Ability of insulin to modulate hepatic glucose production in aging rats is impaired by fat accumulation

Increased total fat mass (FM) and visceral fat (VF) may account in part for age-associated decrease in hepatic insulin action. This study determined whether preventing the changes in body fat distribution abolished this defect throughout aging. We studied the F(1) hybrid of Brown Norway-Fischer 344 rats (n = 29), which we assigned to caloric restriction (CR) or fed ad libitum (AL). CR (55% of the calories consumed by AL) was initiated and used at 2 mo to prevent age-dependent increases in FM and VF. AL rats were studied at 2, 8, and 20 mo; CR rats were studied at 8 and 20 mo. VF and FM remained unchanged throughout aging in CR rats. AL-fed rats at 8 and 20 mo had over fourfold higher FM and VF compared with both CR groups. Insulin clamp studies (3 mU. kg(-1). min(-1) with somatostatin) were performed to assess hepatic insulin sensitivity. Prevention of fat accretion resulted in a marked improvement in insulin action in the suppression of hepatic glucose production (HGP) (6.3 +/- 0.3 and 7.2 +/- 1.2 mg. kg(-1). min(-1) in 8- and 20-mo CR rats vs. 8.3 +/- 0.5 and 10.8 +/- 0.9 mg. kg(-1). min(-1) in 8- and 20-mo AL rats, respectively). The rate of gluconeogenesis (by enrichment of hepatic uridine diphosphate glucose and phosphoenolpyruvate pools by [(14)C]lactate) was unchanged in all groups. The improvement in hepatic insulin action in the CR group was mostly due to effective suppression of glycogenolysis (4.4 +/- 0.3 and 4.9 +/- 0.3 mg. kg(-1). min(-1) in 8- and 20-mo CR rats vs. 5.8 +/- 0.6 and 8.2 +/- 1.0 mg. kg(-1). min(-1) in 8- and 20-mo AL rats, respectively). The results demonstrated the preservation of hepatic insulin action in aging CR rats. Therefore, body fat and its distribution are major determinants of age-associated hepatic insulin resistance.     

Aerobic power declines with aging in rat skeletal muscles perfused at matched convective O2 delivery.

Although it is well established that maximal O(2) uptake (Vo(2 max)) declines from adulthood to old age, the role played by alterations in skeletal muscle is unclear. Specifically, because during whole body exercise reductions in convective O(2) delivery to the working muscles from adulthood to old age compromise aerobic performance, this obscures the influence of alterations within the skeletal muscles. We sought to overcome this limitation by using an in situ pump-perfused hindlimb preparation to permit matching of muscle convective O(2) delivery in young adult (8 mo; muscle convective O(2) delivery = 569 +/- 42 micromol O(2) x min(-1) x 100 g(-1)) and late middle-aged (28-30 mo; 539 +/- 62 micromol O(2) x min(-1) x 100 g(-1)) Fischer 344 x Brown Norway F1 hybrid rats. The distal hindlimb muscles were electrically stimulated for 4 min (60 tetani/min), and Vo(2 max) was determined. Vo(2 max) normalized to the contracting muscle mass was 22% lower in the 28- to 30-mo-old (344 +/- 17 micromol O(2). min(-1) x 100 g(-1)) than the 8-mo-old (441 +/- 20 micromol O(2) x min(-1) x 100 g(-1); P < 0.05) rats. The flux through the electron transport chain complexes I-III was 45% lower in homogenates prepared from the plantaris muscles of the older animals. Coincident with these alterations, the tension at Vo(2 max) and lactate efflux were reduced in the 28- to 30-mo-old animals, whereas the percent decline in tension was greater in the 28- to 30-mo-old vs. 8-mo-old animals. Collectively, these results demonstrate that alterations within the skeletal muscles, such as a reduced mitochondrial oxidative capacity, contribute to the reduction in Vo(2 max) with aging.     

Effect of caloric restriction on Hprt lymphocyte mutation in aging rats

Caloric restriction (CR) reduces tumor incidence and retards aging in laboratory animals, including non-human primates. Because of the relationships among mutation, disease susceptibility, and aging, we investigated whether or not CR affects the accumulation of somatic cell mutations in aging animals. Starting at approximately 2 months of age, male CD rats (Harlan Sprague-Dawley-derived) were placed on different levels of dietary intake: ad libitum (AL) feeding, and 90% (10% CR), 75% (25% CR) and 60% (40% CR) of the total calories consumed by AL animals. At 3, 6, 12, and 24 months after the beginning of CR, Hprt mutant frequencies (MFs) were determined. The MFs measured in spleen lymphocytes from AL and CR rats sacrificed at 3 months of dietary restriction were similar for all dietary groups. However, the MFs at 6, 12, and 24 months of CR were significantly higher in AL-fed rats compared with animals on 40% CR: (4.5+/-0.4)x10(-6) versus (3.3+/-0.3)x10(-6) (P=0.032) in 6 months CR rats; (10.3+/-2.3)x10(-6) versus (7.3+/-1.2)x10(-6) in 12 months CR rats (P=0.04), and (18.3+/-3.2)x10(-6) versus (7.8+/-1.0)x10(-6) (P=0.001) in 24 months CR rats. In addition, rats receiving 25% CR for 24 months had a MF, (10.7+/-2.0)x10(-6), between the 40% CR and AL rats. Multiplex PCR of the Hprt gene in mutant clones from 12 and 24 months 40% CR rats and the corresponding AL rats detected deletions in 42% of CR mutants and 19% of AL mutants. Because of the difference in Hprt MF in the two groups, the estimated MF associated with deletions in CR rats was similar to the deletion MF in AL rats. This observation implies that the lower MF in CR rats is due to a reduction in smaller Hprt mutations (i.e. base substitutions and frameshifts). The pattern of smaller Hprt mutations from AL rats suggests that many were produced by reactive oxygen species (ROS). The results indicate that CR reduces the accumulation of spontaneous somatic cell mutation in aging rats, especially those caused by base substitutions and frameshifts.     

Caloric restriction optimizes the proteasome pathway with aging in rat plantaris muscle: implications for sarcopenia

To gain insight into the significance of alterations in the proteasome pathway for sarcopenia and its attenuation by calorie restriction, we examined protein oxidation and components of the proteasome pathway in plantaris muscle in 8-, 30-, and 35-mo-old ad libitum-fed (AL) rats; and in 8-, 35-, and 40-mo-old calorie-restricted (CR) rats. We hypothesized that CR rats would exhibit a lesser accumulation of protein carbonyls with aging and that this would be associated with a better maintenance of skeletal muscle proteasome activity and function with aging. Consistent with this view, whereas AL rats had a significant increase in protein carbonylation with aging, there was no such increase in CR rats. Protein levels of the ubiquitin ligases MuRF1 and MAFbx increased similarly with aging in both AL and CR rats. On the other hand, chymotrypsin-like activity of the proteasome increased with aging more gradually in CR rats, and this increase was paralleled by increases in the expression of the C2 subunit in both groups, suggesting that differences in activity were not related to differences in proteasome function with aging. Interestingly, the plot of muscle mass vs. proteasome activity showed that the oldest animals in both diets had a lower muscle mass than would be predicted by their proteasome activity, suggesting that other factors explain the acceleration of sarcopenia at advanced age. Since calorie restriction better protects skeletal muscle function than muscle mass with aging (Hepple RT, Baker DJ, Kaczor JJ, Krause DJ, FASEB J 19: 1320-1322, 2005), and our current results show that this protection of function is associated with a prevention of oxidative protein damage accumulation, we suggest that calorie restriction optimizes the proteasome pathway to preserve skeletal muscle function at the expense of modest muscle atrophy.     

Insulin-dependent glycogen synthesis is delayed in onset in the skeletal muscle of food-deprived aged rats

Insulin resistance with aging may be responsible for impaired glycogen synthesis in the skeletal muscle of aged rats and contribute to the well-known decreased ability to respond to stress with aging. For this reason, to assess the ability of the skeletal muscle to utilize glucose for glycogen synthesis during aging, the time course of glycogen synthesis was continuously monitored by 13C nuclear magnetic resonance for 2 h in isolated [13C] glucose-perfused gastrocnemius-plantaris muscles of 5-day food-deprived adult (6-8 months; n=10) or 5-day food-deprived aged (22 months; n=8) rats. [13C] glucose (10 mmol/L) perfusion was carried out in the presence or absence of an excess of insulin (1 micromol/L). Food deprivation only decreased glycogen level in adult rats (8.9+/-2.4 micromol/g in adults vs. 35.6+/-2.4 micromol/g in aged rats; P<.05). In the presence of an excess of insulin, muscle glycogen synthesis was stimulated in both adult and aged muscles, but the onset was delayed with aging (40 min later). In conclusion, this study highlights the important role of glycogen depletion in stimulating glycogen synthesis in muscles. Consequently, the absence of glycogen depletion in response to starvation in aged rats may be the origin of the delay in insulin-stimulated glycogen synthesis in the skeletal muscle. Glycogen synthesis clearly was not impaired with aging.     

Metabolic response to carbohydrate ingestion during exercise in males and females

The present study investigated potential sex-related differences in the metabolic response to carbohydrate (CHO) ingestion during exercise. Moderately endurance-trained men and women (n = 8 for each sex) performed 2 h of cycling at approximately 67% Vo(2 max) with water (WAT) or CHO ingestion (1.5 g of glucose/min). Substrate oxidation and kinetics were quantified during exercise using indirect calorimetry and stable isotope techniques ([(13)C]glucose ingestion, [6,6-(2)H(2)]glucose, and [(2)H(5)]glycerol infusion). In both sexes, CHO ingestion significantly increased the rates of appearance (R(a)) and disappearance (R(d)) of glucose during exercise compared with WAT ingestion [males: WAT, approximately 28-29 micromol x kg lean body mass (LBM)(-1) x min(-1); CHO, approximately 53 micromol x kg LBM(-1) x min(-1); females: WAT, approximately 28-29 micromol x kg LBM(-1) x min(-1); CHO, approximately 61 micromol x kg LBM(-1) x min(-1); main effect of trial, P < 0.05]. The contribution of plasma glucose oxidation to the energy yield was significantly increased with CHO ingestion in both sexes (from approximately 10% to approximately 20% of energy expenditure; main effect of trial, P < 0.05). Liver-derived glucose oxidation was reduced, although the rate of muscle glycogen oxidation was unaffected with CHO ingestion (males: WAT, 108 +/- 12 micromol x kg LBM(-1) x min(-1); CHO, 108 +/- 11 micromol x kg LBM(-1) x min(-1); females: WAT, 89 +/- 10 micromol x kg LBM(-1) x min(-1); CHO, 93 +/- 11 micromol x kg LBM(-1) x min(-1)). CHO ingestion reduced fat oxidation and lipolytic rate (R(a) glycerol) to a similar extent in both sexes. Finally, ingested CHO was oxidized at similar rates in men and women during exercise (peak rates of 0.70 +/- 0.08 and 0.65 +/- 0.06 g/min, respectively). The present investigation suggests that the metabolic response to CHO ingestion during exercise is largely similar in men and women.     

Improved insulin sensitivity with calorie restriction does not require reduced JNK1/2, p38, or ERK1/2 phosphorylation in skeletal muscle of 9-month-old rats

Calorie restriction [CR; ～40% below ad libitum (AL) intake] improves the health of many species, including rats, by mechanisms that may be partly related to enhanced insulin sensitivity for glucose disposal by skeletal muscle. Excessive activation of several mitogen-activated protein kinases (MAPKs), including JNK1/2, p38, and ERK1/2 has been linked to insulin resistance. Although insulin can activate ERK1/2, this effect is not required for insulin-mediated glucose uptake. We hypothesized that skeletal muscle from male 9-mo-old Fischer 344/Brown Norway rats CR (35-40% beginning at 3 mo old) versus AL rats would have 1) attenuated activation of JNK1/2, p38, and ERK1/2 under basal conditions; and 2) no difference for insulin-induced ERK1/2 activation. In contrast to our hypothesis, there were significant CR-related increases in the phosphorylation of p38 (epitrochlearis, soleus, and gastrocnemius), JNK1 (epitrochlearis and soleus), and JNK2 (gastrocnemius). Consistent with our hypothesis, CR did not alter insulin-mediated ERK1/2 activation. The greater JNK1/2 and p38 phosphorylation with CR was not attributable to diet effects on muscle oxidative stress (assessed by protein carbonyls and 4-hydroxynonenal protein conjugates). In muscles from the same rats used for the present study, we previously reported a CR-related increase in insulin-mediated glucose uptake by the epitrochlearis and the soleus (Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Am J Physiol Endocrinol Metab 300: E966-E978, 2011). The present results indicate that the improved insulin sensitivity with CR is not attributable to attenuated MAPK phosphorylation in skeletal muscle.     

NAD(P)H oxidase-generated superoxide anion accounts for reduced control of myocardial O2 consumption by NO in old Fischer 344 rats

We investigated the role of nitric oxide (NO) in the control of myocardial O2 consumption in Fischer 344 rats. In Fischer rats at 4, 14, and 23 mo of age, we examined cardiac function using echocardiography, the regulation of cardiac O2 consumption in vitro, endothelial NO synthase (eNOS) protein levels, and potential mechanisms that regulate superoxide. Aging was associated with a reduced ejection fraction [from 75 +/- 2% at 4 mo to 66 +/- 3% (P < 0.05) at 23 mo] and an increased cardiac diastolic volume [from 0.60 +/- 0.04 to 1.00 +/- 0.10 ml (P < 0.01)] and heart weight (from 0.70 +/- 0.02 to 0.90 +/- 0.02 g). The NO-mediated control of cardiac O2 consumption by bradykinin or enalaprilat was not different between 4 mo (36 +/- 2 or 34 +/- 3%) and 14 mo (29 +/- 1 or 25 +/- 3%) but markedly (P < 0.05) reduced in 23-mo-old Fischer rats (15 +/- 3 or 7 +/- 2%). The response to the NO donor S-nitroso-N-acetyl penicillamine was not different across groups (35%, 35%, and 44%). Interestingly, the eNOS protein level was not different at 4, 14, and 23 mo. The addition of tempol (1 mmol/l) to the tissue bath eliminated the depression in the control of cardiac O2 consumption by bradykinin (25 +/- 3%) or enalaprilat (28 +/- 3%) in 23-mo-old Fischer rats. We next examined the levels of enzymes involved in the production and breakdown of superoxide. The expression of Mn SOD, Cu/Zn SOD, extracellular SOD, and p67phox, however, did not differ between 4- and 23-mo-old rats. Importantly, there was a marked increase in gp91phox, and apocynin restored the defect in NO-dependent control of cardiac O2 consumption at 23 mo to that seen in 4-mo-old rats, identifying the role of NADPH oxidase. Thus increased biological activity of superoxide and not decreases in the enzyme that produces NO are responsible for the altered control of cardiac O2 consumption by NO in 23-mo-old Fischer rats. Increased oxidant stress in aging, by decreasing NO bioavailability, may contribute not only to changes in myocardial function but also to altered regulation of vascular tone and the progression of cardiac or vascular disease.     

Brief food restriction increases FA oxidation and glycogen synthesis under insulin-stimulated conditions

To determine the effects of brief food restriction on fatty acid (FA) metabolism, hindlimbs of F344/BN rats fed either ad libitum (AL) or food restricted (FR) to 60% of baseline food intake for 28 days were perfused under hyperglycemic-hyperinsulinemic conditions (20 mM glucose, 1 mM palmitate, 1,000 microU/ml insulin, [3-(3)H]glucose, and [1-(14)C]palmitate). Basal glucose and insulin levels were significantly lower (P < 0.05) in FR vs. AL rats. Palmitate uptake (34.3 +/- 2.7 vs. 24.5 +/- 3.1 nmol/g/min) and oxidation (3.8 +/- 0.2 vs. 2.7 +/- 0.3 nmol.g(-1).min(-1)) were significantly higher (P < 0.05) in FR vs. AL rats, respectively. Glucose uptake was increased in FR rats and was accompanied by significant increases in red and white gastrocnemius glycogen synthesis, indicating an improvement in insulin sensitivity. Although muscle triglyceride (TG) levels were not significantly different between groups, glucose uptake and total preperfusion TG concentration were negatively correlated (r(2) = 0.27, P < 0.05). In conclusion, our results show that under hyperglycemic-hyperinsulinemic conditions, brief FR resulted in an increase in FA oxidative disposal that may contribute to the improvement in insulin sensitivity.     

Chronic Calorie Restriction Alters Glycogen Metabolism in Rhesus Monkeys

Chronic caloric restriction (CR) prevents the development of obesity and maintains health, slows aging processes, and prevents or substantially delays the development of non-insulin-dependent diabetes. Because changes in energy metabolism could be involved in all of these positive effects of CR, we examined glycogen synthase (GS) and glycogen phosphorylase (GP) activities and glucose 6-phosphate (G6P) and glycogen concentrations in skeletal muscle samples before and during a euglycemic hyperinsulinemic clamp in 6 older aged monkeys in which CR had been continued for 10.4 ± 2.1 years. Basal GS activity (fractional velocity and independent) was significantly higher in the CR monkeys than has been previously shown in normal, hyperinsulinemic and diabetic monkeys. The normal effect of insulin to activate GS was absent in the CR group due to the paradoxical finding in some of these monkeys of a reduction in GS activity by insulin. Insulin also had the unexpected effect of increasing the independent activity of GP above basal activity (p<0.05). There was an inverse relationship between the change (insulin-stimulated minus basal) in GS fractional velocity and GP activity ratio (r=-0.91, p<0.005). The basal independent activities of GS and GP were also inversely correlated (r=-0.79, p<0.05). The insulin-stimulated concentration of G6P tended to be higher than the basal concentration (p<0.06) and was significantly higher than that previously shown in normal monkeys (p<0.05). We suggest that long-term calorie restriction (1) results in alterations in glycogen metabolism that may be important to the anti-diabetogenic and anti-aging effects of CR and (2) unmasks early defects which may indicate the likelihood of ultimately developing obesity and diabetes.     

Impaired fatty acid oxidation in muscle of aging rats perfused under basal conditions

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under conditions of euglycemia with low insulin levels. Male Fischer 344 x Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 8 mM glucose, 1 mM palmitate, 25 microU/ml insulin, [1-(14)C]palmitate, and [3-(3)H]glucose. Glucose and palmitate uptake were similar among age groups. The percent and total palmitate oxidized (nmol.min(-1).g(-1)) were 30-36 and 41-49% lower (P < 0.05) in 15-mo- and 24-mo-old than in 5-mo-old animals. Compared with 5-mo- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly (P < 0.05) elevated 91-305% in red and 118-219% in white muscles of 24-mo-old animals. Fatty acid-binding protein content was 40-64% higher (P < 0.05) in 24-mo- than in 5-mo- or 15-mo-old animals. In red muscle, hormone-sensitive lipase (HSL) content was 28% lower (P < 0.05) in 24-mo- than in 5-mo-old animals. These results indicate that, under euglycemic conditions in the presence of low insulin levels, the reduction in FA disposal to oxidation and the decrease in HSL content may contribute to the accumulation of TG in muscle of old animals.     

Ventilatory long-term facilitation is greater in 1- vs. 2-mo-old awake rats.

Respiratory long-term facilitation (LTF) declines in middle-aged vs. adult male rats. Chronic intermittent hypoxia (CIH; 5 min 11-12% O2/5 min air, 12 h/night, 7 nights) enhances LTF in adult rats. However, LTF in immature rats and the effect of early CIH are unevaluated. The present study compared LTF in 1- and 2-mo-old rats and examined the effect of neonatal CIH (initiated at 2 days after birth) on the LTF. Ventilatory LTF, elicited by 5 (protocol 1) or 10 (protocol 2) episodes of poikilocapnic hypoxia (5 min 12% O2/5 min air), was measured twice by plethysmography on the same male conscious rat when it was 1 and 2 mo old. In untreated (without CIH) rats, both resting ventilation (54.7 +/- 0.6 vs. 43.0 +/- 0.2 ml.100 g(-1).min(-1)) and hypoxic ventilatory response (131 +/- 4 vs. 66 +/- 3% above baseline) were greater in 1- vs. 2-mo-old rats. Protocol 1 elicited LTF in 1-mo-old (12.5 +/- 1.0% above baseline) but not 2-mo-old rats. Protocol 2 elicited a greater LTF in 1-mo-old (24.3 +/- 0.8%) vs. 2-mo-old rats (18.2 +/- 0.5%). In CIH-treated rats, protocol 1 also elicited LTF in 1-mo-old (13.1 +/- 1.5%) but not 2-mo-old rats. Protocol 2 elicited LTF in both age groups, but LTF was enhanced by the CIH only in 1-mo-old rats (28.8 +/- 0.9%). These results suggest that ventilatory LTF and hypoxic ventilatory response are greater in male rats shortly before their sexual maturity and that the neonatal CIH somewhat enhances ventilatory LTF approximately 3 wk after CIH, but this enhancement does not last to adulthood.     

Exercise with calorie restriction improves insulin sensitivity and glycogen synthase activity in obese postmenopausal women with impaired glucose tolerance

Our objective was to compare the effects of in vivo insulin on skeletal muscle glycogen synthase (GS) activity in normal (NGT) vs. impaired glucose-tolerant (IGT) obese postmenopausal women and to determine whether an increase in insulin activation of GS is associated with an improvement in insulin sensitivity (M) following calorie restriction (CR) and/or aerobic exercise plus calorie restriction (AEX + CR) in women with NGT and IGT. We did a longitudinal, clinical intervention study of CR compared with AEX + CR. Overweight and obese women, 49-76 yr old, completed 6 mo of CR (n = 46) or AEX + CR (n = 50) with Vo(2?max), body composition, and glucose tolerance testing. Hyperinsulinemic euglycemic (80 mU·m(-2)·min(-1)) clamps (n = 73) and skeletal muscle biopsies (before and during clamp) (n = 58) were performed before and after the interventions (n = 50). After 120 min of hyperinsulinemia during the clamp, GS fractional activity and insulin's effect to increase GS fractional activity (insulin - basal) were significantly lower in IGT vs. NGT (P < 0.01) at baseline. GS total activity increased during the clamp in NGT (P < 0.05), but not IGT, at baseline. CR and AEX + CR resulted in a significant 8% weight loss with reductions in total fat mass, visceral fat, subcutaneous fat, and intramuscular fat. Overall, M increased (P < 0.01), and the change in M (postintervention - preintervention) was associated with the change in insulin-stimulated GS fractional activity (partial r = 0.44, P < 0.005). In IGT, the change (postintervention - preintervention) in insulin-stimulated GS total activity was greater following AEX + CR than CR alone (P < 0.05). In IGT, insulin-stimulated GS-independent (P < 0.005) and fractional activity (P = 0.06) increased following AEX + CR. We conclude that the greatest benefits at the whole body and cellular level (insulin activation of GS) in older women at highest risk for diabetes are derived from a lifestyle intervention that includes exercise and diet.     

Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle

Skeletal muscle insulin sensitivity improves with short-term reduction in calorie intake. The goal of this study was to evaluate changes in the abundance and phosphorylation of Akt1 and Akt2 as potential mechanisms for enhanced insulin action after 20 days of moderate calorie restriction [CR; 60% of ad libitum (AL) intake] in rat skeletal muscle. We also assessed changes in the abundance of SH2 domain-containing inositol phosphatase (SHIP2), a negative regulator of insulin signaling. Fisher 344 x Brown Norway rats were assigned to an AL control group or a CR treatment group for 20 days. Epitrochlearis muscles were dissected and incubated with or without insulin (500 microU/ml). Total Akt serine and threonine phosphorylation was significantly increased by 32 (P < 0.01) and 30% (P < 0.005) in insulin-stimulated muscles from CR vs. AL. Despite an increase in total Akt phosphorylation, there was no difference in Akt1 serine or Akt1 threonine phosphorylation between CR and AL insulin-treated muscles. However, there was a 30% decrease (P < 0.05) in Akt1 abundance for CR vs. AL. In contrast, there was no change in Akt2 protein abundance, and there was a 94% increase (P < 0.05) in Akt2 serine phosphorylation and an increase of 75% (P < 0.05) in Akt2 threonine phosphorylation of insulin-stimulated CR muscles compared with AL. There was no diet effect on SHIP2 abundance in skeletal muscle. These results suggest that, with brief CR, enhanced Akt2 phosphorylation may play a role in increasing insulin sensitivity in rat skeletal muscles.     

Long-term AT1 receptor blockade improves metabolic function and provides renoprotection in Fischer-344 rats

Fischer-344 (F344) rats exhibit proteinuria and insulin resistance in the absence of hypertension as they age. We determined the effects of long-term (1 yr) treatment with the angiotensin (ANG) II type 1 (AT(1)) receptor blocker L-158,809 on plasma and urinary ANG peptide levels, systolic blood pressure (SBP), and indexes of glucose metabolism in 15-mo-old male F344 rats. Young rats at 3 mo of age (n = 8) were compared with two separate groups of older rats: one control group (n = 7) and one group treated with L-158,809 (n = 6) orally (20 mg/l) for 1 yr. SBP was not different between control and treated rats but was higher in young rats. Serum leptin, insulin, and glucose levels were comparable between treated and young rats, whereas controls had higher glucose and leptin with a similar trend for insulin. Plasma ANG I and ANG II were higher in treated than untreated young or older rats, as evidence of effective AT(1) receptor blockade. Urinary ANG II and ANG-(1-7) were higher in controls compared with young animals, and treated rats failed to show age-related increases. Protein excretion was markedly lower in treated and young rats compared with control rats (young: 8 +/- 2 mg/day vs. control: 129 +/- 51 mg/day vs. treated: 9 +/- 3 mg/day, P < 0.05). Long-term AT(1) receptor blockade improves metabolic parameters and provides renoprotection. Differential regulation of systemic and intrarenal (urinary) ANG systems occurs during blockade, and suppression of the intrarenal system may contribute to reduced proteinuria. Thus, insulin resistance, renal injury, and activation of the intrarenal ANG system during early aging in normotensive animals can be averted by renin-ANG system blockade.     

Diminished feeding responsiveness to orexin A (hypocretin 1) in aged rats is accompanied by decreased neuronal activation

Orexin A is produced in caudal lateral, posterior, perifornical, and dorsomedial hypothalamic areas. Orexin A in the rostro-dorsal lateral hypothalamic area (rLHa) stimulates feeding and activates several feeding-regulatory brain areas. We hypothesized that aging diminishes feeding and c-Fos-immunoreactivity (c-Fos-ir; marker of neuronal activation) response to orexin A. Young (3 mo), middle-aged (12 mo), and old (24 mo) male Fischer 344 rLHa-cannulated rats were injected with orexin A (0.5, 1, and 2 nmol). Food intake was measured at 1, 2, and 4 h. c-Fos-ir in hypothalamic, limbic, and hindbrain regions was measured in two additional sets of rLHa-orexin A injected rats. In a separate study, orexin A effects on feeding and c-Fos-ir were measured in 6-mo-old rats. Orexin A significantly elevated feeding in rats aged 3, 6, and 12 mo in the 0-1 and 1-2- h time intervals, whereas in old rats this was significant in the 1-2 h time interval only. At 1 h, 6-8 (of 14) brain areas showed elevated c-Fos-ir in response to orexin A in 3- and 6-mo-old rats, but 24-mo-old rats exhibited attenuated or absent c-Fos-ir response in all brain regions except the hypothalamic paraventricular nucleus (PVN) and rostral nucleus of the solitary tract (rNTS). Orexin A did not elevate c-Fos-ir in 3-mo-old rats at 2 h after injection, whereas the PVN and mediodorsal thalamic nucleus (MD) showed elevated c-Fos-ir at 2 h in 24-mo-old rats. These data suggest that delayed and diminished feeding responses in old animals may be due to ineffective neural signaling and implicate the orexin A network as one feeding system affected by aging.     

De novo expression of podocyte proteins in parietal epithelial cells in experimental aging nephropathy

A progressive decrease in podocyte number underlies the development of glomerulosclerosis and reduced kidney function in aging nephropathy. Recent data suggest that under certain disease states, parietal epithelial cells (PECs) begin to express proteins considered specific to podocytes. To determine whether this phenomenon increases in aging kidneys, 4-, 12-, and 20-mo ad libitum-fed and 20-mo calorie-restricted (CR) rats were studied. Single and double immunostaining were performed with antibodies to the PEC protein paired box gene 2 (PAX2) and tight junction protein claudin-1, the podocyte-specific protein Wilms' tumor 1 (WT-1), and the proliferating cell protein (Ki-67). ImageJ software measured Bowman's basement membrane (BBM) length and glomerular tuft area in individual glomeruli from each animal to assess glomerular size. The results showed that in aged ad libitum rats, the decrease in number of podocytes/glomerular tuft area was accompanied by an increase in the number of PECs/BBM length at 12 and 20 mo (P < 0.01 vs. 4 mo). The increase in PEC number was due to proliferation (increase in PAX2/Ki-67 double-positive cells). Aging was accompanied by a progressive increase in the number of glomerular cells double staining for PAX2 and WT-1. In contrast, the control 20-mo-old CR rats had no increase in glomerular size, and podocyte and PEC number were not altered. These results suggest that although the number of PECs and PECs expressing podocyte proteins increase in aging nephropathy, they are likely not sufficient to compensate for the decrease in podocyte number.     

Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats

Calorie restriction [CR; ~65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[(3)H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr(308) and Ser(473)) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser(588) and Thr(642)) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.     

Physiological hyperinsulinemia impairs insulin-stimulated glycogen synthase activity and glycogen synthesis

Although chronic hyperinsulinemia has been shown to induce insulin resistance, the basic cellular mechanisms responsible for this phenomenon are unknown. The present study was performed 1) to determine the time-related effect of physiological hyperinsulinemia on glycogen synthase (GS) activity, hexokinase II (HKII) activity and mRNA content, and GLUT-4 protein in muscle from healthy subjects, and 2) to relate hyperinsulinemia-induced alterations in these parameters to changes in glucose metabolism in vivo. Twenty healthy subjects had a 240-min euglycemic insulin clamp study with muscle biopsies and then received a low-dose insulin infusion for 24 (n = 6) or 72 h (n = 14) (plasma insulin concentration = 121 +/- 9 or 143 +/- 25 pmol/l, respectively). During the baseline insulin clamp, GS fractional velocity (0.075 +/- 0.008 to 0.229 +/- 0.02, P < 0.01), HKII mRNA content (0.179 +/- 0.034 to 0.354 +/- 0.087, P < 0.05), and HKII activity (2.41 +/- 0.63 to 3.35 +/- 0.54 pmol x min(-1) x ng(-1), P < 0.05), as well as whole body glucose disposal and nonoxidative glucose disposal, increased. During the insulin clamp performed after 24 and 72 h of sustained physiological hyperinsulinemia, the ability of insulin to increase muscle GS fractional velocity, total body glucose disposal, and nonoxidative glucose disposal was impaired (all P < 0.01), whereas the effect of insulin on muscle HKII mRNA, HKII activity, GLUT-4 protein content, and whole body rates of glucose oxidation and glycolysis remained unchanged. Muscle glycogen concentration did not change [116 +/- 28 vs. 126 +/- 29 micromol/kg muscle, P = nonsignificant (NS)] and was not correlated with the change in nonoxidative glucose disposal (r = 0.074, P = NS). In summary, modest chronic hyperinsulinemia may contribute directly (independent of change in muscle glycogen concentration) to the development of insulin resistance by its impact on the GS pathway.     

Age-related changes in contractile properties of single skeletal fibers from the soleus muscle.

Peak absolute force, specific tension (peak absolute force per cross-sectional area), cross-sectional area, maximal unloaded shortening velocity (Vo; determined by the slack test), and myosin heavy chain (MHC) isoform compositions were determined in 124 single skeletal fibers from the soleus muscle of 12-, 24-, 30-, 36-, and 37-mo-old Fischer 344 Brown Norway F1 Hybrid rats. All fibers expressed the type I MHC isoform. The mean Vo remained unchanged from 12 to 24 mo but did decrease significantly from the 24- to 30-mo time period (from 1.71 +/- 0.13 to 0.85 +/- 0.09 fiber lengths/s). Fiber cross-sectional area remained constant until 36 mo of age, at which time there was a 20% decrease from the values at 12 mo of age (from 5,558 +/- 232 to 4,339 +/- 280 micrometer2). A significant decrease in peak absolute force of single fibers occurred between 12 and 24 mo of age (from 51 +/- 2 x 10(-5) to 35 +/- 2 x 10(-5) N) and then remained constant until 36 mo, when another 43% decrease occurred. Like peak absolute force, the specific tension decreased significantly between 12 and 24 mo by 20%, and another 32% decline was observed at 37 mo. Thus, by 24 mo, there was a dissociation between the loss of fiber cross-sectional area and force. The results suggest time-specific changes of the contractile properties with aging that are independent of each other. Underlying mechanisms responsible for the time-dependent and contractile property-specific changes are unknown. Age-related changes in the molecular dynamics of myosin may be the underlying mechanism for altered force production. The presence of more than one beta/slow MHC isoform may be the mechanism for the altered Vo with age.