Protein-deprived Rats Research Articles

Prolonged protein deprivation differentially affects calretinin- and parvalbumin-containing interneurons in the hippocampal dentate gyrus of adult rats

Protein deprivation is a detrimental nutritional state that induces several deleterious changes in the rat hippocampal formation. In this study, we compared the effects of protein deprivation in the number of parvalbumin (PV)-immunoreactive and calretinin (CR)-immunoreactive interneurons of the dentate gyrus, which are involved in the control of calcium homeostasis and fine tuning of the hippocampal circuits. Two month-old rats were randomly assigned to control and low-protein diet groups. The rats of the latter group were fed with a low-protein diet (8% casein) for 6 months. All animals were perfused at 8 months of age. The number of neurons expressing CR in the molecular layer and in the hilus of dentate gyrus was reduced in protein-deprived rats. Conversely, protein deprivation increased the number of PV-containing interneurons in the dentate granule cell layer and hilus. These results support the view that protein deprivation may disturb calcium homeostasis, leading to neuronal death including GABAergic interneurons expressing CR. In the other hand, the up-regulation of PV cells may reflect a protective mechanism to counteract the calcium overload and protect the remaining neurons of the dentate gyrus.

Prolonged protein deprivation, but not food restriction, affects parvalbumin-containing interneurons in the dentate gyrus of adult rats

Several studies have demonstrated the vulnerability of the hippocampal formation to malnutrition. In this study, we compared the effects of food restriction and protein malnutrition in the total number of neurons of the dentate gyrus and in the number of parvalbumin-immunoreactive (PV-IR) interneurons, which are related to the control of calcium homeostasis and fine tuning of the hippocampal circuits. Two month-old rats were randomly assigned to control, food-restricted and low-protein diet groups. After 6 months, 10 rats from the low-protein diet group were selected at random and fed with a normal protein diet for 2 months. The total number of granule and hilar cells was reduced in protein-deprived rats and the nutritional reestablishment with a normal protein diet did not recover neuron numbers. Protein deprivation increased the number of PV-IR interneurons in the granule cell layer and hilus, but their number returned to values similar to controls after nutritional rehabilitation. Food restriction did not affect the total number of neurons or the density of PV-IR interneurons in the dentate gyrus. These results support the view that protein deprivation may disturb calcium homeostasis, leading to neuronal death. The up-regulation of PV-IR cells may reflect a protective mechanism to counteract the calcium overload and protect the remaining neurons of the dentate gyrus. This imbalance in cell-ratio favoring GABAergic interneurons may justify some learning and memory impairments described in protein-deprived animals. This contrast between the results of food restriction and protein deprivation should be further analyzed in future studies.

Administration of growth hormone in selectively protein-deprived rats decreases BMD and bone strength

Introduction Isocaloric protein undernutrition is associated with decreased bone mass and decreased bone strength, together with lower IGF-I levels. It remains unclear whether administration of growth hormone (GH) corrects these alterations in bone metabolism. Materials and methods Six-month-old female rats were fed isocaloric diets containing either 2.5% or 15% casein for 2 weeks. Bovine growth hormone (bGH, 0.5 or 2.5 mg/kg of body weight) or vehicle was then administered as subcutaneous injections, twice daily, to rats on either diet for 4 weeks. At the proximal tibia, analysis of bone mineral density (BMD), maximal load and histomorphometry were performed. In addition, urinary deoxypyridinoline, plasma osteocalcin and IGF-I concentrations were measured. Weight was monitored weekly. Results bGH caused a dose-dependent increase in plasma IGF-I regardless of the dietary protein content. However, bGH dose-dependently decreased BMD and bone strength in rats fed the low-protein diet. There was no significant effect of bGH on BMD in rats fed the normal protein diet within this short-term treatment period, however bone formation as detected by histomorphometry was improved in this group but not the low-protein group. Osteoclast surface was increased in the low-protein bGH-treated animals only. Changes in bone turnover markers were detectable under both normal and low-protein diets. Conclusion These results emphasize the major importance of dietary protein intake in the bone response to short-term GH administration, and highlight the need for further investigation into the effects of GH treatment in patients with reduced protein intake.

Altered Renal Expression of Na+ Transporters and ROMK in Protein-Deprived Rats

Potassium depletion has been associated with altered sodium reabsorption in tubule segments. We studied if the altered abundance of Na⁺ transporters and ROMK are associated with distal potassium secretion that contributes to the development of hypokalemia in protein-deprived rats. After weaning, Wistar rats were fed with a low-protein diet (8%, LP) for 14 days and then recovered with a normal-protein (NP) diet (24%, RP). An age-matched control group was fed with an NP diet (24%, NP). We showed hypokalemia, lower glomerular filtration rate and higher FEK⁺ in the LP group. Immunoblotting revealed that the type 3 Na⁺/H⁺ exchanger in the cortex was decreased in the LP group. However, the type 2 Na⁺-K⁺-2Cl^– cotransporter was increased in the outer stripe of the outer medulla in the LP group. The abundance of the aldosterone-regulated Na⁺-Cl^– cotransporter (NCC) and epithelial Na⁺ channel (ENaC) was higher in the LP group and was associated with higher plasma aldosterone level. ROMK protein levels were increased. Na⁺/K⁺-ATPase protein levels were the same in both groups. After the recovery period, the expression of Na⁺ transporters and ROMK returned to control values. We conclude that increased expression of NCC, ENaC subunits, and ROMK contributed to distal potassium secretion leading to enhanced potassium excretion, which may explain the hypokalemia resulting from LP feeding. A role of aldosterone may be suggested.

Changes in specific gravity as a sign of disturbed brain maturation in protein-deprived rats.

The specific gravity of the cerebral cortex of normal and protein-deprived rats aged 15, 21 and 25 days was determined. The increase in specific gravity seen in control rats in the course of their development was reduced in protein-deprived rats in the course of the period between 15 and 21 days of age. The results are in agreement with previous reports on delayed maturation of the brain in protein-deprived rats and indicate that determination of brain specific gravity may serve as a suitable screening method in situations when a disturbed brain maturation is suspected.

Malnutrition and Its Relationship with the Recovery of Enzymatic Activity in Rat Thymus

Abstract It is known that the thymus is severely affected by nutritional deficiency. Adenosine deaminase (ADA) and Purine nu-cleoside phosphorylase (PNP) are thought to be important for normal lymphocyte development. The present paper studies the effect on the activity of these thymic enzymes in growing rats after severe protein deprivation and the posterior oral administration of a 20% dietary protein during a short period of time. Statistical differences in the activity of ADA and PNP were observed in severely protein-deprived rats (PFG) when compared to their respective age-matched control (C38) (17.0 ± 2.6 vs. 9.0 ± 4.0; 11.5 ± 4.2 vs. 4.1 ± 1.3, respectively). After 9 days of refeeding with a 20% casein diet (R), activity of ADA and PNP decreased significantly when compared to PFG. No statistical differences in ADA and PNP were observed between R and its control C47 (7.4 ± 2.5 vs. 9.3 ± 1.8; 3.6 ± 0.5 vs. 3.9 ± 0.6, respectively). The results suggest that the refeeding with a 20% protein diet over 9 ...

P-89 Function and regulation of insulin-like growth factor binding protein-2 and -3 in ovarian cancer cell models

for 8 days by pair-feeding. On the 9th day, half of the rats in each group were sacrificed after 16 hours fasting (F) and the other half were sacrificed after 16 hours fasting followed by 6hr re-feeding (R). First, we measured plasma concentrations of insulin and glucocorticoid that are main hormones to regulate IGFBP-1 transcription. Plasma insulin concentrations were higher in the re-fed groups than in the fasted groups but did not differ among the three dietary groups. Plasma corticosterone concentrations were not affected by protein-deprivation or antioxidant intake. In contrast, liver TBARS concentrations, which reflect the magnitude of oxidative stress, were significantly higher in the protein-deprived groups than in the control groups, and returned to the control level in antioxidant-fed groups. As we previously reported, hepatic IGFBP-1 mRNA significantly increased by 16 hours fasting. Six hours re-feeding in the 20C group decreased hepatic IGFBP-1 mRNA levels by 74%; however, this repression was not observed in the 0C group. Interestingly, feeding-dependent suppression of IGFBP-1 mRNA was restored by intake of the antioxidants even in the 0C group. These results demonstrated that oxidative stress attenuates the effects of feeding on reduction of hepatic IGFBP-1 mRNA in proteindeprived rats, probably caused by impairment of insulin signals.

P-88 Role of oxidative stress on the regulation of IGFBP-1 mRNA in protein-deprived rat liver

P-88 Role of oxidative stress on the regulation of IGFBP-1 mRNA in protein-deprived rat liver

Use of social information by sodium- and protein-deficient rats: test of a prediction (Boyd & Richerson 1988)

Boyd & Richerson (1988 Social Learning: Psychological and Biological Perspectives, pp. 29–48) predicted from formal models of the evolution of social learning that an animal should rely more heavily on socially acquired information when seeking a nutrient that it has difficulty learning to select for itself than when seeking a nutrient it can readily identify. We compared the extent to which Norway rats, Rattus norvegicus, seeking either sodium (which they can easily identify) or protein (which they cannot identify), relied on information acquired from recently fed conspecific demonstrators when selecting a food. Consistent with Boyd & Richerson's prediction, when choosing between two unfamiliar, distinctively flavoured foods rich in both protein and sodium, protein-deprived rats showed significantly greater social influence on their food choices than did sodium-deprived rats.

A hierarchical analysis of transcriptome alterations in intrauterine growth restriction (IUGR) reveals common pathophysiological pathways in mammals

Intra-uterine growth restriction (IUGR) is a frequent disease, affecting up to 10% of human pregnancies and responsible for increased perinatal morbidity and mortality. Moreover, low birth weight is an important cause of the metabolic syndrome in the adult. Protein depletion during the gestation of rat females has been widely used as a model for human IUGR. By transcriptome analysis of control and protein-deprived rat placentas, we were able to identify 2543 transcripts modified more than 2.5 fold (1347 induced and 1196 repressed). Automatic functional classification enabled us to identify clusters of induced genes affecting chromosome structure, transcription, intracellular transport, protein modifications and apoptosis. In particular, we suggest the existence of a complex balance regulating apoptosis. Among repressed genes, we noted several groups of genes involved in immunity, signalling and degradation of noxious chemicals. These observations suggest that IUGR placentas have a decreased resistance to external aggression. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites. There was an over-representation of Zn finger (ZNF) proteins and Pdx1 (pancreatic and duodenal homeobox protein 1) putative binding sites. Consistently, Pdx1 and a high proportion of ZNF genes were induced at the transcriptional level. A similar analysis of ZNF promoters showed an increased presence of putative binding sites for the Tata box binding protein (Tbp). Consistently again, we showed that the Tbp and TBP-associated factors (Tafs) were up-regulated in IUGR placentas. Also, samples of human IUGR and control placentas showed that human orthologous ZNFs and PDX1 were transcriptionally induced, especially in non-vascular IUGR. Immunohistochemistry revealed increased expression of PDX1 in IUGR human placentas. In conclusion, our approach permitted the proposition of hypotheses on a hierarchy of gene inductions/repressions leading to massive transcriptional alterations in the IUGR placenta, in humans and in rodents.

Kidney Gene Expression Analysis in a Rat Model of Intrauterine Growth Restriction Reveals Massive Alterations of Coagulation Genes

In this study, low birth weight was induced in rats by feeding the dams with a low-protein diet during pregnancy. Kidneys from the fetuses at the end of gestation were collected and showed a reduction in overall and relative weight, in parallel with other tissues (heart and liver). This reduction was associated with a reduction in nephrons number. To better understand the molecular basis of this observation, a transcriptome analysis contrasting kidneys from control and protein-deprived rats was performed, using a platform based upon long isothermic oligonucleotides, strengthening the robustness of the results. We could identify over 1800 transcripts modified more than twice (772 induced and 1040 repressed). Genes of either category were automatically classified according to functional criteria, making it possible to bring to light a large cluster of genes involved in coagulation and complement cascades. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites, suggesting an overrepresentation of the AP1R binding site, together with the transcription induction of factors actually binding to this site in the set of induced genes. The induction of coagulation cascades in the kidney of low-birth-weight rats provides a putative rationale for explaining thrombo-endothelial disorders also observed in intrauterine growth-restricted human newborns. These alterations in the kidneys have been reported as a probable cause for cardiovascular diseases in the adult.

Palmitic acid increase levels of pancreatic duodenal homeobox-1 and p38/stress-activated protein kinase in islets from rats maintained on a low protein diet

A severe reduction in insulin release in response to glucose is consistently noticed in protein-deprived rats and is attributed partly to the chronic exposure to elevated levels of NEFA. Since the pancreatic and duodenal transcription factor homeobox 1 (PDX-1) is important for the maintenance of beta-cell physiology, and since PDX-1 expression is altered in the islets of rats fed a low protein (LP) diet and that rats show high NEFA levels, we assessed PDX-1 and insulin mRNA expression, as well as PDX-1 and p38/stress activated protein kinase 2 (SAPK2) protein expression, in islets from young rats fed low (6%) or normal (17%; control) protein diets and maintained for 48 h in culture medium containing 5.6 mmol/l glucose, with or without 0.6 mmol/l palmitic acid. We also measured glucose-induced insulin secretion and glucose metabolism. Insulin secretion by isolated islets in response to 16.7 mmol/l glucose was reduced in LP compared with control rats. In the presence of NEFA, there was an increase in insulin secretion in both groups. At 2.8 mmol/l glucose, the metabolism of this sugar was reduced in LP islets, regardless of the presence of this fatty acid. However, when challenged with 16.7 mmol/l glucose, LP and control islets showed a severe reduction in glucose oxidation in the presence of NEFA. The PDX-1 and insulin mRNA were significantly higher when NEFA was added to the culture medium in both groups of islets. The effect of palmitic acid on PDX-1 and p38/SAPK2 protein levels was similar in LP and control islets, but the increase was much more evident in LP islets. These results demonstrate the complex interrelationship between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis by affecting molecular mechanisms and stimulus/secretion coupling pathways.

A panic experimental model: Validation of a complex operant behavioral method in undernourished rats, with desipramine to provide a template effect profile

Introduction Clinical studies have shown that some antidepressants may be more efficient than benzodiazepines to alleviate anxiety associated with panic disorders; however, operant conflict procedures in rats developed so far seem not particularly able to model human anxiety sensitive to antidepressant treatments. Previous panic models with learned responses did not statistically subtract the effect of confounding factors from the variable of interest. Methods Undernourished rats were selected due to their behavioral and neurobiological resemblance to human patients suffering from panic disorder. The Geller–Seifter paradigm represented the stressful environmental condition in adult life. Desipramine (10 mg/kg/day) or saline were administered IP during 7 days under a cross over design ( N = 10). Five daily 15 min-operant sessions were carried out on each experiment. Unpunished, unrewarded and punished operant behavioral periods were identical both in their duration and in their reward system (the FR1 schedule) in order to measure response suppression, which has not been considered in previous studies with the Geller–Seifter paradigm. The dependent variable was the difference between comparable unpunished and punished periods. Results A significant Diet × Drug interaction was observed in the dependent variable, which represented the level of “suppression/suppression release” induced by treatments. Discussion Compared to control rats, deprived rats showed a significant and selective anticonflict effect of desipramine on the stressful and complex operant performance. The animal model of perinatally protein-deprived rats along with the Geller–Seifter's operant behavioral paradigm may represent a more sensitive approach to model human anxiety sensitive to antidepressant treatments by considering the combined impact of both early biological trauma and adult learned experiences under the same design.

Altered serotonergic function of dorsal raphe nucleus in perinatally protein-deprived rats: Effects of fluoxetine administration

We have previously described that perinatally undernourished rats showed increased locus coeruleus activity, a phenomenon reversed by repeated desipramine or fluoxetine administration. Since there is reciprocal modulation between the locus coeruleus and the dorsal raphe nucleus, and because these structures are associated with the pathophysiology of different states of anxiety, we evaluated the activity of serotonergic dorsal raphe neurons from early malnourished animals compared with controls, using in vivo extracellular single-unit recordings. The number of spontaneously active cells/track was significantly higher in protein-deprived animals, although the firing rate and the sensitivity of 5-HT 1A receptors did not differ from those of controls. Five days of fluoxetine administration (5 mg/kg/day i.p.) was able to reverse the increased number of active serotonergic cells without affecting their firing rate. Furthermore, subsensitivity of 5-HT 1A autoreceptors developed in the same way after repeated fluoxetine administration in both control and protein-deprived animals. These results suggest that the increased noradrenergic transmission observed in protein-deprived animals may induce an activation of serotonergic neurons in the dorsal raphe nucleus, and that this effect is normalized following fluoxetine treatment, which normalizes locus coeruleus activity.

H+-ATPase Activity in Collecting Duct Segments in Protein-Deprived Rats – Role of Angiotensin II on Its Regulation

Background: Enhanced expression of the genes that encode for components of the renin-angiotensin system (RAS) has been exhibited in low-protein-fed (LP) rats. We examined distal proton secretion in LP rats through the activity of H⁺-ATPase on microdissected CCD, OMCD and IMCD segments. The effect of angiotensin II AT₁ receptor inhibition and protein recovery (24% protein) on H⁺-ATPase activity was studied. Methods: Bafilomycin-sensitive H⁺-ATPase activity on CCD, OMCD and IMCD segments of LP (protein 8%) and control rats CP (protein 24%) was evaluated. We examined the levels of mRNA expression of RAS components: angiotensin-converting enzyme (ACE), angiotensinogen and angiotensin II AT₁ expression; AT₁receptor binding and distribution were determined by quantitative autoradiography. Results: Increased ACE and AT₁ mRNA expression was found in cortex and medulla of LP compared to NP rats. AT₁ receptor binding density was significantly reduced in renal cortex and inner stripe of the outer medulla of LP compared to NP rats. Minimal radioligand binding was shown in inner medulla of LP. Whole kidney expression of angiotensinogen was unaltered in LP. H⁺-ATPase activity significantly decreased in IMCDs and OMCDs of LP. The inhibitory effect of LP was abolished when OMCD segments were incubated for 60 min in the presence of losartan 10^–6 to 10^–8M. There was no effect of losartan concentrations from 10^–6 to 10^–8 M on IMCDs. Similar results were observed on H⁺-ATPase activity in OMCD and IMCD segments after readministration of 24% protein in the diet. Conclusion: Both the recovery of H⁺-ATPase activity in OMCD segments induced by losartan and the increased expression of AT₁receptor suggest angiotensin II modulation of proton ATPase activity on this duct segments in LP rats. Intense compromise of proton secretion through the continued H⁺-ATPase inhibition in IMCDs from LP was shown.

Effect of prenatal protein malnutrition on numbers of neurons in the principal cell layers of the adult rat hippocampal formation

Malnutrition has been associated with a variety of functional and anatomical impairments of the hippocampal formation. One of the more striking of these is widespread loss of hippocampal neurons in postnatally malnourished rats. In the present study we have investigated the effect of prenatal malnutrition on these same neuronal populations, neurons that are all generated during the period of the dietary restriction. In prenatally protein deprived rats, using design-based stereology, we have measured the regional volume and number of neurons in the hilus of the dentate gyrus and the pyramidal cell layers of CA3, CA2, CA1, and the subiculum of 90-day-old animals. These results demonstrated a statistically significant reduction of 20% in neuron numbers in the CA1 subfield, while numbers in the other subfields were unchanged. There was a corresponding significant reduction of 22% in the volume of the CA1 subfield and a significant 14% decrease in the volume of the pyramidal layer of the subiculum. The change in volume of the pyramidal layer of the subiculum without neuron loss may reflect loss of CA1 afferent input to the pyramidal layer. Although the effect of nutritional deprivation on the neuronal population appears to be different in pre- and postnatal malnutrition, both dietary paradigms highlight the vulnerability of key components of the hippocampal trisynaptic circuit (consisting of the dentate granule cell mossy fibers projection to CA3 pyramids and the CA3 projection to the CA1 pyramids), which is an essential circuit for memory and learning.

Locus coeruleus activity in perinatally protein-deprived rats: effects of fluoxetine administration

We have previously described an increased locus coeruleus activity in perinatally protein-deprived rats. Since locus coeruleus dysfunction has been involved in different types of anxiety disorders and considering the modulating action of serotonergic transmission on locus coeruleus activity, we assessed the effect of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on locus coeruleus activity as measured by the firing rate and the number of spontaneously active cells/track. Repeated fluoxetine administration reduced locus coeruleus activity in both control and protein-deprived rats, although the reduction was greater in protein-deprived rats. Dose–response curves for the inhibitory effect of clonidine showed subsensitivity of α 2-adrenergic autoreceptors in protein-deprived rats, a phenomenon reversed by fluoxetine treatment. Dose–response curves for the inhibitory effect of 2,5-dimethoxy-4-iodoamphetamine (DOI) were similar in both groups of rats. Following fluoxetine administration, subsensitivity to this effect developed in control but not in protein-deprived rats. Extracellular noradrenaline level in the prefrontal cortex, as measured by microdialysis procedure, was higher in protein-deprived rats compared to controls, and this difference was reduced after fluoxetine administration. A challenge with yohimbine increased the extracellular noradrenaline level in control but not in protein-deprived rats, suggesting subsensitivity of α 2-adrenergic autoreceptors in early protein malnourished animals. These results stress the complexity of plastic changes induced by early protein malnutrition and sustain the hypothesis that perinatally protein-deprived rats may represent a useful animal model for screening antipanic agents.

Effects of caloric, protein, and sodium deprivation on the affiliative behavior of Norway rats (Rattus norvegicus).

The tendency of food-deprived, protein-deprived, and sodium-deprived Norway rats (Rattus norvegicus) and their respective controls to affiliate with conspecifics deprived of either food, protein, or sodium was examined. The authors found that (a) independent of internal state, focal rats offered a forced choice between protein-deprived and protein-replete target rats spent more time near replete than deprived target rats; and (b) both food-deprived and sodium-deprived focal rats offered a forced choice between food-deprived and replete target rats spent less time near fasted rats than did well-fed and sodium-replete focal rats. The data indicate that (a) rats can distinguish both food-deprived and protein-deprived rats from replete rats and (b) the deprivation states of rats can affect their willingness to affiliate with deprived conspecifics.

CRH and NPY are not decreased in hypophagic, protein-deprived rats

To test if elevated CRH and decreased NPY might account for pituitary-adrenal activity and hypophagia in dietary protein deprivation, rats received normal or protein-free diet, or were food-or weight-restricted to match effects of protein deprivation. Protein or food restriction increased plasma ACTH. However, hypothalamic CRH mRNA was unchanged by protein deficiency and significantly decreased by food restriction when protein intake was > 50% of normal. Arcuate nucleus NPY mRNA increased in rats given protein-free diet, correlating with leptin rather than decreased feeding. We conclude that CRH and NPY gene expression does not explain adrenocortical axis or feeding activity in protein-deprived rats.

Suppression of Rat Hepatic Cytochrome P450s by Protein-Calorie Malnutrition: Complete or Partial Restoration by Cysteine or Methionine Supplementation

Pharmacokinetic profiles of therapeutic agents are altered by protein-calorie malnutrition (PCM). The current study was designed to determine the expression of hepatic cytochrome P450s in rats after protein restriction and to investigate its molecular basis. Western blot analysis revealed that rats with protein restriction for 4 weeks exhibited marked suppression in the hepatic P450 1A2, 2C11, 2E1, and 3A1/2 levels. Northern blot analysis showed that hepatic P450 1A2, 2C11, and 3A1/2 mRNAs were significantly decreased in the state of PCM. The P450 2E1 mRNA level was slightly decreased in PCM rats, suggesting the possibility that expression of P450 2E1 affected by PCM might result from the transcriptional and/or posttranscriptional regulation. PCM-induced changes in most P450 expression completely or partially returned to control levels by a week of cysteine supplementation. Cysteine also prevented decreases in P450 1A2, 2C11, 2E1, and 3A1/2 mRNA levels by PCM. Methionine was minimally active in restoring the P450 expression. A metabolic change in hepatic ethoxyresorufin dealkylase activity in PCM rats was consistent with the P450 apoprotein and mRNA levels. Although the plasma concentrations of azosemide, a loop diuretic, primarily metabolized by cytochrome P450 1A, increased in protein-deprived rats, cysteine supplementation significantly reduced the increased plasma concentrations of the drug. The altered pharmacokinetic parameters of azosemide in PCM rats returned to those of control after cysteine supplementation, corroborating the conclusion that cysteine was effective in restoring cytochrome P450 expression and metabolic activities.