Choline Replacement Research Articles

Role of Choline Deficiency in the Fatty Liver Phenotype of Mice Fed a Low Protein, Very Low Carbohydrate Ketogenic Diet

Though widely employed for clinical intervention in obesity, metabolic syndrome, seizure disorders and other neurodegenerative diseases, the mechanisms through which low carbohydrate ketogenic diets exert their ameliorative effects still remain to be elucidated. Rodent models have been used to identify the metabolic and physiologic alterations provoked by ketogenic diets. A commonly used rodent ketogenic diet (Bio-Serv F3666) that is very high in fat (~94% kcal), very low in carbohydrate (~1% kcal), low in protein (~5% kcal), and choline restricted (~300 mg/kg) provokes robust ketosis and weight loss in mice, but through unknown mechanisms, also causes significant hepatic steatosis, inflammation, and cellular injury. To understand the independent and synergistic roles of protein restriction and choline deficiency on the pleiotropic effects of rodent ketogenic diets, we studied four custom diets that differ only in protein (5% kcal vs. 10% kcal) and choline contents (300 mg/kg vs. 5 g/kg). C57BL/6J mice maintained on the two 5% kcal protein diets induced the most significant ketoses, which was only partially diminished by choline replacement. Choline restriction in the setting of 10% kcal protein also caused moderate ketosis and hepatic fat accumulation, which were again attenuated when choline was replete. Key effects of the 5% kcal protein diet – weight loss, hepatic fat accumulation, and mitochondrial ultrastructural disarray and bioenergetic dysfunction – were mitigated by choline repletion. These studies indicate that synergistic effects of protein restriction and choline deficiency influence integrated metabolism and hepatic pathology in mice when nutritional fat content is very high, and support the consideration of dietary choline content in ketogenic diet studies in rodents to limit hepatic mitochondrial dysfunction and fat accumulation.

Substrates and Non-transportable Analogues Induce Structural Rearrangements at the Extracellular Entrance of the Glial Glutamate Transporter GLT-1/EAAT2

To explore rearrangements of the reentrant loop HP2 relative to transmembrane domains (TMs) 7 and 8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. The pairs were introduced around 10-15 A "above" the residues, which make contact with substrate in the related archaeal homologue Glt(Ph). Transport by the double mutants M449C/L466C (HP2/TM 8), L453C/I463C (HP2/TM 8), and I411C/I463C (TM 7/TM 8) was inhibited by copper(II)(1,10-phenanthroline)(3) (CuPh) and by Cd(2+). Inhibition was only observed when the two cysteines were present in the same construct, but not with the respective single cysteine mutants or when only one cysteine was paired with a mutation to another residue. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of M449C/L466C by CuPh. The non-transportable analogues kainate and d, l-threo-beta-benzyloxyaspartate are expected to stabilize an outward-facing conformation, but only the latter potentiated the effect of CuPh on M449C/L466C. However, both analogues increased the aqueous accessibility of the cysteines introduced at positions 449 and 466 to a membrane-impermeant sulfhydryl reagent. Inhibition of L453C/I463C by CuPh was protected not only by glutamate but also by the two analogues. In contrast, these ligands had very little effect on the inhibition of I411C/I463C by CuPh. Our results are consistent with the proposal that HP2 serves as the extracellular gate of the transporter and indicate that glutamate and the two analogues induce distinct conformations of HP2.

Choline Replacement for Sodium in Vitrification and Thaw Solutions Improves the Viability and Embryonic Competence of Mouse Oocytes

Although Metaphase II (MII) oocytes survive freeze-thaw in sodium-based vitrification solutions and complete pre-implantation development, the efficiency at which they complete full term development is still low. Replacing Na+ with choline in slow freezing protocols for MII oocytes has been reported to significant improve survival rates and developmental potential. This study examines whether choline replacement also improves these parameters for vitrified MII mouse oocytes.

Phosphate transport via Na+ -Pi cotransport and anion exchange in lactating rat mammary tissue.

Inorganic orthophosphate (Pi) transport, using 32P-labelled orthophosphate as tracer, by lactating rat mammary tissue has been examined using both tissue explants and the intact perfused gland. Pi uptake was predominantly via a Na+ -dependent pathway. Li+, however, unlike choline, was able to partially substitute for Na+. In addition, Pi release from tissue explants preloaded with 32Pi was stimulated by reversing the Na+ gradient. Thus transferring mammary explants from a buffer containing Na+ to one which was Na+ free (choline replacement) doubled the Pi efflux rate constant. The uptake of Pi by tissue explants was saturable with respect to external Pi, having apparent K(m) and V(max) values of 1.13 mM and 3.36 mmol (kg cell water)-1 (15 min)-1, respectively. The stimulation of Pi uptake by tissue explants by external Na+ was also saturable; the K(m) for Na+ was 9.7 mM. These results, taken together, suggest that the Na+ -dependent pathway is a Na+ -Pi cotransport mechanism. The transport of Pi by the perfused lactating rat mammary gland was examined using a rapid, paired-tracer dilution technique. Pi uptake by the perfused gland was found to be Na+ dependent and displayed saturable kinetics. The results suggest that the Na+ -Pi cotransporter is situated at the basolateral aspect of the secretory cells. The release of Pi from preloaded tissue explants was trans-accelerated by external Pi but not by Cl- or SO4(2-). However, external Pi stimulated Pi efflux with low affinity.

Global in vivo replacement of choline by N-aminodeanol. Testing a hypothesis about progressive degenerative dementia: I. Dynamics of choline replacement

Severe disruption of certain cholinergic pathways is a characteristic feature of Alzheimer's disease. Attempts to establish animal models by interfering with cholinergic function have not been very successful. We now present data which show a substantial and progressive replacement of free and phospholipid-bound choline by the novel choline isostere N-amino-N,N-dimethylaminoethanol during its dietary administration in place of choline. Free choline in blood fell to approximately 20% of controls after 10 to 30 days on diet. Phospholipid-bound choline in plasma was reduced to less than 15%, and in erythrocytes to about 22%. After 120 days of diet free and bound choline were reduced in most tissues to approximately 30% of controls. Only liver retained more than 80% of free choline. Acetylcholine was decreased to 33 to 50% of control. Total true and false transmitter in experimental animals was in all tissues less that acetylcholine in controls, suggesting that muscarinic transmission would be impaired. Moderate reduction of choline acetyltransferase activity was seen in striatum and myenteric plexus, and of QNB-binding in hippocampus, striatum and myenteric plexus.

Influence of a carrier transport process on in vivo release and metabolism of dopamine: Dependence of extracellular Na +

In vivo microdialysis was utilized to evaluate the role of extracellular Na+ in regulating dopamine (DA) neurotransmission in the caudate-putamen of halothane-anaesthetized rats. Reduction of the extracellular Na+ concentration by introduction of a perfusion media containing 50mM Na+ (with choline replacement) produced an excessive release of DA that could be effectively blocked by nomifensine and Lu 19-005, potent inhibitors of an amine transport carrier. These results substantiate reports of a carrier-mediated efflux of DA from presynaptic terminals. Pretreatment with amphetamine, considered both a DA uptake inhibitor and releaser, did not, however, influence the efflux of DA induced by the low extracellular Na+ environment. Although the release of DA from an apparent non-granular cytosolic pool was greatly enhanced by the low extracellular Na+ environment, 3,4-dihydrophenylacetic acid (DOPAC) levels, which supposedly reflect metabolism of non-vesicular DA, were minimally effected. In contrast, homovanillic acid (HVA) was sensitive to extracellular Na+ and not directly related to extracellular levels of either DA or DOPAC, suggesting the possibility of a Na+-sensitive (carrier-mediated?) process involved in the formation of HVA. Overall, the results of this paper cannot be completely reconciled with the traditional concept of intracellular organization of DA pools and suggests the possibility of various non-granular pools being differentially sensitive to efflux and metabolism.

Effects of sodium replacement on uptake of the dipeptide glycylsarcosine by hamster jejunum in vitro.

We report observations on transport of the hydrolysis-resistant dipeptide glycylsarcosine by rings of everted hamster jejunum in vitro in the presence and absence of Na+, using several substituents for Na+: Li+, K+, Cs+, Tris, choline and mannitol. At most concentrations, mediated influx of glycylsarcosine was depressed by Li+, K+, Cs+ and Tris, though not abolished. At high concentrations, it was moderately increased by choline and mannitol. Under conditions in which the tissue could concentrate the peptide in the presence of Na+, uptake was greatly depressed by all the substituents and the ability to concentrate was abolished. The Kt of mediated influx was affected in different ways according to the substituent used. Kt was reduced by Li+ replacement of Na+ and increased by choline replacement. Vmax was greatly reduced by all metallic substituents but not by non-metallic substituents. Though the results cannot yet be satisfactorily interpreted, they suggest possible reasons for previous conflicting results and show that it is impossible to make the unqualified statement that transport of glycylsarcosine is 'Na+-dependent'. It is now doubtful whether transport of this peptide into the intestinal cells is by co-transport with Na+, and the whole matter of Na+-dependence of intestinal peptide transport is in question.

Taurine transport associated with cell volume regulation in flounder erythrocytes under anisosmotic conditions.

The taurine transport of flounder erythrocytes is associated with a cell volume regulation in anisosmotic media. An osmolality reduction leads to a cell volume increase, which is followed by a volume readajustment towards the original level. A 75 mosM reduction is accompanied by a 33 mumol g dry wt.-1 reduction in the cellular taurine content. The reduction in osmolality activates the taurine release mechanism by transiently increasing the rate coefficient for taurine efflux. The rate coefficient for taurine influx is similarly stimulated. This influx is mediated by a Na+-independent transport system. The concomitant activation of influx and efflux suggests a coupling between these two systems. Higher taurine efflux and influx rate coefficients which decayed more slowly with time were measured in cells suspended in Na+-free (choline replacement) media than in the presence of Na+. This suggests that Na+ may play a role in the taurine release mechanism. Noradrenaline induced a cellular swelling at normal osmolality (330 mosM), but had only a minor effect on the taurine efflux and influx and the cellular taurine content. Urea-induced cellular swelling at normal osmolality initiated a volume regulatory process and activated the taurine release mechanism, similarly to an osmolality reduction. These results show that osmolality reduction and cellular swelling are no prerequisites for the activation of the taurine release mechanism and the cell volume readajustment. It is suggested that the dimension of an intracellular solute compartment determines the activation level of this mechanism.

Transport characteristics of system A in the rat exocrine pancreatic epithelium analyzed using the specific non-metabolized amino acid analogue α-methylaminoisobutyric acid

The selectivity and kinetics of system A amino acid transport in the rat exocrine pancreatic epithelium were characterized using the specific analogue α-methylaminoisobutyric acid. Unidirectional influx of α-methylaminoisobutyric acid was measured in isolated perfused pancreata by rapid dual tracer dilution. In cross-inhibition experiments dl-methylalanine, l-serine, l-cysteine, glycine, l-phenylalanine and l-glutamine were effective inhibitors of influx, whereas l-glutamate and l-lysine were less effective. In the presence of sodium α-methylaminoisobutyric acid influx was saturable with an apparent K t = 1.7 ± 0.2 mM and V max = 0.49 ± 0.03 μ mol/ min per g ( mean ± S.E., n = 6 ). Influx of α-methylaminoisobutyric acid at 50 μM and 100 μM concentrations was significantly inhibited as the perfusate sodium concentration was gradually decreased from 156 mM to 26 mM by isoosmolar choline replacement. Estimated K t values for sodium at these two methylaminoisobutyric acid concentrations approximated 200 mM. System A activity in the basolateral membrane of the exocrine pancreatic epithelium exhibits a high transport affinity, a wide tolerance for different amino acids and a dependency upon the extracellular sodium concentration.

Calcium Action Potentials in the Developing Oocytes of an Insect, Rhodnius Prolixus

ABSTRACT Action potentials (APs) have been recorded for the first time from the oocytes of an arthropod, the haematophagous insect Rhodnius prolixus. In saline containing 2 mmol 1−1 calcium, APs could be evoked by depolarizing the oocyte membrane from its resting potential of –50 mV to a threshold between –35 and –40 mV. The mean duration and overshoot of the APs were 2·6 s and +16 mV, respectively. APs could not be evoked by depolarization of follicular epithelia that had been separated from the oocyte. It is concluded that the APs are generated at the oocyte cell membrane.. The overshoot of the APs was unaffected by the addition of tetrodotoxin (3 μmol 1−1) or the removal of Na+ (choline replacement) from the bathing saline. APs could be reversibly blocked by 1 mmol l− La or 10 mmol 1−1 Co2+. Verapamil at 50μmoll−1 and 100μmoll−1 reduced AP duration by 30% and 50%, respectively. The overshoot increased by 30 mV when bathing saline Ca2+ concentration was increased from 2 to 20 mmol 1−1. These results suggested the occurrence of a Ca2+ influx during the rising phase of the AP. Addition of Ba2+ increased the overshoot and duration of APs. In Ca2+-free saline, addition of 2 mmol 1−1 Ba2+ resulted in the spontaneous production of a series of action potentials. The duration of APs was as long as 120 s in 20 mmol 1−1 Ba2+ saline. It is suggested that Ba2+ may block a voltage-sensitive potassium conductance. Possible functions of action potentials during fertilization and early development are discussed.

Transmembrane potential of renal papillary epithelial cells: effect of urea and DDAVP.

To define how renal papillary epithelial cells respond to wide changes in the ionic and osmotic composition of their environment, measurements were made of the transmembrane potential differences (PD) of rat renal papillary epithelial cells in vitro in media containing 100 mM NaCl, 100 mM KCl, 1.5 mM CaCl2, and 1 mM MgSO4 plus varying amounts of urea and/or sucrose up to 1,400 mM. Glass microelectrodes (resistance 25-75 M Omega) were used. With added sucrose, no change in PD from the initial value of -9.3 +/- 1.3 (SD) mV (n = 29) (cell interior negative) was found. With added urea, alone or while osmolality was maintained nearly constant with sucrose, the PD changed in a triphasic manner, depolarizing to -5.3 mV at 50 mM urea, hyperpolarizing to -20.0 mV at 150 mM urea, and then depolarizing again to -5.5 mV at 1,400 mM urea. When bath potassium was decreased to 10 meQ/liter (choline replacement) the PD hyperpolarized to -46.9 +/- 5.0 (SD) mV (n = 32). DDAVP, a nonpressor vasopressin analogue, and 8-bromo-cAMP depolarized the membrane to -5 mV in the presence of urea but did not change PD when urea was absent. These observations suggest an interaction between urea and ionic movement or conductance in rat renal papillary epithelial cells.

Unanticipated stimulatory action of glucocorticoids on epithelial calcium absorption. Effect of dexamethasone on rat distal colon.

We studied the action of a glucocorticoid (GC, dexamethasone) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on transepithelial calcium (Ca) transport in rat distal colon. GC 1.2 mg or 1,25(OH)2D3 270 ng were given daily for 4 d and Ca fluxes were measured in vitro in the absence of electrochemical gradients (Ussing technique). (a) Both 1,25(OH)2D3 and GC increased Ca absorptive flux from 24 +/- 3 (SEM) to 50 +/- 1 and from 23 +/- 1 to 38 +/- 4 nmol/cm2 per h, respectively (in each case n = 9, P less than 0.01); both steroid hormones had no effect on Ca secretory flux. (b) GC, but not 1,25(OH)2D3 increased the short-circuit current Isc) from 30 +/- 5; to 111 +/- 13 microA/cm2 (P less than 0.01), reflecting stimulation of electrogenic sodium (Na) transport. Choline replacement of Na in the bathing buffer abolished both the Isc and the active Ca transport induced by GC, but has no effect on the 1,25(OH)2D3-stimulated active Ca absorption. (c) When the buffer Ca concentration ([Ca]) on both sides of the epithelium was reduced from 1.25 to 1.25 X 10(-2) mM, the GC-induced, but not the 1,25(OH)2D3-induced, stimulation in Ca absorption was abolished. This suggests that the GC-stimulated Ca absorption may require a "threshold" Ca gradient across the luminal membrane through which Ca influx occurs. Thus, contrary to the current consensus, this study demonstrates that GC stimulates active Ca transport and that this action is mediated through a mechanism dependent on the presence of Na and a critical [Ca] in the ambient medium.

Isoproterenol-induced current changes in glands of frog skin.

In this study we report on isoproterenol-induced changes in short-circuit current (SCC) that are located in the glands of the frog skin. Following complete inhibition of the SCC with amiloride (10(-4) M), the addition of isoproterenol (9.0 X 10(-7) M) to the serosal side of the skin elicits a significant increase in SCC from 0.6 +/- 0.1 to 3.9 +/- 0.5 microA/cm2 (p less than 0.001). The conductance is doubled, and both effects are blocked completely with the beta-blocking agent propranolol. In the split-skin preparation, in which no glands are present, there is no current response to isoproterenol following amiloride. This indicates that the amiloride-insensitive isoproterenol-stimulated SCC resides in the glands. In the intact skin, removal of sodium (choline replacement) from the serosal (but not the mucosal) medium abolishes this current change. The postisoproterenol current change in amiloride-inhibited skins is also dependent on the presence of chloride in the serosal bath. Preincubation of the amiloride-inhibited skin with ouabain (10(-4) M) for 10 min prior to the addition of isoproterenol reduces the isoproterenol response by 50%. These results indicate that there is a sodium- and chloride-dependent ouabain-sensitive secretory mechanism in the skin glands of the frog that is responsible for the development of the isoproterenol-stimulated SCC observed in the presence of amiloride.

Na+-dependent H+ and Cl- transport in in vitro frog gastric mucosa.

Na+ dependency of H+ and Cl- transport in resting and stimulated gastric mucosae was investigated. We measured transepithelial potential difference (PD), short-circuit current (Isc), conductance, H+ secretion (JH), and unidirectional fluxes of 36Cl- (muscosal (m) to serosal (s), JClm leads to s and JCLs leads to m) during Isc conditions in Ussing-type chambers. Resting tissues: Na+-free serosal solution (choline replacement), but not mucosal, Ringer solution caused PD and Isc to decrease to zero with a time course identical to that observed with Cl--free solutions. Conductance also decreased by more than 50%. Isc = JCLs leads to m--JCLm leads to 8 = JClnet during control ([Na+] = 105 mM), and Na+-free conditions. When [Na+] = 50 mM (choline replacement) Isc (=JClnet) was reduced by approximately 25%; when K+ replaced Na+, Isc (=JClnet) was reduced by approximately 65%. The dependence of Isc on [Na+] (choline replacement) was sigmoidal indicating that there may be two sites at which Na+ activates Cl- secretion. Plots of 1/Isc vs. 1/[Cl-] with different [NA+] indicated, that the affinity of the Cl- "carrier" may be affected by [Na+]. stimulated tissues: Na+-free (choline replacement) solutions caused PD, Isc, and JClnet all to decrease to zero but sometimes Na+-containing mucosal solution caused JH to increase back to 50% of control, whereas Isc increased by only 16%. Na+-free effects were not reversed by 1 mM dibutyryl cAMP plus 0.1 mM isobutyl methylxanthine in the serosal solution. As [Na+] was increased, Isc (sigmoidal) and JH (monotonic) both increased. K+ replacement of Na+ caused a larger decrease in Isc than when choline was used; JH remained constant if [Na+] greater than or equal to mM. We have proposed that the energy-dependent step for active Cl- transport occurs at the serosal membrane in a Na+-coupled process. Cl- that accompanies JH during open-circuit conditions may utilize a separate system.

Carbon Dioxide Anesthesia of the Heart of the Horned Passalus Beetle, Popilius disjunctus1,2

The semi-isolated heart of the beetle Popilius disjunctus (Illiger) (Coleoptera: Passalidae), was used to investigate the mechanism of carbon dioxide anesthesia. The effects of several concentrations of CO2 on characteristics of heartbeat were compared with those produced by HCl, acetic acid, and anoxia. The reduction in heart rate by CO2 was similar, with some differences, to that caused by the other acids. Carbon dioxide anesthesia did not resemble the effects of anoxia. The CO2-anesthetized heart was studied in Ringer solutions with varied concentrations of Ca, K, and Na. Increased K potentiated reduction in rate by CO2; decreased K had no consistent effect on the response of hearts to CO2. There was a tendency for increased Ca to reduce the relative lowering of heart rate by CO2, and for lowered Ca to increase the CO2 effect. Strontium replacement of Ca caused a much greater reduction in rate in CO2. Choline replacement of Na resulted in increased response to CO2 anesthesia.

Sodium exchange and distribution in the arterial wall.

Pieces of dog carotid artery were studied with respect to water and sodium content. Total sodium content averaged 113 ± 1.2 mm/kg fresh tissue; total water 73.6 ± 0.3% and inulin space 36.2 ± 0.5% of tissue wet wt. A total of 94.8 ± 1.3% of sodium exchanged within 6–12 min with Na22, and 97.4 ± 0.7% of the stable sodium was extracted in sodium-free solution (choline replacement). The curve of efflux of Na22 at 37 C could be decomposed into three simple exponentials with half-times of 42.5 ± 2.3 sec ( phase 1), 5.0 ± 0.3 min ( phase 2), and 71.0 ± 7.3 min ( phase 3). Exchange of sodium of phase 1 (extracellular) fitted well with a theoretical diffusion curve, showed low temperature coefficient and no potassium dependency, and was not influenced by ouabain. Exchange of sodium of phase 2 (cellular) showed high temperature coefficient and potassium dependency and was sensitive to ouabain. Calculations based on the compartmental analysis indicate a too-great intracellular sodium concentration. Binding of sodium to polyanions in the extracellular space is suggested as a possible explanation of the results.

The Choline Replacement Value of Ethanolamine in Chickens Kept on a High Fat Ration

PREVIOUS studies (Kummerow et al., 1948 and Hite et al., 1949) have indicated that the skin fat of turkeys which had been fed either ethanolamine or choline was more stable than the fat from unsupplemented birds. Ethanolamine, therefore, seemed to be functional in the metabolism of fat in poultry although it has been reported to be nonfunctional in growth and the prevention of perosis. Jukes (1941) found that ethanolamine could not replace choline when fed to chicks which were kept on a purified ration. On the other hand, McGinnis, Norris and Heuser (1944) found betaine and methoinine prevented perosis on a simplified ration, but not on a purified ration similar to the one used by Jukes.In the present study the choline replacement value of ethanolamine was tested with chickens kept on a ration which contained 25 percent linseed oil. The latter was added to the ration, as Sharpless (1940) .