Spontaneous Age-related Changes of Peripheral Nerves in Cattle: Morphological and Biochemical Studies.

Peripheral nerve function is significantly affected by ageing. During ageing process, multiple changes occur on tissue cells and extracellular matrix. The aim of this work was to study the ageing-associated changes of peripheral nerves in adult and old regularly slaughtered cattle compared with young calves, and correlate them to the features reported in humans and laboratory animals. Samples of axial dorsal metacarpal nerves from 44 cows were collected immediately after slaughtering. Each nerve was dissected and divided into two fragments: one used for morphological evaluation (n = 43) and the other one for biochemical analysis (n = 31). Axonal degeneration, demyelination, thickness of perineurium and endoneurium and increase of mast cells were the most important features detected. The mean amount of glycosaminoglycan quantitative content recorded in the samples increased with the age. Axonal degeneration, demyelination and thickness of endoneurium were positively and significantly correlated with biochemistry. The presence of changes affecting the different elements of the peripheral nerves, similar to that reported in humans and in laboratory species, the easy availability of the nerve tissue in this species, the considerable size of the samples and the life conditions more similar to humans than to laboratory animals, allows the authors to consider cattle as a potential good model for the comparative study of spontaneous ageing nerve lesions.

Synapse-independent and synapse-dependent apoptosis of cerebellar granule cells in postnatal rabbits occur at two subsequent but partly overlapping developmental stages.

It has long been known that cells in the external granular layer die during postnatal development of the cerebellum. More recent findings indicate that at certain developmental stages, cell death occurs upon activation of an apoptotic program. We show that cerebellar granule cells in rabbits undergo programmed cell death at two different stages of maturation. At postnatal day 5 (P5), granule cell precursors and pre-migratory granule cells in the external granular layer incorporate the S-phase markers 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine with a pattern that is dependent upon the interval between the administration of the two tracers. Within 12-24 h after proliferation a significant number of labeled cells show typical ultrastructural alterations of apoptosis. DNA electrophoresis and cleavage of poly-ADP-ribose polymerase confirm the activation of the apoptotic machinery. After Southern blotting and immunodetection, incorporated 5-bromo-2'-deoxyuridine is present at the level of low size DNA oligomers as soon as 12 h after cell division. Therefore, this apoptotic phase is intrinsic to external granular layer neurons and independent of synaptic interactions with targets.Apoptotic cells, although fewer in number, are detected also in the internal granular layer and tend to increase from P5 to P10. It seems unlikely that these cells undergo DNA fragmentation in the external granular layer and subsequently migrate to their final destination, considering the data on cell cycle kinetics and the rapid tissue clearance by the glia. Parallel fiber-Purkinje spine synapses are already present in the molecular layer at P5. Therefore, the post-migratory granule cells likely undergo apoptosis as a failure to make proper synaptic contacts in the forming molecular layer. We conclude that the massive apoptosis of pre-migratory cells likely has a role in regulating the size of this rapidly expanding population of pre-mitotic neurons. The less tumultuous cell death of post-mitotic granule cells in the internal granular layer appears to be linked to the formation of the mature synaptic circuitry of the developing cerebellar cortex.

Acute methylprednisolone administration induces a transient alteration of glucose tolerance and pyruvate dehydrogenase in humans.

BACKGROUND: Glucocorticoid administration induces alteration of glucose tolerance, and impairment of glucose oxidation may contribute to glucocorticoid-induced derangement of glucose metabolism. We investigated glucose tolerance following methylprednisolone administration in humans. In the same model, we evaluated pyruvate dehydrogenase (PDH), the rate limiting enzyme of glucose oxidation, in peripheral blood mononuclear cells. MATERIALS AND METHODS: Methylprednisolone (2 x 40 mg, iv, one dose every 12 h) was administered to six healthy volunteers. Glucose tolerance was evaluated through an oral glucose tolerance test (oGTT, 75 g glucose) at least a week before and after drug administration (2 and 24 h post-drug). To assess modifications of lipid metabolism circulating free fatty acids (FFA) and glycerol were measured, during fasting and oGTT. The active form of PDH (PDHa) was evaluated in peripheral blood mononuclear cells, both as ex vivo activity and as in vitro response to insulin (30 pmol l-1). RESULTS: Methylprednisolone induced an alteration of glucose tolerance 2 h after its administration. Such alteration was completely reversed at 24 h. Alteration of glucose tolerance was accompanied by decreased ex vivo PDHa activity. PDH responsiveness to insulin in vitro was also impaired. Circulating FFA were unmodified, but decreased glycerol levels suggested a slight inhibition of lipolysis. CONCLUSIONS: Acute methylprednisolone administration in humans induced a transient decrease of glucose tolerance 2 h after drug administration, accompanied by hyperinsulinaemia, inhibition of ex vivo PDH activity and its response to insulin in vitro. These alterations were completely abolished at 24 h, suggesting that methylprednisolone can be safely administered acutely. Furthermore, methylprednisolone induced only minor modifications of circulating FFA and glycerol, indicating minimal impact on lipid metabolism.

Molecular effects of sulphonylurea agents in circulating lymphocytes of patients with non-insulin-dependent diabetes mellitus.

AIMS: In circulating lymphocytes of NIDDM patients pyruvate dehydrogenase (PDH), the major determinant in glucose consumption through oxidative pathways, is poorly active. The aim of this study is to examine whether sulphonylurea drug treatment revives PDH activity in circulating lymphocytes from NIDDM patients. METHODS: Twenty normal-weight individuals with NIDDM were enrolled in this study. They had maintained their glycaemic levels close to normal by means of a restricted diet that had no longer been successful in the proceeding 2 months. The treatment protocol consisted in 160 mg gliclazide daily for 5 weeks. Twenty healthy subjects, matched for age, body mass index and gender, were enrolled as a control group. Patients, before and after treatment, as well as controls were tested for PDH activity in their circulating lymphocytes. Nine other untreated patients and nine healthy subjects, with the above mentioned characteristics, were recruited for the assay of PDH activity in their circulating lymphocytes before and after exposure, in vitro, to gliclazide, to insulin, and to gliclazide and insulin in combination. RESULTS: In gliclazide-treated NIDDM patients, PDH activity in circulating lymphocytes recovered. In vitro, in circulating lymphocytes of untreated patients and controls insulin at 5 microU ml(-1) was ineffective and highly effective, respectively, in raising enzyme activity; gliclazide at 10 ng ml(-1) was ineffective on PDH in both groups, but in combination with insulin at 5 microU ml(-1) in both groups PDH was as active as in cells of controls exposed to insulin only. In cells of controls, gliclazide alone at 25-50 ng ml(-1) caused enzyme activation, whereas above 50 ng ml(-1) it caused inhibition; in cells of patients below 50 ng ml(-1) it had no effects, but at 50 ng ml(-1) and above raised enzyme activity to the basal level of controls. CONCLUSIONS: This study suggests that free gliclazide concentrations determine recovery of PDH activity in circulating lymphocytes of treated patients through drug-mediated enhanced insulin control over PDH or through the drug alone.

Insulin resistance in obese subjects and newly diagnosed NIDDM patients and derangements of pyruvate dehydrogenase in their circulating lymphocytes.

BACKGROUND: In circulating lymphocytes of individuals with insulin resistance and overt hyperglycaemia (NIDDM patients), alterations, affecting pyruvate dehydrogenase (PDH), the key enzyme in glucose oxidative breakdown, have been observed. They include below normal enzyme activity and, in vitro, no enzyme response to insulin at low physiological levels (5 microU/ml) as well as activation up to the basal values of controls with insulin at high physiological levels (50 microU/ml), instead of activation and inhibition respectively, as in controls. OBJECTIVE: To investigate whether these alterations characterize circulating lymphocytes of individuals with insulin resistance in whom derangements of glucose homeostasis are absent (obese subjects with normal glucose tolerance), or present but still controllable (nonobese and obese newly diagnosed NIDDM patients on an appropriate diet). SUBJECTS: Thirty obese subjects (BMI 36 +/- 3) responding normally to an oral glucose tolerance (OGT) test; 60 newly diagnosed NIDDM patients (30 nonobese, BMI 22 +/- 4 and 30 obese, BMI 38 +/- 2); 30 nonobese (BMI 21 +/- 5) and nondiabetic subjects, with no family history for NIDDM, served as controls. METHODS: Evaluation of PDH activity in circulating lymphocytes before and after exposure to insulin at 5 and 50 microU/ml, and of clinical parameters before and during an OGT test. RESULTS: 1) in circulating lymphocytes of obese nondiabetic subjects as well as obese and nonobese newly diagnosed NIDDM patients, PDH activity was significantly below normal. In vitro, enzyme response to insulin at 5 microU/ml was reduced in nonobese NIDDM patients with respect to controls, and absent in obese nondiabetic subjects and obese NIDDM patients. Enzyme response to insulin at 50 microU/ml was reversed in all individuals, which allowed enzyme activity to recover up to the basal level of controls. 2) In NIDDM patients and obese nondiabetic subjects, undergoing an OGT test, the area under the glycaemic curve (g-AUC) was as expected; the area under the insulinaemic curve (i-AUC) was increased in both groups with respect to controls, but significantly only in the latter. CONCLUSION: In individuals with insulin resistance PDH activity in their circulating lymphocytes rises up to basal levels of controls, only if these cells are exposed to insulin at high physiological concentrations, and g-AUC is normal only in those subjects who have significantly increased i-AUC. This suggests that with insulin at sufficiently high concentrations both parameters can be corrected. We conclude that the derangements responsible for the alterations of the two parameters share common features and thus the described PDH alterations in circulating lymphocytes reflect systemic insulin resistance whether accompanied by hyperglycaemia or not.

G proteins and regulation of pyruvate dehydrogenase activity by insulin in human circulating lymphocytes.

Pertussis toxin (PT) catalyzes ADP-ribosylation of G protein alpha subunits, thus preventing their role as transducers of external signals targeting metabolic pathways. In vitro, in human circulating lymphocytes insulin at physiological concentrations (5 microU/ml) determines sharp activation of pyruvate dehydrogenase (PDH), the rate limiting enzyme in glucose oxidative breakdown. This study evaluates whether the above-described effects of insulin over PDH are mediated through G proteins. Human circulating lymphocytes (six samples from different donors) were exposed to insulin (5 microU/ml), PT (1-2 micrograms/ml) or PT-9K, a mutated PT void of catalytic activity (1-10 micrograms/ml), and to insulin in combination with the two toxins, and then assessed for PDH activity. Plasma membranes from cells incubated with and without PT or PT-9K were subjected to ADP-ribosylation in the presence of [32P] NAD+ and activated PT. In circulating lymphocytes exposed to PT alone, or in combination with insulin, PDH activity falls significantly below basal values (P < 0.001); PT-9K instead has no effect on basal or on insulin-stimulated PDH activity. ADP-ribosylation of a plasma membrane component with apparent molecular mass (42 kDa) comparable to that of the Gi (inhibitory) protein alpha subunit takes place in cells exposed to PT but not in those exposed to PT-9K. In human circulating lymphocytes Gi proteins or Gi protein-like components appear to be involved in preserving basal PDH activity as well as in the mechanism by which insulin exerts its control over PDH.

Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina.

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin-Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.

Effect of sulfonylurea agents on pyruvate dehydrogenase activity in circulating lymphocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM).

In circulating lymphocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM) subnormal pyruvate dehydrogenase (PDH) activity returns to normal following patient treatment with sulfonylurea (gliclazide, 80 mg twice daily/5 weeks). Moreover, in vitro in cells from diabetic patients exposed to insulin at 50 microU/mL PDH activation also occurs; in cells of controls the same happens for insulin at 5 microU/mL, whereas at 50 microU/mL inhibition takes place. Therefore, the low PDH activity in cells of NIDDM patients might be caused by defective insulin control on the enzyme and its recovery in gliclazide-treated patients by drug-mediated removal of the defect. The validity of the hypothesis was verified in this study where cells of NIDDM patients before and after gliclazide treatment were exposed, in vitro, to insulin at 5 and 50 microU/mL and then tested for PDH activity. In such conditions, the profile of PDH behavior in treated patients was no longer comparable to that in untreated patients but closer to that in euglycemic controls, thus supporting the view that the recovery of PDH activity in NIDDM patients following gliclazide treatment might be the expression of an additional effect that the drug would have in these patients, aimed to renew cell responsiveness to insulin.

Levels of carnitine and glycogen in rabbit tissues during development.

This study attempts to add further light on the development of metabolic pathways in mammalians from fetal to post-natal life, by examining ongoing modifications of carnitine (in terms of total acid soluble, short chain esterified and free carnitine) and glycogen levels in the liver, heart, muscle and brain of rabbit during development.