Recovering the regenerative potential in chronically injured nerves by using conditioning electrical stimulation.

OBJECTIVE: Chronically injured nerves pose a significant clinical challenge despite surgical management. There is no clinically feasible perioperative technique to upregulate a proregenerative environment in a chronic nerve injury. Conditioning electrical stimulation (CES) significantly improves sensorimotor recovery following acute nerve injury to the tibial and common fibular nerves. The authors' objective was to determine if CES could foster a proregenerative environment following chronically injured nerve reconstruction. METHODS: The tibial nerve of 60 Sprague Dawley rats was cut, and the proximal ends were inserted into the hamstring muscles to prevent spontaneous reinnervation. Eleven weeks postinjury, these chronically injured animals were randomized, and half were treated with CES proximal to the tibial nerve cut site. Three days later, 24 animals were killed to evaluate the effects of CES on the expression of regeneration-associated genes at the cell body (n = 18) and Schwann cell proliferation (n = 6). In the remaining animals, the tibial nerve defect was reconstructed using a 10-mm isograft. Length of nerve regeneration was assessed 3 weeks postgrafting (n = 16), and functional recovery was evaluated weekly between 7 and 19 weeks of regeneration (n = 20). RESULTS: Three weeks after nerve isograft surgery, tibial nerves treated with CES prior to grafting had a significantly longer length of nerve regeneration (p < 0.01). Von Frey analysis identified improved sensory recovery among animals treated with CES (p < 0.01). Motor reinnervation, assessed by kinetics, kinematics, and skilled motor tasks, showed significant recovery (p < 0.05 to p < 0.001). These findings were supported by immunohistochemical quantification of motor endplate reinnervation (p < 0.05). Mechanisms to support the role of CES in reinvigorating the regenerative response were assessed, and it was demonstrated that CES increased the proliferation of Schwann cells in chronically injured nerves (p < 0.05). Furthermore, CES upregulated regeneration-associated gene expression to increase growth-associated protein-43 (GAP-43), phosphorylated cAMP response element binding protein (pCREB) at the neuronal cell bodies, and upregulated glial fibrillary acidic protein expression in the surrounding satellite glial cells (p < 0.05 to p < 0.001). CONCLUSIONS: Regeneration following chronic axotomy is impaired due to downregulation of the proregenerative environment generated following nerve injury. CES delivered to a chronically injured nerve influences the cell body and the nerve to re-upregulate an environment that accelerates axon regeneration, resulting in significant improvements in sensory and motor functional recovery. Percutaneous CES may be a preoperative strategy to significantly improve outcomes for patients undergoing delayed nerve reconstruction.

Mutant huntingtin interacts with the sterol regulatory element-binding proteins and impairs their nuclear import.

Brain cholesterol homeostasis is altered in Huntington's disease (HD), a neurodegenerative disorder caused by the expansion of a CAG nucleotide repeat in the HTT gene. Genes involved in the synthesis of cholesterol and fatty acids were shown to be downregulated shortly after the expression of mutant huntingtin (mHTT) in inducible HD cells. Nuclear levels of the transcription factors that regulate lipid biogenesis, the sterol regulatory element-binding proteins (SREBP1 and SREBP2), were found to be decreased in HD models compared to wild-type, but the underlying causes were not known. SREBPs are synthesized as inactive endoplasmic reticulum-localized precursors. Their mature forms (mSREBPs) are generated upon transport of the SREBP precursors to the Golgi and proteolytic cleavage, and are rapidly imported into the nucleus by binding to importin ß. We show that, although SREBP2 processing into mSREBP2 is not affected in YAC128 HD mice, mSREBP2 is mislocalized to the cytoplasm. Chimeric mSREBP2-and mSREBP1-EGFP proteins are also mislocalized to the cytoplasm in immortalized striatal cells expressing mHTT, in YAC128 neurons and in fibroblasts from HD patients. We further show that mHTT binds to the SREBP2/importin ß complex required for nuclear import and sequesters it in the cytoplasm. As a result, HD cells fail to upregulate cholesterogenic genes under sterol-depleted conditions. These findings provide mechanistic insight into the downregulation of genes involved in the synthesis of cholesterol and fatty acids in HD models, and have potential implications for other pathways modulated by SREBPs, including autophagy and excitotoxicity.

PISD is a mitochondrial disease gene causing skeletal dysplasia, cataracts, and white matter changes.

Exome sequencing of two sisters with congenital cataracts, short stature, and white matter changes identified compound heterozygous variants in the PISD gene, encoding the phosphatidylserine decarboxylase enzyme that converts phosphatidylserine to phosphatidylethanolamine (PE) in the inner mitochondrial membrane (IMM). Decreased conversion of phosphatidylserine to PE in patient fibroblasts is consistent with impaired phosphatidylserine decarboxylase (PISD) enzyme activity. Meanwhile, as evidence for mitochondrial dysfunction, patient fibroblasts exhibited more fragmented mitochondrial networks, enlarged lysosomes, decreased maximal oxygen consumption rates, and increased sensitivity to 2-deoxyglucose. Moreover, treatment with lyso-PE, which can replenish the mitochondrial pool of PE, and genetic complementation restored mitochondrial and lysosome morphology in patient fibroblasts. Functional characterization of the PISD variants demonstrates that the maternal variant causes an alternative splice product. Meanwhile, the paternal variant impairs autocatalytic self-processing of the PISD protein required for its activity. Finally, evidence for impaired activity of mitochondrial IMM proteases suggests an explanation as to why the phenotypes of these PISD patients resemble recently described "mitochondrial chaperonopathies." Collectively, these findings demonstrate that PISD is a novel mitochondrial disease gene.

A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt-/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt-/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt-/- mice compared with those of Pemt+/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt-/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice.

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt-/- mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt-/- mice. Treatment with vitamin E (0.5 g/kg) for 3 weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt-/- mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.

LACTB is a tumour suppressor that modulates lipid metabolism and cell state.

Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression.

Fenofibrate, but not ezetimibe, prevents fatty liver disease in mice lacking phosphatidylethanolamine N-methyltransferase.

Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT) are protected from high-fat diet (HFD)-induced obesity and insulin resistance. However, these mice develop severe nonalcoholic fatty liver disease (NAFLD) when fed the HFD, which is mainly due to inadequate secretion of VLDL particles. Our aim was to prevent NAFLD development in mice lacking PEMT. We treated Pemt-/- mice with either ezetimibe or fenofibrate to see if either could ameliorate liver disease in these mice. Ezetimibe treatment did not reduce fat accumulation in Pemt-/- livers, nor did it reduce markers for hepatic inflammation or fibrosis. Fenofibrate, conversely, completely prevented the development of NAFLD in Pemt-/- mice: hepatic lipid levels, as well as markers of endoplasmic reticulum stress, inflammation, and fibrosis, in fenofibrate-treated Pemt-/- mice were similar to those in Pemt+/+ mice. Importantly, Pemt-/- mice were still protected against HFD-induced obesity and insulin resistance. Moreover, fenofibrate partially reversed hepatic steatosis and fibrosis in Pemt-/- mice when treatment was initiated after NAFLD had already been established. Increasing hepatic fatty acid oxidation can compensate for the lower VLDL-triacylglycerol secretion rate and prevent/reverse fatty liver disease in mice lacking PEMT.

Pioglitazone attenuates hepatic inflammation and fibrosis in phosphatidylethanolamine N-methyltransferase-deficient mice.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt(-/-) mice are protected against high-fat diet (HFD)-induced obesity and insulin resistance; however, these mice develop nonalcoholic fatty liver disease (NAFLD). We hypothesized that peroxisomal proliferator-activated receptor-γ (PPARγ) activation by pioglitazone might stimulate adipocyte proliferation, thereby directing lipids from the liver toward white adipose tissue. Pioglitazone might also act directly on PPARγ in the liver to improve NAFLD. Pemt(+/+) and Pemt(-/-) mice were fed a HFD with or without pioglitazone (20 mg·kg(-1)·day(-1)) for 10 wk. Pemt(-/-) mice were protected from HFD-induced obesity but developed NAFLD. Treatment with pioglitazone caused an increase in body weight gain in Pemt(-/-) mice that was mainly due to increased adiposity. Moreover, pioglitazone improved NAFLD in Pemt(-/-) mice, as indicated by a 35% reduction in liver weight and a 57% decrease in plasma alanine transaminase levels. Livers from HFD-fed Pemt(-/-) mice were steatotic, inflamed, and fibrotic. Hepatic steatosis was still evident in pioglitazone-treated Pemt(-/-) mice; however, treatment with pioglitazone reduced hepatic fibrosis, as evidenced by reduced Sirius red staining and lowered mRNA levels of collagen type Iα1 (Col1a1), tissue inhibitor of metalloproteinases 1 (Timp1), α-smooth muscle actin (Acta2), and transforming growth factor-ß (Tgf-ß). Similarly, oxidative stress and inflammation were reduced in livers from Pemt(-/-) mice upon treatment with pioglitazone. Together, these data show that activation of PPARγ in HFD-fed Pemt(-/-) mice improved liver function, while these mice were still protected against diet-induced obesity and insulin resistance.

Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle.

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt(-/-) mice than in Pemt(+/+) mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt(-/-) mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.

Vagus nerve contributes to the development of steatohepatitis and obesity in phosphatidylethanolamine N-methyltransferase deficient mice.

BACKGROUND & AIMS: Phosphatidylethanolamine N-methyltransferase (PEMT), a liver enriched enzyme, is responsible for approximately one third of hepatic phosphatidylcholine biosynthesis. When fed a high-fat diet (HFD), Pemt(-/-) mice are protected from HF-induced obesity; however, they develop steatohepatitis. The vagus nerve relays signals between liver and brain that regulate peripheral adiposity and pancreas function. Here we explore a possible role of the hepatic branch of the vagus nerve in the development of diet induced obesity and steatohepatitis in Pemt(-/-) mice. METHODS: 8-week old Pemt(-/-) and Pemt(+/+) mice were subjected to hepatic vagotomy (HV) or capsaicin treatment, which selectively disrupts afferent nerves, and were compared to sham-operated or vehicle-treatment, respectively. After surgery, mice were fed a HFD for 10 weeks. RESULTS: HV abolished the protection against the HFD-induced obesity and glucose intolerance in Pemt(-/-) mice. HV normalized phospholipid content and prevented steatohepatitis in Pemt(-/-) mice. Moreover, HV increased the hepatic anti-inflammatory cytokine interleukin-10, reduced chemokine monocyte chemotactic protein-1 and the ER stress marker C/EBP homologous protein. Furthermore, HV normalized the expression of mitochondrial electron transport chain proteins and of proteins involved in fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase in Pemt(-/-) mice. However, disruption of the hepatic afferent vagus nerve by capsaicin failed to reverse either the protection against the HFD-induced obesity or the development of HF-induced steatohepatitis in Pemt(-/-) mice. CONCLUSIONS: Neuronal signals via the hepatic vagus nerve contribute to the development of steatohepatitis and protection against obesity in HFD fed Pemt(-/-) mice.

The concentration of phosphatidylethanolamine in mitochondria can modulate ATP production and glucose metabolism in mice.

Phosphatidylethanolamine (PE) N-methyltransferase (PEMT) catalyzes the synthesis of phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected against diet-induced obesity and insulin resistance. We investigated the role of PEMT in hepatic carbohydrate metabolism in chow-fed mice. A pyruvate tolerance test revealed that PEMT deficiency greatly attenuated gluconeogenesis. The reduction in glucose production was specific for pyruvate; glucose production from glycerol was unaffected. Mitochondrial PC levels were lower and PE levels were higher in livers from Pemt(-/-) compared with Pemt(+/+) mice, resulting in a 33% reduction of the PC-to-PE ratio. Mitochondria from Pemt(-/-) mice were also smaller and more elongated. Activities of cytochrome c oxidase and succinate reductase were increased in mitochondria of Pemt(-/-) mice. Accordingly, ATP levels in hepatocytes from Pemt(-/-) mice were double that in Pemt(+/+) hepatocytes. We observed a strong correlation between mitochondrial PC-to-PE ratio and cellular ATP levels in hepatoma cells that expressed various amounts of PEMT. Moreover, mitochondrial respiration was increased in cells lacking PEMT. In the absence of PEMT, changes in mitochondrial phospholipids caused a shift of pyruvate toward decarboxylation and energy production away from the carboxylation pathway that leads to glucose production.

The membrane lipid phosphatidylcholine is an unexpected source of triacylglycerol in the liver.

The increased prevalence of obesity and diabetes in human populations can induce the deposition of fat (triacylglycerol) in the liver (steatosis). The current view is that most hepatic triacylglycerols are derived from fatty acids released from adipose tissue. In this study, we show that phosphatidylcholine (PC), an important structural component of cell membranes and plasma lipoproteins, can be a precursor of ~65% of the triacylglycerols in liver. Mice were injected with [(3)H]PC-labeled high density lipoproteins (HDLs). Hepatic uptake of HDL-PC was ~10 µmol/day, similar to the rate of hepatic de novo PC synthesis. Consistent with this finding, measurement of the specific radioactivity of PC in plasma and liver indicated that 50% of hepatic PC is derived from the circulation. Moreover, one-third of HDL-derived PC was converted into triacylglycerols. Importantly, ~65% of the total hepatic pool of triacylglycerol appears to be derived from hepatic PC, half of which is derived from HDL. Thus, lipoprotein-associated PC should be considered a quantitatively significant source of triacylglycerol for the etiology of hepatic steatosis.

Phosphatidylcholine synthesis for lipid droplet expansion is mediated by localized activation of CTP:phosphocholine cytidylyltransferase.

Lipid droplets (LDs) are cellular storage organelles for neutral lipids that vary in size and abundance according to cellular needs. Physiological conditions that promote lipid storage rapidly and markedly increase LD volume and surface. How the need for surface phospholipids is sensed and balanced during this process is unknown. Here, we show that phosphatidylcholine (PC) acts as a surfactant to prevent LD coalescence, which otherwise yields large, lipolysis-resistant LDs and triglyceride (TG) accumulation. The need for additional PC to coat the enlarging surface during LD expansion is provided by the Kennedy pathway, which is activated by reversible targeting of the rate-limiting enzyme, CTP:phosphocholine cytidylyltransferase (CCT), to growing LD surfaces. The requirement, targeting, and activation of CCT to growing LDs were similar in cells of Drosophila and mice. Our results reveal a mechanism to maintain PC homeostasis at the expanding LD monolayer through targeted activation of a key PC synthesis enzyme.

Impaired de novo choline synthesis explains why phosphatidylethanolamine N-methyltransferase-deficient mice are protected from diet-induced obesity.

Phosphatidylcholine (PC) is synthesized from choline via the CDP-choline pathway. Liver cells can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). The current study investigates whether or not hepatic PC biosynthesis is linked to diet-induced obesity. Pemt(+/+) mice fed a high fat diet for 10 weeks increased in body mass by 60% and displayed insulin resistance, whereas Pemt(-/-) mice did not. Compared with Pemt(+/+) mice, Pemt(-/-) mice had increased energy expenditure and maintained normal peripheral insulin sensitivity; however, they developed hepatomegaly and steatosis. In contrast, mice with impaired biosynthesis of PC via the CDP-choline pathway in liver became obese when fed a high fat diet. We, therefore, hypothesized that insufficient choline, rather than decreased hepatic phosphatidylcholine, was responsible for the lack of weight gain in Pemt(-/-) mice despite the presence of 1.3 g of choline/kg high fat diet. Supplementation with an additional 2.7 g of choline (but not betaine)/kg of diet normalized energy metabolism, weight gain, and insulin resistance in high fat diet-fed Pemt(-/-) mice. Furthermore, Pemt(+/+) mice that were fed a choline-deficient diet had increased oxygen consumption, had improved glucose tolerance, and gained less weight. Thus, de novo synthesis of choline via PEMT has a previously unappreciated role in regulating whole body energy metabolism.

Impaired ganglioside metabolism in Huntington's disease and neuroprotective role of GM1.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine stretch in the protein huntingtin (Htt). HD neurons are dysfunctional at multiple levels and have increased susceptibility to stress and apoptotic stimuli. We have discovered that synthesis of the ganglioside GM1 is reduced in fibroblasts from HD patients and in cell and animal models of HD, and that decreased GM1 levels contribute to heighten HD cell susceptibility to apoptosis. The apoptotic susceptibility is recapitulated through inhibition of ganglioside synthesis in wild-type striatal cells, suggesting that decreased GM1 levels might be one of the key events leading to HD pathogenesis and progression. Administration of GM1 restores ganglioside levels in HD cells and promotes activation of AKT and phosphorylation of mutant Htt, leading to decreased mutant Htt toxicity and increased survival of HD cells. Our data identify GM1 as a potential treatment for HD.

Hepatic CTP:phosphocholine cytidylyltransferase-alpha is a critical predictor of plasma high density lipoprotein and very low density lipoprotein.

CTP:phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for the biosynthesis of phosphatidylcholine (PC). We previously generated a mouse in which the hepatic CTalpha gene was specifically inactivated by the cre/loxP procedure. In CTalpha knock-out mice, plasma high density lipoprotein (HDL) and very low density lipoprotein (VLDL) levels were markedly lower than in wild type mice (Jacobs, R. L., Devlin, C., Tabas, I., and Vance, D. E. (2004) J. Biol. Chem. 279, 47402-47410.) To investigate the mechanism(s) responsible for the decrease in plasma lipoprotein levels, we isolated primary hepatocytes from knock-out and wild type mice. ABCA1 expression was reduced in knock-out hepatocytes and apoAI-dependent cholesterol, and PC efflux was impaired. When knock-out hepatocytes were infected with an adenovirus expressing CTalpha, apoAI-dependent PC efflux returned partially, whereas cholesterol efflux and ABCA1 levels were not restored to normal levels. Adenoviral expression of CTalpha did not increase VLDL secretion in knock-out hepatocytes, even though cellular PC levels returned to normal. However, in vivo adenoviral delivery of CTalpha normalized plasma HDL and VLDL levels in knock-out mice. The observations demonstrate that hepatic PC biosynthesis is a key player in maintaining plasma VLDL and HDL, and further underscores the importance of the liver in HDL formation.

Sp-1 binds promoter elements that are regulated by retinoblastoma and regulate CTP:phosphocholine cytidylyltransferase-alpha transcription.

The retinoblastoma (Rb) protein is implicated in transcriptional regulation of at least five cellular genes. Co-transfection of Rb and truncated promoter constructs has defined a discrete element (retinoblastoma control element (RCE)) within the promoters of each of these genes as being necessary for Rb-mediated transcriptional control. In the present report we demonstrate that two RCEs identified within the CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) proximal promoter are essential to promote transcription. Mutations that abolished each RCE markedly decreased CTalpha transcription. Co-transfection of Rb and truncated promoter constructs demonstrated that Rb regulates CTalpha expression by different mechanisms depending on the phase of the cell cycle. The regulation of CTalpha expression by Rb required both the Sp1 and the RCEs. Maximal expression occurred when both Rb and Sp1 were overexpressed. Moreover, RCEs were required for Rb association with the DNA. This regulatory mechanism alters CTalpha activity and thereafter changes PC availability and cell physiology. This is the first report demonstrating not only that surrounding Sp1 binding sites alter regulation mediated by Rb, but also that the expression of a gene involved in PC biosynthesis shares a common regulatory pathway with genes responsible for cell growth and differentiation.

Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation.

5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK.

Role of histone deacetylase in the expression of CTP:phosphocholine cytidylyltransferase alpha.

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of CTP:phosphocholine cytidylyltransferase alpha (CTalpha) are not fully understood. In this study, we investigated whether or not histone deacetylation is involved in repression of CTalpha expression in quiescent C3H10T1/2 mouse embryo fibroblasts. We have examined the contributions of the Sp1 and E2F binding sites in the repression of CTalpha gene expression. Immunoprecipitation experiments showed that histone deacetylase 1 (HDAC1) and HDAC activity are associated with Sp1 in serum-starved cells or during serum stimulation. However, HDAC1 association with E2F was only detected in serum-starved cells. By chromatin immunoprecipitation assays, we detected both direct and indirect association of HDAC1 with the CTalpha promoter. Treatment with the HDAC inhibitor trichostatin A induced CTalpha expression. Our data suggest that HDAC1 plays a critical role in CTalpha repression and that Sp1 and E2F may serve as key targets for HDAC1-mediated CTalpha repression in fibroblasts.

Localization-independent regulation of homocysteine secretion by phosphatidylethanolamine N-methyltransferase.

Genetic ablation of phosphatidylethanolamine N-methyltransferase (PEMT) in mice causes a 50% reduction in plasma homocysteine (Hcy) levels. Because hyperhomocysteinemia is an independent risk factor for cardiovascular disease, resolution of the molecular basis for this reduction is of significant clinical interest. The PEMT pathway is a metabolically channeled process localized to the endoplasmic reticulum (ER). To assess the importance of PEMT localization for Hcy homeostasis, we identified and ablated the minimal ER targeting motif. Mutagenesis of a conserved, C-terminal lysine residue (197) relocalized the enzyme to the Golgi, demonstrating that Lys-197 is essential for targeting PEMT to the ER. To evaluate the functional significance of PEMT localization, hepatoma cell lines were generated that stably expressed either ER- or Golgi-localized PEMT only. Intriguingly, stable expression of PEMT in either the ER or the Golgi caused increased Hcy secretion. Moreover, PEMT-mediated Hcy secretion correlated with the methyltransferase activity of the enzyme, independently of subcellular localization. Thus, our data suggest that Hcy homeostasis is regulated concomitantly with PEMT activity but independently of PEMT localization.