Correlation between gamma glutamyltransferase fractions and bone quality.

Gamma-glutamyltransferase (GGT) has been recently identified as a bone-resorbing factor. The aim of this study was to investigate the association between plasma GGT fractions levels and bone quality. Plasma GGT fractions were analysed by gel-filtration chromatography. Bone quality was established quantitatively by two micro-CT derived microarchitectural parameters: the BV/TV (mineralised bone volume/total volume), and the SMI (structure model index) that describes the rod-like (low resistant) or plate-like (high-resistant) shape of bone trabeculae. We enrolled 93 patients hospitalised for elective total hip replacement (group Arthrosis, n=46) or for proximal femoral fracture (group Fracture, n=47). Patients within the first quartile of BV/TV (Q1, osteoporotic patients, n=6) showed higher levels of b-GGT fraction [median (min-max): 3.37 (1.42­6.81)] compared to patients with normal bone density (fourth quartile Q4, n=10; 1.40 (0.83­4.36); p=0.0393]. Also, according to SMI, b-GGT value was higher in the subgroup with bone fragility [Q1, n=8: 1.36 (0.43­4.36); Q4, n=8: 5.10 (1.4 ­7.60); p=0.0117]. In conclusion, patients characterised by fragile bone structure showed specifically higher levels of plasma b-GGT activity thus suggesting fractional GGT analysis as a possible biomarker in the diagnosis of osteoporosis.

The antilipolytic agent 3,5-dimethylpyrazole inhibits insulin release in response to both nutrient secretagogues and cyclic adenosine monophosphate agonists in isolated rat islets.

This study intended to test the hypothesis that intracellular lipolysis in the pancreatic beta cells is implicated in the regulation of insulin secretion stimulated by nutrient secretagogues or cyclic adenosine monophosphate (cAMP) agonists. Indeed, although lipid signaling molecules were repeatedly reported to influence beta-cell function, the contribution of intracellular triglycerides to the generation of these molecules has remained elusive. Thus, we have studied insulin secretion of isolated rat pancreatic islets in response to various secretagogues in the presence or absence of 3,5-dimethylpyrazole (DMP), a water-soluble and highly effective antilipolytic agent, as previously shown in vivo. In vitro exposure of islets to DMP resulted in an inhibition (by approximately 50%) of the insulin release stimulated not only by high glucose, but also by another nutrient secretagogue, 2-ketoisocaproate, as well as the cAMP agonists 3-isobutyl-1-methylxanthine and glucagon. The inhibitory effect of DMP, which was not due to alteration of islet glucose oxidation, could be reversed upon addition of sn-1,2-dioctanoylglycerol, a synthetic diglyceride, which activates protein kinase C. The results provide direct pharmacologic evidence supporting the concept that endogenous beta-cell lipolysis plays an important role in the generation of lipid signaling molecules involved in the control of insulin secretion in response to both fuel stimuli and cAMP agonists.

Dexamethasone-induced insulin resistance and pancreatic adaptive response in aging rats are not modified by oral vanadyl sulfate treatment.

OBJECTIVE: To explore the adaptive response of the endocrine pancreas in vivo and in vitro and the possible beneficial effect of the insulino-mimetic agent vanadyl sulfate (VOSO(4)), using glucocorticoid treatment to increase insulin resistance, in aging rats. DESIGN AND METHODS: Dexamethasone (Dex) (0.13 mg/kg b.w.) was administered daily for 13 days to 3- and 18-month old Sprague-Dawley rats and oral VOSO(4) was given from the 5th day. Plasma glucose, insulin and free fatty acids (FFA) concentrations were measured during these treatments and the insulin secretory response of the isolated perfused pancreas was assessed at the end of the experiment. RESULTS AND CONCLUSIONS: In both young and aging rats, particularly in the latter, hyperinsulinemia and increased in vitro insulin responsiveness to glucose were observed in response to Dex treatment, concomitant with an increase in plasma FFA concentrations. Thus, in glucocorticoid-treated animals, the beta-cell adaptive response occurred in both age groups and could possibly be mediated by increased circulating FFA; however, it was insufficient to prevent hyperglycemia in 60% of aging animals. Oral VOSO(4) administration failed to correct Dex-induced alterations in glucose and lipid metabolism, although it influenced in vitro beta-cell responsiveness to stimuli in aging rats.

Effects of tryptophan load on amino acid metabolism in type 1 diabetic patients.

Tolerance to an oral tryptophan load (50 mg/kg body weight) was evaluated in a group of 15 insulin-dependent diabetic patients of both sexes in poor metabolic control. Tryptophan was measured fluorometrically, and the plasma levels of the other physiological amino acids were determined by HPLC. The ratio of the plasma concentration of each large neutral amino acid (LNAA) to the sum of the others was calculated to serve as an index for the competitive transport of these amino acids into the brain. The results show that post-loading plasma tryptophan levels in diabetic patients increased less than in healthy controls, suggesting enhanced liver catabolism of this amino acid (as reported for diabetic animals). Small changes were observed in the post-loading plasma concentrations of other amino acids. Therefore, the increment in the tryptophan/LNAA ratio in controls (basal, 0.12 +/- 0.01; 120 min after the load, 0.89 +/- 0.04; 240 min, 0.51 +/- 0.03) was greatly attenuated in diabetic patients (basal, 0.11 +/- 0.01, NS; 120 min, 0.46 +/- 0.04, p < 0.01; 240 min, 0.31 +/- 0.04, p < 0.01). Post-loading excursions in some other ratios were slightly larger in control than diabetic subjects. These differences, which may occur to a lesser extent after a protein-rich meal, could modify the availability of precursor amino acids to the brain for synthesis of neurotransmitters. Thus, as happens in certain animal species, an impairment of the post-absorptive accumulation of tryptophan and serotonin in the brain may occur in diabetic patients as a result of altered metabolic disposal of tryptophan.

A new method for the investigation of endocrine-regulated autophagy and protein degradation in rat liver.

The effect of the antilipolytic agent 3,5-dimethylpyrazole (DMP) on liver autophagy and protein degradation was studied on male young adult rats (200 g body wt) of the Sprague-Dawley strain by electron microscopy and short-term single-pass liver perfusion and HPLC amino acid assay in the perfusate. Treatment with DMP (12 mg/kg body wt) enlarged the lysosomal-autophagic compartment in liver cells in 30 min and increased significantly the concentrations of valine and total amino acid in blood plasma (taken at sacrifice) and valine concentration in the liver perfusate in 60 min. These effects of DMP stimulating liver were secondary to the metabolic and endocrine effects of the drug (which caused a decrease in FFA, glucose, and insulin and an increase in glucagon and corticosterone plasma levels with a shorter latency, about 15 min). The effects of DMP were compared to those of other treatments inducing liver autophagy and protein degradation in vivo. Alterations after DMP or glucagon injections were similar, but they were larger and lasted for a longer time with DMP administration. Treatment with vinblastine or chloroquine enlarged the lysosomal-autophagic compartment without increasing protein breakdown.

[The enzymatic activity of adenosine metabolism in the rat heart. The effects of cardiac hypertrophy]. / Attività degli enzimi del metabolismo dell'adenosina nel cuore di ratto. Effetti dell'ipertrofia cardiaca.

The transmural distribution of the adenosine metabolizing enzyme activities 5'nucleotidase (5'N) and adenosine deaminase (ADA) across the left ventricular wall was explored in rats with heart hypertrophy induced by aortocaval shunt (FAC), or coarctation of the abdominal aorta (S) or by tireotoxicosis (T). FAC caused largest heart hypertrophy with a very short latency (1 day: + 0%, 3.5 days: + 30%, 7 days: + 41%, 21 days: + 49%). A 30% increase in the left ventricle free wall was observed after 3.5, 14 or 28 days of treatment with FAC, T or S respectively. Different changes in enzyme levels and alteration of transmural distribution profiles were observed with the different types of heart hypertrophy. Level of 5'N was decreased with FAC, did not change with T and increased with S; levels of ADA were always increased. The pattern of the transmural distribution of 5'N was affected by S but not by T and FAC, whereas distribution of ADA was not affected by S and T and was altered by FAC transiently.

Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples.

An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38 degrees C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3-50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 +/- 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.

Exploring the mechanisms of in vivo regulation of liver protein breakdown in the rat by the use of the new antilipolytic-agent model.

In this study, we explored the changes in the rate of protein degradation in liver cells in vivo, using a method based on the physiological stimulation of liver autophagy. Male albino rats 1, 2, 6, 12 and 24 months old were fasted overnight, and then received an injection of the antilipolytic agent 3,5-dimethylpyrazole (DMP) to evoke a sudden shortage of lipid fuel. A comparison was made with the in vivo effects of glucagon by giving the 2-month-old group an intraperitoneal injection of this hormone. Samples of liver were taken after 0, 15, 20, 30, 60 and 150 min and processed for electron microscopy, and groups of rats were subjected to short-term single pass liver perfusion. Results show that in the younger age-groups, the DMP stimulation of liver autophagy and amino acid release is highly significant, and compares favourably with the glucagon model of induction of the autophagic process. With older rats, an age-related longer time-lag of the autophagic response and a decrease in the effect of DMP were observed. In conclusion, hormones may activate autophagy, whereas levels of plasma amino acids may tune down the process to adjust the availability of the substrate to tissue needs.

Protection by 3-aminobenzamide and nicotinamide against streptozotocin-induced beta-cell toxicity in vivo and in vitro.

To increase knowledge about the defense mechanisms of pancreatic beta-cells exposed to acute injury, the patterns of protection exerted by 3-aminobenzamide and nicotinamide against the effects of streptozotocin have been studied in vivo and in vitro. It was found that 3-aminobenzamide, a strong inhibitor of the NAD-consuming nuclear enzyme poly(ADP-ribose) synthetase, is maximally effective against streptozotocin-induced diabetes in the rat when administered 45-60 min after the beta-cytotoxic agent, unlike nicotinamide, which exerts best protection when given very close to streptozotocin. A partial protection is still afforded by 3-aminobenzamide administered as long as 120 minutes after streptozotocin. In isolated islets, each protective agent, if added to the incubation medium 20 or even 40 minutes after the exposure of the islets to streptozotocin, is able to partially prevent the effects of the damaging drug on glucose-stimulated insulin release. However, best protection in vitro is obtained when either 3-aminobenzamide or nicotinamide is added simultaneously with the toxic agent. Our results support the concept that the reversibility of streptozotocin-induced acute damage in beta-cells depends on the preservation of intracellular NAD pools during critical time intervals. This can be achieved by two different partially overwhelming mechanisms: a) early stimulation of NAD biosynthesis (prevailing effect of nicotinamide) and b) strong inhibition of poly ADP-ribosylation activity (main effect of 3-aminobenzamide).

The regional distribution of adenosine-regulating enzymes in the left and right ventricle walls of control and hypertrophic heart.

The transmural distribution of the adenosine-generating enzyme 5'-nucleotidase (5'N) and of the adenosine-degrading enzymes adenosine deaminase (ADA), AMP deaminase (AMP-D) and adenosine kinase (Ado-K) were determined across the walls of left and right ventricles of control and hypertrophic rat hearts. The enzyme distribution across the left ventricle wall (but not across the right wall) of normal hearts was not uniform: 5'N activity shows its highest levels in the subepicardial and in the subendocardial regions, whereas all the other enzyme activities show their lowest levels. A similar pattern of transmural distribution was also detected in other mammalian species (ox and pig). In the experimental cardiac hypertrophy, caused by two different types of chronic cardiac overload, the levels and the profiles of transmural distribution of 5'N and ADA enzyme activities may significantly change across the rat left ventricle wall.

Effects of metformin therapy on plasma amino acid pattern in patients with maturity-onset diabetes.

Plasma amino acid concentrations and metformin levels were measured by high-performance liquid chromatography in a group of diabetic patients on therapy with the biguanide drug. Diabetic patients treated with metformin showed higher concentrations of plasma glutamate, asparagine, alanine, methionine and tryptophan, and lower levels of ornithine in comparison with normal subjects, and higher levels of alanine when compared to diet-treated diabetic patients. No correlation emerged between amino acid concentrations and metformin dose or plasma concentration. It is concluded that besides the various metabolic abnormalities that are known to occur during metformin treatment, patients on therapy with metformin also show multiple changes of plasma amino acid pattern.

Metabolic heterogeneity of the muscle tissue: transmural distribution of glucose metabolizing enzymes across the left ventricular wall of control and hypertrophic rat heart.

Mammalian cardiac muscle is a remarkably heterogeneous tissue, as judged from enzyme analysis of tissue from different myocardial layers of the left ventricle free-wall. Its diversity results from a spectrum of fibres with different metabolic properties and different location across the wall, which may be especially suited to the local range of functional demands.

Transmural distribution of glucose metabolizing enzymes across the left and the right ventricle heart walls in three different mammalian species.

The transmural distribution of five glucose metabolizing enzymes (hexokinase; glucose-6-phosphate dehydrogenase; phosphofructokinase; aldolase; and lactate dehydrogenase) were explored in the left and in the right ventricle wall of rat, ox and pig hearts. The levels of most of these enzyme activities were different in the different animal species and (within the same species) in the two ventricles. Most of these enzyme activities were found to be non-uniformly distributed across the left (but not across the right) ventricle wall. Differences in the transmural distribution of enzyme activities were detected among the three examined mammalian species.