The study of bending properties of monolayer MoS2 in non-collinear electrodes using first principles theory.

In this work, we report the theoretical maximum bending angle of MoS2 devices using the creative non-collinear electrodes method based on the first principles theory. The results show that the device with 1T-phase MoS2 electrodes sandwiching N-type MoS2 in a zigzag direction has a better conducting behavior as compared with P-type in an armchair direction. The conductance decreases less than 15% when the angle between the two electrodes is less than 45° in both the equilibrium state and non-equilibrium state because of the continuous resonant response between the two electrodes and the little deformed band structure. This work provides guidance and a physical mechanism for achieving flexible MoS2 transistors that are reliable at a sub-nm bending radius.

Choline kinase beta is required for normal endochondral bone formation.

BACKGROUND: Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb(-/-) mice display neonatal forelimb bone deformations. METHODS: To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb(-/-) mice. RESULTS: The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb(-/-) mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb(-/-) mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb(-/-) mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb(-/-) mice contained fewer osteoclasts along the cartilage/bone interface. CONCLUSIONS: Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice. GENERAL SIGNIFICANCE: Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.

Phosphatidylcholine metabolism and choline kinase in human osteoblasts.

There is a paucity of information about phosphatidylcholine (PC) biosynthesis in bone formation. Thus, we characterized PC metabolism in both primary human osteoblasts (HOB) and human osteosarcoma MG-63 cells. Our results show that the CDP-choline pathway is the only de novo route for PC biosynthesis in both HOB and MG-63 cells. Both CK activity and CKα expression in MG-63 cells were significantly higher than those in HOB cells. Silencing of CKα in MG-63 cells had no significant effect on PC concentration but decreased the amount of phosphocholine by approximately 80%. The silencing of CKα also reduced cell proliferation. Moreover, pharmacological inhibition of CK activity impaired the mineralization capacity of MG-63 cells. Our data suggest that CK and its product phosphocholine are required for the normal growth and mineralization of MG-63 cells.

Early increase and late decrease of purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures.

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4-5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

Choline supplementation promotes hepatic insulin resistance in phosphatidylethanolamine N-methyltransferase-deficient mice via increased glucagon action.

Biosynthesis of hepatic choline via phosphatidylethanolamine N-methyltransferase (PEMT) plays an important role in the development of type 2 diabetes and obesity. We investigated the mechanism(s) by which choline modulates insulin sensitivity. PEMT wild-type (Pemt(+/+)) and knock-out (Pemt(-/-)) mice received either a high fat diet (HF; 60% kcal of fat) or a high fat, high choline diet (HFHC; 4 g of choline/kg of HF diet) for 1 week. Hepatic insulin signaling and glucose and lipid homeostasis were investigated. Glucose and insulin intolerance occurred in Pemt(-/-) mice fed the HFHC diet, but not in their Pemt(-/-) littermates fed the HF diet. Plasma glucagon was elevated in Pemt(-/-) mice fed the HFHC diet compared with Pemt(-/-) mice fed the HF diet, concomitant with increased hepatic expression of glucagon receptor, phosphorylated AMP-activated protein kinase (AMPK), and phosphorylated insulin receptor substrate 1 at serine 307 (IRS1-s307). Gluconeogenesis and mitochondrial oxidative stress were markedly enhanced, whereas glucose oxidation and triacylglycerol biosynthesis were diminished in Pemt(-/-) mice fed the HFHC diet. A glucagon receptor antagonist (2-aminobenzimidazole) attenuated choline-induced hyperglycemia and insulin intolerance and blunted up-regulation of phosphorylated AMPK and IRS1-s307. Choline induces glucose and insulin intolerance in Pemt(-/-) mice through modulating plasma glucagon and its action in liver.

Choline Deficiency Attenuates Body Weight Gain and Improves Glucose Tolerance in ob/ob Mice.

Previous studies demonstrated that choline supply is directly linked to high-fat-diet-induced obesity and insulin resistance in mice. The aim of this study was to evaluate if choline supply could also modulate obesity and insulin resistance caused by a genetic defect. Eight-week-old male ob/ob mice were fed for two months with either choline-deficient or choline-supplemented diet. Tissue weight including fat mass and lean mass was assessed. Intracellular signaling, plasma glucagon and insulin, and glucose and insulin tolerance tests were also investigated. The choline-deficient diet slowed body weight gain and decreased fat mass. Choline deficiency also decreased plasma glucose level and improved glucose and insulin tolerance although fatty liver was exacerbated. Increased adipose lipolytic activity, decreased plasma glucagon and reduced expression of hepatic glucagon receptor were also observed with the choline-deficient diet. Our results demonstrate that a choline-deficient diet can decrease fat mass and improve glucose tolerance in obese and diabetic mice caused by a genetic defect.

Choline kinase and its function.

Choline kinase (CK) was discovered in 1953. Progress in understanding the function of CK was slow until its purification in 1984. The subsequent cloning and expression of the cDNA led to the description of the gene structures. Two genes encode choline kinase, Chka and Chkb, and 3 isoforms of the enzyme have been identified - CKalpha-1, CKalpha-2, and CKbeta - and the active form of CK is a hetero- or homo-dimer. More recently, gene-disrupted mice have been described. Mice that lack CKalpha die early in embryogenesis. In contrast, mice that lack CKbeta survive to adulthood, but develop hindlimb muscular dystrophy and forelimb bone deformity. It has been shown that this hindlimb muscular dystrophy is due to decreased biosynthesis of phosphatidylcholine and increased catabolism of phosphatidylcholine in the hindlimbs, but not the forelimbs, of mice. CK and its product phosphocholine have also been implicated in development of numerous cancers. Thus, a possible treatment for some kinds of cancer may involve drug inhibition of CK or targeting the expression of CK with RNA interference. In the mid 1950s it was clear that CK was important for the biosynthesis of phosphatidylcholine, but no one predicted a role for CK in muscular dystrophy, bone deformities, or cancer.

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse.

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase beta, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase beta is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase alpha is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb(-/-) mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A(2) in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb(-/-) mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb(-/-) mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb(-/-) mice is due to abundant choline kinase alpha and the stable homeostasis of phosphatidylcholine.

Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice.

Choline kinase in mice is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneously occurring genomic deletion in murine Chkb results in neonatal bone deformity and hindlimb muscular dystrophy. We have investigated the mechanism by which a lack of choline kinase beta, encoded by Chkb, causes hindlimb muscular dystrophy. The biosynthesis of phosphatidylcholine (PC) is impaired in the hindlimbs of Chkb -/- mice, with an accumulation of choline and decreased amount of phosphocholine. The activity of CTP: phosphocholine cytidylyltransferase is also decreased in the hindlimb muscle of mutant mice. Concomitantly, the activities of PC phospholipase C and phospholipase A2 are increased. The mitochondria in Chkb -/- mice are abnormally large and exhibit decreased inner membrane potential. Despite the muscular dystrophy in Chkb -/- mice, we observed increased expression of insulin like growth factor 1 and proliferating cell nuclear antigen. However, regeneration of hindlimb muscles of Chkb -/- mice was impaired when challenged with cardiotoxin. Injection of CDP-choline increased PC content of hindlimb muscle and decreased creatine kinase activity in plasma of Chkb -/- mice. We conclude that the hindlimb muscular dystrophy in Chkb -/- mice is due to attenuated PC biosynthesis and enhanced catabolism of PC.

Early embryonic lethality caused by disruption of the gene for choline kinase alpha, the first enzyme in phosphatidylcholine biosynthesis.

Choline kinase alpha (CK-alpha) is one of two mammalian enzymes that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid, phosphatidylcholine. We created mice lacking CK-alpha with an embryonic stem cell line containing an insertional mutation in the gene for CK-alpha (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-alpha is crucial for the early development of mouse embryos. Heterozygous mutant mice (Chka(+/-)) appeared entirely normal in their embryonic development and gross anatomy, and they were fertile. Although choline kinase activity was decreased by approximately 30%, the amount of phosphatidylcholine in cells and the levels of other enzymes involved in phosphatidylcholine biosynthesis were unaffected. Phosphatidylcholine biosynthesis measured by choline incorporation into hepatocytes was also not compromised in Chka(+/-) mice. Enhanced levels of choline and attenuated levels of phosphocholine were observed in both the livers and testes of Chka(+/-) mice. Triacylglycerol and cholesterol ester were elevated approximately 2-fold in the livers, whereas neutral lipid profiles in plasma were similar in Chka(+/-) and wild-type (Chka(+/+)) mice. Thus, Chka is an essential gene for early embryonic development, but adult mice do not require full expression of the gene for normal levels of phosphatidylcholine.

A transcription-dependent mechanism, akin to that in adipose tissue, modulates lipoprotein lipase activity in rat heart.

The enzyme lipoprotein lipase (LPL) releases fatty acids from lipoprotein triglycerides for use in cell metabolism. LPL activity is rapidly modulated in a tissue-specific manner. Recent studies have shown that in rat adipose tissue this occurs by a shift of extracellular LPL toward an inactive form catalyzed by an LPL-controlling protein whose expression changes in response to the nutritional state. To explore whether a similar mechanism operates in other tissues we injected actinomycin D to block transcription of the putative LPL controlling protein(s). When actinomycin was given to fed rats, heparin-releasable LPL activity increased by 160% in heart and by 150% in a skeletal muscle (soleus) in 6 h. Postheparin LPL activity in blood increased by about 200%. To assess the state of extracellular LPL we subjected the spontaneously released LPL in heart perfusates to chromatography on heparin-agarose, which separates the active and inactive forms of the lipase. The amount of lipase protein released remained relatively constant on changes in the nutritional state and/or blockade of transcription, but the distribution between the active and inactive forms changed. Less of the LPL protein was in the active form in perfusates from hearts from fed compared with fasted rats. When glucose was given to fasted rats the proportion of LPL protein in the active form decreased. Actinomycin D increased the proportion that was active, in accord with the hypothesis that the message for a rapidly turning over LPL-controlling protein was being removed.

Calcium triggers folding of lipoprotein lipase into active dimers.

The active form of lipoprotein lipase (LPL) is a noncovalent homodimer of 55-kDa subunits. The dimer is unstable and tends to undergo irreversible dissociation into inactive monomers. We noted that a preparation of such monomers slowly regained traces of activity under assay conditions with substrate, heparin, and serum or in cell culture medium containing serum. We therefore studied the refolding pathway of LPL after full denaturation in 6 M guanidinium chloride or after dissociation into monomers in 1 M guanidinium chloride. In crude systems, we identified serum as the factor promoting reactivation. Further investigations demonstrated that Ca2+ was the crucial component in serum for reactivation of LPL and that refolding involved at least two steps. Studies of far-UV circular dichroism, fluorescence, and proteolytic cleavage patterns showed that LPL started to refold from the C-terminal domain, independent of calcium. The first step was rapid and resulted in formation of an inactive monomer with a completely folded C-terminal domain, whereas the N-terminal domain was in the molten globule state. The second step was promoted by Ca2+ and converted LPL monomers from the molten globule state to dimerization-competent and more tightly folded monomers that rapidly formed active LPL dimers. The second step was slow, and it appears that proline isomerization (rather than dimerization as such) is rate-limiting. Inactive monomers isolated from human tissue recovered activity under the influence of Ca2+. We speculate that Ca2+-dependent control of LPL dimerization might be involved in the normal post-translational regulation of LPL activity.

Extracellular degradation of lipoprotein lipase in rat adipose tissue.

BACKGROUND: Recent studies in vivo indicate that short-term regulation of lipoprotein lipase (LPL) in rat adipose tissue is post-translational and occurs by a shift of the lipase protein towards an inactive form under the influence of another gene with short-lived message and product. It has not been possible to reproduce this process with isolated adipocytes suggesting that other cells are needed, and perhaps mediate the regulation. The objective of the present study was, therefore, to explore if explants of adipose tissue could be used for studies of the regulatory process. RESULTS: When explants of rat epididymal adipose tissue were incubated, LPL mass and activity decreased rapidly. Mass and activity within adipocytes remained constant for at least six hours, demonstrating that it was the extracellular portion of the enzyme that decreased. Adipocytes isolated from the explants after three or six hours of incubation retained their ability to secrete LPL to the medium. Addition of a cocktail of protease inhibitors to the incubation medium slowed down the decrease of LPL mass. Chloroquine was without effect, indicating that the degradation was not lysosomal. 125I-labeled LPL added to the medium was degraded to acid soluble products, indicating that the degradation occurred extracellularly. Fragmentation of the labelled lipase occurred in conditioned medium and this process was virtually abolished by two MMP inhibitors. CONCLUSIONS: The decrease of LPL mass and activity that occurs when explants of rat adipose tissue are incubated is due to proteolysis of extracellular LPL. The adipocytes continue to produce and secrete the enzyme. The effect of inhibitors indicates, but does not prove, that the degradation is mediated by MMPs. It appears that this process is accelerated in the tissue fragments compared to intact tissue.

Lipoprotein lipase in the kidney: activity varies widely among animal species.

Much evidence points to a relationship among kidney disease, lipoprotein metabolism, and the enzyme lipoprotein lipase (LPL), but there is little information on LPL in the kidney. The range of LPL activity in the kidney in five species differed by >500-fold. The highest activity was in mink, followed by mice, Chinese hamsters, and rats, whereas the activity was low in guinea pigs. In contrast, the ranges for LPL activities in heart and adipose tissue were less than six- and fourfold, respectively. The activity in the kidney (in mice) decreased by >50% on food deprivation for 6 h without corresponding changes in mRNA or mass. This decrease in LPL activity did not occur when transcription was blocked with actinomycin D. Immunostaining for kidney LPL in mice and mink indicated that the enzyme is produced in tubular epithelial cells. To explore the previously suggested possibility that the negatively charged glomerular filter picks up LPL from the blood, bovine LPL was injected into rats and mice. This resulted in decoration of the glomerular capillary network with LPL. This study shows that in some species LPL is produced in the kidney and is subject to nutritional regulation by a posttranscriptional mechanism. In addition, LPL can be picked up from blood in the glomerulus.

Rapid downregulation of adipose tissue lipoprotein lipase activity on food deprivation: evidence that TNF-alpha is involved.

When food was removed from young rats in the early morning, adipose tissue tumor necrosis factor (TNF)-alpha activity increased 50% and lipoprotein lipase (LPL) activity decreased 70% in 6 h. There was a strong negative correlation between the TNF-alpha and LPL activities. Exogenous TNF-alpha further decreased LPL activity. Pentoxifylline, known to decrease production of TNF-alpha, had no effect on LPL activity in fed rats but almost abolished the rise of TNF-alpha and the decrease of LPL activity in rats deprived of food. The specific activity of LPL decreased from 0.92 mU/ng in fed rats to 0.35 and 0.24 mU/ng in rats deprived of food given saline or TNF-alpha, indicating a shift in the LPL molecules toward an inactive state. Lipopolysaccharide increased adipose tissue TNF-alpha and decreased LPL activity. Both of these effects were strongly impeded by pretreatment of the rats with pentoxifylline, or dexamethasone. Pretreatment of the rats with actinomycin D virtually abolished the response of LPL activity to food deprivation or exogenous TNF-alpha. We conclude that food deprivation, like lipopolysaccharide, signals via TNF-alpha to a gene whose product causes a rapid shift of newly synthesized LPL molecules toward an inactive form and thereby shuts down extraction of lipoprotein triglycerides by the adipose tissue.

Nutritional regulation of lipoprotein lipase in mice.

Tissue-specific regulation of lipoprotein lipase (LPL) has been extensively studied in rats. The mouse is now the most used animal in lipoprotein research, and we have therefore explored the regulation of LPL in this species. In C57 black mice, fed ad libitum adipose tissue LPL activity changed about three-fold with the time of day, indicating a circadian rhythm. The highest activity was at midnight and the lowest activity was at noon. Withdrawal of food did not markedly accelerate the drop of activity that occurred from midnight until noon, but prevented the return of activity that occurred during the evening and early night. When food was returned to mice that had been fasted for 24h, adipose tissue LPL activity rose rapidly and returned to the fed level in 2h. LPL mass in adipose tissue changed less than LPL activity, indicating that regulation is mainly post-translational as previously demonstrated for rats. When transcription was blocked in fasted mice, adipose tissue LPL activity increased, as previously observed in rats. LPL activity in heart was highest early in the light period at 9:00h and lowest at 21:00h. The change was, however, only about 30%. Heparin-releasable LPL activity in heart was 1.8-fold higher in mice fasted for 6h compared to fed controls. We conclude that LPL activity responds to the nutritional state in the same direction and by the same mechanisms in mice as in rats, but the magnitude of the changes are less in mice.

Calreticulin promotes folding/dimerization of human lipoprotein lipase expressed in insect cells (sf21).

Lipoprotein lipase (LPL) is a non-covalent, homodimeric, N-glycosylated enzyme important for metabolism of blood lipids. LPL is regulated by yet unknown post-translational events affecting the levels of active dimers. On co-expression of LPL with human molecular chaperones, we found that calreticulin had the most pronounced effects on LPL activity, but calnexin was also effective. Calreticulin caused a 9-fold increase in active LPL, amounting to about 50% of the expressed LPL protein. The total expression of LPL protein was increased less than 20%, and the secretion rates for active and inactive LPL were not significantly changed by the chaperone. Thus, the main effect was an increased specific activity of LPL both in cells and media. Chromatography on heparin-Sepharose and sucrose density gradient centrifugation demonstrated that most of the inactive LPL was monomeric and that calreticulin promoted formation of active dimers. Higher oligomers of inactive LPL were present in cell extracts, but only monomers and dimers were secreted to the medium. Interaction between LPL and calreticulin was demonstrated, and the effect of the chaperone was prevented by castanospermine, an inhibitor of N-glycan glucose trimming. Our data indicate an important role of endoplasmic reticulum-based chaperones for the folding/dimerization of LPL.

The distribution of lipoprotein lipase in rat adipose tissue. Changes with nutritional state engage the extracellular enzyme.

Lipoprotein lipase (LPL) acts at the vascular endothelium. Earlier studies have shown that down-regulation of adipose tissue LPL during fasting is post-translational and involves a shift from active to inactive forms of the lipase. Studies in cell systems had indicated that during fasting LPL might be retained in the endoplasmic reticulum. We have now explored the relation between active/inactive and intra/extracellular forms of the lipase. Within adipocytes, neither LPL mass nor the distribution of LPL between active and inactive forms changed on fasting. Extracellular LPL mass also did not change significantly, but shifted from predominantly active to predominantly inactive. To explore if changes in secretion were compensated by changes in turnover, synthesis of new protein was blocked by cycloheximide. The rates at which intra- and extracellular LPL mass and activity decreased did not change on fasting. To further explore how LPL is distributed in the tissue, heparin (which detaches the enzyme from the endothelial surface) was injected. Tissue LPL activity decreased by about 10% in 2 min and by 50% in 1 h. Heparin released mainly the active form of the lipase. There was no change of LPL activity or mass within adipocytes. The fraction of extracellular LPL that heparin released and the time course were the same in fed and fasted rats, indicating that active, extracellular LPL was distributed in a similar way in the two nutritional states. This study suggests that the nutritional regulation of LPL in adipose tissue determines the activity state of extracellular LPL.

Down-regulation of adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on.

During short term fasting, lipoprotein lipase (LPL) activity in rat adipose tissue is rapidly down-regulated. This down-regulation occurs on a posttranslational level; it is not accompanied by changes in LPL mRNA or protein levels. The LPL activity can be restored within 4 h by refeeding. Previously, we showed that during fasting there is a shift in the distribution of lipase protein toward an inactive form with low heparin affinity. To study the nature of the regulatory mechanism, we determined the in vivo turnover of LPL activity, protein mass, and mRNA in rat adipose tissue. When protein synthesis was inhibited with cycloheximide, LPL activity and protein mass decreased rapidly and in parallel with half-lives of around 2 h, and the effect of refeeding was blocked. This indicates that maintaining high levels of LPL activity requires continuous synthesis of new enzyme protein. When transcription was inhibited by actinomycin, LPL mRNA decreased with half-lives of 13.3 and 16.8 h in the fed and fasted states, respectively, demonstrating slow turnover of the LPL transcript. Surprisingly, when actinomycin was given to fed rats, LPL activity was not down-regulated during fasting, indicating that actinomycin interferes with the transcription of a gene that blocks the activation of newly synthesized LPL protein. When actinomycin was given to fasted rats, LPL activity increased 4-fold within 6 h, even in the absence of refeeding. The same effect was seen with alpha-amanitin, another inhibitor of transcription. The response to actinomycin was much less pronounced in aging rats, which are obese and insulin-resistant. These data suggest a default state where LPL protein is synthesized on a relatively stable mRNA and is processed into its active form. During fasting, a gene is switched on whose product prevents the enzyme from becoming active even though synthesis of LPL protein continues unabated.