A preliminary study on T-786C endothelial nitric oxide synthase gene and renal hemodynamic and blood pressure responses to dietary sodium.

The purpose of the present study was to examine the role of the T-786C endothelial nitric oxide synthase (eNOS) gene polymorphism on changes in renal hemodynamics and blood pressure due to Na(+) loading. Twenty-eight older (63+/-1 years), moderately obese (39+/-2 % fat) hypertensives had their glomerular filtration rate (GFR), renal plasma flow (RPF), blood pressure (BP) and plasma nitric oxide (NO(x)) levels determined after eight days of low (20 mEq) and high (200 mEq) Na(+) diets. The two Na(+) diets were separated by a 1-week washout period. Subjects were genotyped for the eNOS-786 site and were grouped on whether they were homozygous or heterozygous for the C allele (TC+CC, n=13) or only homozygous for the T allele (TT, n=15). The TC+CC genotype group had a significantly greater increase in diastolic (P=0.021) and mean arterial (P=0.018) BP and a significant decline in both RPF (P=0.007) and GFR (P=0.029) compared to the TT genotype group with Na(+) loading. Furthermore, Na(+) loading resulted in a significant (P=0.036) increase in plasma NO(x) in the TT, but not in the TC+CC genotype group as well as a trend (P=0.051) for an increase in urine NO(x) in TC+CC, but not in the TT genotype group. The increase in BP during Na(+) loading in older hypertensives was associated with the eNOS genotype and may be related to changes in renal hemodynamics due to changes in NO metabolism.

Effect of aerobic exercise training on blood pressure sensitivity to dietary sodium in older hypertensives.

Although aerobic exercise training has been shown to lower blood pressure (BP) in older adults, its effect on BP sensitivity to dietary sodium (Na(+)) is unknown. Therefore, the present study was undertaken to examine the effect of aerobic exercise training on BP sensitivity to dietary Na(+) in older hypertensive individuals. Blood pressure was measured after 8 days of low (20 mEq) and high (200 mEq) Na(+) diets in 31 older (63+/-7 years, mean+/-standard deviation), hypertensive (152+/-11/88+/-5 mm Hg) individuals at baseline and following 6 months of aerobic exercise training (at 75% VO(2)max, 3 times/week, 40 min/session). Subjects were grouped on the basis of the difference in mean arterial BP (MAP) between diets (Na(+) sensitive: >or=5 mm Hg increase in MAP on high Na(+), n=20; Na(+) resistant: <5 mm Hg increase in MAP on the high Na(+) diet, n=11). Following 6 months of aerobic exercise training, there was a significant increase in maximal aerobic capacity (VO(2)max: 18.3+/-3.8 vs 20.7+/-4.2 ml/kg/min, P<0.017). Aerobic exercise training had a significant (P=0.02) effect on Na(+) sensitivity status, with the proportion of Na(+)-resistant individuals increasing from 35% at baseline to 61% following the 6-month aerobic exercise training programme. This study demonstrates the importance of physical activity on BP sensitivity to dietary Na(+).

Effect of aging and obesity on insulin responsiveness and glut-4 glucose transporter content in skeletal muscle of Fischer 344 x Brown Norway rats.

This study investigated the metabolic changes with age in the Fischer 344 x Brown Norway rat and its suitability as an animal model of postmaturational insulin resistance. Specifically, we determined whether an age-associated decrease in glucose disposal is associated with diminished whole body insulin responsiveness and/or a decrease in glucose transporter (GLUT-4) protein and mRNA content in medial gastrocnemius muscle of male Fischer 344 x Brown Norway rats of ages 8, 18, and 28 months. Fasting plasma glucose was unchanged with age. There was a significant age effect on visceral adiposity, fasting plasma insulin levels, insulin responsiveness, and GLUT-4 protein content. Insulin responsiveness and GLUT-4 protein were lower in the 18-month-old rats than in the 8-month-old rats. The findings of age-associated increases in visceral adiposity and insulin resistance, and decreases in GLUT-4 in the Fisher 344 x Brown Norway rat, suggest that this rat strain may be an appropriate model for studying the effects of aging on glucose homeostasis.

GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure.

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.

Transient enhancement of GLUT-4 levels in rat epitrochlearis muscle after exercise training.

The purpose of the present study was to examine the effect of detraining on the glucose transport system after short-term swim training (5 days), long-term swim training (5 wk), and treadmill run training (5 wk). Skeletal muscles were isolated from female Wistar rats at 24 or 48 h posttraining. SST produces a 48% increase in GLUT-4 mRNA, a 30% increase in GLUT-4 protein, and a 60% increase in insulin-stimulated glucose transport activity at 24 h posttraining but not at 48 h posttraining. Similar to SST, long-term swim training produces a 60% increase in GLUT-4 mRNA and a 30% increase in GLUT-4 protein content at 24 h posttraining but not at 48 h posttraining. Finally, treadmill run training produces a transient 35% increase in GLUT-4 protein content that is completely reversed at 48 h after the last bout of exercise. These results demonstrate that the increase in GLUT-4 mRNA and GLUT-4 protein occurs during the first week of exercise training and is rapidly lost after training cessation. We believe that the transient enhancement in GLUT-4 protein after exercise training is due to a short GLUT-4 half-life, a process that is primarily regulated by pretranslational mechanisms.

1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle.

Previous studies have indicated a role for calmodulin in hypoxia-and insulin-stimulated glucose transport. However, since calmodulin interacts with multiple protein targets, it is unknown which of these targets is involved in the regulation of glucose transport. In the present study, we have used the calcium-dependent calmodulin protein kinase II (CAMKII) inhibitor 1-[N, O-bis-(5-isoquinolinesulphonyl) -N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) to investigate the possible role of this enzyme in the regulation of glucose transport in isolated rat soleus and epitrochlearis muscles. KN-62 did not affect basal 2-deoxyglucose transport, but it did inhibit both insulin- and hypoxia-stimulated glucose transport activity by 46 and 40% respectively. 1-[N,O-Bis-(1, 5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-04), a structural analogue of KN-62 that does not inhibit CAMKII, had no effect on hypoxia-or insulin-stimulated glucose transport. Accordingly, KN-62 decreased the stimulated cell-surface GLUT4 labelling by a similar extent as the inhibition of glucose transport (insulin, 49% and hypoxia, 54%). Additional experiments showed that KN-62 also inhibited insulin- and hypoxia-stimulated transport by 37 and 40% respectively in isolated rat epitrochlearis (a fast-twitch muscle), indicating that the effect of KN-62 was not limited to the slow-twitch fibres of the soleus. The inhibitory effect of KN-62 on hypoxia-stimulated glucose transport appears to be specific to CAMKII, since KN-62 did not inhibit hypoxia-stimulated 45Ca efflux from muscles pre-loaded with 45Ca, or hypoxia-stimulated glycogen breakdown. Additionally, KN-62 affected neither insulin-stimulated phosphoinositide 3-kinase nor Akt activity, suggesting that the effects of KN-62 are not due to non-specific effects of this inhibitor on these regions of the insulin-signalling cascade. The results of the present study suggest that CAMKII might have a distinct role in insulin- and hypoxia-stimulated glucose transport, possibly in the vesicular trafficking of GLUT4.

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells.

This investigation examined the effects of short-term exercise training on insulin-stimulated GLUT-4 glucose transporter translocation and glucose transport activity in rat adipose cells. Male Wistar rats were randomly assigned to a sedentary (Sed) or swim training group (Sw, 4 days; final 3 days: 2 x 3 h/day). Adipose cell size decreased significantly but minimally (approximately 20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats. Basal 3-O-methyl-D-[14C]glucose transport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIS cell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sed animals, similar to the increases observed in MIS glucose transport activity and total GLUT-4. These results suggest that increases in total GLUT-4 and GLUT-4 translocation to the cell surface contribute to the increase in MIS glucose transport with short-term exercise training. In addition, the results suggest that the exercise training-induced adaptations in glucose transport occur more rapidly than previously thought and with minimal changes in adipose cell size.

Mechanism of hypoxia-stimulated glucose transport in rat skeletal muscle: potential role of glycogen.

We have previously reported that exercise training is associated with enhanced insulin-stimulated glucose transport activity and inhibited hypoxia-stimulated glucose transport activity in rat epitrochlearis muscle. Here we examine the potential role of muscle glycogen in the inhibited glucose transport response to hypoxia. Three days of swim training (2 x 3 h/day) produce a 100% increase in glycogen and a 70% increase in GLUT-4 in epitrochlearis muscle. Glucose transport after 1 h of hypoxia in muscles from fed exercise-trained (ET) rats is not significantly elevated above basal and is 40% lower than that in muscles from fed sedentary (SED) rats. Glycogen levels after 1 h of hypoxia are reduced by 27 and 64% in muscles from fed ET and fed SED rats, respectively. After 2 h of hypoxia, glucose transport is significantly increased above basal in muscles from fed ET rats, but this response is still 55% lower than that in muscles from fed SED rats. After 2 h of hypoxia, glycogen is reduced by 50 and 83% in muscles from fed ET and fed SED rats, respectively. After a modified overnight fast (approximately 4.5 g of chow), the glucose transport and glycogen responses to 1 h of hypoxia are not significantly different between muscles from ET and SED rats. These findings demonstrate a strong inverse relationship between glycogen and hypoxia-stimulated glucose transport activity and that high levels of glycogen contribute to the inhibited glucose transport response to hypoxia. Furthermore, failure of the overexpression of GLUT-4 after exercise training to enhance the glucose transport response to contraction/hypoxia suggests selective targeting of the additional GLUT-4 to the insulin-responsive pool.

GLUT4 glucose transporter expression in rodent brain: effect of diabetes.

This study describes the regional and cellular expression of the insulin-sensitive glucose transporter, GLUT4, in rodent brain. A combination of in situ hybridization, immunohistochemistry and immunoblot techniques was employed to localize GLUT4 mRNA and protein to the granule cells of the olfactory bulb, dentate gyrus of the hippocampus and the cerebellum, with the greatest level of expression being in the cerebellum. Estimates of the concentration of GLUT4 in cerebellar membranes indicate that this transporter isoform is present in significant amounts, relative to the other isoforms, GLUT1 and GLUT3. Cerebellar GLUT4 expression was increased in the genetically diabetic, hyperinsulinemic, db/db mouse relative to the non-diabetic control, and even higher levels were observed in db/db female than db/db male mice. Levels of expression of GLUT4 protein in cerebellum appear to respond to the level of circulating insulin, and are reduced in the hypoinsulinemic streptozotocin-diabetic rat. Exercise training also results in reduced insulin levels and comparably reduced levels of GLUT4 in the cerebellum. These studies demonstrate a chronic insulin-sensitive regulation of GLUT4 in rodent brain and raise the possibility of acute modulations of glucose uptake in these GLUT4 expressing cells.

Effects of exercise training on glucose transport and cell surface GLUT-4 in isolated rat epitrochlearis muscle.

The effects of exercise training on maximal glucose transport activity and cell surface GLUT-4 were examined in rat epitrochlearis muscle. Five days of swim training (2 x 3 h/day) produce a significant increase in citrate synthase activity (24.5 +/- 0.6 vs. 20.1 +/- 0.7 micromol x min(-1) x g(-1)), GLUT-4 content (22.9 +/- 0.8 vs. 17.4 +/- 0.4% GLUT-4 standard), and glycogen levels (54.3 +/- 9.4 vs. 28.6 +/- 9.4 micromol/g). Maximally, insulin-stimulated glucose transport activity and cell surface GLUT-4 are increased by 55 (1.50 +/- 0.11 vs. 0.97 +/- 0.10 micromol x ml(-1) x 20 min(-1)) and 48% [12.0 +/- 0.8 vs. 8.1 +/- 0.9 disintegrations x min(-1) (dpm) x mg(-1)], respectively, in exercise-trained epitrochlearis muscles. In contrast, hypoxia-stimulated glucose transport activity and cell surface GLUT-4 are reduced by 38 (0.78 +/- 0.08 vs.1.25 +/- 0.14 micromol x ml(-1) x 20 min(-1)) and 40% (5.7 +/- 0.9 vs. 9.4 +/- 1.2 dpm/mg), respectively, in exercise-trained epitrochlearis muscles. These results demonstrate that changes in insulin- and hypoxia-stimulated glucose transport activity after exercise training are fully accounted for by the appearance of cell surface GLUT-4 and support the concept of two intracellular pools of GLUT-4. Finally, we propose that high levels of muscle glycogen with exercise training may contribute to the decrease in hypoxia-stimulated glucose transport activity.

Regulation of cell surface GLUT4 in skeletal muscle of transgenic mice.

Marked overexpression of the glucose transporter GLUT4 in skeletal muscle membrane fractions of GLUT4 transgenic (TG) mice is accompanied by disproportionately small increases in basal and insulin-stimulated glucose transport activity. Thus we have assessed cell surface GLUT4 by photolabelling with the membrane-impermeant reagent 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1, 3-bis(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA) and measured the corresponding glucose transport activity using 2-deoxyglucose in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice in the absence and presence of 13.3 nM (2000 mu units/ml) insulin, without or with hypoxia as a model of muscle contraction. TG mice displayed elevated rates of glucose transport activity under basal and insulin-stimulated conditions, and in the presence of insulin plus hypoxia, compared with NTG mice. Photoaffinity labelling of cell surface GLUT4 indicated corresponding elevations in plasma membrane GLUT4 in the basal and insulin-stimulated states, and with insulin plus hypoxia, but no difference in cell surface GLUT4 during hypoxia stimulation. Subcellular fractionation of hindlimb muscles confirmed the previously observed 3-fold overexpression of GLUT4 in the TG compared with the NTG mice. These results suggest that: (1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, regardless of the levels observed in the corresponding subcellular membrane fractions, (2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the response to hypoxia/ contraction-stimulated glucose transport is unchanged, and (3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.

Effects of chromium and resistive training on muscle strength and body composition.

Sixteen untrained males (23 +/- 4 yr), were studied to determine the effects of chromium (Cr) supplementation (200 micrograms.d-1) and a 12-wk resistive exercise training program on muscle strength, body composition, and Cr excretion. The subjects trained 3 times per week with two sets of 8-10 repetitions at 90% of 1 repetition maximum using Keiser variable resistance machines. Food records were used to estimate Cr intake (approximately 36 micrograms.d-1), energy intake, and the percent kJ from protein. The resistive training program resulted in significant increases in total body muscular strength in both the Cr and placebo groups (24% and 33%; P < 0.05). Body weight, percent body fat, lean body mass, and skinfold thicknesses were unchanged in either group after resistive training. Cr excretion increased in the Cr group after 6 wk of Cr supplementation (0.15 +/- 0.08 vs 1.52 +/- 1.26 micrograms.d-1; P < 0.01) and remained higher at 12 wk of training (2.03 +/- 1.73). These results indicate that Cr supplementation, in conjunction with a progressive, resistive exercise training program, does not promote a significant increase in strength and lean body mass, or a significant decrease in percent body fat. Cr supplementation results in a significant increase in Cr excretion that is not altered by resistive training.