Differential effects of dietary caloric and protein restriction in the aging rat

Numerous studies have shown caloric restriction retards the physiological decline and increases the life span of animals. However, in these studies protein consumption was also reduced; thus, whether the beneficial effects were due to caloric or to protein restriction is unclear. To examine independently the effects of caloric and protein restriction on growth, renal function, and survival, caloric restricted male rats were fed 18, 30 or 42 percent casein diets that provided two-thirs of the quantity of diet consumed by groups fed 12, 20, or 28 percent casein diets ad libitum, respectively. Hence, caloric restricted groups consumed the same amount of protein as their paired ad libitum fed groups but one-third fewer calories. The results showed that caloric restriction decreased mature body weight, increased the rate of attaining mature body weight, retarded the age-associated decline in renal function, and increased survival. Protein restriction had no effect on mature body weight, decreased maturation rate, improved renal function, and decreased survival. Thus, protein restriction did not contribute to the survival-promoting effects of caloric restriction in rats.     

短期能量限制对正常大鼠胰岛素敏感性的影响

目的探讨短期能量限制（CR）对正常大鼠胰岛素敏感性的影响及其机制。方法将24只F344/NSIc系雄性大鼠随机分为对照组（AL组）和能量限制组（CR组）各12只。AL组自由摄入食物及水,CR组限制食物摄入（不禁水）8周（饲料总摄入量为对照组的64%）。采用高胰岛素—正常血糖钳夹试验测定两组葡萄糖输注率（GIR）判断其胰岛素敏感性（GIR越高,胰岛素敏感性越高）;比较两组体质量增加及内脏脂肪重量;Western blot法检测骨骼肌葡萄糖转运体4（Glu T4）、蛋白激酶B底物蛋白160（AS160）及磷酸化AS160（p-AS160）蛋白表达。结果CR组GIR明显高于对照组,体质量及内脏脂肪重量明显低于对照组;两组Glu T4、AS160及p-AS160蛋白表达无显著差异。结论短期CR可提高大鼠正常状态下的胰岛素敏感性,其机制可能与AS160上游的胰岛素信号转导蛋白有关。     

Effects of caloric restriction on insulin pathway gene expression in the skeletal muscle and liver of normal and long-lived GHR-KO mice

Growth hormone receptor/binding protein knockout (GHR-KO) mice are characterized by resistance to growth hormone (GH), reduced insulin like growth factor 1 (IGF1) levels and enhanced insulin sensitivity and markedly increased lifespan. Findings in these and other long-lived mutant mice, and in normal animals subjected to caloric restriction (CR) indicate that insulin signaling is importantly involved in the control of longevity. We have examined the mRNA expression level of genes involved in insulin/IGF1 action in the skeletal muscle and liver of normal and GHR-KO mice fed ad libitum or subjected to long term 30% CR. The levels of IR, IRS1, IRS2, GLUT4 and IGF1 message in the skeletal muscle were reduced by CR in both normal and GHR-KO mice. In the liver, the results indicate that in GHR-KO mice mRNA expression of genes related to early steps of insulin signaling is up-regulated in the liver but not in the muscle. The results also show that improved insulin sensitivity in response to CR is not due to increased mRNA expression of the above genes in either normal or GHR-KO animals.     

Effects of long-term caloric restriction on early steps of the insulin-signaling system in mouse skeletal muscle

In this study, we analyzed the effects of long-term (14 months) caloric restriction (CR) on the first steps of the insulin signaling system in skeletal muscle of normal mice. CR induced a significant decrease in serum insulin and glucose levels, indicating an enhancement of insulin sensitivity. CR reduced the in vivo insulin-induced phosphorylation of the insulin receptor substrate (IRS)-1 by 27%, but this difference was not significant (p =.298). CR reduced insulin receptor (IR) abundance by 34% from the ad libitum values, but this difference did not reach significance (p =.246). The abundance of the p85 regulatory subunit of PI3K and glucose transporter 4 was unaltered after CR. However, IRS-1 abundance was significantly increased by 42% in muscle of mice exposed to CR. These findings indicate that the CR-induced improvement of insulin action in mice is not related to changes in glucose transporter 4, the p85 regulatory subunit of PI3K, or IR abundance in skeletal muscle but might be related to an increase in IRS-1 abundance in this tissue.     

In vivo insulin resistance in individual peripheral tissues of the high fat fed rat: assessment by euglycaemic clamp plus deoxyglucose administration

Summary We have examined peripheral insulin action in conscious rats chronically fed high fat (60% calories as fat) or high carbohydrate (lab chow) diets using the euglycaemic clamp plus 3 H-2-deoxyglucose technique. A response parameter of individual tissue glucose metabolic rate (the glucose metabolic index, based on tissue deoxyglucose phosphorylation) was used to assess diet effects in eight skeletal muscle types, heart, lung and white and brown adipose tissue. Comparing high fat with high carbohydrate fed rats, basal glucose metabolism was only mildly reduced in skeletal muscle (only diaphragm was significant,p<0.05), but was more substantially reduced in other tissues (e.g. white adipose tissue 61% and heart 33%). No evidence of basal hyperinsulinaemia was found. In contrast, widespread insulin resistance was found during the hyperinsulinaemic clamp (150 mU/l) in high fat fed animals; mean whole body net glucose utilization was 34% lower (p<0.01), and the glucose metabolic index was lower in skeletal muscle (14 to 56%,p< 0.05 in 6 out of 8 muscles), white adipose (27%,p<0.05) and brown adipose tissue (76%,p<0.01). The glucose metabolic index was also lower at maximal insulin levels in muscle and fat, suggesting the major effect of a high fat diet was a loss of insulin responsiveness. White adipose tissue differed from muscle in that incremental responses (maximal insulin minus basal) were not reduced by high fat feeding. The heart showed an effect opposite to other tissues, with an increase in insulin-stimulated glucose metabolism in high fat versus chow fed rats. We conclude that high fat feeding, without a major increase in body weight or basal hyperinsulinaemia, causes widespread but varying degrees of in vivo insulin resistance in peripheral tissues, with major effects in principally oxidative skeletal muscle.     

Calorie restriction initiated at middle age improved glucose tolerance without affecting age-related impairments of insulin signaling in rat skeletal muscle

Calorie restriction (CR) may affect glucose tolerance via modulation of the insulin action in skeletal muscle. The present study investigated the effect of CR initiated at middle age in rats bearing glucose intolerance, in comparison with CR at a younger age. Male F344 rats at 2.5 and 18months (mo) of age were fed ad libitum (AL) or 30% CR diets for 4-4.5mo, subjected to glucose tolerance testing, and then sacrificed 15min after intraperitoneal glucose or saline injection to evaluate glucose-stimulated insulin response and subsequent activation of insulin signaling molecules. The protein abundance of phosphorylated (p) insulin receptors, p-Akt, and p-atypical PKC and the membrane fraction of glucose transporter 4 in quadriceps femoris muscle (QFM) were analyzed by EIA or immunoblotting. CR initiated either at young or middle age improved glucose tolerance with a lower serum insulin response to glucose. However, middle-aged CR did not improve aging-related impairments in insulin signaling in QFM. The present results emphasized the possibilities of CR activation of an insulin-independent mechanism in skeletal muscle and also of the involvement of non-muscle tissues in glucose uptake.     

Normal insulin receptor tyrosine kinase activity and glucose transporter (GLUT 4) levels in the skeletal muscle of hyperinsulinaemic hypertensive rats

Summary The spontaneous hypertensive rat is an animal model characterized by a syndrome of hypertension, insulin resistance and hyperinsulinaemia. To elucidate whether in analogy to other insulin resistant animal models an inactivity of the insulin receptor kinase or an alteration of the glucose transporter (GLUT 4) level in the skeletal muscle might contribute to the pathogenesis of insulin resistance we determined insulin receptor kinase activity and GLUT 4 level in the hindlimbs of spontaneous hypertensive rats and normotensive control rats. Normotensive normoinsulinaemic Lewis and Wistar rats were used as insulin sensitive controls, obese Zucker rats were used as an insulin resistant control with known reduced skeletal muscle insulin receptor kinase activity. Binding of ¹²⁵I-insulin, crosslinking of ¹²⁵I-B²⁶-insulin, autophosphorylation in vitro with ³²P-ATP and phosphorylation of the synthetic substrate Poly (Glu 4: Tyr 1) were performed after partial purification of solubilized receptors on wheat germ agglutinin columns. GLUT 4 levels were determined by Western blotting of subcellular muscle membranes. Insulin receptors from spontaneous hypertensive rats compared to those from Lewis and Wistar rats showed no difference of the binding characteristics or the in vitro auto- and substrate phosphorylation activity of the receptor, while in the Zucker rats the earlier described insulin receptor kinase defect was clearly evident. Western blots of subcellular muscle membrane fractions with antibodies against GLUT 4 revealed no difference in transporter levels. These data suggest that insulin resistance in spontaneous hypertensive rats is caused neither by an insulin receptor inactivity nor by a decreased number of glucose transporters in the skeletal muscle.     

Chronic caloric restriction alters muscle membrane fatty acid content

Chronic caloric restriction (CR) has been demonstrated to increase longevity in lower species and studies are ongoing to evaluate its effect in higher species. A consistent metabolic feature of CR is improved insulin sensitivity and lowered lifetime glycemia, yet the mechanism responsible is currently unknown. However, the membrane's physiochemical properties, as determined by phospholipid composition, have been related to insulin action in animal and human studies and CR has been reported to alter membrane lipid content. We evaluated muscle membrane fatty acid content in rodents randomized to CR versus control diets for up to 29 months. CR was observed to increase the membrane content of C22:6 (docosahexaenoate) and to decrease C18:2 content. The membrane lipid content was related to insulin levels but not to parameters assessing glycemic control. This study suggests that membrane lipids, in particular 22:6, may contribute to the variation in insulin sensitivity seen with age.     

Caloric restriction in obese pre-diabetic rats prevents beta-cell depletion,loss of beta-cell GLUT 2 and glucose incompetence

Summary Pre-diabetic male Zucker diabetic fatty rats (ZDF) become diabetic between 8 and 10 weeks of age. At that time their beta cells exhibit high basal insulin secretion, absent insulin response to glucose and loss of GLUT 2 glucose transporter. Beta-cell volume, which is increased at the onset of non-insulin-dependent diabetes, declines precipitously by age 18 weeks. To determine if expression of this diabetic phenotype was dependent upon the increased food intake of these rats, they were diet-matched to lean littermates for 12 weeks beginning at 6 weeks of age. Untreated control ZDF rats received an unrestricted diet for 3 months. All of the controls became hyperglycaemic by 8 weeks of age, whereas all diet-matched rats remained euglycaemic throughout the 3 months, despite the fact that at 18 weeks of age their mean body weight equaled that of obese rats on an unrestricted diet. In the former rats glucose-stimulated insulin secretion was absent at 12 weeks of age and GLUT-2-positive beta cells had fallen below 30%. The volume fraction of their beta cells was 2.6 times normal at this age but by 18 weeks of age it had declined by 75%. Diet restriction for 3 months prevented the loss of glucose-stimulated insulin secretion and the reduction of beta-cell GLUT-2 and beta-cell volume fraction. However, neither the elevated basal insulin secretion nor the exaggerated arginine-stimulated insulin secretion of the obese rats was reversed or prevented by caloric restriction. We conclude that in diabetic ZDF rats the glucose incompetence of beta cells and the reduction of beta-cell GLUT 2, which coincide with the onset of hyperglycaemia, and the subsequent loss of beta-cell volume, occur only when the caloric intake is excessive. The increased basal insulin secretion and exaggerated insulin response to arginine appear to be relatively independent of caloric intake.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - ZDF Zucker diabetic fatty rats - GSIS glucose-stimulated insulin secretion - GLUT-2 glucose transporter     

Glut-1 translocation in FRTL-5 thyroid cells: role of phosphatidylinositol 3-kinase and N-glycosylation

It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached the cell surface. Likewise, a decrease in glucose consumption (-50%) after treatment of cells with tunicamycin was accompanied by a decrease (-70% vs. control) in the membrane expression of a 50-kDa form of Glut-1 and an increase in its unglycosylated 41-kDa form. These results suggest that carbohydrate moiety is essential for the biological activity of glucose transport but is not required for the translocation of Glut-1 from the intracellular membrane pool to the plasma membrane.     

Muscle insulin resistance may not be a primary etiological factor in the genetically obese fa/fa rat.

It is not known whether hyperinsulinemia of the genetically obese fa/fa rat occurs before insulin resistance and abnormal glucose handling or vice versa. Therefore, it was decided to study, as a function of age, the evolution of the insulin-stimulated glucose uptake measuring the in vitro uptake of its analog, 2-deoxy-D-glucose (2DG), by diaphragm. The expression of the insulin-sensitive glucose transporter (GLUT 4) mRNA and protein were also investigated in muscles. The maximum increase over baseline in 2DG uptake in response to increasing insulin concentrations in the medium was upward shifted in diaphragm from preweaned 21-day-old preobese rats relative to that in lean controls (increased responsiveness). By 31 days of age the maximum increase over baseline diaphragm 2DG uptake in response to insulin was similar in young lean and obese rats. At 70 days of age, the 2DG uptake muscle dose response to insulin was significantly downward shifted, i.e. clearly insulin resistant (decreased responsiveness). Muscle (diaphragm and extensor digitorum longus) expression of GLUT 4 mRNA and protein revealed no intergroup difference at any of the ages studied. Hyperinsulinemia was moderate in preobese animals and progressively increased with the duration of the obesity syndrome. Based on the observation that diaphragm glucose uptake of 21-day-old preobese rats was overresponsive to insulin, normoinsulin responsive at 31 days, and insulin resistant at a later time, it is concluded that muscle insulin resistance is not a primary etiological defect, but must be secondary to other pathological alterations, the nature of which remains to be elucidated.     

Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with NIDDM

Summary We investigated the response of the glucose transport system to insulin, in the presence of ambient glucose concentrations, in isolated skeletal muscle from seven patients with non-insulin-dependent diabetes mellitus (NIDDM) (age, 55±3 years, BMI 27.4±1.8 kg/m²) and seven healthy control subjects (age, 54±3 years, BMI 26.5±1.1 kg/m²). Insulin-mediated whole body glucose utilization was similar between the groups when studied in the presence of ambient glucose concentrations (approximately 10 mmol/l for the NIDDM patients and 5 mmol/l for the control subjects). Samples were obtained from the vastus lateralis muscle, by means of an open muscle biopsy procedure, before and after a 40-min insulin infusion. An increase in serum insulin levels from 54±12 to 588±42 pmol/l, induced a 1.6±0.2-fold increase in glucose transporter protein (GLUT4) in skeletal muscle plasma membranes obtained from the control subjects (p<0.05), whereas no significant increase was noted in plasma membrane fractions prepared from NIDDM muscles, despite a similar increase in serum insulin levels. At concentrations of 5 mmol/l 3-O-methylglucose in vitro, insulin (600 pmol/l) induced a 2.2-fold (p<0.05) increase in glucose transport in NIDDM muscles and a 3.4-fold (p<0.001) increase in the control muscles. Insulin-stimulated 3-O-methylglucose transport was positively correlated with whole body insulin-mediated glucose uptake in all participants (r=0.78,p<0.001) and negatively correlated with fasting plasma glucose levels in the NIDDM subjects (r=0.93,p<0.001). Muscle fibre type distribution and capillarization were similar between the groups. Our results suggest that insulin-stimulated glucose transport in skeletal muscle from patients with NIDDM is down-regulated in the presence of hyperglycaemia. The increased flux of glucose as a consequence of hyperglycaemia may result in resistance to any further insulin-induced gain of GLUT4 at the level of the plasma membrane.     

Moderate caloric restriction during gestation results in lower arcuate nucleus NPY‐ and αMSH‐neurons and impairs hypothalamic response to fed/fasting conditions in weaned rats

Aim: We aimed to characterize the developmental programming effects of moderate caloric restriction during early pregnancy on factors involved in hypothalamic control of energy balance. Methods: Twenty‐five‐days‐old offspring Wistar rats from 20% caloric restricted dams (from 1 to 12 days of pregnancy) (CR) and from control dams were studied under fed and 12 h fasting conditions. Morphometric studies on arcuate nucleus (ARC) and determinations of circulating parameters and hypothalamic levels of neuropeptide Y (NPY), proopiomelanocortin (POMC), long‐form leptin receptor (ObRb), insulin receptor (InsR) and suppressor of cytokine signalling‐3 (SOCS‐3) mRNA were performed. Results: CR animals did not show different body weight with respect to their controls, but presented higher food intake. They exhibited lower neuropeptide Y‐ and α‐melanocyte‐stimulating hormone‐neurons (decreases of 18 and 13% in males, and 10 and 18% in females respectively) and lower total cells (decrease of 3% in males and 18% in females) in ARC. Under fed conditions, CR animals presented lower circulating leptin and ghrelin levels (decreases of 37 and 43% in males, and 15 and 34% in females respectively); furthermore, hypothalamic POMC, NPY (only in females), ObRb and InsR mRNA levels were reduced (39, 16 and 26% in males, and 112, 33, 61 and 56% in females), and those of SOCS‐3 were increased (86% in males and 74% in females). Unlike control animals, under fasting conditions, ObRb, InsR and POMC mRNA levels did not decrease in CR females, and NPY mRNA decreased instead of increase in CR males. Conclusions: Moderate caloric restriction during gestation affects offspring hypothalamic structure and function, impairing its response to fed/fasting conditions, which suggests a predisposition to insulin and leptin resistance.     

Influence of severe diabetes mellitus early in pregnancy in the rat: effects on insulin sensitivity and insulin secretion in the offspring

Summary We studied the influence of severe diabetes early in pregnancy on insulin sensitivity and insulin secretion in the offspring. Diabetes (blood glucose >20 mmol/l) was induced in female Sprague-Dawley rats before mating. Diabetic dams were insulin treated during the second half of pregnancy (mean blood glucose 10.6 mmol/l). The offspring were reared by foster mothers. Offspring of both sexes were insulin resistant at four and seven months of age as evidenced by normal glucose tolerance after glucose (2 g/kg body weight intraperitoneally) concomitant with higher than normal rises in insulin levels. Regardless of fetal environment the male rats had higher glucose and insulin levels than the female rats. Insulin responses to glucose (27 mmol/l) in vitro in perfused pancreases were not increased by maternal diabetes, male gender or higher age. Conversely responses to 3-isobutyl-1-methylxanthine (1.0 mmol/l) were enhanced by all three conditions. The pancreatic content of insulin was only marginally affected by maternal diabetes. We conclude that severe diabetes during early pregnancy affects glucose homeostasis in the offspring primarily by diminishing insulin sensitivity and that susceptibility to this effect is not sex- or age-dependent.     

Involvement of insulin-like growth factor-1 in the effect of caloric restriction: regulation of plasma adiponectin and leptin

Insulin-like growth factor (IGF)-1 signaling might partly mediate effects of caloric restriction (CR), an experimental intervention for increasing longevity in mammals. The present study evaluated effects of recombinant human (rh)IGF-1 infusion on adipokine levels in CR and transgenic (Tg) dwarf rats with the reduced growth hormone-IGF-1 axis, which shared similar body weight and food intake. At 9 months of age, each rat received a continuous infusion of rhIGF-1 for 14 days, and rats received an injection of glucose after overnight fasting. Infusion of rhIGF-1 had metabolic effects in all rat groups although it did not affect insulin sensitivity in any of the groups. In addition, plasma adiponectin was decreased to the control group levels and plasma leptin was further reduced in CR and Tg rats. The similarity of phenotypes and adipokine responses to rhIGF-1 between CR and Tg rats supports a role for reduced IGF-1 signaling in the CR effect.     

Heart glucose transport and transporters in rat heart: regulation by insulin,workload and glucose

Summary Aspects of the regulation of the glucose transport by perfused hearts of normal rats have been studied by measuring glucose transport (via the efflux of labelled 3-O-methyl-D-glucose) and glucose transporters (via the labelled cytochalasin B binding assay). Similary to what is observed with insulin, increasing workload (by raising perfusion pressure from 50 to 100 mm Hg) stimulated glucose transport 7 to 8-fold. Glucose (via its analog 3-O-methylglucose, used at 15 mmol/l) stimulated its own transport 4-fold. The three stimuli favored the translocation of glucose transporters from an intracellular pool (microsomes) to the plasma membrane. Insulin increased the apparent affinity (decreased dissociation constant values) of plasma membrane transporters for cytochalasin, as well as the Hill coefficient, indicating the occurrence of a positive cooperativity amongst plasma membrane transporters. Workload increased only the Hill coefficient, glucose only the apparent affinity for cytochalasin of plasma membrane transporters. This study shows that insulin, workload and glucose itself stimulate glucose transport by favouring the translocation process of glucose transporter as well as by changing, albeit by a different mechanism, the functional properties of the transporters once translocated to the plasma membrane.     

Influence of age and long-term dietary restriction on plasma insulin-like growth factor-1 (IGF-1), IGF-1 gene expression, and IGF-1 binding proteins.

The purpose of these studies was to determine more accurately the relationship between IGF-1 and life span, and to determine whether moderate dietary caloric restriction alters the age-related changes in IGF-1. Studies included an assessment of plasma IGF-1, hepatic IGF-1 mRNA, and plasma IGF-1 binding proteins (IGF-1-BP). Rats (6, 12, 22, and 28 months of age) were fed ad libitum or maintained on a diet 60% of ad libitum. In ad libitum fed animals, plasma IGF-1 decreased by 20% between 6 and 28 months of age. Similar age-related declines were observed in dietary restricted animals but levels were generally 14-25% lower at each age group. IGF-1 mRNA levels demonstrated similar decreases with age in ad libitum fed animals, but in dietary restricted animals, levels plateaued at 22 and 28 months. IGF-1 binding protein analysis revealed 3 bands at approximate molecular weights of 40k, 30k, and 24k. All bands demonstrated a decrease in relative IGF-1-BP concentration with age, as well as a decrease in the 40k and 30k binding proteins after dietary restriction. These results indicate that (a) aging in ad libitum fed animals is associated with decreases in plasma IGF-1, IGF-1-BP, and IGF-1 mRNA levels; and (b) long-term dietary restriction decreases plasma IGF-1 and IGF-1-BP levels in each age group although the age-associated decreases in IGF-1 mRNA levels are prevented by dietary restriction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caloric restriction protects mitochondrial function with aging in skeletal and cardiac muscles

In skeletal muscles and heart in vitro complex IV activity is lower in young adult caloric restricted (CR) animals despite normal aerobic function in situ and in vivo. On the other hand, whereas markers of oxidative capacity decline 25% to 46% between 8 and 10 months and 35 months in ad libitum fed (AL) animals, in most muscles there is no decline in CR across the same absolute age (35 mo old) or relative age (35% survival rate) span and PGC-1alpha gene expression in gastrocnemius muscle declines more slowly with aging. The present results show that CR largely prevents the age-associated decline in mitochondrial function in heart and skeletal muscles, and suggest that this is secondary to a better-maintained drive on mitochondrial biogenesis.     

Age and diet alter skeletal muscle tubular aggregates

Tubular aggregates (TA’s) may comprise the major histopathologic finding in hyperkalemic and normokalemic periodic paralysis. They also constitute a conspicuous morphologic abnormality in a number of myopathies, and have been associated with non-specific muscle myalgias/cramps. Caloric restriction (CR) is an experimental manipulation that increases longevity and reduces lesions in mice as compared to animals fed ad libitum (AL). However, the impact of CR on the dynamics of muscle tissue has not been previously established. In this paper we week to characterize the relationship between tubular aggregates and age and determine whether they are modulated by caloric restriction. We examined the effects of 40% caloric restriction (CR) on fiber damage formation in middle-aged (19 months of age) and older (27 months) mice. We also examined the effects of age and diet restriction on tubular aggregate formation in 12, 24, and 30 month old mice. Tissue blocks from the 19 and 27 month animals were also examined using electron microscopy for qualitative differences in tubular aggregates which may suggest a diet and age effect. There appeared to be no fiber or Z-disc damage attributable to age as assessed by quantitative light microscopy (LM). Age and diet had significant effects on the percent of fibers occupied by tubular aggregates. Qualitative LM revealed that many TA’s may not have yet penetrated the sarcolemma in the young animals, and in particular, the young CR mice. The presence of round fibers was evident in the young CR mice whereas fibers were more angular in the AL mice. Fiber splitting was also evident in the CR mice, perhaps associated with new fiber formation. Other observations especially prevalent in mice with higher degrees of tubular aggregates included centrally displaced nuclei, nuclear chains among the sarcolemma borders, interfasicular wedging, longitudinally split fibers and cellular infiltrates. Electron microscopic examination of these areas revealed non distinction of the TA ultrastructure as a result of aging and diet. Large clusters of TA’s were observed to be in close approximation with mitochondria and in some instances, mitochondria with internal vesicles were present. We conclude that diet restriction may reduce the accumulation of age-related tubular aggregates in skeletal muscle without altering their ultrastructure.