Ricardo R. Rodríguez

Modified oscillation behavior and decreased d-3-hydroxybutyrate dehydrogenase activity in diabetic rat liver mitochondria☆

Abstract One month after induction of diabetes in adult white rats with streptozotocin or 4–10 months after its induction by pancreatectomy (in every case glycemia was over 3 g/liter), the following alterations were observed in liver mitochondria: (a) a decrease of amplitude and an increase of the damping factor of volume oscillations induced by potassium ions and valinomycin; (b) a 50% decrease of d -3-hydroxybutyrate dehydrogenase (HBD) activity in mitochondria disrupted by repeated freeze-thawing; (c) a similar decrease in the rate of d -3-hydroxybutyrate oxidation by intact mitochondria; (d) a significant increase of cytochrome oxidase activity and cytochrome aa 3 content. Measurement of succinate dehydrogenase and NADH dehydrogenase activity, the cytochrome b , c 1 , and c content, and the P:O ratio for mitochondria oxidizing d -3-hydroxybutyrate did not reveal significant differences between control and diabetic rat mitochondria. In the streptozotocin-injected rats, the variation of HBD activity and the modification of the mitochondrial oscillation pattern were time-dependent phenomena, both effects reaching their maximal expression about 1 month after the onset of diabetes. The variation of HBD activity followed a biphasic course, since it rose to above the control level during the first 2 weeks of diabetes, then fell progressively to about half the control value after the third week. Treatment of diabetic rats with NPH insulin (5 IU twice daily, for 3 days, reinforced by the same dose 45 min before sacrifice) restored the mitochondrial oscillation pattern, HBD activity, and rate of d -3-hydroxybutyrate oxidation by intact mitochondria to their normal values.

Melatonin restores endothelium-dependent relaxation in aortic rings of pancreatectomized rats.

Abstract:  In rats turned hyperglycemic by a subtotal pancreatectomy, a decreased relaxation response of aortic rings to acetylcholine (ACh) was found; this effect was amplified by preincubation in a high glucose medium (44 mmol/L). The relaxation response to ACh did not occur in endothelium‐denuded rings or after the aortic rings were exposed to l‐nitro‐arginine methyl ester [l‐NAME, a nitric oxide (NO) synthase inhibitor]. Incubation with the NO donor sodium nitroprusside (SNP) restored the impaired relaxation response seen in endothelium‐denuded or l‐NAME‐treated aortic rings. Pancreatectomy decreased the vasorelaxation of aortic rings caused by SNP. Only in pancreatectomized rats, incubation in a high glucose medium impaired the relaxation effect of SNP. To assess whether melatonin preincubation reversed the impaired relaxation response to ACh (intact endothelium aortic rings) or to SNP (endothelium‐denuded or l‐NAME‐treated rings) in hyperglycemic rats, cumulative dose–response curves were performed in the presence of 10−5 mol/L melatonin. Melatonin preincubation did not modify ACh‐induced relaxation of aortic rings in a normal glucose concentration but was highly effective in preventing the impairment of relaxation caused by a high glucose solution. Melatonin was also effective in restoring the impaired SNP‐induced vasorelaxation seen in endothelium‐denuded or l‐NAME‐treated aortic rings from hyperglycemic rats. The results further support the improvement by melatonin of the endothelial‐mediated relaxation in blood vessels of diabetic rats.

Adrenocorticotrophic hormone, cortisol and catecholamine concentrations during insulin hypoglycaemia in dogs anaesthetized with thiopentone

Glucose homeostasis is maintained by complex neuroendocrine control mechanisms. Increases in plasma concentrations of various glucose-raising hormones such as glucagon, catecholamines, adrenocorticotrophic hormone (ACTH), and cortisol are observed under certain conditions associated with stress (haemorrhage and hypoglycaemia). The purpose of this study was to determine the effect of thiopentone anaesthesia on the cathecholamine, ACTH and cortisol response to insulin hypoglycaemia in dogs. Blood sugar (BS), plasma cathecholamine, and ACTH, and serum cortisol concentrations were measured during the course of (1) an intravenous insulin test (ITT) and (2) an ACTH test in conscious and in anaesthetized fasted dogs. During the ITT, the anaesthetized dogs showed a moderate resistance, compared with conscious dogs, to the hypoglycaemic action induced by insulin (blood sugar concentration 30 min after insulin injection: 2.91 ± 0.25 vs 1.93 ± 0.12 mM · L−1; P < 0.01). In addition, decreased epinephrine (220 ± 27 vs 332 ± 32 pg · ml−1 ACTH (65 ± 6 vs 90 ± 5 pg · ml−1) and cortisol (4.48 ± 0.3 vs 6.25 ± 0.5 μg · ml−1) concentrations were detected 60 min after insulin injection (P < 0.01). The norepinephrine response to hypoglycaemia was not altered by anaesthesia (273 ± 33 vs 325 ± 25 pg · ml−1). Anaesthetized dogs showed a decreased cortisol response to ACTH at 45 min (5.68 ± 0.54 vs 8.87 ± 0.47 μg · ml−1) when compared with control dogs (P < 0.001). Haemodynamic variables during anaesthesia showed little changes (P < NS); while respiratory rate was altered (P < 0.01 between 60 and 105 min). Arterial pH was decreased (7.29 ± 0.03 vs 7.36 ± 0.04; P < 0.05) and PaCO2 was increased (6.8 ± 0.3 vs 5.2 ± 0.3; P < 0.01) at 30 min from induction of anaesthesia but little change was seen after the beginning of the ITT and ACTH tests. We conclude that thiopentone anaesthesia provokes a moderate resistance to the hypoglycaemic action of insulin. This does not appear to be related to increases in plasma concentrations of cathecholamines, cortisol or ACTH. Since the hyperglycaemic effects of cathecholamines and glucagon are synergistic it is possible that glucagon plays an important role in the altered blood sugar response to insulin administration.RésuméL’homéostase du glucose sanguin est maintenue par des mécanismes de contrôle neuroendocriniens complexes. On observe une augmentation des concentrations plasmatiques (ou sériques) de plusieurs hormones hyperglycémiantes telles que le glucagon, l’ACTH et le cortisol sous certaines conditions liées au stress chirurgical comme l’hémorragie et l’hypoglycémie. Le but principal de cette étude est de déterminer les effets de l’anesthésie au thiopental sur la réponse, à l’hypoglycémie insulinique, des catécholamines, de l’ACTH et du cortisol. On détermine la glycémie et les concentrations plasmatiques des catécholamines et de l’ACTH, et les niveaux sériques du cortisol pendant 1) le test de tolérance à l’insuline, 2) l’épreuve à l’ACTH chez des chiens éveillés et anesthésiés. Lorsqu’on compare les chiens anesthesias avec les chiens éveillés, on constate que l’injection iv d’insuline provoque sur des mesures aux 30 minutes une insulino-résistance (glycémie: 2,91 ± 0,25 vs 1,93 ± 0,12 mM · L−1; P < 0,01) et une baisse de concentration plasmatique mesurée aux 60 minutes de l’épinéphrine (220 ± 27 vs 332 ±32 pg · ml−1), de l’ACTH (65 ±6 vs 90 ± 5 pg · ml−1) et du cortisol (4,48 ± 0,3 vs 6,25 ± 0,5 μg · ml−1) (P < 0,01). La réponse de la norépinéphrine plasmatique n’est pas modifiée par l’anesthésie (273 ± 33 vs 325 ± 25 pg · ml−1). Quarantecinq minutes après l’injection iv d’ACTH, on constate chez les chiens anesthésiés une diminution de la réponse au cortisol (5,68 ± 0,54 vs 8,87 ± 0,47) comparativement aux contrôles (P < 0,001). Les paramètres hémodynamiques pendant l’anesthésie demeurent inchangés (P < NS). Par contre, la fréquence respiratoire change (P < 0,01 entre 60 et 105 min). Trente minutes après l’induction de l’anesthésie, le pH artériel diminue (7,29 ± 0,03 vs 7,36 ± 0,04: P < 0,05), la PaCO2 augmente (6,8 ± 0,3 vs 5,2 ± 0,3; P < 0,01). On note peu de changements après le début des test de tolérance au glucose et l’épreuve à l’ACTH. A partir de ces données on peut conclure que l’anesthésie au thiopental produit une insulino-résistance modérée. Cet effet ne résulte pas d’une augmentation plasmatique des catécholamines, de l’ACTH ou du cortisol. Au contraire, le niveau sanguin de ces hormones diminue. On énonce comme postulat l’intervention probable du glucagon dans les mécanismes de régulation aigus de la glycémie chez les chiens anesthésiés.

Blood sugar, serum insulin and serum non- esterified fatty acid levels during thiopentone anaesthesia in dogs

The purpose of this study was to determine the effect of thiopentone anaesthesia on glucose metabolism. Blood sugar (BS), serum immunoreactive insulin (IRI) and serum non-esterified fatty acid (NEFA) concentrations were measured during the course of (1) an intravenous glucose tolerance test (IVGTT), and (2) an intravenous insulin test (ITT), in conscious and anaesthetized fasted dogs. The IVGTTs were repeated in dogs under alpha-or beta-adrenergic blockade, induced by phentolamine or propranolol. During the IVGTT, the anaesthetized dogs showed glucose intolerance (blood sugar levels were higher than in the control group) and little serum IRI response to hyperglycaemia was detected. An attenuated initial decrease and a slower rebound of NEFA concentration was observed in anaesthetized animals than in controls. Phentolamine administration (5 mg · kg−1 iv) partly restored the IRI response without affecting the BS levels; propanolol (1 mg · kg−1 iv) had no effect. Anaesthetized dogs showed a moderate resistance to insulin induced hypoglycaemic action and a lack of serum NEFA response during counter-regulation of hypoglycaemia, while in conscious controls an intense rebound was observed. Hyperinsulinaemia after iv insulin administration was longer in anaesthetized dogs than in controls. The insulin distribution space was 78% of body weight and insulin t1/2 in blood group compared with 54% and 16 min, in controls. We conclude that thiopentone provokes disturbances in glucose and serum NEFA metabolisms and abolishes the serum IRI response to hyperglycaemia. These effects are influenced by extrapancreatic factors regulating serum IRI levels and by an alpha-adrenergic mechanism, via the inhibition of insulin secretion.RésuméCette étude porte sur les effets de l’anesthésie au thiopental sur la glycémie, l’insulinémie et la concentration des acides gras non-estérifiés (AGNE) sur des chiens en réponse à un test d’hyperglycémie provoquée et à une injection intraveineuse d’insuline. Le groupe contrôle se compose de chiens éveillés. Chez les chiens anesthésiés, on constate après le test d’hyperglycémie provoquée, une intolérance au glucose, une absence de réponse insulinique 5 min après l’injection du glucose (29 ± 15 μU · ml−1 comparativement à 85 ± 8 μU · ml−1 pour les contrôles) et une baisse moins importante des AGNE. Un bloc β- adrénergique au propanolol n’a pas modifié ces résultats. Cependant, l’administration de phentolamine (blocage α- adrénergique) rétablit partiellement la réponse insulinique sans modifier la glycémie. Chez le chien anesthesié, l’injection intraveineuse d’insuline entraîne une résistance aux actions hypoglycémiantes de l’insuline et lipolytique des hormones antiinsuliniques. L’insuline injectée lors du test d’hyperglycémie provoquée avait une demi-vie plus longue (27,5 min contre 16 min) et un volume de distribution plus grand (78% contre 54%) chez les chiens anesthésiés que chez les contrôles. On peut conclure que la réponse insulinique à l’hyperglycémie diminue chez les chiens anesthésiés au thiopental. En toute probabilité, on peut attribuer ces résultats à une association de facteurs extrapancréatiques auxquels s’ajoute un facteur α-adrénergique inhibitoire de la sécrétion insulinique.

Effect of estrogens on blood sugar, serum insulin and serum free fatty acids, and pancreas cytology in female dogs

SummaryWe analyzed the changes in blood sugar (BS), serum immunoreactive insulin (IRI), circulating free fatty acids (FFA) and pancreatic cytology caused by estrogenization at low pharmacological dosage in female dogs. Vehicle-injected and untreated controls (anestrus) were studied as well. Neither mean basal BS nor basal serum IRI was modified by the treatments, while the mean basal serum FFA value was raised. Glucose tolerance was not modified by the estrogens while glucosey-mean was significantly raised. Hyperglycemia was higher for a longer time in estrogenized animals compared to both controls, while the profiles of hyperinsulinemia coincided. In the estrogen-treated bitches, the pancreatic B-cells contained scarse brown-stained granules near their vascular pole, as shown by an immunochemical method. In the peripheral part of the pancreas, near the acini, some solitary, poorly β-granulated B-cells were present. During the IVGTT, serum FFA reached lower values for a longer time in the estrogenized bitches as compared to those found in both control groups. Insulin-induced hypoglycemia in the estrogenized animals coincided with the one evoked in the vehicle controls; in the semilog relationship of serum IRI and time,y-mean was lower than that observed in oil-injected controls, and insulin space was larger. The serum FFA levels of the estrogenized bitches, very high in the basal conditions, did not respond to insulin administration, and were above those found in untreated controls and also in vehicle-injected controls just at the beginning of the test. These results are discussed. We came to the conclusion that estrogenization causes some glucose intolerance in bitches while insulin sensitivity remains normal in the IVITT as studied measuring BS. The glucose intolerance is thought to be related to a reduction in glucose space and occurs despite the normality of the serum IRI response. The pancreas must have an intense secretory responsein vivo so as to maintain normal IRI activity despite degranulation of the islets of Langerhans and poor islet hypertrophy and neoformation. The serum FFA changes are thought to contribute towards the tendency to adiposity in these animals.

Offspring of streptozotocin diabetic rats: size changes in Langerhans islets with time after birth.

It has been demonstrated that, in the diabetic rat, pregnancy and lactation are severely altered: in this study, we have measured the size of Langerhans islets of rat pups, the offspring of experimental diabetic mothers and nondiabetic controls. Diabetes was induced through streptozotocin administration (dose, 60 mg/kg body wt.). This drug was injected in every animal; their blood sugar was measured 1 week later (Haemo-Glukotest, Boehringer Mannheim), and they were then separated into three groups according to their fasting blood sugar levels: (a) severe diabetics (above 16.5 mM/l); (b) mild diabetics (6.5-16.5 mM/l); and (c) nondiabetic normals. They received insulin therapy (2-4 I.U./day) as the mild diabetics exhibited a slightly higher than normal fasting blood sugar, and the diabetic ones, above 15 mM/l. The areas of Langerhans islets of pups were measured 1 and 5 days after parturition; pancreas sections were dyed (haematoxylin-eosin) and morphometry was then performed using a digitalized magnetic tabloid connected to a Zeiss Morphomat 30 (Kontron). On the first day after parturition, the pancreas section areas in pups from mildly and severely diabetic mothers were smaller than those in neonates from nondiabetic controls (P < 0.001). The areas in neonates from severely diabetic mothers showed a more intense decrease than those from mildly diabetic animals (P < 0.01). On day 5 after delivery, the areas of Langerhans islets in offspring from normal mothers decreased and those in pups from diabetic mothers tended to normalize (P < 0.01), particularly those from the severely sick group (P < 0.01). We conclude that after parturition the offspring is no longer exposed to the high blood sugar levels found in both diabetic groups of mothers, thereby no hyperinsulinemia is needed; as time elapses, then, the area of their Langerhans islets tends to normalization.

Effect of ovarian hormones upon liver mitochondrial function in diabetic rats

In the present study it is shown that streptozotocin (SZ)-induced chronic diabetes of female albino rats produced significant alterations in liver mitochondrial function after 30-35 days of diabetes. The disturbances were as follows: (1) a significant fall of the mean values of the respiratory control ratio and of state 3 of respiration using three substrates, 3-hydroxybutyrate, malate-glutamate and succinate, and (2) a significant increase of the mean damping factor of the oscillatory osmotic variations (with valinomycin as K+ ionophore and succinate as substrate). The same mitochondrial function parameters were analyzed for comparison in control non-diabetic rats (group N) and in the following groups of female rats with chronic diabetes: intact (group I), oophorectomized (6 days after the injection of SZ) (group O), and oophorectomized with restitution therapy of 17 beta-estradiol (from the operation until the day before killing) (group O + Eol). The O group showed significantly higher values of the respiratory control ratio and of state 3 of respiration and significantly lower damping factors than group I. The restitution treatment in the O + Eol group restored the mitochondrial functions assayed to values similar to those of group I. These data provide strong evidence that estrogens exert a negative effect at the molecular level upon impaired liver mitochondrial functions in SZ-induced diabetes.

Effects of short-term thyroxine treatment on pancreatic cytology and responses of blood sugar, serum insulin and serum free fatty acids to epinephrine infusion

The influence of short-term treatment with l-thyroxine on pancreatic histology and on the responses of glycemia, insulinemia and serum free fatty acids to a continuous l-epinephrine infusion in the absence or presence of alpha- and beta-adrenergic blockade was studied in male dogs. l-Epinephrine dosage: 0.06 microgram/kg body weight/min for 55 min. Two experimental groups were studied, one treated for 10 days with sodium l-thyroxine, one daily dose of 100 micrograms/kg body weight, the other of untreated controls. Three alternative treatments were applied to dogs of both groups: 1) no treatment; 2) alpha-adrenergic blockade with phentolamine (2.0 mg/kg body weight, 35 min before starting the l-epinephrine infusion); 3) beta-adrenergic blockade with propranolol (0.3 mg/kg body weight, 20 min before starting the l-epinephrine infusion). Body weight, rectal temperature, heart and respiratory rates were used as guidelines to assess experimental hyperthyroidism. Insulin immunocytolocalization was also studied in the Langerhans islets of T4-treated and control dogs. Body weight decreased and rectal temperature did not vary as a result of thyroxine administration which had no significant effect on respiratory and heart rate. The mean number of breaths from 0 to 120 min from the start of l-epinephrine infusion decreased in both T4-treated and control dogs submitted to propranolol blockage compared to non-blocked animals; phentolamine had no effect on the respiratory rate. Thyroxine treatment did not modify the number of heart beats, but phentolamine blockade had a different effect in T4-treated compared to control dogs whereas propranolol had similar effects in these two groups. Histological examination of the Langerhans islets of dogs submitted to short-term thyroxine treatment showed degranulation though no vacuolation. Most of the beta-granules contained in the B-cells of these islets were found near the cell membrane, thus forming a dark brown line after the immunochemical reaction. Since negative images of B-cell nuclei and vascular spaces were predominant in these specimens, the pancreas of T4-treated dogs presented a mesh structure. In dogs submitted to short-term thyroxine treatment, the hyperglycemic response to l-epinephrine was enhanced and prolonged as compared to untreated controls. In normal dogs, this response is mainly mediated by alpha-adrenergic receptors while beta-receptors hardly influence this response.(ABSTRACT TRUNCATED AT 400 WORDS)