteinases, whereas other inhibitors did not. Preincubation with the substrates carnitine, palmitoyl-CoA, or coenzyme A prior to proteolysis showed no protective effects against the loss of inhibition or loss of activity; however, preincubation with these substrates enhanced proteinase effects to more seriously diminish activity and inhibition by malonyl-CoA. Proteinases were also found to act on purified mitochondrial outer membranes to reduce inhibition by malonyl-CoA with little effect on activity. Using these outer membrane preparations it was found that the very potent inhibition of CPT-I by the active-site-directed substrate analog (+)-hemipalmitoylcarnitinium was not altered by proteinase treatment; however, inhibition by the malonyl-CoA analog Ro 25-0187, which is a more potent inhibitor than malonyl-CoA, was drastically reduced by proteinase treatment of mitochondrial outer membranes, confirming the different locations for the malonyl-CoA site and the active site. Coenzyme A and malonyl-CoA both act as competitive inhibitors with respect to the acyl-CoA substrate, but coenzyme A lacks cooperative effects seen with malonyl-CoA. For ligand binding to the malonyl-CoA regulatory site, there appears to be a requirement for two carbonyl groups in close juxtaposition, but there is apparently no requirement for the coenzyme A moiety per se. Current evidence, including the recently deduced primary structure for CPT-I, favors the hypothesis that (a) inhibitors of CPT-I may act at two distinct sites, (b) malonyl-CoA binds primarily to a regulatory site that is distinct from the active site of carnitine palmitoyltransferase-I, and (c) the two inhibitory sites are located on opposite sides of the mitochondrial outer membrane.

Cholate extracts of mitochondrial outer membranes increase inhibition by malonyl-CoA of carnitine palmitoyltransferase-I by a mechanism involving phospholipids.

It has been reported that sodium cholate can separate the catalytic component of carnitine palmitoyltransferase-I (CPT-I) from a putative malonyl-CoA-binding regulatory protein capable of conferring sensitivity to malonyl-CoA on CPT-II. We found that cholate preferentially extracted a contaminating malonyl-CoA-sensitive CPT from mitochondrial inner membranes. When cholate extracts of outer membranes were incubated either with cholate extracts of inner membranes or with osmotically swollen mitochondria, inhibition of CPT by malonyl-CoA was increased. Treatment of intact mitochondria with subtilisin abolished the increased inhibition by malonyl-CoA, suggesting that the outer-membrane CPT-I was responsible for the increased inhibition. Incubation of cholate extracts with proteinase K did not prevent the increased inhibition. Fractionation of the cholate extract indicated the presence of phospholipids. Addition of cardiolipin or phosphatidylglycerol to osmotically swollen mitochondria increased sensitivity of CPT to malonyl-CoA, but several other phospholipids did not. When cardiolipin was added to intact mitochondria from either starved or fed rats, there were large increases in inhibition by malonyl-CoA; sensitivity in mitochondria from starved rats increased to that normally observed with mitochondria from fed rats. These results suggest that phospholipids are responsible for the increased inhibition of CPT by malonyl-CoA with added cholate extracts and that changes in membrane composition may be involved in the physiological regulation of CPT-I.