qRT-PCR Analysis of GLUT-4 and Assessment of Trolox as an Effective Antioxidant in Diabetic Cardiomyoblasts.

A high global prevalence of diabetes and its implications on the heart in vivo and in vitro tools have been pursued to alleviate the complications of high glucose. This chapter oulines the methods used for maintaining H9C2 cardiomyoblasts in vitro and for stimulating hyperglycemic situation. In addition, we present a method to assess cellular GLUT-4 expression using qRT-PCR. This cellular model also allows us to examine the therapeutic approach of an antioxidant, Trolox, for upregulating GLUT-4 and uptake of glucose under hyperglycemic condition.

Flow Cytometric Analysis of Hyperglycemia-Induced Cell Death Pathways in Cardiomyoblasts.

Type-2 diabetes, characterized by hyperglycemia causing various symptoms of metabolic disorders in the heart, kidneys, and brain, has many underlying molecular mechanisms leading to functional insufficiency of these organs. We describe protocols wherein we have optimized conditions for maintenance of hyperglycemic H9c2 cell lines and design to assess the effect of a water-soluble vitamin, Trolox, on the apoptotic pathway. Primarily, the design provides researchers to analyze apoptosis by flow cytometry.

Protection against oxidative stress caused by intermittent cold exposure by combined supplementation with vitamin E and C in the aging rat hypothalamus.

This study investigated the effects of combined supplementation with vitamin E and C against oxidative stress (OS) caused by intermittent cold exposure (ICE) in the hypothalamus (HY) of aging male Wistar rats [adult (3-months), middle-aged (18-months) and old (24-months)]. Each age was divided into sub-groups: control (CON), cold-exposed at 10 °C (C10), cold-exposed at 5 °C (C5), supplemented control (CON+S) and supplemented cold-exposed at either 5 °C (C5+S) or 10 °C (C10+S). The supplement was a daily dose of 400 mg vitamin C and 50 IU of vitamin E/kg body weight. Cold exposure lasted 2 h/day for 4 weeks. All age groups exposed to cold showed increase in body mass and feeding efficiency. Feeding efficiency in the supplemented old group showed a statistically significant increase in the cold (p < 0.001). Age-related increases in levels of hydrogen peroxide (H2O2), protein carbonyl (PrC), advanced oxidation protein products and thiobarbituric acid reactive substances (TBARS) were further increased by cold in the HY. Cold reduced thiol(P-SH) levels and increased superoxide dismutase (SOD) and, catalase (CAT) activities as well as Hsp72 levels. However, supplementation lowered H2O2, PrC and TBARS with decreases in Hsp72 levels and in SOD and CAT activities. These changes were concomitant with elevations in P-SH, vitamin E and C levels. The results show that the OS caused by ICE in the HY and its subsequent protection following supplementation is related to the intensity of ICE as well as age of the animal. Immunohistochemical studies are underway to examine the findings on ICE-induced oxidative injury in the HY, and the prospects for vitamin E and C supplementation in the senescent.

Protective role of vitamins E and C against oxidative stress caused by intermittent cold exposure in aging rat's frontoparietal cortex.

This study examined the role of vitamins E and C in combating oxidative stress (OS) caused by intermittent cold exposure (ICE) in the frontoparietal cortex (FPC) of adult (3 months), late-adult (12 months), middle-aged (18 months) and old (24 months) male Wistar rats. Each age group was divided into sub-groups, control (CON), cold-exposed at 5°C (C5), control supplementees (CON+S) and cold-exposed supplementees (C5+S). The supplement was a daily dose of 400mg vitamin C and 50I.U.of vitamin E/kg body weight. Cold exposure lasted 2h/day for 4 weeks. All age groups except the old showed an increase in the final body mass in the cold-exposed. The feeding efficiency was higher in the cold-exposed irrespective of age. OS as reflected in age-related increased levels of hydrogen peroxide, protein carbonyl, advanced oxidation protein products and malondialdehyde showed further increase with ICE in the FPC. However, vitamins E and C supplementation attenuated the ICE-induced OS. ICE depleted the levels of tissue vitamins E and C while supplementation resulted in increased levels. Further age emerged as a significant factor in ICE-induced stress and also the response to vitamins E and C supplementation. Behavioral studies are underway to examine the findings on ICE-induced oxidative injury in the FPC, and the prospects for using vitamins E and C in cold exposures in the aged.

Peroxyl-induced oxidative stress in aging erythrocytes of rat.

This study aims at determining the possible changes in intracellular calcium (Ca (i) (2+) ), plasma membrane calcium ATPase (PMCA) activity and phosphatidylserine (PS) along with glutathione (GSH) level in response to an oxidant challenge in vitro. Erythrocytes were isolated on Percoll and incubated with 2, 2'azobis (2-aminopropane) hydrochloride (AAPH) as well as with vitamin C preceding AAPH incubation. Membrane integrity in terms of hemolysis was negatively related to acetylcholine esterase (AChE) activity with the extent of reduction under OS being higher in the old erythrocyte than in the young. A divergent pattern was seen towards lower PMCA and higher (Ca (i) (2+) ) in the young and old cells. However, the PMCA activity in the stressed young cell was high when pre-treated with vitamin C. PS externalization in the young under OS is perhaps analogous to normal aging, with vitamin C preventing premature death. These findings suggest that young erythrocytes may benefit from vitamin C in therapies addressed towards the mechanisms underlying the reduced effects of OS.

Oxidative stress and intracellular pH in the young and old erythrocytes of rat.

The effects of oxidative stress (OS) on the rat erythrocytes (RBCs) that were fractionated on the percoll/BSA gradient into young and old cells were studied to find out if the altered Na+/H+ and Cl⁻/ HCO3⁻ antiporters and in turn the intracellular pH (pHi) could act as one of the promoters of cell death. Old cells were more spherical with lesser surface area, more fragile osmotically and had lesser protein sulphydryl content than the young cells. OS was induced in RBCs by 2,20-azobis (2-amidinopropane) dihydrochloride (AAPH). AAPH increased the superoxide dismutase (SOD) activity and MDA level and, the changes between the young and old. Interestingly, vitamin C was effective in reducing MDA in the old. Further, in the old a rapid Na+-dependent acidification in the presence of AAPH and a marginal acidosis in the presence of vitamin C were evident. Old RBCs exhibited higher acidosis and vitamin C was less effective in lowering the stress-induced acidosis compared to the young. Our studies suggest that increased acidosis followed by low intracellular pH could be one of the determinant factors for the disappearance of old RBCs from circulation, and perhaps of the young too under OS.

Age-related responses of the rat cerebral cortex: influence of vitamin E and exercise on the cholinergic system.

In this study, we have assessed the impact of vitamin E and exercise on acquisition and retention of spatial memory for a given task in aging rats, using a T-maze. Acetylcholine esterase (AChE) and cholineacetyl transferase (ChAT) activities and acetylcholine (ACh) were measured in the cerebral cortex (CC) of male Wistar rats of 4- (adult), 12- (middle-aged) and 18-months (old) of age. Animals were categorized into sedentary [(SEC (N)], sedentary supplemented [SEC (+E)], swim trained [SWT (N)] and swim trained supplemented [SWT (+E)]. In the old, ChAT activity increased in the SEC (+E). AChE activity was highest in the adults, irrespective of training or supplementation. By contrast, ACh concentration remained unaltered with age, exercise and supplementation. Middle-aged and old rats were benefited in terms of a better acquisition and retention in the case of those that were trained and supplemented with Vitamin E. Adults showed better retention in all the groups after 7 and 15 days, while in the middle-aged, training was beneficial after 15 days. We observed decreased AChE activity when old rats were trained with the supplement. Our results also suggest that this regimen may be analogous to the AChE inhibitors that are widely advocated to derive positive benefits in up-regulating the possible reduction in ACh and in turn age-associated memory deficits.

In vitro models of oxidative stress in rat erythrocytes: effect of antioxidant supplements.

The present study was designed to induce oxidative stress in lipid and aqueous phases through azo bis(2-amidinopropane)dihydrochloride (AAPH), 2,2'-azobis 2,4-dimethylvaleronitrile (ADVN) and hydrogen peroxide (H(2)O(2)) either alone or in combination with vitamin C or vitamin El and to assess the vulnerability of rat erythrocytes to oxidative stress. While AAPH acted equally on cell membrane and cytosol, ADVN increased OS in the membrane. The extent of hemolysis and increased membrane fragility caused was more in the case of azo compounds than of H(2)O(2). While vitamin E (2mM) reduced oxidative stress in the membrane, vitamin C (60mM) was more effective in the lysates. The concentration of malondialdehyde and advanced oxidation protein products was lowered by antioxidants. The level of lipofuscin, a product of lipid peroxidation was also increased by ADVN and H(2)O(2). Antioxidants, did, however, reduce the accumulation of protein carbonyl content in cells exposed to azo compounds although they were ineffective in inhibiting oxidation of membrane band 3 protein and sulphydryl content. Taken together, our study demonstrated the antioxidative property of vitamin E and vitamin C in reducing oxidative stress in aqueous as well as lipid phases of erythrocytes and further suggested the feasibility of in vitro models in evaluating the mechanisms of oxidative injury.

Intermittent hypobaric hypoxia-induced oxidative stress in rat erythrocytes: protective effects of vitamin E, vitamin C, and carnitine.

This study was aimed at determining the effect of vitamin E, vitamin C, and carnitine on intermittent hypobaric-hypoxia-induced oxidative stress (OS) in erythrocytes. For this purpose, male Wistar rats of 4 months of age were orally supplemented with one of the antioxidants prior to exposure to altitudes of 5700 m or 6300 m. Hemoglobin (Hb) and OS indices such as osmotic fragility and hemolysis were measured together with lipid peroxidation (LPO) and protein oxidation. The increase in Hb was accompanied by increase in activities of antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) during exposure to both the altitudes without any further elevation by supplements. The extent of reduction in osmotic fragility and hemolysis by vitamin E and carnitine was greater at 6300 m than at 5700 m. Increase in LPO products, for example, malondialdehyde (MDA) and lipofuscin-like autofluorescent substances (AFS) was noticeable at both the altitudes, and vitamin E and carnitine were effective in reducing LPO. While protein oxidation products such as carbonyl content (PrC) and advanced oxidation protein products (AOPP) increased at 6300 m, protein sulphydryl (P-SH) content decreased. P-SH levels were restored on supplementation of antioxidants. Hence, our results indicate that vitamin E, vitamin C, and carnitine may be beneficial in overcoming OS and hemolysis under situations such as intermittent hypobaric hypoxia (IHH) and hypobarotherapy wherein hypoxia is used to correct many pathological situations in humans. Further, this study suggests that supplementation of vitamin E, vitamin C, and L-carnitine alone and not in combination can be beneficial in attenuating the OS associated with IHH compared to the unsupplemented rats exposed to two different altitudes.

Modification by vitamin E and exercise of oxidative stress in regions of aging rat brain: studies on superoxide dismutase isoenzymes and protein oxidation status.

This study was aimed at determining the effect of exercise and vitamin E on age-associated changes in the superoxide dismutase (SOD), lipid (LPO) and protein oxidations (PO) in the cerebral cortex (CC), cerebellum (CB) and hippocampus (HC) of rat brain. For this, male Wistar albino rats of 4- (adult), 12- (middle-age) and 18-month (old) of age were orally supplemented with vitamin E and swim trained at 3% intensity for 30 min/day, 5 days/week, and for a period of 30 days. Reduced total SOD was evident with age in the CC while it was highest in the HC of old rats. Vitamin E elevated SOD in the old trainees. Mn-SOD increased in the middle-age and old trainees and Cu Zn-SOD increased in the supplemented and trained adults. Age-related and region-specific increase in protein carbonyl (PrC) content with decreased sulphydryl (P-SH) was seen. Vitamin E reduced PrC and advanced oxidation of protein products (AOPP) in all ages, and appreciably in the HC and CB. Our study emphasizes a correlation between mitochondrial H(2)O(2) generation, Mn-SOD activity and MDA level, and reveals in part an age-related increase in lipid peroxidation and protein oxidation, and that may occur under conditions such as vitamin E deficiency.

Blood lipid profile and myocardial superoxide dismutase in swim-trained young and middle-aged rats: comparison between left and right ventricular adaptations to oxidative stress.

Region-wise interactive effects of age, swim intensity, and duration on exercise performance in the myocardium and serum lipid profile in young (4 months) and middle-aged (12 months) rats were examined. Animals were allocated to the sedentary control (SE-C) or one of the nine trainee groups. Swim training was for 6 days/week and for 4 weeks at 3 durations (20, 40, and 60 min/day) and intensities (2%, low; 3%, medium; 5%, high). Swim velocity and external work showed an age-related decline with low-intensity of 20 min/day in the middle aged. Reduction in serum cholesterol, low-density lipoproteins (LDLs), and triglycerides were accompanied by elevated levels in high-density lipoprotein in the low-to-moderately trained ones for 20 and 40 min/day. Training at 2%, intensity for 20 min/day was sufficient to alter the blood lipid profile and improve swim performance, and endurance in terms of blood lactate. A concomitant increase in Mn-superoxide dismutase (Mn-SOD) activity and reduced malondialdehyde in the left ventricle (LV) and right ventricle (RV) were evident. Lipofuscin was higher in the LV compared to RV. Our results reflect the minimization of free radical generation through appropriate exercise protocols. Our findings on improved blood lipid profile could be related to lower free radicals, which would otherwise oxidize LDLs. Further, swim training when initiated in the young and middle age for as low as 20 min/day at 2% intensity improves the Mn-SOD in the LV and RV. However, the adaptive response of the LV was weaker when compared to the RV, more so in the middle aged.

Adaptations of the antioxidant system in erythrocytes of trained adult rats: impact of intermittent hypobaric-hypoxia at two altitudes.

We have investigated the effects of daily exposure to intermittent hypobaric-hypoxia to two simulated altitudes (5700 m and 6300 m) in adult male rats that had been regularly swim trained in normoxia at sea level prior to exposures. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) along with the oxidative stress (OS) indices, malondialdehyde (MDA) and protein carbonyl content were measured in erythrocytes and their membranes. Hemoglobin increased in the trained animals exposed to 5700 m and in untrained rats exposed to 6300 m. Osmotic fragility in terms of hemolysis increased in altitude exposed animals. SOD increased in those exposed to 6300 m, while CAT increased in trained rats exposed to 5700 m and to 6300 m unlike in untrained rats where CAT increased only at 6300 m. GSH-Px showed varying degrees of elevation in all animals exposed to both altitudes. Erythrocyte membranes showed significant elevations in malondialdehyde (MDA) at 6300 m, while elevated protein carbonyls were noticeable at both altitudes in whole cells and membranes. These results suggest a positively associated elevation in protein oxidation with altitude in trained rats. At 5700 m, animals were less stressed, unlike at 6300 m, as seen from the magnitude of elevations in the OS indices and from the responses of the antioxidant enzymes.

Swim exercise training and adaptations in the antioxidant defense system of myocardium of old rats: relationship to swim intensity and duration.

We examined a suitable swim program of different intensities and durations that could evoke changes in the myocardial antioxidant capacity in 22-month-old rats. Male rats (Rattus norvegicus) were assigned to either a sedentary control (SE-C) group or one of six trainee groups. Animals were swim-exercised for 4 weeks with either 20 min or 40 min/day, and three intensities, low, moderate and high. Low-intensity at 20 min/day elicited maximum swim velocity (Sv) and endurance capacity (P<0.05). While serum total cholesterol, triglyceride and low-density lipoprotein (LDL-C) levels were significantly reduced, high-density lipoprotein (HDL-C) showed an increase (P<0.05) in low-intensity trained rats (20 min/day) over SE-C. Notable reduction in blood lactate was also evident. Exercise training significantly increased superoxide dismutase (Mn-SOD), decreased lipid peroxidation products, malondialdehyde and lipofuscin in the left and right ventricles. Increased Mn-SOD with concomitant decrease in lipofuscin in left ventricle was significantly greater than in right ventricle. Moderate- to high-intensity exercise was not effective in either reducing lipid peroxidation products or elevating Mn-SOD activity. These data suggest that swim training at low-intensity of 20 min/day is beneficial as a major protective adaptation against oxidative stress in old myocardium.

Dietary vitamin E and physical exercise: I. Altered endurance capacity and plasma lipid profile in ageing rats.

The effect of vitamin E on the exercise performance and plasma lipid profile was studied in male Wistar rats of 4-(young adults), 8-(old adults), 12-(middle-age) and 22-months (old) of age. Animals were orally supplemented with vitamin E and allowed to swim for 30 min/day, 5 days/week and for a total period of 60 days. Swim velocity (S(v)), external work done (W(ext)) and endurance (E) capacity were the parameters that were used to assess the exercise performance of the trained rats that were either supplemented or non-supplemented with the dietary antioxidant. Plasma lipid profile analyses were in terms of low-density lipoprotein (LDL-C), high-density lipoprotein, (HDL-C) cholesterol and total cholesterol (C). Age-related decline in S(v) was noticeable in the 22-months old rats. However, the effect of vitamin E on the S(v) between the trained groups was not evident in any of the age groups. W(ext) increased linearly with age with no significant variations between the trainees. Trainee rats, when allowed to swim to exhaustion, showed a higher endurance capacity when supplemented with vitamin E. However, this capacity declined with age. There was a significant age-associated elevation in plasma C with corresponding increase in LDL-C. Exercise training in conjunction with vitamin E supplementation was most effective in elevating HDL-C levels in all age groups. These changes were accompanied by significant reductions in cholesterol/HDL-C ratios in animals receiving vitamin E, sedentary or otherwise. Our data suggests that it may be important to consider vitamin E while attempting to derive the benefits of swim training, both in terms of favorably altering the plasma lipid profile as well as enhancing the endurance capacity of exercise trainees. Dietary supplementation by vitamin E could attenuate the early onset of fatigue in the old.

Dietary vitamin E and physical exercise: II. Antioxidant status and lipofuscin-like substances in aging rat heart.

The heart faces a high risk of free radical injury owing to a slow generation of antioxidant (AO) enzymes by its cells. A general decline in this system may be another reason for the development of age-related diseases. Although the correlation between aging and exercise has been studied extensively, these studies have produced conflicting data on the effects of vitamin E on the aging heart, when it is introduced as an intervening factor. To investigate these effects, we determined the activities of antioxidant enzymes (AOEs) such as superoxide dismutase (SOD) and catalase (CAT), lipid peroxidation (LP), lipofuscin (LF)-like autofluorescent substances and vitamin E content in the left and right ventricles (LV and RV) of the heart in male Wistar albino rats of 4-(young adults), 8-(old adults), 12-(middle-age) and 22-mos(old) of age. Animals were orally supplemented with vitamin E and allowed to swim for 30 min/day, 5 days/week and for a total period of 60 days. Exercise training in all the age groups except the old was effective in upregulating the SOD activity. Old trainees showed an increase in SOD activity when supplemented with vitamin E. In the 22-mo-olds, a remarkable decrease in CAT activity was seen. Exercise by itself upregulated the CAT as well as SOD activity in all age groups except the old wherein vitamin E was effective in increasing the activities of AOEs. Supplementation significantly reduced LP as evidenced by lowered malondialdehyde (MDA) and LF-like autofluorescent substances in the trained as well as sedentary rats. Tissue vitamin E content was low in the swim trainees that were not supplemented. This change, well emphasized in the trainee groups of 22-mo-old suggests the probable utilization of vitamin E in keeping free radicals at bay. Our results suggest that vitamin E can stand out as a significant tool in ameliorating the declining AO defense in the old rats.

Effect of fenvalerate-20EC on sericigenous insects. I. Food utilization in the late-age larva of the silkworm, Bombyx mori L.

The effect of the common pyrethroid insecticide fenvalerate-20EC (Sumicidine-20EC) on food utilization parameters in the pure multivoltine (Pure Mysore) and pure bivoltine (NB4D2) races of silkworm, Bombyx mori, fed ad libitum with Morus alba, was studied in the final two instars. Insecticide treatment was found to reduce the rates of feeding, assimilation, and conversion along with the efficiencies of conversion of ingested and digested food into body substance. This was due to the toxic effects, which interfered with the silkworms' feeding behavior, and physiology of digestion, and the magnitude of changes increased with the increase in the concentration of fenvalerate used. To overcome the deleterious effects of such pyrethroid insecticides, it has been suggested that the safe periods for use of mulberry leaves contaminated with fenvalerate for summer and winter.

Effect of fenvalerate-20EC on sericigenous insects. II. Digestive enzymes in the nutritive physiology of silkworm, Bombyx mori L.

Changes in the activities of a few digestive enzymes after fenvalerate-20EC treatment were studied during the late stages of multivoltine and bivoltine silkworms, Bombyx mori. Insecticide treatment induced a reduction in the activity of such enzymes as amylase, sucrase, and protease, whereas the trehalase activity was enhanced when the midgut was used as the source of enzyme. The changes recorded in the activity of these enzymes indicated probable adverse effects of fenvalerate on silkworm metabolism through interference with the functional abilities of digestive enzymes.