Decreased insulin secretion in islets from rats fed a low protein diet is associated with a reduced PKAalpha expression

A low protein diet has been shown to affect the amount and activity of several enzymes and to decrease insulin secretion by islets isolated from rats fed such a diet. To understand the mechanisms involved in this phenomenon, we investigated the effects of forskolin, a stimulator of adenylyl cyclase, on insulin secretion by pancreatic islets from rats fed a normal (17%; NP) or low (6%; LP) protein diet for 8 wk. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 8.3 mmol glucose/L, with or without 10 micromol forskolin/L. The forskolin-induced insulin secretion was higher in islets from NP rats than in those from LP rats (P<0.05). Western blotting revealed that the amount of the alpha catalytic subunit of protein kinase A (PKAalpha) was 35% lower in islets from LP rats than in islets from NP rats (P<0.05). Moreover, PKAalpha mRNA expression was reduced by 30% in islets from LP rats (P<0.05). Our results indicated a possible relationship between a low protein diet and a reduction in PKAalpha expression. These alterations in PKAalpha may be responsible in part for the decreased insulin secretion by islets from rats fed a low protein diet.     

Emergence of ageing-related changes in insulin secretion by pancreatic islets of male rat offspring of mothers fed a low-protein diet

Maternal low-protein (LP) diets programme β-cell secretion, potentially altering the emergence of ageing of offspring pancreatic function. We hypothesised that isolated pancreatic islet β-cell secretory responses are blunted in offspring exposed to LP during development and age-related reduction is influenced by the developmental stage of exposure to decreased nutrition. We studied male offspring of rats fed control (C) or LP protein (R) diets in pregnancy, first letter and/or lactation second letter of CC, RR, CR or RC groups. Serum glucose, insulin and homeostatic model assessment (HOMA) were measured. Pancreatic islets were isolated and in vitro insulin secretion quantified in low (LG - 5 mM) or high glucose (HG - 11 mM). Body weight and serum values between groups were similar at all ages. Insulin and HOMA rose with age and were highest at postnatal day (PND) 450 in all groups. At PND 36, insulin secretion was greatest in RR and RC. Only CC increased insulin secretion to HG. By PND 110, restricted groups responded less to LG but increased secretion to HG. By PND 450, CC offspring alone increased secretion to HG. Despite minimal differences in circulating insulin and glucose, reduced maternal protein intake affected insulin secretion at all ages. In addition, ageing reduced function in all R groups compared with CC by PND 110 and further by PND 450 most markedly in RC. We conclude that maternal LP diet during pregnancy and/or lactation impairs offspring insulin secretory response to a glucose challenge and alters the trajectory of ageing of pancreatic insulin secretion.     

A protein-restricted diet during pregnancy alters in vitro insulin secretion from islets of fetal Wistar rats

Previous studies indicate that insulin secretion from the fetuses of dams fed a low protein (LP) diet is reduced in response to leucine or arginine. The aim of this study was to locate the defect in the insulin secretion pathway induced by a LP diet during gestation. The effects of various secretagogues acting at different levels of the insulin secretion cascade were investigated in vitro in fetal islets from dams fed either a normal or a LP diet during pregnancy. Insulin content, insulin secretion and the cAMP content were then measured. Although insulin content of LP islets did not differ from that of control islets, insulin secretion from LP fetal islets was reduced when challenged by amino acids or cAMP enhancers. This reduction did not appear to be related solely to an altered islet cAMP content. An impairment of insulin secretion remained after stimulation of fetal LP islets with either metabolic or nonmetabolic secretagogues. The insulin secretion by LP islets was restored to normal, however, with barium or cytochalasin-B. These findings demonstrate that an in utero isocaloric LP diet impairs insulin secretion of the fetus. This alteration is located at the exocytosis step in the insulin secretion cascade and not in the insulin pool of the beta cell.     

Decreased cholinergic stimulation of insulin secretion by islets from rats fed a low protein diet is associated with reduced protein kinase calpha expression

Undernutrition has been shown to affect the autonomic nervous system, leading to permanent alterations in insulin secretion. To understand these interactions better, we investigated the effects of carbamylcholine (CCh) and phorbol 12-myristate 13-acetate (PMA) on insulin secretion in pancreatic islets from rats fed a normal (17%; NP) or low (6%; LP) protein diet for 8 wk. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 8.3 mmol glucose/L, with or without PMA (400 nmol/L) and CCh. Increasing concentrations of CCh (0.1-1000 micro mol/L) dose dependently increased insulin secretion by islets from both groups of rats. However, insulin secretion by islets from rats fed the NP diet was significantly higher than that of rats fed the LP diet, and the dose-response curve to CCh was shifted to the right in islets from rats fed LP with a 50% effective concentration (EC(50)) of 2.15 +/- 0.7 and 4.64 +/- 0.1 micro mol CCh/L in islets of rats fed NP and LP diets, respectively (P < 0.05). PMA-induced insulin secretion was higher in islets of rats fed NP compared with those fed LP. Western blotting revealed that the protein kinase (PK)Calpha and phospholipase (PL)Cbeta(1) contents of islets of rats fed LP were 30% lower than those of islets of rats fed NP (P < 0.05). In addition, PKCalpha mRNA expression was reduced by 50% in islets from rats fed LP. In conclusion, a reduced expression of PKCalpha and PLCbeta(1) may be involved in the decreased insulin secretion by islets from LP rats after stimulation with CCh and PMA.     

Low-protein diets reduce PKAalpha expression in islets from pregnant rats

We investigated the effect of protein restriction on insulin secretion and the expression of protein kinase (PK)Aalpha and PKCalpha in islets from control and pregnant rats. Adult control nonpregnant (CN) and control pregnant (CP) rats were fed a normal-protein diet (17%), whereas low-protein nonpregnant (LPN) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) for 15 d. In the presence of 2.8 and 8.3 mmol glucose/L, insulin secretion by islets of CP rats was higher than that by islets of CN rats. Compared with the CN groups, insulin secretion by islets of LPN rats was lower with 8.3 but not with 2.8 mmol glucose/L. The insulin secretion by islets of LPP rats was higher than by LPN rats at both glucose concentrations. IBMX (1 mmol/L), a phosphodiesterase inhibitor, increased insulin secretion by islets from pregnant rats, and this effect was greater in islets of CP rats than in LPP rats. Forskolin (0.01-100 micromol/L), a stimulator of adenylyl cyclase, increased insulin secretion only in islets of CN and CP rats, with a higher 50% effective concentration in islets of CP rats compared with CN rats. The insulin secretion induced by phorbol 12-myristate 13-acetate (a stimulator of PKC) was higher in islets of LPN and LPP rats than in the respective controls, especially at 8.3 mmol glucose/L. PKAalpha, but not PKCalpha, expression was lower in islets of rats fed low protein than in the controls, regardless of the physiological status of the rats. All endocrine cells of the islets, including beta-cells, expressed the PKAalpha isoform. The cytoplasmic distribution of this enzyme in beta-cells was not modified by pregnancy and/or protein restriction. In conclusion, our results indicate that the response of islets from rats fed low protein during pregnancy is similar to that of control rats, at least for physiologic glucose concentration. However, the decreased response to IBMX and forskolin indicates decreased production and/or sensitivity to cAMP; this was associated with a decrease in PKA expression, which may result in lower PKA activity.     

Moderate physical training attenuates muscle-specific effects on fibre type composition in adult rats submitted to a perinatal maternal low-protein diet

Aim

To verify whether moderate physical training affects the muscle fibre composition of adult rats subjected to a low protein diet during the perinatal period.

Methods

Male Wistar rats were divided into two groups according to their mother’s diet during gestation and lactation: control (17% casein, C) and low-protein (8% casein, LP). On postnatal day 60, half of each group was submitted to moderate physical training (8?weeks, 5?days/week^?1, 60?min/day^?1, at 70% of VO_2max, T) or not. After the physical training period, soleus and extensor digitorum longus (EDL) muscles were removed. Myofibrillar ATPase staining was used to classify muscle fibres as type I, IIa, IIb, and intermediate.

Results

In the EDL muscle, LP rats showed no changes in the fibre type proportion. Both the C?+?T and LP?+?T groups showed a higher percentage of fibres of type IIa, and a lower proportion of fibres of type IIb. In the soleus muscle, LP animals showed a reduction in the proportion of fibre types I and intermediate. C?+?T rats showed an increase in the fibre type I and IIa. In the LP?+?T rats, the proportions of the fibre types remained similar to control rats.

Conclusions

Moderate physical training acts as a positive environmental stimulus that reverts the effects of a perinatal low-protein diet on the proportion of fibre types in skeletal muscle.     

Plasma proteins in growing analbuminaemic rats fed on a diet of low-protein content

1. Analbuminaemic and Sprague-Dawley (control) rats were fed on low- (60 g/kg) protein and control (200 g protein/kg) diets ad lib. from weaning. Males and females were studied separately. Body-weight and plasma protein concentrations were determined at 10 d intervals from 25 to 75 d of age. Electrophoresis of plasma proteins was performed in samples from day 75. Extracellular fluid volume was measured at 10 d intervals from day 45 onwards. Colloid osmotic pressure was measured in plasma and interstitial fluid (wick technique) at the start and end of the trial. 2. Body-weight increased much less on the low-protein diet than on the normal diet in both strains and sexes. The growth retardation was slightly more pronounced in the male analbuminaemic rats than in the male Sprague-Dawley controls. 3. Plasma protein concentration increased during normal growth in all groups, particularly in the female analbuminaemic rats. This increase was reduced by the 60 g protein/kg diet in all groups, with the exception of the male analbuminaemic rats. 4. Differences in plasma colloid osmotic pressure were similar to those seen in plasma protein concentration. Interstitial colloid osmotic pressure was higher in the control rats than in the analbuminaemic ones. The interstitial colloid osmotic pressure increased during growth in the control but not in the analbuminaemic rats. The difference in interstitial colloid osmotic pressure between the strains was maintained during low-protein intake, but at a lower level than during normal protein intake. 5. Subtracting interstitial from plasma colloid osmotic pressure, resulted in a rather similar transcapillary oncotic gradient in the various groups at 75 d, both on the control protein diet (11-14 mmHg), and on the low-protein diet (9-11 mmHg). 6. All protein fractions were reduced to a similar extent by the low-protein diet in the control rats, whereas in the analbuminaemic rats protein fractions produced in the liver were more severely depressed. 7. Extracellular fluid volume as a percentage of body-weight was similar in all groups, and decreased with increasing age. 8. In conclusion, the analbuminaemic rats were able to maintain the transcapillary oncotic gradient on both diets by reducing the interstitial colloid osmotic pressure. Oedema was not observed. 9. Despite the absence of albumin, the protein-malnourished analbuminaemic rat is no more susceptible to hypoproteinaemia and oedema than its normal counterpart.     

Evidence of progressive deterioration of renal function in rats exposed to a maternal low-protein diet in utero

Intrauterine growth retardation associated with maternal undernutrition is proposed to play a significant role in the aetiology of hypertension and CHD. Animal experiments suggest that the kidney, which is extremely vulnerable to the adverse effects of growth-retarding factors, may play an important role in the prenatal programming of hypertension. Maintenance of renal haemodynamic functions following structural impairment in fetal life is proposed to require adaptations which raise systemic blood pressure and promote a more rapid progression to renal failure. Rats were fed on diets containing 180 g casein/kg (control) or 90 g casein/kg (low protein) during pregnancy. The offspring were studied in terms of blood pressure, creatinine clearance, blood urea N, plasma and urinary albumin, renal morphometry and metabolic activity at 4, 12 and 20 weeks of age. Blood pressure was elevated at all ages in the low-protein-exposed offspring, relative to control rats. Rats (4 weeks old) exposed to the low-protein diet had smaller kidneys which were shorter and wider than those of control animals. Creatinine clearance was significantly reduced in 4-week-old rats exposed to the low-protein diet. Renal morphometry and creatinine clearance at older ages were not influenced by prenatal diet. Blood urea N, urinary output and urinary albumin excretion were, however, significantly greater in low-protein-exposed rats than in control rats at 20 weeks of age. These findings are suggestive of a progressive deterioration of renal function in hypertensive rats exposed to mild maternal protein restriction during fetal life. This is consistent with the hypothesis that adaptations to maintain renal haemodynamic functions following impairment of fetal nephrogenesis result in an accelerated progression towards glomerulosclerosis and increased intrarenal pressures mediated by rising vascular resistance.     

Effects of low-protein diet on the baroreflex and Bezold-Jarisch reflex in conscious rats

The present study evaluated the effects of a low-protein diet (LP, 6% protein) on cardiovascular reflexes of Male Fisher rats. Three experimental groups, and their respective controls (15% protein), were used: (1) Baroreceptor reflex (BAR); (2) Bezold-Jarisch reflex (BJR); and (3) Prazosin treated. Dietary restriction began after weaning (three weeks) and lasted for a period of five weeks, after which animals were subjected to the experimental protocols. The BAR group was evaluated through injections of phenylephrine (0.5-5.0 microgram/Kg, i.v.) and sodium nitroprusside (0.7-7.0 microgram/Kg, i.v.) while the BJR was evaluated through injections of serotonin (2.5-10 microgram/Kg, i.v.). Our results showed an increased baroreflex gain bradycardia for the LP group (-0.96+/-0.34 vs. -2.12+/-1.06 bpm/mmHg) and a larger bradycardia for the BJR the LP group (160+/-18% greater than controls). Basal cardiovascular parameters were not different between LP and control rats, however LP animals treated with prazosin resulted in a larger fall of blood pressure (-19+/-3 vs. -28+/-5 mmHg). In conclusion, LP rats present an increased responsiveness of BAR and BJR, which could contribute to their normal levels of cardiovascular parameters, in spite of the possible increase in the sympathetic vasomotor tonus observed with the prazosin protocol.     

Muscle lipid metabolism and insulin secretion are altered in insulin-resistant rats fed a high sucrose diet

Feeding rats a sucrose rich diet (SRD) induces hypertriglyceridemia and insulin resistance. The purposes of this study were to determine the time course of changes in lipid and glucose metabolism in the gastrocnemius muscle, both in the basal state and after the euglycemic hyperinsulinemic clamp, in rats fed a SRD for 3, 15 or 30 wk, and to analyze the changes in glucose-stimulated insulin secretion from perifused isolated islets from SRD-fed rats and their relationships to peripheral insulin insensitivity. A control group of rats was fed a control diet (CD) for the same period of time. After 3 wk of consuming the SRD, long-chain acyl CoA (LCACoA) levels in muscle were greater than in rats fed the CD, an early indication of the disturbance of lipid metabolism. Neither glycogen storage nor glucose oxidation were impaired at this time. Moreover, the biphasic patterns of glucose-stimulated insulin secretion showed a marked increase in the first peak, which helped maintain normoglycemia in SRD-fed rats. After 15 or 30 wk of consuming the SRD, triglyceride and LCACoA levels in muscles were greater than in rats fed the CD. Glucose oxidation as well as insulin-stimulated glycogen synthase activity and glycogen storage were lower than in rats fed the CD. Moreover, the altered pattern of insulin secretion further deteriorated. This was accompanied by peripheral insulin resistance and moderate hyperglycemia. Our results indicate that the dyslipemia present in rats chronically fed a SRD may play an important role in the progressive deterioration of insulin secretion and sensitivity in this animal model.     

Quantitative ultrastructural evidence of myelin malformation in optic nerves of rats submitted to a multideficient diet

Pups were subjected to malnutrition by feeding the lactating mothers a multi-deficient (8% protein content) diet, known as regional basic diet (RBD), from birth up to weaning. The weanings were fed the same diet until 60 days of age. Ultrastructure of the optic nerve was analyzed at postnatal (P) day P8, P13, P21, P30 and P60. Electron microscopy revealed that at P8 the process of myelination has not started yet in neither groups. At P 13 different stages of myelination were observed and, in the experimental group, the optic nerve showed non-organized axon bundles and empty spaces. Control optic nerve at P21 exhibited axons with fully developed myelin sheath; whereas malnourished group had many axons being enveloped by myelin with anomalous alteration. These alterations were present in malnourished group at P30 and P60. Quantitative analysis showed statistically significant difference between control and malnourished groups when compared to the percentage of myelinated axons, axons with myelin anomalous alterations (MAA) and non-myelinated axons. Also, the myelin area was significantly smaller in malnourished groups when compared to control group. Finally, a high percentage of large non-myelinated fibers were found in the malnourished group. In conclusion, our results show that early malnutrition by a multideficient diet (RBD) affects permanently the optic nerve organization and myelination, indicating an impairment of nerve transmission and a probable dysfunction in the visual ability.     

Short- and long-term effects of a neonatal low-protein diet in rats on the morphology of the larynx

ObjectiveThe aim of this study was to investigate the short- and long-term effects of a maternal low-protein diet during lactation on offspring laryngeal morphology. Our hypothesis was that a neonatal low-protein diet during the critical period of development alters micro- and macroscopic structures of the larynx in adult rats.MethodsMale Wistar rats were assigned to a control (casein 17%, n = 24) or low-protein (casein 8%, n = 24) group according to their mother's diet during lactation. Body weight gain and growth rate were recorded throughout the experiment. The larynx was removed from offspring at days 22 and 60 of life. The anteroposterior and laterolateral lengths of the cartilages epiglottis, thyroid, and cricoid were measured by a digital caliper. The supraglottis, glottis, infraglottis, and vocal cords were stained by hematoxylin–eosin and their structures were analyzed by a Scion Image Beta 4.0.2 program.ResultsPups from mothers fed a low-protein diet showed a lower body weight gain. The laterolateral and anteroposterior lengths of the larynx were shorter in undernourished offspring at 22 d old. There were no differences in the structure of the supraglottis, glottis, and infraglottis between groups except for keratinization in pups from undernourished mothers. The microstructure of the vocal cords was changed only at 60 d old.ConclusionMacroscopic structures of the larynx are sensitive to short-term effects of a neonatal low-protein diet. Vocal cord development can be studied within the context of programming because their microscopic structures are sensitive to the long-term effects of a low-protein diet during lactation.     

Transient hypophagia in rats switched from high-fat diets with different fatty-acid pattern to a high-carbohydrate diet

The present study investigates the mechanisms underlying the transient hypophagia occurring when rats adapted to high-fat, carbohydrate-free diets are switched to high-carbohydrate, low-fat diets. The hypophagia after the high-fat, carbohydrate-free to high-carbohydrate, low-fat diet shift seems to depend on the amount of carbohydrate in the diet, since an attenuation of hypophagia was observed when high-fat, carbohydrate-free-adapted rats were switched to a medium-carbohydrate, medium-fat diet. A role of glucose intolerance in the hypophagia is supported by the attenuation of carbohydrate anorexia in rats adapted to a high-fat diet containing n -3 polyunsaturated fatty acids from fish oil (60% of fat as fish oil), which has been shown to improve glucose tolerance in rats. Furthermore, the increased plasma glucose concentration in the high-fat, carbohydrate-free diet to high-carbohydrate, low-fat shifted rats despite the suppression in food intake also suggests an involvement of glucose intolerance in the hypophagia. The failure of the inhibitor of hepatic-fatty-acid oxidation mercaptoacetate (400 micromol/kg, i.p.) to counteract carbohydrate anorexia in the HF-adapted rats argues against an involvement of fatty-acids oxidation in the inhibition of eating after high-fat, carbohydrate-free to high-carbohydrate, low-fat diet shift. This is also supported by the failure to demonstrate a relationship between plasma beta-hydroxybutyrate and the severity of the hypophagia. A role of leptin in the hypophagia seems unlikely, since plasma leptin after diet shift was unchanged. Ingestion of the high-carbohydrate, low-fat diet also produced an aversion towards this diet in high-fat, carbohydrate-free-adapted rats. It is concluded that the transient hypophagia induced by switching rats from a high-fat to a high-carbohydrate diet is not related to fatty acid oxidation but to transiently impaired carbohydrate utilization.     

L-threonine supplementation increases the amplitude of the feed intake cycle of rats fed a low-protein zinc-deficient diet

Rats fed a Zn-deficient diet show characteristic variations in feed intake. These variations were followed by means of a personal computer. The specific feed intake patterns in rats fed a zinc-deficient diet before and after supplementation with protein and several essential amino acids were determined. The high-protein diet decreased the amplitude of feed intake under zinc deficiency, probably because of a decrease in sensitivity to the deficiency. Furthermore, the zinc-deficient diet was supplemented with essential amino acids, and of them L-threonine showed the most marked effect on the increased variability of feed intake.     

Effect of a high-protein diet on insulin and glucagon secretion in ventromedial hypothalamic (VMH) lesioned rats

The effects of a high-protein diet on insulin and glucagon secretion in ventromedial hypothalamic (VMH) lesioned and sham-operated (sham) rats were studied in vivo as well as in perfusate from isolated pancreas. Two weeks after VMH destruction or sham operation, the rats were given either a balanced diet (protein 27%, carbohydrate 61%, fat 12%) or a high-protein diet (protein 55%, carbohydrate 30%, fat 15%) for the following 2 weeks. The calorie intake and body weight changes after the commencement of the diets were almost the same in the groups of VMH lesioned rats, but these were much greater than those in the two sham-operated groups. Fasting blood glucose, plasma insulin, and plasma glucagon concentrations were also similar between the two VMH groups, but in the sham-operated rats fasting blood glucose and plasma insulin concentrations of those rats on high-protein diet were significantly increased when compared to those on balanced diet. In the isolated, perfused pancreas, an arginine-induced excess insulin and glucagon secretion was not significantly different between the VMH lesioned rats. An arginine-induced rise in insulin concentration in the sham-operated rats on high-protein diet was significantly higher than for rats on balanced diet. We therefore suggest that hyperinsulinemia already produced in the VMH lesioned rats may not be influenced by the change in the composition of the dietary protein and carbohydrate.     

Fetal exposure to a maternal low-protein diet during mid-gestation results in muscle-specific effects on fibre type composition in young rats

This study assessed the impact of reduced dietary protein during specific periods of fetal life upon muscle fibre development in young rats. Pregnant rats were fed a control or low-protein (LP) diet at early (days 0-7 gestation, LPEarly), mid (days 8-14, LPMid), late (days 15-22, LPLate) or throughout gestation (days 0-22, LPAll). The muscle fibre number and composition in soleus and gastrocnemius muscles of the offspring were studied at 4 weeks of age. In the soleus muscle, both the total number and density of fast fibres were reduced in LPMid females (P = 0.004 for both, Diet x Sex x Fibre type interactions), while both the total number and density of glycolytic (non-oxidative) fibres were reduced in LPEarly, LPMid and LPLate (but not LPAll) offspring compared with controls (P < 0.001 for both, Diet x Fibre type interaction). In the gastrocnemius muscle, only the density of oxidative fibres was reduced in LPMid compared with control offspring (P = 0.019, Diet x Fibre type interaction), with the density of slow fibres being increased in LPAll males compared with control (P = 0.024, Diet x Sex x Fibre type interaction). There were little or no effects of maternal diet on fibre type diameters in the two muscles. In conclusion, a maternal low-protein diet mainly during mid-pregnancy reduced muscle fibre number and density in 4-week-old rats, but there were muscle-specific differences in the fibre types affected.     

Effect of a neonatal low-protein diet on the morphology of myotubes in culture and the expression of key proteins that regulate myogenesis in young and adult rats

Aim  

To investigate the effects of a neonatal low-protein diet on the morphology of myotubes in culture and the expression of key proteins that regulate myogenesis in young and adult rats.