Pleiotropic Alterations in Lipid Metabolism in Yeast sac1 Mutants: Relationship to “Bypass Sec14p” and Inositol Auxotrophy

SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect “bypass Sec14p” will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene.

Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion.

Chlamydia trachomatis undergoes its entire life cycle within an uncharacterized intracellular vesicle that does not fuse with lysosomes. We used a fluorescent Golgi-specific probe, (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]) aminocaproylsphingosine (C6-NBD-Cer), in conjunction with conventional fluorescence or confocal microscopy to identify interactions between the Golgi apparatus and the chlamydial inclusion. We observed not only a close physical association between the Golgi apparatus and the chlamydial inclusion but the eventual presence of a metabolite of this fluorescent probe associated with the chlamydiae themselves. Sphingomyelin, endogenously synthesized from C6-NBD-Cer, was specifically transported to the inclusion and incorporated into the cell wall of the intracellular chlamydiae. Incorporation of the fluorescent sphingolipid by chlamydiae was inhibited by brefeldin A. Chlamydiae therefore occupy a vesicle distal to the Golgi apparatus that receives anterograde vesicular traffic from the Golgi normally bound for the plasma membrane. Collectively, the data suggest that the chlamydial inclusion may represent a unique compartment within the trans-Golgi network.

Increased CNS levels of apolipoprotein D in schizophrenic and bipolar subjects: Implications for the pathophysiology of psychiatric disorders

Chronic administration of the atypical antipsychotic drug, clozapine, to rodents has been shown to increase the concentration of apolipoprotein D (apoD) in several area of the brain, suggesting that apoD could be involved in the therapeutic effects of antipsychotic drugs and/or the pathology of psychotic illnesses. Here, we measured a significant decrease in the concentration of apoD in serum samples from schizophrenic patients. In contrast, apoD levels were significantly increased (92–287%) in dorsolateral prefrontal cortex (Brodmann's area 9) of schizophrenic and bipolar subjects. Elevated levels of apoD expression were also observed in the caudate of schizophrenic and bipolar subjects (68–89%). No differences in apoD immunoreactivity were detected in occipital cortex (Brodmann's area 18) in either group, or in the hippocampus, substantia nigra, or cerebellum of the schizophrenic group. The low serum concentrations of apoD observed in these patients supports recent hypotheses involving systemic insufficiencies in lipid metabolism/signaling in schizophrenia. Elevation of apoD expression selectively within central nervous system regions implicated in the pathology of these neuropsychiatric disorders suggests a focal compensatory response that neuroleptic drug regimens may augment.

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin’s biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin’s effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.

The “Spot 14” gene resides on the telomeric end of the 11q13 amplicon and is expressed in lipogenic breast cancers: Implications for control of tumor metabolism

Enhanced long chain fatty acid synthesis may occur in breast cancer, where it is necessary for tumor growth and predicts a poor prognosis. “Spot 14” (S14) is a carbohydrate- and thyroid hormone-inducible nuclear protein specific to liver, adipose, and lactating mammary tissues that functions to activate genes encoding the enzymes of fatty acid synthesis. Amplification of chromosome region 11q13, where the S14 gene (THRSP) resides, also predicts a poor prognosis in breast tumors. We localized the S14 gene between markers D11S906 and D11S937, at the telomeric end of the amplified region at 11q13, and found that it was amplified and expressed in breast cancer-derived cell lines. Moreover, concordant expression of S14 and a key lipogenic enzyme (acetyl-CoA carboxylase) in a panel of primary breast cancer specimens strongly supported a role for S14 as a determinant of tumor lipid metabolism. S14 expression provides a pathophysiological link between two prognostic indicators in breast cancer: enhanced lipogenesis and 11q13 amplification.

Circadian rhythms in Neurospora crassa: Lipid deficiencies restore robust rhythmicity to null frequency and white-collar mutants

The conidiation rhythm in the fungus Neurospora crassa is a model system for investigating the genetics of circadian clocks. Null mutants at the frq (frequency) locus (frq9 and frq10) make no functional frq gene products and are arrhythmic under standard conditions. The white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants, cel (chain-elongation) and chol-1 (choline-requirer), which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains chol-1 frq9, chol-1 frq10, chol-1 wc-1, chol-1 wc-2, cel frq9, cel frq10, and cel wc-2. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the frq, wc-1, or wc-2 gene products. The rhythms in the double mutant strains are similar to the cel and chol-1 parents, except that they are less sensitive to light. This suggests that the frq, wc-1, and wc-2 gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered.

Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes

Hepatic glucokinase plays a key role in glucose metabolism as underlined by the anomalies associated with glucokinase mutations and the consequences of tissue-specific knock-out. In the liver, glucokinase transcription is absolutely dependent on the presence of insulin. The cis-elements and trans-acting factors that mediate the insulin effect are presently unknown; this is also the case for most insulin-responsive genes. We have shown previously that the hepatic expression of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) is activated by insulin. We show here in primary cultures of hepatocytes that the adenovirus-mediated transduction of a dominant negative form of SREBP-1c inhibits the insulin effect on endogenous glucokinase expression. Conversely, in the absence of insulin, the adenovirus-mediated transduction of a dominant positive form of SREBP-1c overcomes the insulin dependency of glucokinase expression. Hepatic fatty acid synthase and Spot-14 are insulin/glucose-dependent genes. For this latter class of genes, the dominant positive form of SREBP-1c obviates the necessity for the presence of insulin, whereas glucose potentiates the effect of SREBP-1c on their expression. In addition, the insulin dependency of lipid accumulation in cultured hepatocytes is overcome by the dominant positive form of SREBP-1c. We propose that SREBP-1c is a major mediator of insulin action on hepatic gene expression and a key regulator of hepatic glucose/lipid metabolism.

Plasminogen deficiency accelerates vessel wall disease in mice predisposed to atherosclerosis

A critical link between hemostatic factors and atherosclerosis has been inferred from a variety of indirect observations, including the expression of procoagulant and fibrinolytic factors within atherosclerotic vessels, the presence of fibrin in intimal lesions, and the cellular infiltration of mural thrombi leading to their incorporation into developing plaques. To directly examine the role of the key fibrinolytic factor, plasminogen, in atherogenesis, plasminogen-deficient mice were crossed to hypercholesterolemic, apolipoprotein E-deficient mice predisposed to atherosclerosis. We report that the loss of plasminogen greatly accelerates the formation of intimal lesions in apolipoprotein E-deficient animals, whereas plasminogen deficiency alone does not cause appreciable atherosclerosis. These studies provide direct evidence that circulating hemostatic factors strongly influence vessel wall disease in the context of a disorder in lipid metabolism.

A new class of obesity genes encodes leukocyte adhesion receptors

Obesity is a complex disease, and multiple genes contribute to the trait. The description of five genes (ob, db, tub, Ay, and fat) responsible for distinct syndromes of spontaneous monogenic obesity in mice has advanced our knowledge of the genetics of obesity. However, many other genes involved in the expression of this disease remain to be determined. We report here the identification of an additional class of genes involved in the regulation of adipose tissue mass. These genes encode receptors mediating leukocyte adhesion. Mice deficient in intercellular adhesion molecule-1 became spontaneously obese in old age on normal mouse chow or at a young age when provided with a diet rich in fat. Mice deficient in the counterreceptor for intercellular adhesion molecule-1, the leukocyte integrin αMβ2 (Mac-1), showed a similar obesity phenotype. Since all mice consumed approximately the same amount of food as controls, the leukocyte function appears to be in regulating lipid metabolism and/or energy expenditure. Our results indicate that (i) leukocytes play a role in preventing excess body fat deposition and (ii) defects in leukocyte adhesion receptors can result in obesity.

Generation and reproductive phenotypes of mice lacking estrogen receptor β

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERα and ERβ. We previously generated and studied knockout mice lacking estrogen receptor α and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor β (ERβ −/−) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERβ −/− mice lack normal ERβ RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERβ is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERβ in bone and cardiovascular homeostasis.

The transcriptional program of a human B cell line in response to Myc

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity

Perilipin coats the lipid droplets of adipocytes and is thought to have a role in regulating triacylglycerol hydrolysis. To study the role of perilipin in vivo, we have created a perilipin knockout mouse. Perilipin null (peri−/−) and wild-type (peri+/+) mice consume equal amounts of food, but the adipose tissue mass in the null animals is reduced to ≈30% of that in wild-type animals. Isolated adipocytes of perilipin null mice exhibit elevated basal lipolysis because of the loss of the protective function of perilipin. They also exhibit dramatically attenuated stimulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity. Plasma leptin concentrations in null animals were greater than expected for the reduced adipose mass. The peri−/− animals have a greater lean body mass and increased metabolic rate but they also show an increased tendency to develop glucose intolerance and peripheral insulin resistance. When fed a high-fat diet, the perilipin null animals are resistant to diet-induced obesity but not to glucose intolerance. The data reveal a major role for perilipin in adipose lipid metabolism and suggest perilipin as a potential target for attacking problems associated with obesity.

Glial reduction in the subgenual prefrontal cortex in mood disorders

Mood disorders are among the most common neuropsychiatric illnesses, yet little is known about their neurobiology. Recent neuroimaging studies have found that the volume of the subgenual part of Brodmann’s area 24 (sg24) is reduced in familial forms of major depressive disorder (MDD) and bipolar disorder (BD). In this histological study, we used unbiased stereological techniques to examine the cellular composition of area sg24 in two different sets of brains. There was no change in the number or size of neurons in area sg24 in mood disorders. In contrast, the numbers of glia were reduced markedly in both MDD and BD. The reduction in glial number was most prominent in subgroups of subjects with familial MDD (24%, P = 0.01) or BD (41%, P = 0.01). The glial reduction in subjects without a clear family history was lower in magnitude and not statistically significant. Consistent with neuroimaging findings, cortical volume was reduced in area sg24 in subjects with familial mood disorders. Schizophrenic brains studied as psychiatric controls had normal neuronal and glial numbers and cortical volume. Glial and neuronal numbers also were counted in area 3b of the somatosensory cortex in the same group of brains and were normal in all psychiatric groups. Glia affect several processes, including regulation of extracellular potassium, glucose storage and metabolism, and glutamate uptake, all of which are crucial for normal neuronal activity. We thus have identified a biological marker associated with familial mood disorders that may provide important clues regarding the pathogenesis of these common psychiatric conditions.

A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in cftr−/− mice

A deficiency in essential fatty acid metabolism has been reported in plasma from patients with cystic fibrosis (CF). However, its etiology and role in the expression of disease is unknown. The objective of this study was to determine whether alterations in fatty acid metabolism are specific to CF-regulated organs and whether they play a role in the expression of disease. A membrane lipid imbalance was found in ileum, pancreas, and lung from cftr−/− mice characterized by an increase in phospholipid-bound arachidonic acid and a decrease in phospholipid-bound docosahexaenoic acid (DHA). This lipid imbalance was observed in organs pathologically affected by CF including lung, pancreas, and ileum and was not secondary to impaired intestinal absorption or hepatic biosynthesis of DHA. As proof of concept, oral administration of DHA to cftr−/− mice corrected this lipid imbalance and reversed the observed pathological manifestations. These results strongly suggest that certain phenotypic manifestations of CF may result from remediable alterations in phospholipid-bound arachidonic acid and DHA levels.

The lipid phosphatase activity of PTEN is critical for its tumor supressor function

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.