Kinetics of entry of galactolipids and phospholipids into myelin.

Abstract— Brain slices from 17 day rats were incubated with [³H]galactose and [³⁵S]sulphate to label cerebroside and sulphatide. Myelin was isolated by centrifugation on a sucrose density gradient. Following lipid extraction and alkaline methanolysis, cerebroside and sulphatide were isolated by tic, and radioactivity was measured. Appearance of [³H]cerebroside and [³H]sulphatide in myelin showed a lag of less than 15min, while appearance of [³⁵S]sulphatide in myelin showed a longer lag of about 30min. In chase experiments, the rate of appearance of [³H]cerebroside and [^3SS]sulphatide in the non-myelin fraction and of [³H]cerebroside in the myelin fraction slowed markedly after the chase. In contrast, [³⁵S]sulphatide continued to increase in myelin at a normal rate for 30min after the chase, then stopped, while ³H from galactose continued to accumulate in myelin sulphatides for 60 min. These data are interpreted to demonstrate an interval of 30 min between synthesis of cerebroside and its sulphation in the non-myelin fraction, and another delay of 30 min between sulphation and appearance in myelin. The distribution of newly synthesized cerebroside and sulphatide between myelin and non-myelin fractions also supported the concept that a complex metabolic pool of cerebroside in the non-myelin fraction is precursor to sulphatide of myelin. For comparison, entry of phosphatidyl choline and phosphatidyl ethanolamine into myelin was followed with [2-³H]glycerol as precursor. Like cerebroside, both phospholipids showed little delay in their initial appearance in myelin, and prompt cessation of their addition after a chase with unlabeled precursor. These results are consonant with either rapid entry of these three lipids into myelin after synthesis at an extra-myelin site, or synthesis of the lipids within myelin itself.     

Hydrolase activities in brain of neurological mutants: cerebroside galactosidase, nitrophenyl galactoside hydrolase, nitrophenyl glucoside hydrolase and sulphatase

Abstract—

• 1 The brains of 17-day-old quaking and jimpy mice were compared with those of the corresponding normal phenotypes. The concentrations of cerebroside and sulphatide were found to be markedly lower in the affected mutants, while the relative amounts of ceramide and free fatty acid appeared normal.
• 2 The concentration of cerebroside glactosidase was not significantly abnormal in the jimpy mice but was about 17 per cent lower in quaking mice. In contrast, the relative amount of the enzyme that could be dispersed by sonication was considerably higher in the jimpy animals. It is suggested that this increase is a causative factor in the aetiology of the latter disease.
• 3 The concentrations of other acid hydrolases were determined, as well as the relative amounts dispersible by sonication. No difference was seen between the phenotypes with NPGalH, NPGluH, and nitrocatechol sulphate hydrolase.
• 4 An improved solvent system for the TLC detection of ceramide in brain lipids is described.

     

The characterization of sphingolipids from neurons and astroglia of immature rat brain

Abstract— Isolated neuronal cell bodies and astroglia of young (15–20-day-old) rat brains were both found to contain small concentrations of a variety of glycosphingolipids, including glucosylceramide, galactosylceramide, sulphatide, dihexosylceramide and gangliosides. These sphingolipids, plus sphingomyelin, were isolated, quantitated and their fatty acid and long chain base patterns determined. These data were compared to similar data obtained on these lipids isolated from whole brain and myelin of rats of the same age range. Glucosylceramide was found in an amount equal to galactosylceramide in neurons, and accounted for 35 per cent of the total monohexosylceramide in astroglia. Dihexosylceramide was present in nearly the same amount as sulphatide in both cell types. The sphingolipids of each cell type had characteristic fatty acid patterns. Generally the whole brain fatty acid patterns resembled those of astroglial lipids rather than neuronal lipids. In no case did the cell sphingolipid fatty acids resemble those of myelin. However, the galactosylceramide and sulphatides of both cells had unsubstituted and α-hydroxy acids, both of which had appreciable quantities of C₂₄ acids. The ganglioside fatty acids of each cell type were similar and not unusual, but were quite different from those of glucosylceramide and dihexosylceramide; the latter having appreciable quantities of 16:0 and acids longer than 18:0. The ganglioside patterns of these cells were similar and only slightly different from that of whole brain. Long chain bases of sphingolipids were mainly C₁₈-sphingosine in both cell types, and those of ganglioside and sphingomyelin contained small amounts of C₂₀-sphingosine.     

Synthesis of sulphatide-containing lipoproteins in rat brain

Abstract—

• 1 Puromycin inhibits [¹⁴C]leucine Hincorporation into brain proteins, but has no effect on the incorporation of [³⁵S]sulphate into sulphatide. These effects of puromycin are observed not only with the proteins and sulphatide of whole brain, but also with the protein and sulphatide portion of water-soluble lipoprotein complexes.
• 2 Microsomes can be separated into three subfractions which differ chemically, morphologically and metabolically. Protein synthesis and sulphatide synthesis are located in different submicrosomal fractions.
• 3 The addition of water-soluble brain proteins to the incubation medium causes release of newly synthesized [³⁵S]sulphatide and formation of soluble sulphatide protein complexes. One acceptor protein is identified as the lipoprotein previously shown to bind [³⁵S]sulphatide in vivo (Herschkowitz , Mc Khann , Saxena and Shooter , 1968b).
• 4 These results suggest that protein and sulphatide synthesis can function independently and that association of newly synthesized lipid to preformed protein is possible.

     

Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA⁺RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T₃-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T₄ intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

METABOLIC RELATIONSHIPS BETWEEN MYELIN SUBFRACTIONS: ENTRY OF GALACTOLIPIDS AND PHOSPHOLIPIDS 1

Abstract— Partially purified myelin from the brains of 17-day-old rats was separated into 4 subfractions on a three-step sucrose gradient by virtue of heterogeneity in density and particle size. Precursor-product relationships between different membrane fractions were investigated by determining the specific radioactivity of individual lipids in each subcellular fraction 15 min after intracranial injection of an appropriate precursor. Rats were injected with [2-³H]glycerol. myelin subfractions prepared, and individual lipids separated by TLC. For choline and ethanolamine phospholipids, specific radioactivity was highest in the densest fraction (D), intermediate in the next densest fraction (C), and lowest in the lighter fractions (B and A). Similar results were observed for cerebroside and sulphatide when [³H]galactose was the precursor. These data are consistent with (but do not prove) a precursor-product relationship for individual lipids from the densest to the lightest subfraction. Another experimental design involving time staggered injections of [³H] and [¹⁴C] precursors was developed which enables a more definitive result with regard to precursor-product relationships to be obtained. A precursor-product relationship between a given lipid in a dense myelin membrane fraction, and the same lipid in a lighter subfraction, would be indicated by a change in isotope ratio. If there is no precursor-product relationship. Ihe isotope ratio should be constant. Such experiments were done with [³H] and [¹⁴C]glycerol. The data indicated that phosphatidyl ethanolamine and its plasmalogen analog were added first to the densest subfraction and then in turn to the lighter subfractions. In contrast, phosphatidyl choline and its plasmalogen analog were added “simultaneously” (i.e. with delays of much less than 15min) to each of the subfractions. Similar experiments with [³H] and [¹⁴C]galactose showed that cerebroside, sulphatide and galactosyl diglyceride also entered the subfractions simultaneously rather than in sequential order. Thus the assembly of the myelin sheath involves an obligate order of addition of certain lipids. while other lipids are probably added in a random order.     

Effect of Malnutrition and Subsequent Rehabilitation on the Development of Mouse Brain Myelin

Abstract: Malnutrition in mice from birth resulted in myelin of brain having higher than normal molar proportions of cholesterol and phospholipids relative to a molar unit of cerebroside + sulphatide. This was found at all ages between 20 and 60 days, and the molar ratio of these lipids in older animals was comparable to that in the younger controls. The phospholipid and the ganglioside patterns were also immature for age. The phospholipid composition was characterized by lower molar proportions of ethanolamine phosphoglyceride (EPG) and sphingomyelin (SPh) and higher proportion of choline phosphoglyceride (CPG), and the ganglioside pattern was characterized by higher molar proportions of the disialogangliosides G_Dla and G_Dlb and markedly lower proportion of the monosialoganglioside G_M1. Malnutrition imposed from 30 days of age did not affect the contents of the major lipids (and so their molar ratio), but within the phospholipids there was a small but significant deficit of SPh, which was compensated by a higher content of CPG. The ganglioside pattern was as if the animals were malnourished from birth. Nutritional rehabilitation up to 60 days of age subsequent to malnutrition for the first 30 days fully corrected the ganglioside pattern, but not the molar ratio, of the major lipids (because of persistent deficit in cerebroside + sulphatide) and the composition of the phospholipids (because of small but significant deficit of SPh). The results indicate that malnutrition instituted at any time during the entire programme of myelination can affect one or other aspect of myelin development, and nutritional rehabilitation of animals malnourished in early life cannot fully correct this developmental gap.     

SULPHATE METABOLISM IN ACUTE EAE1 RATS USING ISOLATED BRAIN PERFUSION TECHNIQUE

—Metabolism of glycolipids and glycosaminoglycans were studied in rats in the acute stage of experimental allergic encephalomyelitis (EAE) using isolated brain perfusion technique. It was observed that there was a significant decrease in the concentration of cerebroside, sulphatide and GAG (hyaluronic acid and low sulphated GAG) when compared to normal and pairfed control rats. The radioactive sulphate incorporation into the cerebroside sulphate and sulphated GAG was significantly higher in the case of rats in the acute stage of EAE than the normal and pairfed control rats.     

Composition of myelin lipids and synthesis of 3-ketodihydrosphingosine in the vitamin B 6 -deficient developing rat

Abstract— Recoverable myelin in 20-day-old, Vitamin B₆-deficient rats was reduced from that in control animals. The composition of myelin lipids from deficient animals was nearly identical to that for controls except for a striking reduction in phospholipids polyunsaturated fatty acids. Enzymatic synthesis in vitro of 3-ketodihydrosphingosine was impaired in the deficient animals; addition of the vitamin Bs cofactor to these incubation mixtures restored activity of the condensing enzyme to control levels.     

KINETIC ANALYSIS OF PHENYLALANINE-INDUCED INHIBITION IN THE SATURABLE INFLUX OF TYROSINE, TRYPTOPHAN, LEUCINE AND HISTIDINE INTO BRAIN CORTEX SLICES FROM ADULT AND 7-DAY-OLD RATS

—An attempt was made to isolate the saturable uptake from the unidirectional influx of amino acids into tissue slices and to estimate the transport constants and maximal velocities of saturable transport. The method was applied to studies on the inhibition of phenylalanine in the saturable influx of tyrosine, tryptophan, histidine and leucine into brain cortex slices from adult and 7-day-old rats. In both age groups phenylalanine inhibited the influx of the other amino acids, and vice versa. The apparent transport constants of the other amino acids increased in the presence of phenylalanine more noticeably in the slices from 7-day-old rats than in those from adult rats, whereas the concomitant influx of phenylalanine was inhibited less in the slices from 7-day-old rats. In immature animals in vivo competition between amino acids may play a more marked role in the supply of amino acids from plasma to brain, as the transport systems in brain slices from 7-day-old rats become saturated with extracellular amino acids more readily than do the transport systems in brain slices from adult rats.     

The fatty acid composition of sphingolipids from bovine CNS axons and myelin

Abstract— Cerebrosides, sulphatides and sphingomyelin were isolated from bovine CNS myelin and from myelin-free axons derived from myelinated axons. The fatty acid composition of each sphingolipid was determined by gas-liquid chromatography of the fatty acid methyl esters. In each case the fatty acids of the axonal sphingolipids were of shorter average chain length than those from the corresponding myelin lipids. These differences, however, were small and the fatty acids of the axonal cerebrosides and sulphatides were similar in average chain length to those reported previously for bovine myelin. The principal unsubstituted acid of both cerebroside and sulphatide from axons was 24: 1, with the total long chain acids (> C₁₈) amounting to 80 and 85 per cent, respectively. The corresponding figures for myelin galactolipids were 94 and 95 per cent long chain acids. The principal α-hydroxy acid of both axonal galactolipids was 24 h:0, with cerebroside having 80 per cent and sulphatide 92 per cent long chain acids, compared to the figures of 87 and 97 per cent for the corresponding myelin lipids. In axonal sphingomyelin the major acid was 18:0 (compared to 24:1 in myelin) and the long chain acids were 61 per cent of the total vs 76 per cent of the total for myelin sphingomyelin. The non-identity of axonal and myelin sphingolipid fatty acids substantiates the belief that they are intrinsic axonal constituents. These findings do not rule out the possibility of a close metabolic relationship between the sphingolipids of the axon and its myelin sheath.     

QUANTITATION AND CHARACTERIZATION OF BRAIN TUBULIN (COLCHICINE-BINDING ACTIVITY) IN DEVELOPING HYPOTHYROID RATS

The influence of early hypothyroidism on the concentration and biochemical properties of soluble and particulate tubulin from the cerebral cortex and cerebellum was investigated during development in the rat. Cellular soluble tubulin concentration (pmol colchicine bound/μg DNA) was approx 16% lower in both brain areas of hypothyroid animals compared to controls at 25 days of age. No effect of thyroid hormone deficiency was observed when tubulin concentration was expressed in terms of tissue protein or weight. The particulate tubulin concentration was approx 20% lower in the cerebral cortex of 25-day-old hypothyroid rats although the distribution of tubulin between soluble and particulate fractions was similar to controls. The incorporation of [¹⁴C]leucine into cerebral cortical tubulin in vitro (c.p.m. in tubulin/c.p.m. in total protein) was not significantly altered by the hormonal deficiency. Thus there was no apparent evidence of a selective defect in tubulin synthesis. Tubulin from hypothyroid rats behaved similarly to control samples with respect to the effects of pharmacological agents and temperature, lability of binding, chromatographic profile and electrophoretic mobility on sodium dodecyl sulfate polyacrylamide gels.     

BIOSYNTHESIS AND COMPOSITION OF BRAIN GALACTOLIPIDS IN NORMAL AND HYPOTHYROID RATS

Abstract— Newborn rats were rendered hypothyroid by methimazole treatment. Incorporation of [1-¹⁴C]galactose both in vivo and in vitro into brain cerebrosides of hypothyroid rats was significantly lower than in normals. Biosynthesis of sulphatides was affected by hypothyroidism to a smaller extent than cerebrosides. Assay of cerebroside biosynthesis from [1-¹⁴C]galactose or UDP-[1-¹⁴C]galactose by brain preparations revealed that incorporation of the sugar in both cases is affected to the same extent by methimazole treatment, suggesting that the phenomenon is not due to impairment of the nucleotide biosynthesis. A radioactive galactolipid tentatively characterized as glycerogalactolipid was synthesized in vitro and its biosynthesis was reduced to a large extent in the brain preparations from hypothyroid rats. The fatty acid composition of cerebrosides and sulphatides from the brains of hypothyroid rats was found to be different from that of normal rats. The percentage of normal C₂₄ fatty acids was significantly decreased in the methimazole-treated rats. Brain sphingomyelin fatty acids did not differ between normal and hypothyroid rats.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

Sulpholipid metabolism in developing chicken retina.

Glycolipid analysis of chicken retina and brain indicated the presence of cerebroside, cerebroside 3-sulphate and sulphogalactosylglycerolipid In retina, the ratio of cerebroside to cerebroside 3-sulphate was approximately half compared to brain. During chicken retina ontogenesis the ratio of cerebroside 3-sulphate to sulphogalactosylglycerolipid increased rapidly and in the adult animal, the amount of cerebroside 3-sulphate was 14 times higher than that of sulphogalactosylglycerolipid. The activity of PAPS: cerebroside sulphotransferase and arylsulphatase A in developing chicken retina indicated that the general ontogenic profiles of retinal PAPS: cerebroside sulphotransferase and arylsulphatase A were similar to those obtained for the brain. Both the enzymes showed the highest activity just before hatching. The significance of occurrence of sulpholipids in retina is discussed.     

BRAIN LIPID COMPOSITION OF IMMATURE THIAMINE-DEFICIENT AND UNDERNOURISHED RATS1

The brain lipid composition of 25-day-old offspring of rats exposed to dietary thiamine (vitamin B₁) deficiency from the 14th day of gestation was examined and compared to normal and pair-fed (undernourished) controls. Thiamine-deprived rats displayed neurological signs and a marked diminution of growth at 25 days of age. No changes in brain lipids of either whole brain or selected brain areas (brain stem, cerebellum, diencephalon) which were distinct from the effects of undernutrition (pair-fed controls) were observed in the thiamine-deficient group. Undernutrition, as exemplified by the pair-fed control group produced a highly significant depression of all lipids expressed per total brain and a significant deficit of whole brain and regional lipid, cerebroside and cholesterol concentrations indicating a deficiency in myelinogenesis. Ganglioside NeuNAc concentration was shown to be significantly greater in whole brain and certain brain areas of the same group while no changes were evident in total phospholipid concentration and the distribution of individual phospholipids. The implications of these findings in terms of the pathophysiology of thiamine-deficiency encephalopathy and undernutrition in early life are discussed.     

The activator of cerebroside-sulphatase. A model of the activation.

The activator of cereboroside-sulphatase (cerebroside-3-sulphate-3-sulphohydrolase, EC 3.1.6.8) is necessary for the enzymic hydrolysis of sulphatides (cerebroside sulphates) at ionic concentrations in the physiological range. The pH optimum of the reaction is 4.5--4.8. Under similar incubation conditions, a complex is formed between activator and sulphatides which is partially inhibited, due to competitive binding in the presence of cerebrosides of phosphatidylserine. Inhibition depends upon the concentration of the lipids and is of the same order of magnitude as the inhibition (by these lipids) of enzymic sulphatide hydrolysis in the presence of activator. Complex formation between activator and sulphatides is reversible since the complex dissociates partially when certain concentrations of phosphatidylserine are added. Moreover, the rate of sulphatide hydrolysis increases with the concentration of the activator.sulphatide complex in the reaction mixture. This indicates that the activator.sulphatide complex is the substrate for the enzyme and a model for this activation is presented.     

Myelin synthesis in peripheral nerve in vitro: sulphatide incorporation requires a transport lipoprotein

Abstract— Sciatic nerves from 18-day-old chick embryos incorporated ³⁵SO₄ into myelin sulphatide in vitro. Sulphatide in a microsomal subfraction of the nerve was rapidly labelled with ³⁵SO₄, and a lipoprotein fraction in the nerve served to transfer the [³⁵S]sulphatide from the microsomal subfraction to myelin. Puromycin and cycloheximide inhibited the incorporation of [³⁵S]sulphatide into myelin after a lag period of about 2 h. These agents did not alter the rate of appearance of [35S]sulphatide in the microsomal subfraction, and did not diminish the capacity of myelin to take up [³⁵S]sulphatide from the lipoprotein fraction; instead, they appeared to interfere with the incorporation of [35S]sulphatide into myelin by decreasing the available pool of the transport lipoprotein. Partial characterization of the [³⁵S]labelled lipoprotein fraction indicated that it had a density of 1.06–1.08. The lipoprotein was highly aggregated, but, after incubation with SDS and mercaptoethanol, it was dissociated into sulphatide-containing micelles and proteins.     

Evaluation ofin vivo insulin action and glucose metabolism in milk-fed rats

Milk diet has long been recommended in the management of gastrointestinal pathologies. Since milk feeding represents a high fat-low carbohydrate diet and it is acknowledged that insulin resistance is one of the consequences of high fat feeding, it is important to know whether or not chronic milk feeding leads to an impairment of the insulin-mediated glucose metabolism. To examine this question, adult female rats were given raw cow's milk (50% of total calories as lipids) for 18 days. They were compared to rats raised in parallel and fed the standard laboratory diet (15% of total calories as lipids). At the end of the 18 day period, body weight, daily caloric intake, basal plasma glucose and insulin levels in the milk-fed rats were similar to those in the control rats.In vivo insulin action was assessed with the euglycemichyperinsulinemic clamp technique in anesthetized animals. These studies were coupled with the 2-deoxyglucose technique allowing a measurement of glucose utilization by individual tissues. In the milk fed rats: 1) the basal rate of endogenous glucose production was significantly (p<0.01) reduced (by 20%); 2) their hepatic glucose production was however normally suppressed by hyperinsulinemia; 3) their basal glucose utilization rate was significantly (p<0.01) reduced (by 20%); 4) their glucose utilization rate by the whole-body mass or by individual tissues was normally increased by hyperinsulinemia. These results indicate that insulin action in adult rats is not grossly altered after chronic milk-feeding, at least under the present experimental conditions.