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The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.

Syntrophins are a family of proteins forming membrane-anchored scaffolds and serving as adaptors for various transmembrane and intracellular signaling molecules. To understand the physiological roles of β1 syntrophin, one of the least characterized members, we generated mouse models to eliminate β1 syntrophin specifically in the endocrine or exocrine pancreas. β1 syntrophin is dispensable for the morphology and function of insulin-producing β cells. However, mice with β1 syntrophin deletion in exocrine acinar cells exhibit increased severity of cerulein-induced acute pancreatitis. Reduced expression of cystic fibrosis transmembrane conductance regulator and dilation of acinar lumen are potential predisposition factors. During the disease progression, a relative lack of autophagy is associated with deficiencies in both actin assembly and endoplasmic reticulum nucleation. Our findings reveal, for the first time, that β1 syntrophin is a critical regulator of actin cytoskeleton and autophagy in pancreatic acinar cells and is potently protective against cerulein-induced acute pancreatitis.

This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca2+-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca2+ sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE-Biotin) and Cy5-streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored. This protocol is based on a method developed to study yeast vacuolar fusion. In contrast to other protocols used to study the release machinery, this assay incorporates N-ethylmaleimide sensitive factor (NSF) and α-SNAP, which disassemble syntaxin-1 and SNAP-25 heterodimers. As a result, fusion requires Munc18-1, which binds to the released syntaxin-1, and Munc13-1, which, together with Munc18-1, orchestrates SNARE complex assembly. The protocol can be readily adapted to investigation of other types of intracellular membrane fusion by using appropriate alternative proteins. Total time required for one round of the assay is 4 d.

Munc13–1 acts as a master regulator of neurotransmitter release, mediating docking-priming of synaptic vesicles and diverse presynaptic plasticity processes. It is unclear how the functions of the multiple domains of Munc13–1 are coordinated. The crystal structure of a Munc13–1 fragment including its C1, C2B and MUN domains (C1C2BMUN) reveals a 19.5 nm-long multi-helical structure with the C1 and C2B domains packed at one end. The similar orientations of the respective diacyglycerol- and Ca2+-binding sites of the C1 and C2B domains suggest that the two domains cooperate in plasma-membrane binding and that activation of Munc13–1 by Ca2+ and diacylglycerol during short-term presynaptic plasticity are closely interrelated. Electrophysiological experiments in mouse neurons support the functional importance of the domain interfaces observed in C1C2BMUN. The structure imposes key constraints for models of neurotransmitter release and suggests that Munc13–1 bridges the vesicle and plasma membranes from the periphery of the membrane-membrane interface. DOI: http://dx.doi.org/10.7554/eLife.22567.001, eLife digest The human brain contains billions of cells called neurons that communicate with each other using molecules called neurotransmitters. An electrical signal in one neuron triggers the release of neurotransmitters from the cell, which then activate or inhibit electrical signals in neighboring neurons. Inside the cell, neurotransmitters are stored in small bubble-like structures called synaptic vesicles. The vesicles fuse with the membrane that surrounds the cell to release the neurotransmitters. This process must be tightly controlled to ensure that neurotransmitters are released rapidly and at the right time. A protein called Munc13 is a key component of the machinery that regulates the fusion of synaptic vesicles. It helps the synaptic vesicle to dock onto the cell membrane and get ready for fusion. Munc13 is a large protein and contains several different regions, including three domains called C1, C2B and MUN. These three domains control the release of neurotransmitters, but how they do so is poorly understood. Xu, Camacho et al. used a technique called X-ray crystallography to analyse the three-dimensional shape of the part of Munc13 that contains the three domains. The experiments reveal that the MUN domain forms a long rod-like shape with the C1 and C2B domains packed at one end. Several mutations that reduce the ability of the domains to interact with each other altered the release of neurotransmitters from mouse neurons to different extents. These findings suggest that the overall architecture of the region containing the C1, C2B and MUN domains is important for the normal activity of Munc13. The structure revealed by Xu, Camacho et al. sets a framework for understanding how Munc13 controls neurotransmitter release, and thus mediates diverse forms of information processing in the brain. DOI: http://dx.doi.org/10.7554/eLife.22567.002

Adenosine triphosphate (ATP) is released from cholangiocytes into bile and is a potent secretogogue by increasing intracellular Ca2+ and stimulating fluid and electrolyte secretion via binding purinergic (P2) receptors on the apical membrane. Although morphological differences exist between small and large cholangiocytes (lining small and large bile ducts, respectively), the role of P2 signaling has not been previously evaluated along the intrahepatic biliary epithelium. The aim of these studies therefore was to characterize ATP release and P2-signaling pathways in small (MSC) and large (MLC) mouse cholangiocytes. The findings reveal that both MSCs and MLCs express P2 receptors, including P2X4 and P2Y2. Exposure to extracellular nucleotides (ATP, uridine triphosphate, or 2′,3′-O-[4-benzoyl-benzoyl]-ATP) caused a rapid increase in intracellular Ca2+ concentration and in transepithelial secretion (Isc) in both cell types, which was inhibited by the Cl− channel blockers 5-nitro-2-(-3-phenylpropylamino)-benzoic acid (NPPB) or niflumic acid. In response to mechanical stimulation (flow/shear or cell swelling secondary to hypotonic exposure), both MSCs and MLCs exhibited a significant increase in the rate of exocytosis, which was paralleled by an increase in ATP release. Mechanosensitive ATP release was two-fold greater in MSCs compared to MLCs. ATP release was significantly inhibited by disruption of vesicular trafficking by monensin in both cell types. Conclusion: These findings suggest the existence of a P2 signaling axis along intrahepatic biliary ducts with the “upstream” MSCs releasing ATP, which can serve as a paracrine signaling molecule to “downstream” MLCs stimulating Ca2+-dependent secretion. Additionally, in MSCs, which do not express the cystic fibrosis transmembrane conductance regulator, Ca2+-activated Cl− efflux in response to extracellular nucleotides represents the first secretory pathway clearly identified in these cholangiocytes derived from the small intrahepatic ducts. (HEPATOLOGY 2010)

OBJECTIVE—Fluxes through mitochondrial pathways are defective in insulin-resistant skeletal muscle, but it is unclear whether similar mitochondrial defects play a role in the liver during insulin resistance and/or diabetes. The purpose of this study is to determine whether abnormal mitochondrial metabolism plays a role in the dysregulation of both hepatic fat and glucose metabolism during diabetes. RESEARCH DESIGN AND METHODS—Mitochondrial fluxes were measured using 2H/13C tracers and nuclear magnetic resonance spectroscopy in ZDF rats during early and advanced diabetes. To determine whether defects in hepatic fat oxidation can be corrected by peroxisome proliferator–activated receptor (PPAR-)-α activation, rats were treated with WY14,643 for 3 weeks before tracer administration. RESULTS—Hepatic mitochondrial fat oxidation in the diabetic liver was impaired twofold secondary to decreased ketogenesis, but tricarboxylic acid (TCA) cycle activity and pyruvate carboxylase flux were normal in newly diabetic rats and elevated in older rats. Treatment of diabetic rats with a PPAR–α agonist induced hepatic fat oxidation via ketogenesis and hepatic TCA cycle activity but failed to lower fasting glycemia or endogenous glucose production. In fact, PPAR-α agonism overstimulated mitochondrial TCA cycle flux and induced pyruvate carboxylase flux and gluconeogenesis in lean rats. CONCLUSIONS—The impairment of certain mitochondrial fluxes, but preservation or induction of others, suggests a complex defect in mitochondrial metabolism in the diabetic liver. These data indicate an important codependence between hepatic fat oxidation and gluconeogenesis in the normal and diabetic state and potentially explain the sometimes equivocal effect of PPAR-α agonists on glycemia.

The initial response of liver cells to insulin is mediated through exocytosis of Cl− channel-containing vesicles and a subsequent opening of plasma membrane Cl− channels. Intracellular accumulation of fatty acids leads to profound defects in metabolism, and is closely associated with insulin resistance. It is not known whether the activity of Cl− channels is altered in insulin resistance and by which mechanisms. We studied the effects of fatty acid accumulation on Cl− channel opening in a model liver cell line. Overnight treatment with amiodarone increased the fat content by ∼2-fold, and the rates of gluconeogenesis by ∼5-fold. The ability of insulin to suppress gluconeogenesis was markedly reduced indicating that amiodarone treatment induces insulin resistance. Western blot analysis showed that these cells express the same number of insulin receptors as control cells. However, insulin failed to activate exocytosis and Cl− channel opening. These inhibitory effects were mimicked in control cells by exposures to arachidonic acid (15 μm). Further studies demonstrated that fatty acids stimulate the PKC activity, and inhibition of PKC partially restored exocytosis and Cl− channel opening in insulin-resistant cells. Accordingly, activation of PKC with PMA in control cells potently inhibited the insulin responses. These results suggest that stimulation of PKC activity in insulin resistance contributes to the inhibition of cellular responses to insulin in liver cells.

Carnitine palmitoyltransferase I (CPT I) catalyzes the formation of acylcarnitine, the first step in the oxidation of long- chain fatty acids in mitochondria. The enzyme exists as liver (L-CPT I) and muscle (M-CPT I) isoforms that are encoded by separate genes. Genetic deficiency of L-CPT I, which has been reported in 16 patients from 13 families, is characterized by ep- isodes of hypoketotic hypoglycemia beginning in early child- hood and is usually associated with fasting or illness. To date, only two mutations associated with L-CPT I deficiency have been reported. In the present study we have identified and characterized the mutations underlying L-CPT I deficiency in six patients: five with classic symptoms of L-CPT I deficiency and one with symptoms that have not previously been associ- ated with this disorder (muscle cramps and pain). Transfection of the mutant L-CPT I cDNAs in COS cells resulted in L-CPT I mRNA levels that were comparable to those expressed from the wild-type construct. Western blotting revealed lower levels of each of the mutant proteins, indicating that the low enzyme activity associated with these mutations was due, at least in part, to protein instability. The patient with atypical symptoms had � 20% of normal L-CPT I activity and was homozygous for a mutation (c.1436C → T) that substituted leucine for proline at codon 479. Assays performed with his cultured skin fibroblasts indicated that this mutation confers partial resistance to the inhibitory effects of malonyl-CoA. The demonstration of L-CPT I deficiency in this patient suggests that the spectrum of clinical sequelae associated with loss or alteration of L-CPT I function may be broader than was previously recognized .— Brown, N. F., R. S. Mullur, I. Subramanian, V. Esser, M. J. Ben- nett, J-M. Saudubray, A. S. Feigenbaum, J. A. Kobari, P. M. Macleod, J. D. McGarry, and J. C. Cohen. Molecular character- ization of L-CPT I deficiency in six patients: insights into func- tion of the native enzyme. J. Lipid Res. 2001. 42: 1134-1142.

Because we had found whole testis from adult rats to be much richer in the messenger RNA for the muscle (M) than for the liver (L) form of mitochondrial carnitine palmitoyltransferase I (CPT I), we sought to determine which cell type(s) accounts for this expression pattern and how it might relate to reproductive function. Studies with immature (14-day-old) and adult animals included 1) Northern blot analysis of testis mRNA; 2) in situ hybridization with slices of testis; 3) enzyme assays for CPT I, CPT II, and carnitine acetyltransferase (CAT) in testicular germ cells and nongerm cells, together with measurement of the malonyl-coenzyme A (CoA) sensitivity and affinity for carnitine of CPT I; 4) labeling of testicular CPT I with [3H]etomoxir, a covalent inhibitor of the enzyme; and 5) the response of testicular and nontesticular CPT I to dietary etomoxir. The data established the following: 1) L-CPT I was the sole isoform detected in immature testis. 2) Expression of the M-CPT I gene was associated only with meiotic and postmeiotic germ cells. 3) Adult testis contains a mixture of the L- and M-CPT I enzymes, the L and M form dominating in extratubular cells and spermatids, respectively. Mature epididymal spermatozoa appear to be devoid of CPT I activity while possessing abundant levels of CPT II and CAT. 4) Five days of dietary etomoxir treatment at a dose that resulted in essentially complete inhibition of CPT I in liver, heart, skeletal muscle, and kidney was totally without effect on either the L- or M-type enzyme in the testis of mature rats. The data point to an important role for transient expression of M-CPT I, coupled with sustained activity of CAT, in the maturation and/or function of rat sperm. They also suggest that, at least in the case of one CPT I inhibitor (etomoxir), the testis is unusually resistant to the agent when given orally.

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPT I) represents the initial and regulated step in the β-oxidation of fatty acids. It exists in at least two isoforms, denoted L (liver) and M (muscle) types, with very different kinetic properties and sensitivities to malonyl-CoA. Here we have examined the relative expression of the CPT I isoforms in two different models of adipocyte differentiation and in a number of rat tissues. Adipocytes from mice, hamsters and humans were also evaluated. Primary monolayer cultures of undifferentiated rat preadipocytes expressed solely L-CPT I, but significant levels of M-CPT I emerged after only 3 days of differentiation in vitro; in the mature cell M-CPT I predominated. In sharp contrast, the murine 3T3-L1 preadipocyte expressed essentially exclusively L-CPT I, both in the undifferentiated state and throughout the differentiation process in vitro. This was also true of the mature mouse white fat cell. Fully developed adipocytes from the hamster and human behaved similarly to those of the rat. Thus the mouse white fat cell differs fundamentally from those of the other species examined in terms of its choice of a key regulatory enzyme in fatty acid metabolism. In contrast, brown adipose tissue from all three rodents displayed the same isoform profiles, each expressing overwhelmingly M-CPT I. Northern blot analysis of other rat tissues established L-CPT I as the dominant isoform not only in liver but also in kidney, lung, ovary, spleen, brain, intestine and pancreatic islets. In addition to its primacy in skeletal muscle, heart and fat, M-CPT I was also found to dominate in the testis. The same inter-tissue isoform pattern (with the exception of white fat) was found in the mouse. Taken together, the data bring to light an intriguing divergence between white adipocytes of the mouse and other mammalian species. They also raise a cautionary note that should be considered in the choice of animal model used in further studies of fat cell physiology.

We asked whether the well known starvation-induced impairment of glucose-stimulated insulin secretion (GSIS) seen in isolated rat pancreas preparations also applies in vivo. Accordingly, fed and 18-24-h-fasted rats were subjected to an intravenous glucose challenge followed by a hyperglycemic clamp protocol, during which the plasma-insulin concentration was measured. Surprisingly, the acute (5 min) insulin response was equally robust in the two groups. However, after infusion of the antilipolytic agent, nicotinic acid, to ensure low levels of plasma FFA before the glucose load, GSIS was essentially ablated in fasted rats, but unaffected in fed animals. Maintenance of a high plasma FFA concentration by coadministration of Intralipid plus heparin to nicotinic acid-treated rats (fed or fasted), or further elevation of the endogenous FFA level in nonnicotinic acid-treated fasted animals by infusion of etomoxir (to block hepatic fatty acid oxidation), resulted in supranormal GSIS. The in vivo findings were reproduced in studies with the perfused pancreas from fed and fasted rats in which GSIS was examined in the absence and presence of palmitate. The results establish that in the rat, the high circulating concentration of FFA that accompanies food deprivation is a sine qua non for efficient GSIS when a fast is terminated. They also serve to underscore the powerful interaction between glucose and fatty acids in normal beta cell function and raise the possibility that imbalances between the two fuels in vivo could have pathological consequences.

A cDNA encoding full-length carnitine palmitoyltransferase I (CPT I) from rat liver was expressed in Saccharomyces cerevisiae, a system devoid of endogenous CPT activity. The recombinant enzyme was of the expected size (as deduced from immunoblots), membrane-bound, and detergent-labile. It was also potently inhibited by malonyl-CoA, with an I50 value (concentration causing 50% inhibition) of approximately 5 microM, similar to that of the native enzyme in rat liver mitochondria. A truncated variant of the enzyme that lacked the amino-terminal 82 residues encompassing the first hydrophobic domain retained catalytic function but was much less sensitive to malonyl-CoA (I50 > 80 microM). Deletion of the cDNA segment encoding amino acids 31-148 (which includes both first and second hydrophobic stretches) resulted in no detectable product. The data establish unequivocally that a single polypeptide possesses both catalytic and malonyl-CoA binding domains, as well as the other properties previously attributed by us to native CPT I in mammalian mitochondria, and should thus put to rest the controversy surrounding this issue (Kerner, J., Zaluzec, E., Gage, D., and Bieber, L. L. (1994) J. Biol. Chem. 269, 8209-8219). In addition, the results strengthen the view that one site of interaction of malonyl-CoA with the rat liver enzyme involves the NH2-terminal region of the molecule.

To begin to explore the basis for the tissue-specific expression of mitochondrial carnitine palmitoyltransferase I (CPT I), we focused on three rat tissues (liver, heart, and skeletal muscle) in which the enzyme was known to display very different properties. In Northern blot analysis, a cDNA probe corresponding to liver CPT I readily hybridized to a 4.5-kilobase species of mRNA in liver and heart, but not in skeletal muscle. Using the same probe to screen a neonatal rat heart cDNA library, a full-length clone, surprisingly having 100% sequence identity to the liver CPT I cDNA, was isolated. The paradox was resolved by two additional experiments. First, in Western blots of mitochondrial membranes, an antibody raised against liver CPT I recognized the 88-kDa protein in heart, as well as in liver, but not in skeletal muscle. Second, high specific activity [3H]deschloroetomoxir (a covalent ligand for CPT I) reacted with a single form of CPT I in liver (approximately 88 kDa) and skeletal muscle (approximately 82 kDa), while proteins of both sizes were labeled in the cardiac myocyte. Tritiated ligand binding to the two heart proteins was blocked by excess unlabeled malonyl-CoA. It is concluded that liver and skeletal muscle each contains a single and distinct isoform of CPT I with monomeric size of approximately 88 and 82 kDa, respectively. The heart contains a CPT I protein of approximately 82 kDa in size (probably identical to the skeletal muscle protein) but, importantly, also expresses the liver-type enzyme. The results likely explain why previous studies of heart CPT I yielded an apparent Km for carnitine and I50 value for malonyl-CoA inhibition that were intermediate between those of the liver and skeletal muscle enzymes.

G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma β-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.

Our objective was to isolate from rat liver mitochondria the malonyl-CoA-regulated and detergent-labile enzyme, carnitine palmitoyltransferase I (CPT I), whose properties and relationship to CPT II have been the subject of debate. After exposure of mitochondria to the dinitrophenol derivative of etomoxir-CoA (DNP-Et-CoA, a covalent inhibitor of CPT I), followed by detergent solubilization and blue Sepharose chromatography, the DNP-Et-labeled CPT I could be readily visualized on immunoblots using an anti-DNP monoclonal antibody. This material was used to raise a rabbit polyclonal antibody that recognized CPT I regardless of whether it was carrying a covalent ligand. Exposure of membranes from untreated mitochondria to a mixture of trypsin and chymotrypsin caused rapid loss of CPT I activity with a concomitant disappearance of immunodetectable protein. However, inclusion of malonyl-CoA in such incubations afforded major protection of CPT I activity. Under these conditions CPT I simply underwent truncation from approximately 90 to approximately 82 kDa. This was also true if CPT I had first been labeled with Et-CoA or DNP-Et-CoA prior to protease treatment. Thus, the presence of an inhibitor, whether reversible or irreversible, at the active site of CPT I limited the action of trypsin/chymotrypsin to removal of a small portion of the protein which was probably not necessary for catalytic function. These and other experiments with antibodies and proteases provided additional insight into the membrane topology of CPT I. They also strengthened our conviction that CPT I and CPT II are distinct proteins and that the former exists as tissue-specific isoforms. Finally, the 82-kDa truncated form of rat liver CPT I was isolated and subjected to partial amino acid analysis. Four unambiguous peptide sequences were obtained.

We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase I (CPT I). Oligonucleotides corresponding to two tryptic peptides derived from the malonyl-CoA/etomoxir-CoA-binding protein of rat liver mitochondria (Esser, V., Kuwajima, M., Britton, C. H., Krishnan, K., Foster, D. W., and McGarry, J. D. (1993) J. Biol. Chem. 268, 5810-5816) were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone obtained consisted of a 102-nucleotide 5'-untranslated region, a single open reading frame of 2,319 bases predicting a protein of 773 amino acids (M(r) = 88,150), and a 3'-untranslated segment of 1,957 nucleotides followed by the poly(A)+ tail. A 0.9-kilobase fragment of the cDNA recognized a single species of mRNA (approximately 4.7 kilobases in size) in rat liver. The identity of the cDNA was confirmed by the findings that (i) the open reading frame encoded all four peptides found in the original protein; (ii) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV6, resulted in a selective and 10-20-fold induction of a malonyl-CoA- and etomoxir-CoA-sensitive CPT activity; and (iii) the overexpressed product was readily detected on Western blots by an antibody raised against the starting material. It seems likely that the de novo synthesized enzyme is targeted to the mitochondrial outer membrane via a leader peptide and that the mature protein achieves membrane anchoring through a stretch of 20 amino acids present near its amino terminus. The predicted amino acid sequence of the protein shows regions of strong identity with those of three other rat acyltransferases, namely, liver CPT II, liver carnitine octanoyltransferase, and brain choline acetyltransferase. The findings provide the first insight into the structure of a CPT I isoform. They also establish unequivocally that CPT I and CPT II are distinct proteins and that inhibitors of CPT I interact within its catalytic domain, not with an associated regulatory component.

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.

In the liver, adenosine triphosphate (ATP) is an extracellular signaling molecule that is released into bile and stimulates a biliary epithelial cell secretory response via engagement of apical P2 receptors. The molecular identities of the ion channels involved in ATP-mediated secretory responses have not been fully identified. Intermediate-conductance Ca2+-activated K+ channels (IK) have been identified in biliary epithelium, but functional data are lacking. The aim of these studies therefore was to determine the location, function, and regulation of IK channels in biliary epithelial cells and to determine their potential contribution to ATP-stimulated secretion. Expression of IK-1 mRNA was found in both human Mz-Cha-1 biliary cells and polarized normal rat cholangiocyte (NRC) monolayers, and immunostaining revealed membrane localization with a predominant basolateral signal. In single Mz-Cha-1 cells, exposure to ATP activated K+ currents, increasing current density from 1.6 ± 0.1 to 7.6 ± 0.8 pA/pF. Currents were dependent on intracellular Ca2+ and sensitive to clotrimazole and TRAM-34 (specific IK channel inhibitors). Single-channel recording demonstrated that clotrimazole-sensitive K+ currents had a unitary conductance of 46.2 ± 1.5 pS, consistent with IK channels. In separate studies, 1-EBIO (an IK activator) stimulated K+ currents in single cells that were inhibited by clotrimazole. In polarized NRC monolayers, ATP significantly increased transepithelial secretion which was inhibited by clotrimazole. Lastly, ATP-stimulated K+ currents were inhibited by the P2Y receptor antagonist suramin and by the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB. Together these studies demonstrate that IK channels are present in biliary epithelial cells and contribute to ATP-stimulated secretion through a P2Y-IP3 receptor pathway.

It is well established that medium and long chain (+)-acylcarnitines (i.e. fatty acid esters of the unnatural d-isomer of carnitine) inhibit the oxidation of long chain fatty acids in mammalian tissues by interfering with some component(s) of the mitochondrial carnitine palmitoyltransferase (CPT) system. However, whether their site of action is at the level of CPT I (outer membrane), CPT II (inner membrane), carnitine-acylcarnitine translocase (CACT, inner membrane), or some combination of these elements has never been resolved. We chose to readdress this question using rat liver mitochondria and employing a variety of assays that distinguish between the three enzyme activities. The effect on each of (+)-acetylcarnitine, (+)-hexanoylcarnitine, (+)-octanoylcarnitine, (+)-decanoylcarnitine, and (+)-palmitoylcarnitine was examined. Contrary to longstanding belief, none of these agents was found to impact significantly upon the activity of CPT I or CPT II. Whereas (+)-acetylcarnitine also failed to influence CACT, both (+)-octanoylcarnitine and (+)-palmitoylcarnitine strongly inhibited this enzyme with a similar IC(50) value ( approximately 35 microm) under the assay conditions employed. Remarkably, (+)-decanoylcarnitine was even more potent (IC(50) approximately 5 microm), whereas (+)-hexanoylcarnitine was far less potent (IC(50)200 microm). These findings resolve a 35-year-old puzzle by establishing unambiguously that medium and long chain (+)-acylcarnitines suppress mitochondrial fatty acid transport solely through the inhibition of the CACT component. They also reveal a surprising rank order of potency among the various (+)-acylcarnitines in this respect and should prove useful in the design of future experiments in which selective blockade of CACT is desired.

Objective: To validate magnetic resonance spectroscopy (MRS) as a tool for non-invasive quantification of pancreatic triglyceride (TG) content and to measure the pancreatic TG content in a diverse human population with a wide range of body mass index (BMI) and glucose control.Methods: To validate the MRS method, we measured TG content in the pancreatic tissue of 12 lean and 12 fatty ZDF rats (ages 5–14 weeks) both by MRS and the gold standard biochemical assay. We used MRS to measure pancreatic TG content in vivo in 79 human volunteers. Additionally, to assess the reproducibility of the method, in 33 volunteers we obtained duplicate MRS measurements 1–2 weeks apart.Results: MRS quantifies pancreatic TG content with high reproducibility and concordance to the biochemical measurement (Spearman’s rank correlation coefficient = 0.91). In humans, median pancreatic TG content was as follows: (1) normal weight and normoglycemic group 0.46 f/w%, (2) overweight or obese but normoglycemic group 3.16 f/w%, (3) impaired fasting glucose or impaired glucose tolerance group (BMI matched with group 2) 5.64 f/w%, and (4) untreated type 2 diabetes group (BMI matched with group 2) 5.54 f/w% (Jonckheere-Terpstra trend test across groups p < 0.001).Conclusions: Human pancreatic steatosis, as measured by MRS, increases with BMI and with impaired glycemia. MRS is a quantitative and reproducible non-invasive clinical research tool which will enable systematic studies of the relationship between ectopic fat accumulation in the pancreas and development of type 2 diabetes.Pancreatic triglyceride content as measured by Magnetic Resonance Spectroscopy in humans is elevated prior to development of type 2 diabetes, early in the natural history of the disease.

[35S]Methionine-labeled porcine heart citrate synthase (used here as a positive control) and rat liver carnitine palmitoyltransferase II (CPT II) were generated by in vitro transcription and translation of their cDNA constructs in appropriate Bluescript plasmids. Each product was imported into rat liver mitochondria in an energy-dependent manner to yield an immunoprecipitable protein of smaller size that comigrated with the corresponding purified enzyme. The size shift occurring with citrate synthase was consistent with the removal of the postulated 27-amino acid leader peptide. To determine the amino terminus of mature CPT II, [35S]methionine- or [3H]leucine-labeled material (after import and processing) was subjected to Edman degradation, followed by counting of the radioactivity released on each cycle. The results established that the precursor targeting peptide was cleaved between leucine 25 and serine 26 in the previously deduced amino acid sequence. Taken in conjunction with the recent report of Finocchiaro et al. (Finocchiaro, G., Taroni, F., Rocchi, M., Martin, A. L., Colombo, I., Tarelli, G. T., and DiDonato, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 661-665), the present results establish three key points concerning the rat and human forms of CPT II. First, it appears that in both species the initial translation product contains 658 amino acids and, upon mitochondrial import, is reduced in length by 25 residues through cleavage at an identical site. Second, the difference in electrophoretic mobility between the two mature proteins (documented earlier) presumably reflects either anomalous behavior of one of them on polyacrylamide gels or differential covalent modification. Finally, the recent suggestion by Brady et al. (Brady, P. S., Liu, J. S., Park, E. A., Hanson, R. W., and Brady, L. J. (1991) FASEB J. 5, A817) that our CPT II cDNA construct is incomplete in the 5'-coding region is refuted.

5'-AMP-activated kinase (AMPK) plays a key role in the regulation of cellular lipid metabolism. The contribution of vesicular exocytosis to this regulation is not known. Accordingly, we studied the effects of AMPK on exocytosis and intracellular lipid content in a model liver cell line. Activation of AMPK by metformin or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased the rates of constitutive exocytosis by about 2-fold. Stimulation of exocytosis by AMPK occurred within minutes, and persisted after overnight exposure to metformin or AICAR. Activation of AMPK also increased the amount of triacylglycerol (TG) and apolipoprotein B (apoB) secreted from lipid-loaded cells. These effects were accompanied by a decrease in the intracellular lipid content indicating that exocytosis of lipoproteins was involved in these lipid-lowering effects. While AMPK increased the rates of fatty acid oxidation (FAO), the lipid-lowering effects were quantitatively significant even after inhibition of FAO with R-etomoxir. These results suggest that hepatic AMPK stimulates constitutive exocytosis of lipoproteins, which may function in parallel with FAO to regulate intracellular lipid content.

Properties of the carnitine palmitoyltransferase (EC 2.3.1.21) (CPT) enzyme system were compared in isolated mitochondria from a range of tissues in rodents, monkey, and man. Common features were as follows: (a) while membrane-bound, CPT I, but not CPT II, was inhibited reversibly by malonyl-coenzyme A (CoA) and irreversibly by CoA esters of certain oxirane carboxylic acids; (b) the detergent, Tween-20, readily solubilized CPT II in active form while leaving CPT I membrane associated and catalytically functional; (c) octyl glucoside and Triton X-100 released active CPT II but caused essentially complete loss of CPT I activity. Use of [3H]tetradecylglycidyl-CoA, a covalent ligand for CPT I, yielded estimates of the enzyme's monomeric molecular size: approximately 86 kDa in non-hepatic tissues and approximately 90-94 kDa in liver, depending upon species. A polyclonal antibody to purified rat liver CPT II recognized a single protein in each tissue; its apparent molecular mass was approximately 70 kDa in all rat tissues and approximately 68 kDa in all mouse tissues as well as monkey and human liver. On Northern blot analysis a rat liver CPT II cDNA probe detected a single approximately 2.5-kilobase mRNA in all rat and mouse tissues examined. The following points are emphasized. First, CPT I and II are different proteins. Second, within a species CPT II, but not CPT I, is probably conserved across tissue lines. Third, slight variations in size of both enzymes were found in different species, although, at least in the case of CPT II, significant amino acid identity exists among the various isoforms. Fourth, CPT I, unlike CPT II, requires membrane integrity for catalytic function. Finally, the strategic use of detergents provides a simple means of discriminating between the two enzyme activities.

We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase II (CPT II). Beginning with the purified protein CNBr fragments were generated and sequenced. Corresponding oligonucleotides were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone ultimately obtained consisted of a 62 nucleotide 5'-untranslated region, a single open reading frame of 1,974 bases predicting a protein of 658 amino acids (Mr = 74,119), and a 3'-untranslated segment of 260 nucleotides followed by the poly (A) tail. The identity of the cDNA was confirmed by the findings that (a) the open reading frame encoded all three peptides found in the original protein; (b) a fourth peptide synthesized from a portion of the deduced amino acid sequence and used to immunize a rabbit resulted in the generation of an antibody that recognized pure CPT II on a Western blot; (c) in vitro transcription and translation of the cDNA (ligated into pBlue-script KS (+] generated a protein that was specifically immunoprecipitated by anti-CPT II antibody and having a Mr slightly greater than that of mature CPT II; (d) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV4, resulted in a 6-fold induction of mitochondrial CPT II catalytic activity. It seems likely that the de novo synthesized enzyme gains entry into the mitochondrion via a targeting peptide that is subsequently cleaved. The mature protein probably associates (relatively loosely) with the inner membrane through a limited number of membrane spanning domains. The predicted amino acid sequence of CPT II shows strong identity with those of two other acyltransferases, namely, rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase.

Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates the utilization of fat as an energy source during starvation and is the molecular target for the fibrate dyslipidemia drugs. Here, we identify the endocrine hormone fibroblast growth factor 21 (FGF21) as a mediator of the pleiotropic actions of PPARalpha. FGF21 is induced directly by PPARalpha in liver in response to fasting and PPARalpha agonists. FGF21 in turn stimulates lipolysis in white adipose tissue and ketogenesis in liver. FGF21 also reduces physical activity and promotes torpor, a short-term hibernation-like state of regulated hypothermia that conserves energy. These findings demonstrate an unexpected role for the PPARalpha-FGF21 endocrine signaling pathway in regulating diverse metabolic and behavioral aspects of the adaptive response to starvation.

Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the beta-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9A resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.

Genetic predisposition and environmental influences insidiously converge to cause glucose intolerance and hyperglycemia. Beta-cell compensates by secreting more insulin and when it fails, overt diabetes mellitus ensues. The need to understand the mechanisms involved in insulin secretion cannot be stressed enough. Phosphorylation of proteins plays an important role in regulating insulin secretion. In order to understand how a particular cellular process is regulated by protein phosphorylation the nature of the protein kinases and protein phosphatases involved and the mechanisms that determine when and where these enzymes are active should be investigated. While the actions of protein kinases have been intensely studied within the beta-cell, less emphasis has been placed on protein phosphatases even though they play an important regulatory role. This review focuses on the importance of protein phosphatase 2A in insulin secretion. Most of the present knowledge on protein phosphatase 2A originates from protein phosphatase inhibitor studies on islets and beta-cell lines. The ability of protein phosphatase 2A to change its activity in the presence of glucose and inhibitors provides clues to its role in regulating insulin secretion. An aggressive approach to elucidate the substrates and mechanisms of action of protein phosphatases is crucial to the understanding of phosphorylation events within the beta-cell. Characterizing protein phosphatase 2A within the beta-cell will certainly help us in understanding the mechanisms involved in insulin secretion and provide valuable information for drug development.

The initial response of liver cells to insulin is mediated through exocytosis of Cl- channel-containing vesicles and a subsequent opening of plasma membrane Cl- channels. Intracellular accumulation of fatty acids leads to profound defects in metabolism, and is closely associated with insulin resistance. It is not known whether the activity of Cl- channels is altered in insulin resistance and by which mechanisms. We studied the effects of fatty acid accumulation on Cl- channel opening in a model liver cell line. Overnight treatment with amiodarone increased the fat content by approximately 2-fold, and the rates of gluconeogenesis by approximately 5-fold. The ability of insulin to suppress gluconeogenesis was markedly reduced indicating that amiodarone treatment induces insulin resistance. Western blot analysis showed that these cells express the same number of insulin receptors as control cells. However, insulin failed to activate exocytosis and Cl- channel opening. These inhibitory effects were mimicked in control cells by exposures to arachidonic acid (15 microm). Further studies demonstrated that fatty acids stimulate the PKC activity, and inhibition of PKC partially restored exocytosis and Cl- channel opening in insulin-resistant cells. Accordingly, activation of PKC with PMA in control cells potently inhibited the insulin responses. These results suggest that stimulation of PKC activity in insulin resistance contributes to the inhibition of cellular responses to insulin in liver cells.

Udgivelsesdato: 2003-Aug Lipid perturbations associated with triglyceride overstorage in beta-cells impair insulin secretion, a process termed lipotoxicity. To assess the role of hormone-sensitive lipase, which is expressed and enzymatically active in beta-cells, in the development of lipotoxicity, we generated transgenic mice overexpressing hormone-sensitive lipase specifically in beta-cells. Transgenic mice developed glucose intolerance and severely blunted glucose-stimulated insulin secretion when challenged with a high-fat diet. As expected, both lipase activity and forskolin-stimulated lipolysis was increased in transgenic compared with wild-type islets. This was reflected in significantly lower triglycerides levels in transgenic compared with wild-type islets in mice receiving the high-fat diet, whereas no difference in islet triglycerides was found between the two genotypes under low-fat diet conditions. Our results highlight the importance of mobilization of the islet triglyceride pool in the development of beta-cell lipotoxicity. We propose that hormone-sensitive lipase is involved in mediating beta-cell lipotoxicity by providing ligands for peroxisome proliferator-activated receptors and other lipid-activated transcription factors, which in turn alter the expression of critical genes. One such gene might be uncoupling protein-2, which was found to be upregulated in transgenic islets, a change that was accompanied by decreased ATP levels.

Cross-sectional studies in human subjects have used 1 H magnetic resonance spectroscopy (HMRS) to demonstrate that insulin resistance correlates more tightly with the intramyocellular lipid (IMCL) concentration than with any other identified risk factor. To further explore the interaction between these two elements in the rat, we used two strategies to promote the storage of lipids in skeletal muscle and then evaluated subsequent changes in insulin-mediated glucose disposal. Normal rats received either a low-fat or a high-fat diet (20% lard oil) for 4 weeks. Two additional groups (low-fat + etoxomir and lard + etoxomir) consumed diets containing 0.01% of the carnitine palmitoyltransferase-1 inhibitor, R-etomoxir, which produced chronic blockade of enzyme activity in liver and skeletal muscle. Both the high-fat diet and drug treatment significantly impaired insulin sensitivity, as measured with the hyperinsulinemiceuglycemic clamp. Insulin-mediated glucose disposal (IMGD) fell from 12.57 ± 0.72 in the low-fat group to 9.79 ± 0.59, 8.96 ± 0.38, and 7.32 ± 0.28 μmol · min -1 · 100 g -1 in the low-fat + etoxomir, lard, and lard + etoxomir groups, respectively. We used HMRS, which distinguishes between fat within the myocytes and fat associated with contaminating adipocytes located in the muscle bed, to assess the IMCL content of isolated soleus muscle. A tight inverse relationship was found between IMGD and IMCL, the correlation ( R = 0.96) being much stronger than that seen between IMGD and either fat mass or weight. In conclusion, either a diet rich in saturated fat or prolonged inhibition of fatty acid oxidation impairs IMGD in rats via a mechanism related to the accumulation of IMCL.

The onset of NIDDM in obese Zucker diabetic fatty (fa/fa) rats is preceded by a striking increase in the plasma levels of free fatty acids (FFAs) and by a sixfold rise in triglyceride content in the pancreatic islets. The latter finding provides clear evidence of elevated tissue levels of long-chain fatty acyl CoA, which can impair β-cell cell function. To determine if the triglyceride accumulation is entirely the passive consequence of high plasma FFA levels or if prediabetic islets have an increased lipogenic capacity that might predispose to NIDDM, the metabolism of long-chain fatty acids was compared in islets of obese prediabetic and nonprediabetic Zucker diabetic fatty (ZDF) rats and of lean Wistar and lean ZDF rats. When cultured in 1 or 2 mmol/l FFA, islets of both female and male obese rats accumulated, respectively, 7 and 15 times as much triglyceride as islets from lean rats exposed to identical FFA concentrations. The esterification of [14C]palmitate and 9,10-[3H]palmitate was increased in islets of male obese rats and could not be accounted for by defective oxidation of 9,10-[3H]-palmitate. Glycerol-3-PO4 acyltransferase (GPAT) activity was 12 times that of controls. The mRNA of GPAT was increased in islets of obese rats. We conclude that, in the presence of comparable elevations in FFA concentrations, the islets of obese prediabetic rats have a higher lipogenic capacity than controls. This could be a factor in their high risk of diabetes.

Fatty acids are important metabolic substrates for the pancreatic beta-cell, and long term exposure of pancreatic islets to elevated concentrations of fatty acids results in an alteration of glucose-induced insulin secretion. Previous work suggested that exaggerated fatty acid oxidation may be implicated in this process by a mechanism requiring changes in metabolic enzyme expression. We have therefore studied the regulation of carnitine palmitoyltransferase I (CPT I) gene expression by fatty acids in the pancreatic beta-cell line INS-1 since this enzyme catalyzes the limiting step of fatty acid oxidation in various tissues. Palmitate, oleate, and linoleate (0.35 mM) elicited a 4-6-fold increase in CPT I mRNA. The effect was dose-dependent and was similar for saturated and unsaturated fatty acids. It was detectable after 1 h and reached a maximum after 3 h. The induction of CPT I mRNA by fatty acids did not require their oxidation, and 2-bromopalmitate, a nonoxidizable fatty acid, increased CPT I mRNA to the same extent as palmitate. The induction was not prevented by cycloheximide treatment of cells indicating that it was mediated by pre-existing transcription factors. Neither glucose nor pyruvate and various secretagogues had a significant effect except glutamine (7 mM) which slightly induced CPT I mRNA. The half-life of the CPT I transcript was unchanged by fatty acids, and nuclear run-on analysis showed a rapid (less than 45 min) and pronounced transcriptional activation of the CPT I gene by fatty acids. The increase in CPT I mRNA was followed by a 2-3-fold increase in CPT I enzymatic activity measured in isolated mitochondria. The increase in activity was time-dependent, detectable after 4 h, and close to maximal after 24 h. Fatty acid oxidation by INS-1 cells, measured at low glucose, was also 2-3-fold higher in cells cultured with fatty acid in comparison with control cells. Long term exposure of INS-1 cells to fatty acid was associated with elevated secretion of insulin at a low (5 mM) concentration of glucose and a decreased effect of higher glucose concentrations. It also resulted in a decreased oxidation of glucose. The results indicate that the CPT I gene is an early response gene induced by fatty acids at the transcriptional level in beta- (INS-1) cells. It is suggested that exaggerated fatty acid oxidation caused by CPT-1 induction is implicated in the process whereby fatty acids alter glucose-induced insulin secretion.

In the rat, the gene for liver mitochondrial carnitine palmitoyltransferase I (CPT I), though dormant prior to birth, is rapidly activated postnatally. We sought to elucidate which hormonal and/or nutritional factors might be responsible for this induction. In cultured hepatocytes from 20-day-old rat fetus, the concentration of CPT I mRNA, which initially was very low, increased dramatically in a dose-dependent manner after exposure of the cells to dibutyryl cAMP (Bt2cAMP). Similar results were obtained when long-chain fatty acids (LCFA), but not medium-chain fatty acids, were added to the culture medium. The effects of Bt2cAMP and LCFA were antagonized by insulin, also dose dependently. In contrast, CPT II gene expression, which was already high in fetal hepatocytes, was unaffected by any of the above manipulations. Bt2cAMP stimulated CPT I gene expression even when endogenous triacylglycerol breakdown was suppressed by lysosomotropic agents suggesting that the actions of cAMP and LCFA were distinct. Moreover, half-maximal concentrations of Bt2cAMP and linoleate produced an additive effect CPT I mRNA accumulation. While linoleate and Bt2cAMP stimulated CPT I gene transcription by twofold and fourfold, respectively, the fatty acid also increased the half-life of CPT I mRNA (50%). When hepatocytes were cultured in the presence of 2-bromopalmitate, (which is readily converted by cells into its non-metabolizable CoA ester) CPT I mRNA accumulation was higher than that observed with oleate or linoleate. Similarly, the CPT I inhibitor, tetradecylglycidate, which at a concentration of 20 microM did not itself influence the CPT I mRNA level, enhanced the stimulatory effect of linoleate. The implication is that induction of the CPT I message by LCFA does not require mitochondrial metabolism of these substrates; however, formation of their CoA esters is a necessary step. Unlike linoleate, the peroxisome proliferator, clofibrate, increased both CPT I and CPT II mRNA levels and neither effect was offset by insulin. It thus appears that the mechanism of action of LCFA differs from that utilized by clofibrate, which presumably works through the peroxisome proliferator activated receptor. We conclude that the rapid increase in hepatic CPT I mRNA level that accompanies the fetal to neonatal transition in the rat is triggered by the reciprocal change in circulating insulin and LCFA concentrations, coupled with elevation of the liver content of cAMP.