DI Rats Research Articles

Exogenous central angiotensin fails to stimulate a sodium appetite in diabetes insipidus Brattleboro rats

We investigated the effect of central administration of angiotensin II (AngII) on a specific salt appetite (SSA) in homozygous diabetes insipidus Brattleboro (DI) rats because this stimulus induces such a response in all other rat strains. DI rats have a deficiency in the synthesis of arginine vasopressin (AVP) and a reduced content of pituitary oxytocin (OT). They are characterized also by polyuria, polydipsia, and they seldom ingest high concentrations of NaCl solutions. We also tested if the appetite can be influenced by neurohypophyseal hormones especially oxytocin (OT) because it inhibits SSA in other animals. DI rats and Long Evans (LE) controls were fed ad libitum and given a choice between water, and either 0.9% or 1.8% NaCl.The data showed a significant increase of daily spontaneous water intake in DI compared with LE rats. Both DI and LE ingested similar small spontaneous volumes of the isotonic NaCl solution, but DI rats drank significantly less hypertonic NaCl than the LE controls. I.c.v infusion of AngII induced significant sodium intake in LE rats, but only raised water intake in DI rats. When combined with i.c.v. Ang II, OVT enhanced salt intake in LE animals while AVP attenuated water intake in both groups of rats and blocked NaCl intake completely in LE rats. In conclusion, DI rats did not demonstrate a SSA in response to central administration of AngII, although the drinking of water was enhanced. In combination with i.c.v. AngII, AVP inhibits water drinking in both DI and LE rats. In the LE controls OT attenuates AngII-induced SSA but has no effect in DI rats.

INSIGHT OF METHANOLIC EXTRACT OF VENTILAGO MADERASPATANA LEAVES ON HEPATOPROTECTIVE ACTIVITY UNDER STREPTOZOTOCIN-INDUCED DIABETIC RATS

Objective: The methanolic leaf extract of Ventilago maderaspatana (MEVML, 200 mg/kg body weight [b.w.]) was investigate to hepatoprotective activity under streptozotocin-induced (45 mg/kg b.w.) diabetic (Di) rats.
 Methods: In the present study, we determined the blood glucose levels, b.w., insulin, creatinine, and bilirubin levels in normal, Di, Di treated with MEVML and positive control rats.
 Results: The Di rats shown adverse changes in blood glucose levels, b.w., insulin, creatinine, and bilirubin levels when compared to other group rats. Reverse the adverse changes in the above parameters when treated the Di rats with MEVML
 Conclusion: The MEVML shown anti-diabetic activity and reverse the adverse changes in the above parameters in liver so that the MEVML supported the traditional claim of the hepatoprotective activity under Di condition.

ВЛИЯНИЕ ИНТРАНАЗАЛЬНО ВВОДИМЫХ ИНСУЛИНА И С-ПЕПТИДА НА АКТИВНОСТЬ АМФ-АКТИВИРУЕМОЙ ПРОТЕИНКИНАЗЫ, МИТОХОНДРИАЛЬНУЮ ДИНАМИКУ И МАРКЕРЫ АПОПТОЗА В ГИПОТАЛАМУСЕ КРЫС СО СТРЕПТОЗОТОЦИНОВЫМ ДИАБЕТОМ

Most important factors leading to the development of diabetic encephalopathy in type 1 diabetes mellitus (T1DM) are impaired energy metabolism and mitochondrial dynamics, as well as activation of apoptosis in brain neurons, which is largely due to insulin and C-peptide deficiency in the CNS. In T1DM, hypothalamic neuronal dysfunctions lead to disturb both feeding behavior and central regulation of energy metabolism. One of the approaches to compensate for insulin and C-peptide deficiency in the brain is their intranasal administration, how- ever its influence on the functional state of hypothalamic neurons has not yet been studied. The aim of the work was to study the effect of intranasally administered insulin and C-peptide on the activity of AMP-activated protein kinase (AMPK) and expression of Drp-1 and mitofusins (Mfn-1 and -2) responsible for the biogenesis of mitochondria, pro- and anti-apoptotic proteins Bax and Bcl-2, autophagy-associated protein Beclin-1, and melanocortin receptors (MCR) in the hypothalamus of male rats with T1DM induced by 50 mg/kg of streptozotocin. We studied a 7-day intranasal administration to diabetic rats (D) of insulin (20 μ g/rat/day, DI), C-peptide (36 μ g/rat/day, DC), and insulin combined with C-peptide at two doses of 12 and 36 μ g/rat/day (DIC12, DIC36). In the hypothalamus of DI and DIC36 rat groups, there were observed normalization of AMPK activity and the Bax/Bcl-2 ratio (typically increased in T1DM), restoration of Drp-1, Mfn-2 and Beclin-1 expression, and enhancement of type 4 MCR expression responsible for transduction of anorexigenic signals, with all changes being associated with attenuated hyperphagia in these groups. In the DIС12 and DС rat groups, the restorative effects of the intranasal treatment were less pronounced. Thus, intranasal administration of insulin and C-pep- tide, to the greatest extent at a 1:3 molar ratio, restores energetic balance and mitochondrial dynamics, as well as suppresses pro-apoptotic processes in the hypothalamus of rats with severe T1DM. These effects appear to underlie their neuroprotective action.

THE EFFECT OF THE TRAMADOL ACCUMULATED IN RAT LIVER ON THE DEVELOPMENT OF THE IMMATURE STAGES OF THE FLESH FLY SARCOPHAGA ARGYROSTOMA (ROBINEAU-DESVOIDY, 1830) (DIPTERA: SARCOPHAGIDAE).

The present study investigated the effects of the accumulation of tramadol on the development rates of the larvae and pupae. Larvae of the flesh fly Sarcophaga argyrostoma (Robineau-Desvoidy, 1830) were reared on three groups of Wister albino rat livers. One group was administered the recommended or normal dose (Dl) and the second group was injected with higher over or double dose (D2) of tramadol. Tramadol was administered by stomach tube once a day, for 3 months. The third group was injected with normal saline solution as the control. Using GC-Ms analysis, Rat livers contained 0.72 and 1.62mg/g of tramadol, in case of Dl and D2, respectively. Larvae of S. argyrostoma fed on DI and D2 rat livers contained (0.11 & 0.1 8mg/g), respectively. The corresponding tramadol concentrations persisted in the produced pupae of S. argyrostoma were (0.07 & 0.09mg/g), respectively. The accumulation of tramadol in tissues of S. argyrostoma larvae reduced the durations of larval stadia from (7.0408 1.0198 days) in case of the control to (6.6383±0.4857 days) when fed on DI liver and (6.3438±0.4826 days) when fed on D2 livers. The pupal durations was altered to (14.3750±0.4919 days) for D2 and (14.9574±0.6580 days) for DI, compared to (13.9167±1.0071 days), in case of the control. The average weight of the 3rd day larva of S. argyrostoma decreased from 18.1807±1.4949mg, in case of the control to 14.7279±1.3366mg and 14.9560±3.8210mg, when fed on Dl and D2 tramadol treated livers. In contrast, corresponding weight produced pupae increased from 14.1750±0.4667mg (control). to 15.2449±0.302mg & 15.9062±0.2888mg, in case of D1 and D2, respectively.

The Effects of Lycopene and Insulin on Histological Changes and the Expression Level of Bcl-2 Family Genes in the Hippocampus of Streptozotocin-Induced Diabetic Rats

The aim of this study was to evaluate the effects of antioxidants lycopene and insulin on histological changes and expression of Bcl-2 family genes in the hippocampus of streptozotocin-induced type 1 diabetic rats. Forty-eight Wistar rats were divided into six groups of control (C), control treated with lycopene (CL), diabetic (D), diabetic treated with insulin (DI), diabetic treated with lycopene (DL), and diabetic treated with insulin and lycopene (DIL). Diabetes was induced by an injection of streptozotocin (60 mg/kg, IP), lycopene (4 mg/kg/day) was given to the lycopene treated groups as gavages, and insulin (Sc, 1-2 U/kg/day) was injected to the groups treated with insulin. The number of hippocampus neurons undergoing cell death in group D had significant differences with groups C and DIL (p < 0.001). Furthermore, insulin and lycopene alone or together reduced the expression of Bax, but increased Bcl-2 and Bcl-xL levels in DI, DL, and DIL rats, especially when compared to group D (p < 0.001). The ratios of Bax/Bcl-2 and Bax/Bcl-xL in DI, DL, and DIL rats were also reduced (p < 0.001). Our results indicate that treatment with insulin and/or lycopene contribute to the prevention of cell death by reducing the expression of proapoptotic genes and increasing the expression of antiapoptotic genes in the hippocampus.

Oral Magnesium Treatment Reduces Anemia and Levels of Inflammatory Markers in Experimental Diabetes

ABSTRACT Magnesium has been reported to improve glucose utilization in diabetes mellitus. However, information on its effects on anemic and inflammatory markers in diabetes mellitus is limited. This study investigated the effect of oral magnesium (Mg) treatment on some markers of anemia and inflammation in 25 male Wistar rats. Rats (200 ± 15 g) were randomly divided into five groups (n = 5). Group 1 was control (received orally 0.2 mL distilled water daily), group 2 (Diabetic Untreated), group 3 (Diabetic Mg treated—100 mg/kg bw), group 4 (Diabetic Mg treated—250 mg/kg bw), group 5 (Diabetic Insulin treated—1 IU/kg bw). Diabetes was induced with a single dose of alloxan (100 mg/kg intraperitoneal (i.p.)). All treatments were done for 14 days. Anemic and inflammatory markers were investigated on blood samples obtained from each animal using standard laboratory methods. Significant increase (p < 0.05) in total white blood cell (WBC) count was observed in diabetic untreated rats (7.67 ± 0.397 × 109/L) compared to control (5.88 ± 0.25 × 109/L), DMg 100 (5.86 ± 0.74 × 109/L) and DMg 250 (5.06 ± 0.78 × 109/L). Hemoglobin concentration, packed cell volume (PCV) and red blood cell (RBC) count was decreased (p < 0.05) in DU compared to control, DMg 100, and DI rats. Erythrocyte sedimentation rate (ESR) was significantly increased (p < 0.05) in DU compared to control, DMg 100, DMg 250, and DI groups. Fibrinogen level was increased (p < 0.05) in DU rats (0.44 ± 0.02 g/dL) compared to control(0.26 ± 0.02 g/dL). Values obtained in DMg 100 (0.30 ± 0.03 g/dL), DMg 250 (0.22 ± 0.04 g/dL), and DI (0.36 ± 0.02 g/dL) rats were comparable to control (0.26 ± 0.02 g/dL). Total protein, albumin, and globulin levels were decreased in DU rats compared to normal control, DMg 100, DMg 250, and DI rats. In conclusion, anemia and increased hematologic and metabolic inflammatory markers may be associated with untreated diabetes mellitus. Treatment of alloxan-induced diabetic rats with magnesium improved the anemic state and reduced hematologic and metabolic inflammatory markers.

SLC2A2 gene is upregulated in diabetes and dowregulated in insulin treatment: participation of HNFs (1α, 3β and 4α)

ObjectiveInvestigate the role of HNFs (1á, 3â, 4á) at SLC2A2 (GLUT2 gene) in liver of diabetic and insulin treated diabetic rats.Methods3 month old Wistar rats, allocated into non diabetic (C), diabetic (D), diabetic treated with saline (DS) or insulin (DI) for 1, 4 and 6 days (d). The mRNA of GLUT2 and HNFs was analyzed by RT‐PCR. The HNFs binding activity into SLC2A2 promoter was analyzed by EMSA.ResultsIn comparison to C, D rats showed: hyperglycemia, polyuria, glycosuria without ketonuria, less gain of weight; increase of: I) GLUT2 (P&lt;0,0001), HNF1á (P&lt;0,05), HNF3â (P&lt;0,01) and HNF4á (P&lt;0,05) mRNA expression, II) bindig activity of HNF1á (P&lt;0,05), HNF3â (P&lt;0,01) and HNF4á (P&lt;0,05) into SLC2A2. In comparison to DS, DI rats showed: reduction of glycemia, increased of body weight; decrease of: I) mRNA expression: GLUT2 after 4 and 6d (P&lt; 0,001), HNF1á after 6d (P&lt;0,05), HNF3â after 4 and 6d (P&lt;0,001) and HNF4á after 6d (P&lt;0,05), II) binding activity into SLC2A2: HNF1á after 4 and 6d (P&lt;0,01), HNF3â after 1, 4 (P&lt;0,05) and 6d (P&lt;0,01) and HNF4á after 6d (P&lt;0,05).ConclusionThe SLC2A2 overexpression is related to expression and activity of HNFs (1á, 3â, 4á). Furthermore, insulin therapy‐induced regulation of SLC2A2 expression also involves HNFs. Considering the importance of the GLUT2, we hope that the knowledge of the SLC2A2 transcriptional regulation may contribute to control gene expression.FAPESP 2009/03871‐7

Effect of Vasopressin on the renal distribution of Annexin A2 in Brattleboro rats

Aim of the present study was to elucidate the renal expression pattern of Annexin A2 (ANXA2) during steady state and during activation of the urine concentration mechanism. To this end, adult Brattleboro rats (DI rats) were treated for 3d with the selective V2 vasopressin receptor agonist desmopressin (dDAVP, 5ng/h) or vehicle via osmotic minipump. Renal ANXA2 distribution was studied by double-labelling immunofluorescence using two different antibodies against ANXA2 and antibodies against rat endothelial cell antigen for endothelial cells, Tamm-Horsfall protein for thick ascending limbs (TAL) and aquaporin-2 for collecting ducts (CD). In untreated DI rats strong ANXA2 immunoreactivity was detected in cells of the Bowman capsule and in endothelial cells. Cells of the CD showed intermediate staining throughout the cytosol. In addition we observed a weak but consistent ANXA2 signal in the apical membrane of TAL profiles. Treatment of DI rats with dDAVP caused a marked shift of ANXA2 protein from the cytosol of CD cells to the apical and the basolateral membrane. In summary, we have shown that ANXA2 is expressed in cells of the Bowman capsule, the renal endothelium, the TAL and in the collecting duct where it may be involved in vasopressin signalling. This study was supported by the German Research Foundation (FOR 667).

Vasopressin treatment causes widespread hypoxia in the renal medulla of Brattleboro rats

In the present study we aimed to elucidate the effects of chronic vasopressin (AVP) administration on renal medullary oxygen levels in AVP-deficient Brattleboro rats (DI rats). To this end, adult DI rats were treated for 3 days with the V2 AVP receptor agonist desmopressin (dDAVP, 5ng/h; 3d) or its vehicle via osmotic minipump. Pimonidazole immunostaining was employed to study oxygen distribution throughout the kidney. In addition, outer medullary gene expression was determined by microarray analysis. dDAVP treatment resulted in a significant drop in urine output (−94 +/− 24% compared to controls, p < .05). Pimonidazole staining was detected in medullary rays of the cortex and in the outer as well as in the inner medulla of dDAVP-treated rats whereas signal in control animals was weak or absent. Gene expression analysis revealed an upregulation of several known target genes for hypoxia-inducible factors which included hexokinase 2, phosphofructokinase, heme oxygenase 1, insulin like growth factor (IGF) 1 and IGF binding proteins 1 and 3. Our results suggest that an activation of the renal urine concentration mechanism by dDAVP causes renal medullary hypoxia and an upregulation of hypoxia-inducible target gene expression. This study was supported by the German Research Foundation (FOR 667)

Vasopressin treatment lowers renal outer medullary epoxyeicosatrienoic acid levels in Brattleboro rats

Epoxyeicosatrienoic acids (EETs) have been shown to exert an inhibitory effect on the type 2 Na-K-2Cl−-cotransporter (NKCC2) in the thick ascending limb (TAL; He H et al. Am J Physiol Renal Physiol. 2003 284(6):F1235-44). Tissue levels of bioactive EETs are determined in part by the activity of soluble epoxid hydrolase 2 (sEH) which metabolizes EETs to their corresponding, less active, diols (DHETs). We here tested the hypothesis that vasopressin (AVP), a major regulator of NKCC2 activity, decreases EET levels within the outer medulla (OM) of AVP-deficient Brattleboro (DI) rats through activation of sEH. To test this, adult DI rats were treated for 3 days with the V2 AVP receptor agonist desmopressin (dDAVP, 5ng/h; 3d) or its vehicle via osmotic minipump. OM EET- levels were measured by mass spectrometry and sEH levels were determined by quantitative real time PCR. dDAVP treatment caused significant reductions in OM EET levels as compared to controls (−56±3% for 5,6-EET, −50±3.4% for 11,12-EET and −60±3.7% for 14,15-EET; p < .05 each). Concomitantly, OM sEH mRNA levels were increased (+160±37%, p < .05). In summary, we have shown that an activation of AVP signalling causes an upregulation of sEH which is accompanied by a reduction in tissue levels of free EET. Regulation of sEH expression may therefore be an important mechanism of AVP mediated urine concentration.

Phospholipase A2 group VIA regulates Na‐K‐2Cl cotransporter activity in the thick ascending limb

In the present study we attempted to characterize the renal expression pattern of phospholipase A2 group VI isoforms in response to vasopressin (AVP) treatment. To this end we used microarray analysis, PCR, immunohistochemistry, and Western blot in control rats and in Brattleboro rats (DI rats) treated with the selective V2 AVP receptor agonist desmopressin (dDAVP, 5ng/h; 3d) or vehicle via osmotic minipump. Cultured rat medullary TAL cells (mTAL cells) and the R‐ and S‐enantiomere of the iPLA inhibitor, bromoenol lactone (BEL), were employed to study the effect of iPLA on NKCC2 phosphorylation. Microarray analysis and quantitative real time PCR showed a significant downregulation of iPLA2 group VIA mRNA in the outer medulla of treated animals whereas other iPLA2 isoforms were not affected. Immunohistochemistry demonstrated iPLA2 expression in the entire distal nephron. Western blotting revealed a dominant band at 70kDa which was reduced to 50±7% by dDAVP (p&lt;0.05). Treatment of mTAL cells with 5μM S‐BEL for 1h markedly increased phospho‐NKCC2 abundance and membrane localization without effect on total NKCC2. R‐BEL was less effective. In summary, we have shown that the group VIA‐2 isoform of iPLA2 is expressed in the distal nephron of rat kidney and has an inhibitory effect on NKCC2 phosphorylation. Downregulation of iPLA may therefore be an important mechanism of AVP mediated urine concentration.

Effects of Insulin Treatment without and with Recurrent Hypoglycemia on Hypoglycemic Counterregulation and Adrenal Catecholamine-Synthesizing Enzymes in Diabetic Rats

Untreated diabetic rats show impaired counterregulation against hypoglycemia. The blunted epinephrine responses are associated with reduced adrenomedullary tyrosine hydroxylase (TH) mRNA levels. Recurrent hypoglycemia further impairs epinephrine counterregulation and is also associated with reduced phenylethanolamine N-methyltransferase mRNA. This study investigated the adaptations underlying impaired counterregulation in insulin-treated diabetic rats, a more clinically relevant model. We studied the effects of insulin treatment on counterregulatory hormones and adrenal catecholamine-synthesizing enzymes and adaptations after recurrent hypoglycemia. Groups included: normal; diabetic, insulin-treated for 3 wk (DI); and insulin-treated diabetic exposed to seven episodes (over 4 d) of hyperinsulinemic-hypoglycemia (DI-hypo) or hyperinsulinemic-hyperglycemia (DI-hyper). DI-hyper rats differentiated the effects of hyperinsulinemia from those of hypoglycemia. On d 5, rats from all groups were assessed for adrenal catecholamine-synthesizing enzyme levels or underwent hypoglycemic clamps to examine counterregulatory responses. Despite insulin treatment, fasting corticosterone levels remained increased, and corticosterone responses to hypoglycemia were impaired in DI rats. However, glucagon, epinephrine, norepinephrine, and ACTH counterregulatory defects were prevented. Recurrent hypoglycemia in DI-hypo rats blunted corticosterone but, surprisingly, not epinephrine responses. Norepinephrine and ACTH responses also were not impaired, whereas glucagon counterregulation was reduced due to repeated hyperinsulinemia. Insulin treatment prevented decreases in basal TH protein and increased PNMT and dopamine beta-hydroxylase protein. DI-hypo rats showed increases in TH, PNMT, and dopamine beta-hydroxylase. We conclude that insulin treatment of diabetic rats protects against most counterregulatory defects but not elevated fasting corticosterone and decreased corticosterone counterregulation. Protection against epinephrine defects, both without and with antecedent hypoglycemia, is associated with enhancement of adrenal catecholamine-synthesizing enzyme levels.

Temporal response pattern of biochemical analytes in experimental diabetes.

The activities of the enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), creatine kinase (CK), amylase (AMS) and angiotensin converting enzyme (ACE) have been used to assess the toxic effects of xenobiotics that have hypoglycaemic action in hepatic, pancreatic, renal and muscle tissue. Using a validated experimental model of diabetes mellitus in rats, we ascertained whether this syndrome itself affected the serum activities of these enzymes over a 53-day period. Levels of hepatic enzymes AST, ALT and ALP were higher in the streptozotocin (STZ)-diabetic rats (group D), but were controlled by insulin therapy (group DI). AMS was reduced in group D and unchanged in group DI rats. Proteinuria was detected 1 day after STZ administration and partially controlled by insulin (group DI); its early presence in group D rats, and the lack of any change in serum ACE in this group, indicates that proteinuria is the better marker for microangiopathy. Microscopic examination of liver, kidney, heart and skeletal muscles (soleus and extensor digitorum longus) revealed various alterations in group D rat tissues, which were less pronounced in group DI. The liver, pancreas and kidney tissue-damage was consistent with the altered serum levels of AST, ALT, ALP and AMS and proteinuria. We conclude that: (i) rigorous control is required when these serum-enzyme levels are used as indicators of tissue toxicity in experimental diabetes, and (ii) LD, CK and bilirubin serum levels, which are unaffected by diabetes, can be used when testing effects of xenobiotics on tissues.

Sodium and potassium clearance rhythmicity in diabetic rats with insulin treatment

The effect of insulin treatment on the daily distribution of the urinary volume and urinary sodium and potassium excreted, as well as their clearance rhythms in rats with streptozotocin (STZ)-induced diabetes was investigated. Normal(C), uncontrolled (D) and controlled insulin diabetic rats (DI), were studied during a light-dark (12 h:12 h) cycle and given food and water ad libitum. The DI rats showed a significant reduction in the urinary sodium and potassium excreted during 24 h with respect to the D rats, though these values were significantly higher than the C ones. A loss of the normal circadian rhythmicity of diuresis and both sodium and potassium clearance was observed in the D rats, together with higher values of M (MESOR) than in the C rats. These rhythms could be reestablished with continuous insulin infusion, their orthophases occurring near the C ones. However the M values of sodium and potassium clearance in DI rats are greater than C, showing higher values than this group during the rest phase. These results in DI rats may suggest that the constant rate infusion of insulin can be responsible for the high values of clearance of both ions at the rest phase and so for the incomplete renal rhythms restoration.

Effects of recurrent hyperinsulinemia with and without hypoglycemia on counterregulation in diabetic rats.

To understand the mechanisms whereby recurrent hypoglycemia increases the risk of subsequent hypoglycemia, it was necessary to differentiate the effects of recurrent hyperinsulinemia from those of hyperinsulinemic hypoglycemia. We examined basal and hypoglycemic endocrine function in normal rats, streptozotocin-diabetic controls, and diabetic rats exposed to 4 days of 2 episodes/day of hyperinsulinemic hypoglycemia (DH) or hyperinsulinemic hyperglycemia (DI). DH and DI rats differentiated the effects of hyperinsulinemia from those of hypoglycemia. In diabetic controls, basal plasma ACTH tended to be increased, and plasma corticosterone, plasma somatostatin, and pancreatic prosomatostatin and proglucagon mRNA were increased (P < 0.05) vs. normal rats. These parameters were normalized in DH and DI rats. In diabetic controls, glucagon, epinephrine, norepinephrine, corticosterone, and peak glucose production responses to hypoglycemia were reduced (P < 0.05) vs. normal rats. In DI rats, epinephrine responses were normalized. Conversely, DH rats displayed marked further impairment of epinephrine and glucose production responses and increased peripheral insulin sensitivity (P < 0.05 vs. diabetic controls). Both insulin regimens partially normalized glucagon and fully normalized norepinephrine and corticosterone responses. In summary, recurrent hyperinsulinemia in diabetic rats normalized most pituitary-adrenal, sympathoadrenal, and pancreatic parameters. However, concurrent hypoglycemia further impaired epinephrine and glucose production responses and increased insulin sensitivity. We conclude that 1) recurrent hypoglycemia may increase the risk of subsequent hypoglycemia by increasing insulin sensitivity, and 2) epinephrine counterregulation is particularly sensitive to impairment by recurrent hypoglycemia.

Dietary preferences of Brattleboro rats correlated with an overexpression of galanin in the hypothalamus.

Galanin (GAL) is a neuropeptide cosynthesized with vasopressin (AVP) in neurons of the hypothalamo-neurohypophysial system. It increases food intake when injected into the brain and elicits an overconsumption of fat. The Brattleboro rat (DI) is genetically unable to produce AVP; the AVP-deficient-producing neurons of the hypothalamo-neurohypophysial system of DI rats are chronically stimulated and DI rats suffer from diabetes insipidus. We studied the central expression of GAL and the dietary preferences in the DI rat. GAL was overexpressed in the hypothalamus of the DI rat. GAL mRNA was higher by 1.8-fold in the supraoptic (P < 0.05) and by four-fold in the paraventricular nuclei (P < 0.001) of male and female DI rats compared with those of control Long Evans (LE) rats. However, GAL mRNA was lower in the arcuate nuclei of DI rats and equal to that of LE rats in the dorsomedian nuclei. We also measured a high preference for a lipid diet (45% of the daily consumption) when DI rats ate from a choice of the three macronutrients. Chronic infusion with deamino-8D-AVP (agonist of AVP V2 receptors) prevented the diabetes insipidus and the chronic stimulation of the hypothalamo-neurohypophysial system of the DI rats. However, the treatment did not suppress the overexpression of GAL, nor did it affect the rats' preference for a lipid diet. We conclude that the DI rat provides a novel animal model in which a spontaneous dietary preference correlates with the overexpression of one of the hypothalamic peptides, GAL.

Heat stress fails to protect myocardium of streptozotocin-induced diabetic rats against infarction.

Protection conferred by heat stress (HS) against ischaemia-reperfusion injury, in term of mechanical function and myocardial necrosis, has been extensively studied. In contrast, the effects of disease states on this HS-induced cytoprotective response are less known. Therefore, we investigated the effects of prior heat stress on the infarct size in the isolated heart and on the myocardial heat stress protein (HSP) 72 synthesis, in a model of insulin-dependent diabetic rats. Three groups of animals were studied: D rats were rendered diabetic by 55 mg/kg streptozotocin i.v. injection. DI rats received the same treatment plus a daily injection of insulin started 2 weeks after and V rats received the vehicle of streptozotocin plus a daily injection of saline. Eight weeks later, D, DI and V rats were either heat-stressed (42 degrees C for 15 min) or sham-anaesthetised. Twenty-four hours later, their hearts were isolated, perfused using the Langendorff technique, and subjected to a 30 min occlusion of the left coronary artery followed by 120 min of reperfusion. Myocardial HSP72 content was measured 24 h after HS or sham treatment using an electrophoresis coupled with a Western blot analysis. Infarct-to-risk ratio (I/R) was significantly reduced in hearts from heat-stressed (11.7 +/- 2.0%) compared to sham (30.0 +/- 3.2%) V rats. This cardioprotection was not observed in hearts from D (I/R: 31.4 +/- 3.3 vs. 34.3 +/- 3.5%) and DI (I/R: 28.7 +/- 1.6 vs. 30.3 +/- 1.6%) rats. Risk zones were similar between all experimental groups. The incidence of ventricular arrhythmias during ischaemia and reperfusion periods was not different between the six experimental groups. Western blot analysis of the myocardial HSP72 content showed a comparable heat stress-induced increase of this protein, in V, D and DI animals. These results demonstrate that myocardial protective effect induced by heat stress could not extend to a pathological animal model like the diabetic rat and seems to be unrelated to the HSP72 level. Further investigations are required to elucidate the precise role of the heat stress proteins in this adaptive response.

Blood brain barrier permeability and acute inflammation in two models of traumatic brain injury in the immature rat: a preliminary report.

We sought to investigate the course and magnitude of blood brain barrier (BBB) permeability following focal and diffuse traumatic brain injury (TBI) in immature rats and examine the time course of markers of acute inflammation (neutrophil accumulation and E-selectin [E-sel] expression) following these two types of injury. We measured BBB permeability using i.v. injection Evans Blue (EB) and the extent of inflammation using immunohistochemical techniques identifying neutrophils (monoclonal antibody RP-3) and the endothelial adhesion molecule, E-selectin. Male Sprague-Dawley immature (17 day-old) rats (30-45 g, n = 80) were subjected to a controlled cortical impact (CCI: 2 mm, 4 m/s), a closed head diffuse injury (DI: 150 g/2m) or a corresponding sham procedure (with or without craniotomy). EB was injected i.v. at 30 min before sacrifice, which occurred at 1 h, 4 h, or 24 h after injury. BBB permeability was observed in both the CCI and DI rats at 1 h after injury which largely resolved by 24 h. In the CCI, EB extravasation was seen within and around the contusion. In DI, diffuse BBB permeability was seen. DI was not associated with acute inflammation since there was neither neutrophil accumulation nor E-selectin expression. The CCI rats though had 5.1 +/- 2.2 neutrophils/hpf and 3.0 +/- 0.4 endothelial cells/hpf expressing E-selectin (mean +/- SEM) (both p < 0.05 vs sham and DI). These data suggest that BBB breakdown occurs in the immature rat after both focal and diffuse TBI. This early BBB permeability was not associated with acute inflammation in DI but was in CCI. These data also suggest that contusion is a key factor in the development of a traditional acute inflammatory response after TBI in the immature rat.

Role of Vasopressin in Long-Term Preferences of Sucrose and Polycose UnderAd-Libitumand Food-Restricted Conditions

Vasopressin-deficient (DIand vasopressin-containing (LE) rats were given continuous access to 32% sucrose and 32% Polycose solutions underad-libitumand food-restricted conditions in a long-term preference test. Although all animals preferred Polycose to sucrose in both conditions, food restriction introduced a stress that significantly increased the consumption of Polycose in DI rats. Considering total caloric intake, Polycose was preferred by both strains only under food restriction and the effect was greatly exacerbated in DI animals compared to LE animals. The differences observed between DI and LE animals in response toad-libitumfeeding and food-restriction stress indicate that vasopressin, directly and/or indirectly, influences the physical and metabolic functions and processes of the animals, which, in turn, affect their intake of Polycose.

Arginine vasopressin mediates the chloroquine induced increase in renal sodium excretion

We postulated that chloroquine increases plasma arginine vasopressin (AVP) concentrations thus altering renal Na+ clearance. Therefore, we studied a relationship between plasma AVP concentrations and urinary Na+ output in separate groups of Sprague-Dawley (SD) rats administered chloroquine (3 micrograms/min) for 1 h 20 min. We also monitored Na+ excretion rates in Brattleboro AVP-deficient Di rats challenged with hypotonic saline load and administered chloroquine for 1 h 20 min. To establish whether chloroquine-induced changes in renal Na+ excretion were mediated via AVP V1 receptors, we studied Na+ excretion rates in groups of SD rats administered chloroquine or AVP in the presence of AVP V1 receptor antagonist (1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid)-2-O-methyltyrosine arginine vasopressin (d(CH2)5(Tyr(Me)2) AVP) at 11 pmol/min for 1 h 20 min. The Na+ excretion rate rose significantly (P < 0.01) from a pretreatment level of 9.8 +/- 1.0 mumol/min to a peak of 14.1 +/- 0.9 mumol/min in SD rats (n = 7) administered chloroquine. The Na+ excretion rate remained unaltered around 8.5 mumol/min in rats simultaneously administered chloroquine and the AVP V1 receptor antagonist. This compared with control rats (8.1 +/- 0.5 mumol/min, n = 7) and animals administered AVP V1 receptor antagonist alone (8.7 +/- 0.6 mumol/min, n = 7). Chloroquine did not affect urine flow, Na+ or K+ excretion rates in Brattleboro AVP-deficient Di rats. Administration of AVP alone was associated with significant increases in renal Na+ excretion rate. Blockade of AVP V1 receptors abolished the AVP-dependent increase in urinary Na+ loss. We conclude that at least part of the chloroquine-induced increase in Na+ excretion is mediated by chloroquine stimulating an increase in plasma AVP concentration.