Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.     

EFFECTS OF DENERVATION ON THE INCORPORATION OF 32P INTO PHOSPHATIDYL INOSITOL AND OTHER PHOSPHOLIPIDS OF RAT DIAPHRAGM

Abstract— At 24 h after denervation of the rat hemidiaphragm, incorporation of ³²P into phosphatidyl inositol was depressed relative to incorporation of ³²P into phosphatidyl choline (measured 75 min after injection of the isotope intraperitoneally). The ratio of the specific radioactivity of phosphatidyl choline to the specific radioactivity of P_i was unaffected by denervation which implies that denervation had depressed incorporation of isotope into phospatidyl inositol. Denervation did not cause a measurable change in the pool size of phosphatidyl inositol relative to that of phosphatidyl choline. The effect of denervation on incorporation of ³²P into phosphatidyl inositol was not entirely a direct consequence of the cessation of ACh release at the motor end-plate since the effect was clearly manifest in strips of muscle not containing motor end-plates, but the magnitude of the denervation effect was slightly greater in the strips of denervated hemidiaphragm which contained motor end-plates.     

The effect of dietary protein on the excretion of alpha2u, the sex-dependent protein of the adult male rat.

Adult male rats were maintained on normal (20% casein), protein-free (0% casein), high protein (50% casein), decicient protein (20% zein), and a supplemented, deficient protein (20% zein plus L-lysine and L-tryptophan) diets. Rats on a protein-free diet excreted approximately 1 mg alpha2u/24 h compared with a normal of 10-15 mg/24 h. Depleted rats placed on a 20% casein diet showed a rapid restoration of the normal alpha2u excretion as well as total urinary proteins. Accumulation of alpha2u in the blood serum was measured in nep-rectomized rats. Rats on a 0% casein diet accumulated only 30% of the alpha2u compared to normals. On a 50% casein diet, rats excreted 30-50 mg alpha2u/24 h. However, the accumulation was normal in the serum of nephrectomized rats. A high protein diet did not stimulate alpha2u synthesis but probably increased the renal loss of all urinary proteins. The excretion of alpha2u on a zein diet was reduced to the same degree as with the protein-free diet. Supplementation with lysine and tryptophan restored the capacity to eliminate alpha21 to near normal levels. Accumulation of alpha2u in the serum of nephrectomized rats kept on the zein diets showed that the effect to suppress the synthesis of the ahpha2u. Supplementation restored the biosynthesis of alpha2u. We conclude that the effect of dietary protein on the excretion of urinary proteins in the adult male rat is caused in large part by an influence on the hepatic biosynthesis of alphay2u. The biosynthesis of this protein, which represents approximately 30% of the total urinary proteins, is dependent on an adequate supply of dietary protein.     

Phosphoinositides of sheep platelets plasma membranes.

Phospholipid composition of sheep blood platelets and its various plasma membrane fractions have been analyzed. Based on their flotation rates in discontinuous sucrose density gradient centrifugation, three membrane fractions were isolated. 5'-Nucelotidase and alkaline phosphatase were distributed nearly equally in all the three membrane fractions. However these membrane fractions showed differences in the distribution of phosphatidyl ethanolamine, phosphatidyl choline and phosphoinositides. Phosphatidyl ethanolamine was predominant in fraction I (11.05 micrograms PLP/mg protein) while phosphatidyl choline was predominant in fractions II and III (110.10 and 68.30 micrograms PLP/mg protein respectively). Phosphatidyl inositol (Ptd-InsP) was equally distributed in all three membrane fractions. However, both Ptd-InsP and phosphatidyl inositol 4,5-bisphosphate were about 4-fold higher in fraction II (73.55 and 89.89 micrograms PLP/mg protein respectively).     

Synthesis of phosphatidylcholine by rat lung during choline deficiency

The effect of choline deficiency on the de novo pathway for phosphatidylcholine (PC) synthesis in the lung was investigated in rats fed a washed soy protein (lipotrophic) diet deficient in choline and methionine for 2-3 wk. Lungs from lipotrophic rats showed a decreased content of choline and choline-phosphate (P less than 0.05) compared with control but no change in content of cytidine 5'-diphosphocholine or PC. Isolated perfused lungs from lipotrophic rats were evaluated for choline and fatty acid utilization for PC synthesis. Lipotrophic lungs perfused with 5 microM [14C-methyl]-choline chloride showed increased incorporation into PC while there was no significant effect at saturating levels of choline (100 microM). There was increased incorporation of [1-14C]-palmitic acid into PC and diglyceride and increased incorporation of D-[U-14C]glucose into fatty acids of PC. Increased choline and glucose incorporation was not due to alteration of intracellular specific activity of these substrates. This study indicates the utilization of choline and fatty acid for PC synthesis is stimulated as a result of choline deficiency while lung CDP-choline concentration is maintained, possibly through regulation of choline phosphate cytidyl transferase activity. These mechanisms compensate for decreased choline availability to maintain the PC content of lungs.     

Hepatic plasma membrane fluidity and dietary proteins.

Effect of protein deficient diet on hepatic plasma membrane fluidity has been studied in rats using (i) steady state fluorescence polarization and anisotropy, (ii) phospholipid and cholesterol contents, (iii) phospholipid fatty acid composition, (iv) turnover of phosphatidyl choline (PC), and (v) activities of membrane-bound enzymes as parameters and rats fed casein (20%) diet as standard group. A significant increase in steady state fluorescence and anisotropy values was registered in the deficient group, indicating increased resistance and hence decrease in fluidity of the plasma membrane. Supplementation of the diet with lysine and threonine improved these values, thereby suggesting the significance of diet for membrane fluidity. Simultaneous significant alterations in other parameters, viz. (i) decrease in PC, PE and free cholesterol and increase in esterified cholesterol contents, (ii) decrease in unsaturation of fatty acids of PC, (iii) decrease in incorporation of NaH2 32PO4, [CH3-14C]choline and [CH3-14C]methionine into plasma membrane PC, and (iv) decrease in activities of plasma membrane 5'-nucleotidase and phosphodiesterase along with increase of (Na(+)-K+)ATPase and adenyl cyclase, were observed in the deficient group which on supplementation with lysine and threonine showed improvement over alterations.     

Phosphatidyl inositol inhibition of a sperm cyclic AMP-independent protein kinase

Phosphatidyl inositol has been found to inhibit strongly the activity of a cyclic AMP-independent protein kinase located on the external surface of goat epididymal intact spermatozoa. Phosphatidyl inositol at a concentration as low as 10 micrograms/ml inhibited nearly 50% of the ecto-kinase activity for the phosphorylation of the exogenous protein substrate: casein. Phosphatidyl ethanolamine at a relatively high concentration (125 micrograms/ml) inhibited slightly (approx 25%) the activity of the enzyme whereas other phospholipids: phosphatidyl serine and choline, diacyl glycerol, phosphatidic acid and myo-inositol-2-phosphate had no appreciable effect on the kinase activity. Phosphatidyl inositol has also served as a potent inhibitor of the phosphorylation of sperm ecto-phosphoproteins by the endogenous kinase activity of intact spermatozoa. By thin layer chromatography it has been shown that the observed inhibitory effect of the phospholipid was not due to any impurities or degraded products of phosphatidyl inositol. Phosphatidyl inositol inhibited the kinase activity noncompetitively with respect to casein and Mg2+ but uncompetitively with respect to ATP. The results raised the possibility that phosphatidyl inositol-mediated high affinity inhibition of protein kinase(s), may constitute a novel mechanism for the regulatory actins of the phospholipid in mammalian cells.     

Activation of phosphorylating vesicles by net transfer of phosphatidyl choline by phospholipid transfer protein

Vesicles formed with phosphatidyl ethanolamine, phosphatidyl choline, cardiolipin, coupling factors and hydrophobic proteins from bovine heart mitochondria catalyzed a rapid³²P_i-ATP exchange. When phosphatidyl choline was deleted during the assembly of the vesicles, little³²P_i-ATP exchange was observed. Exchange activity was induced by incubating such deficient vesicles with phosphatidyl choline liposomes in the presence of a phosphatidyl choline transfer protein isolated from bovine heart. Transfer of [³²P] phosphatidyl choline was demonstrated by isolation of the activated vesicles by sucrose density centrifugation.     

Hepatic mitochondrial membrane lipid environment and protein nutrition

Effect of feeding rice diet with and without lysine and threonine supplementation on hepatic mitochondria and its inner and outer membrane proteins, enzymes and phospholipids has been studied. The exchange of phosphatidylcholine and phosphatidylethanolamine between microsomes and mitochondria has also been studied under these conditions. Deficient diet lead to significant decrease in proteins as well as activities of monoamine oxidase, succinate dehydrogenase, cytochrome a + a3 and cytochrome c in mitochondria and its inner and outer membranes. Feeding of the deficient diet also significantly reduced total phospholipids and PC in mitochondria and its outer mitochondrial membrane. In the inner mitochondrial membrane, only PE and cardiolipin were reduced. The incorporation (DPM/microgram PLP) of [methyl-3H]choline and [methyl-14C]methionine into PC of mitochondria and its outer membrane and that of 32Pi into PC and PE of outer mitochondrial membrane but only into PC of inner mitochondrial membrane were significantly reduced in the deficient group. The exchange rates of PC and PE between microsomes and mitochondria were reduced in the deficient group. Supplementation of the deficient diet with lysine and threonine profoundly improved the above biochemical lesions as compared to casein fed rats.     

Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C.

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.     

Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in 32Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of 32Pi into this pool is slow. Results are quite different when [3H]inositol is the precursor utilized. Incorporation of [3H]inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of [3H]phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the [3H]inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of [3H]inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing [3H]inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of [3H]inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.     

Hepatic Subcellular Fractions Lipids in Rats Fed Millet (Sorghum vulgarie) at Different Protein Levels

Effect of feeding defatted millet (Sorghum vulgarie) flour at 5, 10 and 14.5% protein levels respectively for six weeks has been studied on rat liver mitochondrial, microsomal and supernatant fractions total lipids, cholesterol, triglycerides, total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine. The results have been compared with rats fed casein at 10% level for the same period. The metabolism of liver subcellular fractions lipids of millet diet and casein diet fed rats has been studied by the incorporation of acetate-1-¹⁴C and . A significant increase in mitochondrial triglycerides of rats fed millet diet at 5 and 10% protein level, in microsomes of rats fed millet diet at 5, 10 and 15% protein levels and in supernatant fractions of rats fed millet diet at 5 and 15% protein levels was observed. A significant increase in total cholesterol in mitochondria and microsomes and a significant decrease in supernatant fraction of rats fed millet diet at 10% protein level was observed. A significant increase in mitochondrial total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine in rats fed millet diet at 10% protein level and a decrease in these in rats fed millet diet at 5 per cent protein level was observed. In microsomes total phospholipids were increased in rats millet diet at 10% protein level and phosphatidyl choline was increased in rats fed millet diet at 15% protein level. Total phospholipids, phosphatidyl choline and phosphatidyl ethanolamine were significantly reduced in the supernatant fraction of rats fed millet at 10% protein level.

Incorporation of acetate-1-¹⁴C into nonsaponifiable fraction of mitochondria, microsomes and supernatant fractions of rats fed millet diet at 5 and 15 % protein levels was significantly greater, and in saponifiable fractions of the above subcellular fractions was greater in rats fed millet diet at 5 per cent protein level. The specific activity (counts/min/mg) of free cholesterol in mitochondria, microsomes and supernatant fractions of millet diet fed rats was significantly greater, whereas the specific activity of triglycerides was not significantly different from the controls. The acetate-1-¹⁴C specific activity of phosphatidyl choline and phosphatidyl ethanolamine was significantly greater in all the above subcellular fractions of millet diet fed rats (except of phosphatidyl choline in rats fed millet diet at 5 % protein level). The specific activities of phosphatidyl choline were significantly greater in mitochondria of rats fed millet diet at 5 % protein level and of phosphatidyl choline and phosphatidyl ethanolamine in microsomes and supernatant fractions of rats fed millet diet at 5 and 15% protein levels. The specific activities of phosphatidyl choline were significantly decreased in mitochondria and microsomes of rats fed millet diet at 10% protein level. The total acetate-1-¹⁴C activities (counts/min/g equivalent wet liver) of free and esterified cholesterol triglycerides, phosphatidyl choline and phosphatidyl ethanolamine showed that their synthesis from acetate-1-¹⁴C was either enhanced in millet diet fed rats or was comparable to the controls. The total activity of (counts/min/g equivalent wet liver) into phosphatidyl choline and phosphatidyl ethanolamine showed that their synthesis was decreased in microsomes of rats fed millet diet at 10% protein level, increased in rats fed millet diet at 5 and 15% protein levels.     

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.     

Lipocortins are major substrates for protein kinase C in extracts of human neutrophils.

We have identified two major proteins in human neutrophils that are phosphorylated in vitro by protein kinase C (PKC) as lipocortins III and a fragment of a lipocortin-like 68-kDa protein. In electroporated cells, the 68-kDa protein was phosphorylated during stimulation of the cells with either FMLP or PMA. Lipocortins are of interest because of their Ca2(+)- and phospholipid-dependent actin binding properties and ability to inhibit phospholipase A2. Two crude fractions of enzymes and proteins exposed to [gamma-32]PATP in the presence of Ca2+, Mg2+, phosphatidylserine and 1,2-oleoyl-acetyl-rac-glycerol were analyzed by gel electrophoresis and autoradiography. A number of proteins in a detergent-free fraction, including proteins at 36 and 32 kDa, were phosphorylated in the presence of these cofactors. In contrast, only two major proteins (35 and 32 kDa) were phosphorylated in a detergent-extracted fraction. Phosphorylation of the 36, 35, and 32 kDa proteins required the presence of Ca2+, Mg2+, and phosphatidylserine in our soluble fraction and detergent extract, indicating PKC-dependent phosphorylation. The 32-kDa protein phosphorylated in both the soluble fraction and detergent extract was identified as lipocortin III by immunoprecipitation with a cross-reactive antibody that recognized lipocortin I and comparison of cyanogen bromide (CNBr) cleavage patterns of this protein with a lipocortin III standard. The 68-kDa protein was identified as a lipocortin VI-like protein by immunoprecipitation with anti-calelectrin. Additionally, the CNBr cleavage pattern of the 68-kDa protein was similar to that of the 36-kDa protein phosphorylated in our soluble fraction. Autoradiograms of the 68- and 36-kDa fragments immunoprecipitated from our soluble fraction with anticalelectrin and cleaved with CNBr showed that both of these proteins were phosphorylated in this sample. Because phosphorylation is known to change the functional characteristics of the lipocortins, the potential exists to link PKC and lipocortins in neutrophils to regulation of granulemembrane interactions or mediation of inflammation.     

Spatial-temporal patterning of metabotropic glutamate receptor-mediated inositol 1,4,5-triphosphate, calcium, and protein kinase C oscillations: protein kinase C-dependent receptor phosphorylation is not required.

The metabotropic glutamate receptors (mGluR), mGluR1a and mGluR5a, are G protein-coupled receptors that couple via G(q) to the hydrolysis of phosphoinositides, the release of Ca(2+) from intracellular stores, and the activation of protein kinase C (PKC). We show here that mGluR1/5 activation results in oscillatory G protein coupling to phospholipase C thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and intracellular Ca(2+) concentrations. The mGluR1/5-stimulated Ca(2+) oscillations are translated into the synchronized repetitive redistribution of PKCbetaII between the cytosol and plasma membrane. The frequency at which mGluR1a and mGluR5a subtypes stimulate inositol 1,4,5-triphosphate, Ca(2+), and PKCbetaII oscillations is regulated by the charge of a single amino acid residue localized within their G protein-coupling domains. However, oscillatory mGluR signaling does not involve the repetitive feedback phosphorylation and desensitization of mGluR activity, since mutation of the putative PKC consensus sites within the first and second intracellular loops as well as the carboxyl-terminal tail does not prevent mGluR1a-stimulated PKCbetaII oscillations. Furthermore, oscillations in Ca(2+) continued in the presence of PKC inhibitors, which blocked PKCbetaII redistribution from the plasma membrane back into the cytosol. We conclude that oscillatory mGluR signaling represents an intrinsic receptor/G protein coupling property that does not involve PKC feedback phosphorylation.     

A moderate threonine deficiency differently affects protein metabolism in tissues of early-weaned piglets

A moderate threonine deficiency may affect differently tissue protein metabolism. In this study, we compared protein metabolism in the small and large intestines, the liver, and the carcass of piglets (Sus scrofa) pair-fed either a control well-balanced diet (C: 9.3 g threonine/kg diet) or a low threonine diet (LT: 6.5 g threonine/kg diet) for 2 weeks. In the small intestine, the LT diet did not modify protein deposition, fractional protein synthesis rate (K_S) and AA protein composition. Ubiquitin mRNA level, a component of the ubiquitin-dependent proteolytic pathway, was significantly decreased in the jejunum of the LT piglets. Protein deposition measured in the carcass and the colon, and K_S measured in the semitendinosus muscle and the colon, did not differ between LT and C piglets. Nevertheless, in these compartments, threonine content was reduced indicating deposition of proteins less rich in threonine. In the liver, protein retention was reduced, K_S was increased and AA protein composition was modified in the LT compared to the C piglets. In conclusion compared to the other compartments, small intestinal protein metabolism seems to be less sensitive to a moderate dietary threonine deficiency. This indicates that dietary threonine extraction by the small intestine may reduce threonine availability for the other tissues when young piglets were fed a diet marginally deficient in threonine.     

Phosphorylation of myocardial sarcolemma proteins during induction of the phosphatidylinositol cycle in isolated perfused rabbit heart]

Phosphorylation of cardiac sarcolemma proteins under stimulation of M-receptors by agonist carbacholine used to stimulate phosphatidylinositide cycle, was investigated in the isolated, rabbit heart perfused with 32Pi. Carbacholine (10(-7) stimulates the polyphosphoinositide metabolism which is expressed in the activated incorporation of 32P from [gamma-32P]ATP in polyphosphoinositide as well as in the increased content of the labelled inositol trisphosphate released through phosphatidylinositol-4,5-bisphosphate break-down by phospholipase C. The diacylglycerol produced simultaneously with inositol triphosphate as a second messenger activates the protein kinase C. This was confirmed by considerable activation of phosphorylation sarcolemma proteins-substrates of protein kinase C, with Mr 94, 87, 78, 51 and 46 kDa.     

Insulin secretion and protein phosphorylation in PKC-depleted islets of Langerhans.

Protein kinase C (PKC)-dependent phosphorylation of endogenous substrates was measured in electrically permeabilised rat islets of Langerhans. The PKC-activating phorbol ester, 4 beta-phorbol myristate acetate (PMA), caused a slow but prolonged increase in insulin secretion from permeabilised islets, which was accompanied by increased 32P incorporation into several islet proteins of apparent M.W. 30-50 kDa. Depletion of islet PKC by prolonged exposure to PMA abolished subsequent secretory and phosphorylating responses to the phorbol ester. However, PKC-depleted islets did not show diminished responses to glucose, suggesting that PKC-mediated phosphorylation of these proteins is not essential for nutrient-induced insulin secretion.     

Diaphragmatic fatigue in normoxia and hyperoxia

The effect of choline deficiency on the lung lipids of actively growing male Sprague-Dawley rats was investigated using a washed soy protein diet deficient in choline and methionine (lipotrophic). The livers from deficient animals had a significantly increased total lipid content and decreased phosphatidylcholine (PC) content and PC-to-phosphatidylethanolamine ratio (P less than 0.01). Although lung free choline levels were decreased 40% compared with controls (P less than 0.05), the PC content of the whole lung homogenate was unchanged. However, disaturated phosphatidylcholine from animals receiving the lipotrophic diet was significantly increased in the lavage and proportionally decreased in the lavaged lung tissue compared with controls (P less than 0.01). This study indicates that, despite decreased lung choline levels as a result of ingesting a lipotrophic diet, and unlike the liver, lung PC content is maintained at normal values. Although the lung total PC levels are maintained, there is a change in the partition of this lipid pool between the tissue and the alveolar space.     

Dietary choline intake is necessary to prevent systems-wide organ pathology and reduce Alzheimer's disease hallmarks

There is an urgent need to identify modifiable environmental risk factors that reduce the incidence of Alzheimer's disease (AD). The B-like vitamin choline plays key roles in body- and brain-related functions. Choline produced endogenously by the phosphatidylethanolamine N-methyltransferase protein in the liver is not sufficient for adequate physiological functions, necessitating daily dietary intake. ~90% of Americans do not reach the recommended daily intake of dietary choline. Thus, it's imperative to determine whether dietary choline deficiency increases disease outcomes. Here, we placed 3xTg-AD, a model of AD, and non-transgenic (NonTg) control mice on either a standard laboratory diet with sufficient choline (ChN; 2.0 g/kg choline bitartrate) or a choline-deficient diet (Ch-; 0.0 g/kg choline bitartrate) from 3 to 12 (early to late adulthood) months of age. A Ch- diet reduced blood plasma choline levels, increased weight, and impaired both motor function and glucose metabolism in NonTg mice, with 3xTg-AD mice showing greater deficits. Tissue analyses showed cardiac and liver pathology, elevated soluble and insoluble Amyloid-β and Thioflavin S structures, and tau hyperphosphorylation at various pathological epitopes in the hippocampus and cortex of 3xTg-AD Ch- mice. To gain mechanistic insight, we performed unbiased proteomics of hippocampal and blood plasma samples. Dietary choline deficiency altered hippocampal networks associated with microtubule function and postsynaptic membrane regulation. In plasma, dietary choline deficiency altered protein networks associated with insulin metabolism, mitochondrial function, inflammation, and fructose metabolic processing. Our data highlight that dietary choline intake is necessary to prevent systems-wide organ pathology and reduce hallmark AD pathologies.