IGF‐I stimulates neurogenesis in the hypothalamus of adult rats

In the brain of adult rats neurogenesis persists in the subventricular zone of the lateral ventricles and in the dentate gyrus of the hippocampus. By contrast, low proliferative activity was observed in the hypothalamus. We report here that, after intracerebroventricular treatment with insulin‐like growth factor I (IGF‐I), cell proliferation significantly increased in both the periventricular and the parenchymal zones of the whole hypothalamus. Neurons, astrocytes, tanycytes, microglia and endothelial cells of the local vessels were stained with the proliferative marker 5‐bromo‐2′‐deoxyuridine (BrdU) in response to IGF‐I. Conversely, we never observed BrdU‐positive ciliated cubic ependymal cells. Proliferation was intense in the subventricular area of a distinct zone of the mid third ventricle wall limited dorsally by ciliated cubic ependyma and ventrally by tanycytic ependyma. In this area, we saw a characteristic cluster of proliferating cells. This zone of the ventricular wall displayed three cell layers: ciliated ependyma, subependyma and underlying tanycytes. After IGF‐I treatment, proliferating cells were seen in the subependyma and in the layer of tanycytes. In the subependyma, proliferating glial fibrillary acidic protein‐positive astrocytes contacted the ventricle by an apical process bearing a single cilium and there were many labyrinthine extensions of the periventricular basement membranes. Both features are typical of neurogenic niches in other brain zones, suggesting that the central overlapping zone of the rat hypothalamic wall could be considered a neurogenic niche in response to IGF‐I.     

Arachidonic acid metabolism in gerbil cerebra: effects of ischemia and pentobarbital

Pentobarbital pretreatment may be used to predict biochemical events involved in ischemic brain damage following bilateral carotid artery ligations in the gerbil, since it reduces the subsequent edema and mortality. The effects of this anesthetic on the ischemia-induced modifications of cerebral arachidonic acid metabolism were investigated, in order to correlate observed alterations with tissue damage. Cerebral lipids were radiolabeled in vivo with [3H]arachidonic acid prior to 10 min of cerebral ischemia and 0-120 min of perfusion. Ischemia stimulated a 97.3% increase in unesterified [3H]arachidonate, which was due to the loss of label from choline, inositol, and ethanolamine glycerophospholipids. Tissue reperfusion stimulated further reductions in [3H]choline and [3H]inositol glycerophospholipids, while ethanolamine glycerophospholipid and triglyceride labeling increased. Inositol glycerophospholipid, but not choline glycerophospholipid, labeling returned to control level by 60 min of reperfusion. Pentobarbital pretreatment reduced the accumulation of [3H]arachidonate by 56.2% during ischemia. It increased the recovery of [3H]ethanolamine glycerophospholipids during the ischemic period and [3H]choline glycerophospholipids during the first 5 min of reperfusion. These effects accounted for the reduction of unesterified [3H]arachidonate observed during ischemia and reperfusion.     

Non-canonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish

Protein expression in the nervous system undergoes regulated changes in response to changes in behavioral states, in particular long-term memory formation. Recently, methods have been developed (BONCAT and FUNCAT), which introduce non-canonical amino acids bearing small bio-orthogonal functional groups into proteins using the cells' own translational machinery. Using the selective 'click reaction', this allows for the identification and visualization of newly synthesized proteins in vitro. Here we demonstrate that non-canonical amino acid labeling can be achieved in vivo in an intact organism capable of simple learning behavior, the larval zebrafish. We show that azidohomoalanine is metabolically incorporated into newly synthesized proteins, in a time- and concentration-dependent manner, but has no apparent toxic effect and does not influence simple behaviors such as spontaneous swimming and escape responses. This enables fluorescent labeling of newly synthesized proteins in whole mount larval zebrafish. Furthermore, stimulation with a GABA antagonist that elicits seizures in the larval zebrafish causes an increase in protein synthesis throughout the proteome, which can also be visualized in intact larvae.     

Effect of essential fatty acid deficiency on the synthesis and turnover of myelin lipid

The metabolism of myelin glycerophospholipids as a function of essential fatty acid (EFA) deficiency was studied in rats. Mothers were fed a lipid-free diet from the eighth day of pregnancy throughout the suckling period. Pups were continued on the diet after weaning. Control mothers and pups were fed the same diet as experimental rats plus 3% corn oil for the same periods. Pups fed the lipid-free diet had lower total body weight, brain weight, and total myelin phospholipid levels than controls. At 17 days of age, each rat pup was injected with a mixture of [³H]-glycerol and [1-¹⁴C]-acetate and were killed up to 80 days after injection. Lipids were extracted from isolated myelin. The ³H/¹⁴C ratio for choline, serine, and ethanolamine glycerophospholipids decreased more slowly in experimental rats than in controls, suggesting an alteration in either the turnover of myelin phospholipids or the recycling of phospholipids into myelin. Radiolabelled acyl moieties were isolated from myelin lipids and separated according to the degree of unsaturation. Acyl groups with three double bonds, presumably 20:3 (ω?9), isolated from EFA-deficient rats appeared to be synthesized from a long-lived pool of precursor. The relative synthesis of other unsaturated acyl moieties appeared to be the same in both groups of rats. These results indicate that EFA deficiency affects myelin metabolism.     

Glucose, free fatty acid and ketone body metabolism in Duchenne muscular dystrophy

We examined how the substances, especially glucose, free fatty acids (FFA) and ketone bodies, and hormones associated with energy metabolism change with the disease progress in Duchenne muscular dystrophy (DMD). Serum creatine kinase (CK) activity was used as an index of the stage of DMD, because this activity is exponentially decreases with the progress of the disease. The glucose concentration in DMD patients with CK activity of less than 1,000 U/l (low CK) was significantly lower than that in controls, although there was no significant difference between that in DMD patients with CK activity of more than 1,00 U/l (high CK) and that in controls. The FFA concentration in both high CK and low CK patients was significantly higher than that in controls. The FFA concentration in low CK patients tended to be higher than that in high CK patients. The ketone body concentration in low CK patients was significantly higher than that in controls and that in high CK patients. The [glucagon]:[insulin] ratio in low CK patients was significantly higher than that in controls and that in high CK patients. It was also observed in a correlational study that the glucose concentration decreased with the age and the decrease in CK activity, i.e., with the progress of DMD. The FFA and ketone body concentrations increased with the decrease in the glucose concentration. The decrease in the glucose concentration may be due to a caloric shortage and/or degenerated muscle, which cannot supply enough gluconeogenic substrates, such as alanine. The kinetics of insulin and glucagon in DMD may help to maintain the glucose metabolism. Increased concentrations of FFA and ketone bodies may be helpful in the advanced stage of DMD, as energy sources and as substrates, sparing muscle protein.     

Muscle carnitine deficiency: fatty acid metabolism in cultured fibroblasts and muscle cells

L-carnitine, as little as 25nM, greatly increased oxidation of palmitate by carnitine-depleted cultured human skin fibroblasts from normal subjects, and from two patients with muscle carnitine deficiency. Carnitine stimulated oxidation of labeled palmitate by cultured muscle cells from rat, normal humans, or patients with muscle carnitine deficiency. Carnitine reduced incorporation of palmitate into glycerides in normal fibroblasts, and it may thereby counteract cellular accumulation of glycerides in cells. Fibroblasts from patients with muscle carnitine deficiency took up labeled carnitine at a normal rate. Dexamethasone increased palmitate oxidation by normal human fibroblasts and muscle cells of rat, and normal and muscle carnitine-deficient humans. The results parallel the reported effectiveness of carnitine, glucocorticoids, or medium-chain triglycerides in treatment of these patients.     

Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area

Recent studies have identified several neuropeptide systems in the hypothalamus that are critical in the regulation of body weight. The lateral hypothalamic area (LHA) has long been considered essential in regulating food intake and body weight. Two neuropeptides, melanin-concentrating hormone (MCH) and the orexins (ORX), are localized in the LHA and provide diffuse innervation of the neuraxis, including monosynaptic projections to the cerebral cortex and autonomic preganglionic neurons. Therefore, MCH and ORX neurons may regulate both cognitive and autonomic aspects of food intake and body weight regulation. The arcuate nucleus also is critical in the regulation of body weight, because it contains neurons that express leptin receptors, neuropeptide Y (NPY), α-melanin-stimulating hormone (α-MSH), and agouti-related peptide (AgRP). In this study, we examined the relationships of these peptidergic systems by using dual-label immunohistochemistry or in situ hybridization in rat, mouse, and human brains. In the normal rat, mouse, and human brain, ORX and MCH neurons make up segregated populations. In addition, we found that AgRP- and NPY-immunoreactive neurons are present in the medial division of the human arcuate nucleus, whereas α-MSH-immunoreactive neurons are found in the lateral arcuate nucleus. In humans, AgRP projections were widespread in the hypothalamus, but they were especially dense in the paraventricular nucleus and the perifornical area. Moreover, in both rat and human, MCH and ORX neurons receive innervation from NPY-, AgRP-, and α-MSH-immunoreactive fibers. Projections from populations of leptin-responsive neurons in the mediobasal hypothalamus to MCH and ORX cells in the LHA may link peripheral metabolic cues with the cortical mantle and may play a critical role in the regulation of feeding behavior and body weight. J. Comp. Neurol. 402:442–459, 1998. © 1998 Wiley-Liss, Inc.     

Phospholipid and phospholipid fatty acid composition of mixed murine spinal cord neuronal cultures

The phospholipid and phospholipid fatty acid compositions of mixed murine spinal cord neuronal cultures are reported. The phospholipid composition was primarily comprised of ethanolamine glycerophospholipids (44.8%) and choline glycerophospholipids (43.5%). Plasmalogens made up 29.1% of the ethanolamine glycerophospholipids (13.0% of the total phospholipids) and 4.5% of the choline glycerophospholipids (1.9% of the total phospholipids). Other phospholipids ranged from 2.9% for sphingomyelin to 1.0% for phosphatidylinositol 4-phosphate. The fatty acid compositions of the ethanolamine glycerophospholipids, choline glycerophospholipids, phosphatidylserine, and phosphatidylinositol were also determined. The choline glycerophospholipids were the most saturated and contained the smallest amount of polyunsaturated fatty acids. The ethanolamine glycerophospholipids were the most unsaturated and contained the highest amount of polyunsaturated fatty acids. The phospholipids contained minimal amounts of 20:3 n-9 (Mead acid) and are not considered polyunsaturated fatty acid deficient. Thus, for the mixed neuronal spinal cord cultures, the phospholipid fatty acid compositions were not polyunsaturated fatty acid deficient and contained a large amount of polyenoic fatty acids of both the n-3 and n-6 series. © 1993 Wiley-Liss, Inc.     

Systemic triglyceride storage disease with normal carnitine: a putative defect in long-chain fatty acid metabolism

A 45-year-old Japanese man presented with lipid storage myopathy, fatty liver, cardiomyopathy, vacuolated leukocytes (Jordans' anomaly) and perceptive deafness. His parents were consanguineous and his younger sister was also affected. Histopathological and biochemical studies revealed an abnormal accumulation of triglyceride in muscle, liver, leukocytes, gastrointestinal endothelial cells and cultured skin fibroblasts. On electron microscopy, the vacuoles lacked limiting membranes and were adjacent to the mitochondria. Total and free carnitines in muscle were normal levels. Production rate of 14CO2 or acid-soluble [14C]metabolites from [1-14C]palmitate in the patient's cells was decreased to about 50% of that in control cells, whereas that from [1-14C]butyrate was normal. Long-chain fatty acyl esterase activities in the patient's leukocytes were normal at both pH 4.0 and pH 8.0. Despite the strong suggestion of an impaired metabolism of long-chain fatty acids, there were no evidences of abnormalities in carnitine metabolism or uptake of fatty acids into cells. The disorder is clinically different from defects in carnitine metabolism, defects in the carnitine-acylcarnitine translocase system or in mitochondrial beta-oxidation enzymes. Although the underlying metabolic defect has not been elucidated, this disease seems to be an autosomal-recessively inherited disorder of systemic triglyceride storage, probably due to an impaired regulation of lipolysis and triacylglycerol synthesis.     

Involvement of membrane-associated proteins in the acute regulation of cellular fatty acid uptake

The transport of long-chain fatty acids across cellular membranes most likely occurs to some extent by passive diffusion and additionally is facilitated by a number of membrane-associated and cytoplasmic proteins. In this overview we focus on the involvement of the membrane proteins fatty acid translocase (FAT/CD36), plasma membrane fatty acid-binding protein (FABP_pm) and fatty acid-transport protein (FATP). Newly obtained evidence is presented that in skeletal muscle, fatty acid uptake is subject to short-term regulation by translocation of FAT/CD36 from intracellular stores to the plasma membrane, analogous to the regulation of muscular glucose uptake by GLUT-4 translocation. These new findings establish a significant role of membrane-associated proteins in the cellular fatty acid-uptake process. Possible implications for the uptake and transport of long-chain fatty acids by the brain are discussed.     

四种抗精神病药对糖代谢及脂代谢的不良影响

目的研究4种抗精神病药对糖代谢、脂代谢的不良影响。方法112例精神分裂症患者根据临床治疗需要分为氯氮平组（30例）、奥氮平组（24例）、利培酮组（29例）和舒必利组（29例），均治疗观察4周。每组患者于治疗前后测空腹血糖、甘油三酯、胆固醇、胰岛素、C肽，量身高、体质量、腰围、臀围，并计算体质量指数（BMI）及胰岛素抵抗指数（IR）。结果（1）治疗后4组患者的空腹胰岛素、C肽及IR均升高，与治疗前的差异有统计学意义（P〈0．05）；治疗后氯氮平组和奥氮平组患者的甘油三酯及胆固醇均明显高于治疗前（P〈0．05）。（2）治疗后BMI的升高程度为：氯氮平〉奥氮平〉舒必利〉利培酮，差异均有统计学意义（P〈0．05）。空腹胰岛素、C肽、甘油三酯、胆固醇及IR的升高程度为：氯氮平和奥氮平〉舒必利和利培酮，差异均有统计学意义（P〈0．05）。（3）氯氮平组、奥氮平组的甘油三酯及IR升高程度均为男性大于女性，胆固醇升高程度为女性大于男性；舒必利组的变化则相反。结论氯氮平和奥氮平对糖代谢及脂代谢的影响大于利培酮和舒必利，并存在性别差异。     

Automated tandem mass spectrometry for mass newborn screening for disorders in fatty acid, organic acid, and amino acid metabolism

Development of acylcarnitine and amino acid profiling using tandem mass spectrometry, and its application for use with dried blood specimens collected on filter-paper cards, has introduced an innovative new technology for detecting inborn errors of fatty acid, organic acid, and amino acid metabolism. From November 1, 1992 through June 30, 1999 we screened more than 700,000 newborns in Pennsylvania, Ohio, North Carolina, and Louisiana. We have prospectively detected 163 inborn errors of metabolism. Eighty-six patients have amino acid metabolism errors. Among them are phenylketonuria, hyperphenylalaninemia, maple syrup urine disease, and several urea cycle disorders. Thirty-two have organic acid metabolism errors, including glutaric aciduria type 1; 3-methylcrotonyl coenzyme A (CoA) carboxylase deficiency, propionic acidemia, methylmalonic acidemia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency; and 45 have fatty acid oxidation errors, including 36 with medium-chain acyl-CoA dehydrogenase deficiency. Details of the methodology are presented and the potential of this screening technology is discussed.     

Tanycytes in the sunfish brain: NADPH-diaphorase histochemistry and regional distribution

NADPH-diaphorase histochemistry has been shown to be a useful method for identifying cells that synthesize and release nitric oxide, which is implicated in the modulation of a variety of neural functions, including synaptic transmission, cerebral blood flow, and excitotoxicity. In the sunfish brain, NADPH-diaphorase histochemistry stains tanycytes specifically and almost exclusively, allowing for a thorough examination of the morphology and distribution of this type of cell. Tanycytes are nonciliated, process-bearing ependymal and extraependymal cells that contact the ventricular surface via apical processes, and the pial surface via basal processes. Ependymal tanycytes are located at the ventricular surface, and project basal processes into the parenchyma of the brain. Extraependymal tanycytes are found away from the ventricular matrix. Some extraependymal tanycytes are small, bipolar, and tend to be associated with bundles of basal processes. Isolated extraependymal tanycytes are larger, darkly stained, and multipolar. Their basal processes terminate in specialized endfeet on blood vessels, neuronal somata, or the pial surface. Specialized types of tanycytes are found in the optic tectum, the epineurial septum between axonal bundles along the midline in the medulla, and in restricted regions on the pial surface in the medulla. The only NADPH-diaphorase-positive neurons are found in the commissural nucleus of area ventralis telencephali. Injection of horseradish peroxidase into the ventricles shows that tanycytes lining the third and fourth ventricles are capable of taking up the tracer and transporting it into their basal processes. Tanycytes are unevenly distributed in the brain. There is a rough rostrocaudal gradient of cell density: tanycytes are sparse in the telencephalon and dense in the isthmus and medulla, although cell density is low in the spinal cord. Not all ventricular linings contain tanycytes: cell density is low in the medial ventricle of the telencephalon and in the infundibular recess, and high along the fourth ventricle. The function of tanycytes in the sunfish is not known. The association of tanycytes with both the ventricles and blood vessels raises the possibility that they play some role in sampling the biochemical constituents of both compartments and communicating the information to neural elements. It is proposed that tanycytes react to the biochemical composition in the ventrile and plasma by increasing or decreasing nitric oxide synthesis and release, which in turn influence neuronal activity or cerebral blood flow. © 1993 Wiley-Liss, Inc.     

The effect of phenytoin on free fatty acid liberation and mononucleotide metabolism in transient ischemia

Ischemia gives rise to severe energy depletion and influx of Ca from the extracellular space, and it is suggested that increased intracellular Ca leads to the activation of phospholipase C and A, and to liberation of free fatty acids (FFA) in particular arachidonic acid. Phenytoin has been reported not only to maintain the intra- and extracellular cation balance but blockade the Ca channel. The purpose of the present study is to investigate the effect of phenytoin on the liberation of FFA, energy metabolism and mononucleotide metabolism in ischemic brain. Male Wistar rats were subjected to global cerebral ischemia induced by the occlusion of basilar and bilateral common carotid arteries. The brains were frozen in situ by the funnel technique after 5 or 30 min of ischemia or after 10, 30, or 60 min of recirculation following 30 min of ischemia. Purine and pyrimidine nucleotides, FFA, and glycolytic intermediates were measured by HPLC, GLC, and fluoro-enzymatic method. In non-treated rats, ATP reached a nadir after 5 and 30 min of ischemia. Phenytoin significantly attenuated ATP depletion after 5 and 30 min of ischemia. And also E.C. is higher in phenytoin treated rats than in non-treated rats in ischemia. After 60 min of recirculation, ATP recovered to 1.93 +/- 0.02 mumol (72.3% of pre-ischemia) in treated rats but 1.60 +/- 0.07 mumol/g (60% of pre-ischemia) in non treated rats. In E.C., there are significant differences between non-treated and treated rats after 10 and 30 min of recirculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for differential function of neuronal and glial cell in protein metabolism and amino acid transport

Amino acid incorporation in neuronal and glial cells has been investigated in several laboratories employing bulk-separation techniques to obtain cell-enriched fractions. The relative rates of incorporation into the proteins of both cell types vary substantially with the method of isotope administration. Through the use of single-pulse perfusions with a duration of 30–40 sec the early time course of labeling has been studied. The difference between neuronal and glial cells with respect to indicating cell interactions will be discussed. The in vitro amino acid incorporation has been measured in neurons and glia after slice incubation. In material from animals developing experimental allergic encephalitis the rate of ³H-leucine incorporation more than doubles in the unfractionated brain. Glial cells increased their rate of incorporation by approximately 400%under the same conditions. The involvement of specific proteins in the cells and organelles has been studied by gel electrophoresis. The high uptake capacity of glial cells for certain amino acids with possible transmitter functions has been further characterized. The release of these substances is measured in a superfusion system where beds of cells, preloaded with the labeled substance, are used. High potassium pulses stimulate release of, for example GABA in both neuronal and glial cells.     

Glucagon injected in the lateral hypothalamus stimulates sympathetic activity and suppresses monoamine metabolism

Glucagon injected in the lateral hypothalamus stimulates sympathetic activity and suppresses monoamine metabolism. The central hypothesis underlying this study is that there is a reciprocal relationship between food intake and sympathetic activity to IBAT. This hypothesis was tested by using intrahypothalamic microinjections of glucagon, a peptide that has been reported to decrease food intake. Sympathetic nerve activity to interscapular brown adipose tissue (IBAT) was measured as electrophysiological discharges of sympathetic nerves to IBAT. The microinjection of glucagon into the lateral hypothalamus (LH) increased sympathetic nerve activity by + 103.8 ± 35.0% (mean±S.E.M.) from pre-injection basal level by 30 min after injection. There was a gradual return to baseline. Micro-injection of glucagon into the LH depressed food intake. Monoamine metabolism was measured by using a microdialysis probe attached to a guide cannula for microinjection of glucagon into the LH. After microinjection of glucagon, the dialysates were collected over 30 min intervals and assayed for norepinephrine (NE), serotonin (5-HT), dopamine (DA) and their metabolites (3-methoxy-4-hydroxyphenylglyucol (MHPG); 5-hydroxyindole-3-acetic acid (5-HIAA); and 3,4-dihydroxyphenylacetic acid (DOPAC). Glucagon suppressed both NE and MHPG concentrations in the lateral hypothalamus (LH), and the concentration of DOPAC was also decreased. There was no change of 5-HT concentration but 5-HIAA levels were reduced by glucagon treatment. These data show that glucagon injected in the LH stimulates sympathetic activity and suggest that this may have occurred by suppression of norepinephrine, dopamine and serotonin turnover in the LH of freely moving rats. These data support the hypothesis of a reciprocal relationship between food intake and sympathetic activity.     

Comparison of electrolytic and ibotenic acid lesions in the lateral hypothalamus

Electrolytic lesions in the lateral hypothalamus (LH) seriously affect ingestive behavior and sensorimotor functions in the rat. We here report that bilateral infusions of the neurotoxin, ibotenic acid (IBO) in the LH yield a decrease in body weight, but not to the same extent as electrolytic lesions. The sensorimotor impairments were most severe after electrolytic lesions. When tested in a residential maze on days 5-7 and 18-20 after surgery, both lesioned groups showed no lack of motivation to seek food and water. Histological examination of the LH following IBO exposure revealed extensive degeneration of neuronal cell bodies with little evidence of non-specific damage. Biochemical analysis of the rostral forebrain content of norepinephrine (NE) and serotonin (5-HT) revealed that the fibers passing through the LH remained largely intact in the IBO treated rats. The results suggest that the observed aphagia and adipsia is not due to a lack of motivation, but rather reflects changes in the process which operate to initiate eating and drinking. Furthermore, selective neuronal degeneration induced the same behavioral changes as the electrolytic ones, though not to the same extent.     

Re-exposure to endotoxin induces differential cytokine gene expression in the rat hypothalamus and spleen

This study was designed to investigate whether the pattern of hypothalamic and splenic cytokine expression induced by peripheral administration of a bacterial lipopolysaccharide (LPS) is affected by prior exposure to LPS derived from another bacterial strain. Injection of LPS from Salmonella enteritidis (LPS₂) alone resulted in increased hypothalamic gene expression of IL-1β, IL-6, TNFα, IL-1ra and IL-10. However, pre-exposure to LPS derived from Escherichia coli (LPS₁) 3 weeks before, significantly attenuated hypothalamic IL-1ra, IL-6 and IL-10 expression. IL-1β expression also tended to be lower. This pattern contrasted with the robust cytokine expression in the spleen of LPS₂-treated rats previously exposed to LPS₁, since pre-treatment with endotoxin resulted in a significantly greater response of IL-1β and IL-1ra to LPS₂. Expression of TNFα and IL-10 also tended to be higher. Pre-treatment with LPS₁ did not significantly affect the marked increase in corticosterone and adrenaline blood levels induced by LPS₂. Thus, while endotoxin pre-exposure seemed not to induce a “tolerant” state in the periphery as judged by the immune and endocrine parameters evaluated upon re-stimulation, expression of four of the six cytokines measured was decreased in the hypothalamus. This is the first demonstration that endotoxin priming can differentially affect cytokine expression in the central nervous system and peripheral tissues when a host is confronted with a second, acute, pro-inflammatory stimulus. These results may provide new evidence for the involvement of cytokine pathways in the central nervous system in modulating peripheral inflammation and mediating cognitive and behavioural alterations during inflammatory diseases.