Sodium orthovanadate suspended in a lichee black tea decoction effectively regulates blood glucose levels in rats with insulin-dependent, streptozotocin (STZ)-induced diabetes. The primary advantage of vanadate delivery with the tea decoction over conventional systems that use water suspensions of vanadate is a significant reduction in the toxic side effects of vanadate. It is unknown if the tea alters the bioavailability of vanadate. Male Sprague-Dawley rats were administered an intravenous injection of STZ to induce diabetes. Four days later, the diabetic rats were treated by oral gavage with 40 mg of Na-orthovanadate suspended in double-distilled, deionized water (V/H2O), tea/vanadate (TV) decoction, or were treated with the tea decoction alone. Vanadium concentrations were measured in blood and various tissues at 1 to 24 hours posttreatment using graphite furnace atomic absorption spectrophotometry. With the exception of bone, maximal vanadium concentration in plasma and tissue samples were observed 2 hours after ingestion, but steadily decreased after that. Plasma vanadium levels continued to decrease until 16 hours. In contrast, vanadium steadily accumulated in bone over the 24-hour period. Overall, rats treated with V/H2O contained similar or significantly higher concentrations of vanadium in all tissues compared with TV treatment. The pattern of vanadium accumulation was also similar over time in both treatment groups. Vanadium levels were highest in bone > kidney > liver > pancreas > lung > heart > muscle > brain in both TV- and V/H2O-treated animals. This study demonstrates that the accumulation of vanadium in diabetic rats is reduced when coadministered with a black tea decoction in comparison to administration of vanadium in water. However, this effect is unlikely to be of a magnitude to explain the full capacity of TV to reduce the toxic side effects of vanadate.

A novel black tea decoction containing vanadate has successfully replaced insulin in a rat model of insulin-dependent diabetes but is untested in non-insulin-dependent diabetic animals. A tea-vanadate decoction (TV) containing 30 or 40 mg sodium orthovanadate was administered by oral gavage to two groups of Zucker diabetic fatty rats and a conventional water vehicle containing 30 or 40 mg of sodium orthovanadate to two others. In the latter group receiving the 30-mg dose, vanadate induced diarrhea in 50% of the rats and death in 10%. In contrast, TV-treated rats had no incidence of diarrhea and no deaths. Symptoms were more severe in both groups with higher vanadate doses, so these were discontinued. After approximately 16 weeks, the level of vanadium in plasma and tissue extracts was negligible in a further group of untreated rats but highly elevated after vanadate treatment. Vanadium levels were not significantly different between the TV-treated diabetic rats and the diabetic rats given vanadate in a water vehicle. Over the 115 days of the study, blood glucose levels increased from approximately 17 to 25 mmol/L in untreated diabetic rats. This was effectively lowered (to <10 mmol/L) by TV treatment. Fasting blood glucose levels were 5, 7, and 20 mmol/L in control (nondiabetic, untreated), TV-treated and untreated diabetic rats, respectively. Rats required treatment with TV for only approximately 50% of the days in the study. Increase in body mass during the study was significantly lower in untreated diabetic rats (despite higher food intake) than the other groups. Body mass gain and food intake were normal in TV-treated rats. Water intake was 28 mL/rat daily in control rats, 130 mL/rat daily in untreated diabetic rats, and 52 mL/rat daily in TV-treated diabetic rats. Plasma creatinine and aspartate aminotransferase levels were significantly depressed in untreated diabetic rats, and TV treatment normalized this. Our results demonstrate that a novel oral therapy containing black tea and vanadate possesses a striking capacity to regulate glucose and attenuates complications in a rat model of type II diabetes.

Oral administration of vanadate has a strong hypoglycemic effect but results in toxic side effects like life-threatening diarrhea. Tea is known to have potent antidiarrhea effects. We investigated the potential of suspending the vanadate in a tea decoction to reduce the diarrheatic action of vanadate. A concentrated extract of Lichee black tea was, therefore, added to sodium orthovanadate. Streptozotocin (STZ)-induced diabetic rats were orally gavaged with vanadate suspended in water or in the tea decoction, or with the tea extract alone. Blood glucose levels were assessed daily over 11 weeks with levels greater than 10 mmol/L warranting therapeutic intervention. Both the vanadate/water and vanadate/tea solutions acutely reduced blood glucose. The tea extract alone had no effect. The majority of vanadate/water-treated rats developed diarrhea and mortality rates approached 40%. Vanadate/tea-treated diabetic rats experienced no diarrhea or mortality and liver and kidney analyses (plasma ALT and creatinine, blood urea nitrogen [BUN], and urine-specific gravity) were normal. Animals treated with vanadate/tea retained blood glucose levels less than 10 mmol/L for an average of 24 consecutive days without subsequent treatments. Cataract formation was completely prevented. The mechanism of action of vanadate may have involved beta-cell stimulation because vanadate/tea-treated diabetic rats exhibited normal plasma insulin levels. In summary, because of its long-lasting effects, oral administration, and lack of side effects, vanadate/tea represents a potentially important alternative therapy for an insulin-deficient diabetic state.

We examined the effects of insulin on fatty acid uptake in L-cell fibroblasts, usingcis-parinaric acid to measure uptake rates in the absence of esterification and [3H]oleic acid to measure uptake rates in the presence of esterification. L-cells exhibited both high and low affinity insulin binding sites withKdof 23 nMand 220 nMand a cellular density of 1.4 and 6.8 × 105sites/cell, respectively. Insulin in the range 10−9to 10−7Msignificantly decreased both the initial rate and maximal extent ofcis-parinaric acid uptake by 24 to 30%. Insulin also reduced [3H]oleic acid uptake up to 35%, depending on insulin concentration and decreased the amount of fatty acid esterified into the phospholipids and neutral lipids by 28 and 70%, respectively. In contrast, glucagon or epinephrine stimulated both the initial rate and extent ofcis-parinaric acid uptake 18 and 25%, respectively. Because L-cells lack β-adrenergic receptors, the epinephrine effect was not the result of β-receptor stimulation. Hence, insulin altered not only fatty acid uptake, as determined bycis-parinaric and oleic acid uptake, but also altered the intracellular oleic acid esterification.

Membrane dielectric as a primary basis for effects of ethanol was examined in synaptic plasma membranes (SPM) of genetically selected ethanol-sensitive long-sleep (LS) and ethanol-resistant short-sleep (SS) mice. Multifrequency phase and modulation fluorometry of diphenylhexatriene (DPH) was used to resolve structural and dielectric differences in the membrane interior core. Fluorescence spectral peak ratios, fluorescence lifetime analysis, and initial rates of photoreaction of DPH in SPM provided sensitive measures of SPM interior core dielectric properties. The membrane microenvironment sensed by DPH was more polar in SPM from SS mice than in SPM from LS mice. Physiological concentrations of ethanol in vitro (25-75 mM) increased the SPM interior core dielectric and potentiated photoreaction of DPH with other membrane components of SPM from LS, but not SS, mice. These effects of ethanol in vitro were maximal by 75 mM ethanol and/or exacerbated at higher ethanol. In addition, ethanol in vitro increased the fraction of DPH associated with photoreaction products with lipids from SPM of ethanol-sensitive LS mice. The data were consistent with ethanol in vitro increasing the polar molecules (ethanol and/or water) of SPM from LS but not SS mice. It is suggested that ethanol alters the polarity and increases reactivity of the interior core lipid-protein interface.

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.

It is well known that Ca2+ plays a crucial role in determining the heart function and cellular integrity.1–5 While the interaction of Ca2+ with myofibrillar proteins is of prime importance in eliciting the contractile events, different membrane systems such as sarcolemma, sarcoplasmic reticulum and mitochondria are considered to be involved in the delivery as well as removal of Ca2+ from the contractile apparatus. The superficial store of Ca2+, which is represented as ATP-independent Ca2+-binding with the sarcolemmal membrane, is believed to be a buffer site between extracellular and intracellular compartments of Ca2+ and serves as an immediate source of Ca2+ for contraction. Although most of the Ca2+ enters the myocardial cell through Ca2+-channels located in the sarcolemmal membrane, the biochemical basis for opening Ca2+-channels is far from clear. Recently, the involvement of a sarcolemmal Ca2+/Mg2+ ATPase, which is activated by millimolar concentrations of Ca2+, has been suggested in a gating mechanism for the influx of Ca2+ into the cardiac cell.1,3,6,7 While Ca2+-stimulated ATPase (activated by micromolar concentration of Ca2+ in the presence of Mg2+ and ATP-dependent Ca2+-binding in the sarcolemmal membrane are believed to serve as a Ca2+-pump mechanism for the efflux of Ca2+, the sarcolemmal Na+-Ca2+ exchange mechanism has been implicated both in entry and removal of Ca2+ from the cell.1,3,8–10

The stepwise N-methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) (phospholipid methylation) was assessed in cardiac sarcolemma and sarcoplasmic reticulum of aging rats. This phenomenon was depressed in aging hearts relative to young ones. A decrease in activity of catalytic sites appears to be involved in the depressed phospholipid methylation of aging myocardium.

Numerous experimental studies have implied a link between diabetes-induced abnormal lipid buildup in the myocardium and the development of cardiomyopathy. Because the diabetic state in rats is associated with lowered T3 (triiodothyronine) and T4 levels and because diabetic patients excrete large amounts of myo-inositol, a lipotropic agent, we investigated the effects of myo-inositol and T3 on the elevated myocardial lipid levels and depressed cardiac performance of streptozocin (55 mg/kg i.v.)-induced diabetic (STZ-D) rats, myo-inositol (2.5 g kg−1 day−1 in the drinking water) and T3 (30 μg · kg−1 · day−1 s.c.) were given for an 8-wk period 3 days after diabetes induction. Untreated diabetic rats were characterized by a decreased rate of body weight gain, hyperglycemia, and hypoinsulinemia, which were not altered after myo-inositol and/or T3 treatment. Thyroid status of diabetic animals was normalized by T3 alone or in combination with myo-inositol but not by myo-inositol alone. The elevations in plasma and myocardial lipids associated with the diabetic state were prevented by myo-inositol treatment. However, the plasma lipid and myocardial cholesterol levels in diabetic rats remained elevated or were further increased with treatment with T3 or myo-inositol plus T3. myo-inositol treatment partially improved cardiac performance in STZ-D rats. T3 treatment alone did not prevent cardiac dysfunction in diabetic rats. There was, however, some improvement in heart function in the groups treated with both myo-inositol and T3, coinciding with a significant decrease in the myocardial triacylglycerol level. The data indicate that a possible correlation may exist between elevated myocardial triacylglycerol levels and cardiac dysfunction in diabetic rats.

Heart hypertrophy in response to increased workload is a complex process in which this organ adapts to the environment by increasing the muscle mass in terms of additional contractile units and formation of different types of contractile proteins (myosin isozymes). In addition, augmentation of membrane function with respect to calcium transport activities of sarcolemma and sarcoplasmic reticulum occurs at early stages of cardiac hypertrophy associated with hyperfunction of the myocardium. However, if cardiac hypertrophy is left unattended beyond a certain period, physiological hypertrophy is converted to pathological hypertrophy whereby the cardiac muscle is unable to generate an adequate amount of contractile activity. It appears that the sympathetic nervous system is activated for producing beneficial effects at early stages but an elevated level of sympathetic tone for a prolonged period could result in dysfunction of the cardiac muscle. The transition of physiological hypertrophy to pathological hypertrophy seems to be due to the occurrence of intracellular calcium overload in the myocardial cell as a consequence of defects in the membrane calcium transport systems. It is suggested that careful attention should be paid not only to removal of the stimulus responsible for cardiac hypertrophy but also to lowering sympathetic tone. Efforts should also be made to prevent the occurrence of intracellular calcium overload due to membrane defects.

Diabetes mellitus is frequently associated with a primary cardiomyopathy. The mechanisms responsible for this heart disease are not clear, but an alteration in myocardial Ca2+ transport is believed to be involved in its development. Even though sarcolemma plays a crucial role in cellular Ca2+ transport, little appears to be known about its Ca2+ transporting capability in the diabetic myocardium. In this regard, we have examined the status of the cardiac sarcolemmal Ca2+ pump during diabetes mellitus. Purified sarcolemmal membranes were isolated from male Wistar diabetic rat hearts 8 wk after streptozotocin injection (55 mg/kg iv). Ca2+ pump activity assessed by measuring its Ca2+-stimulated adenosine triphosphatase and Ca2+-uptake ability in the absence and presence of calmodulin was significantly depressed in the diabetic myocardium relative to controls. These results did not appear to have been influenced by the minimal sarcoplasmic reticular and mitochondrial contamination of this membrane preparation. Hence, it appears that the sarcolemmal Ca2+ pump is defective in the diabetic myocardium and may be involved in the altered Ca2+ transport of the heart during diabetes mellitus.

The subcellular distribution of the 5'-nucleotidase activity was investigated in normal and hypertrophied pig hearts; normal rat hearts were used for comparison. The left ventricular hypertrophy was induced in pigs by banding the supravalvular aorta for 4, 8 and 12 weeks. By employing different procedures for the isolation of cardiac membranes, a major catalytic site for 5'-nucleotidase was found to be located at sarcolemma in rat heart and microsomes (sarcoplasmic reticulum) in pig heart. A progressive decrease in the homogenate and microsomal 5'-nucleotidase activity occurred upon the development of myocardial hypertrophy in pigs. This reduction in microsomal 5'-nucleotidase activity was characterized by a depression in both apparent Vmax and Km values. These results indicate that a primary 5'-nucleotidase pool is present in the intracellular compartment of the pig heart and is altered during the development of hypertrophy.

There is an abnormal lipid accumulation in the myocardium that might be involved in the congestive heart failure frequently associated with individuals with diabetes mellitus. Because choline and methionine have been reported to modify the incidence of myocardial necrosis in rats fed various fat diets, we decided in our study to assess their effect on the cardiac dysfunction of diabetic rats. Female Wistar rats were made diabetic with streptozocin (STZ, 55 mg/kg i.v.). One week after diabetes induction, one group received choline (0.3 mg/ml), another received methionine (0.25 mg/ml), a third received a combination of the same doses of choline and methionine, and a fourth received neither of these agents; all were administered in the drinking water. Animals were treated for 7 wk. Insulin levels were lower and glucose values higher in the serum of STZ rats relative to controls. Serum cholesterol and triglycerides were significantly elevated in all diabetic animals, and they were also elevated (P less than .05) in the hearts of untreated diabetics. In contrast, the myocardial values of the same lipids were drastically reduced in the treated diabetic animals. Cardiac performance was depressed in all STZ animals, but there was a significant improvement in heart function in treated diabetics relative to untreated ones. Thus, it appears that the buildup of cholesterol and triglycerides in the myocardium are important contributors to the cardiac dysfunction that frequently accompanies diabetes mellitus.

The trace element vanadium has an unclear biological function. Vanadate, an oxidized form of vanadium, appears to have an insulin-like action. The effect of vanadate on blood glucose and cardiac performance was assessed in female Wistar rats 6 weeks after they were made diabetic with streptozotocin. When vanadate was administered for a 4-week period to the diabetic rats, their blood glucose was not significantly different from that of nondiabetic controls despite a low serum insulin. In contrast, blood glucose was increased about threefold in the diabetic rats that were not treated with vanadate; these rats also had low insulin levels. Cardiac performance was depressed in the untreated diabetic animals, but the cardiac performance of the vanadate-treated diabetic animals was not significantly different from that of nondiabetic controls. Thus vanadate controlled the high blood glucose and prevented the decline in cardiac performance due to diabetes.

In order to examine changes in Ca2+ transport in heart sarcolemma, cardiac hypertrophy was induced in rabbits by stenosis of the abdominal aorta and hearts were removed 18-20 weeks later; sham-operated normal rabbits were used as controls. Sarcolemmal vesicles were isolated from the left ventricular tissue by a sucrose density gradient method and the membrane composition as well as activities of certain marker enzymes were monitored to determine the purity of control and experimental fractions; Na+-Ca2+ exchange and Ca2+-pump activities were assessed by the Millipore filtration technique. No changes in Ca2+-influx were observed in Na+-loaded vesicles from the hypertrophied hearts when studied in the presence of different concentrations of calcium as well as at different times of incubation. In contrast, Na+-induced Ca2+-efflux from Ca2+-loaded vesicles was enhanced in the hypertrophied heart at different times of incubation and at different concentrations of sodium. ATP-dependent Ca2+-binding activity of sarcolemma from hypertrophied heart, when measured at different times of incubation and at several concentrations of calcium, was more than the control. Minimal but an equal amount of cross contamination was seen in both control and experimental preparations; however, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid contents were increased in sarcolemma from hypertrophied hearts. These results suggest that the sarcolemmal Ca2+-transport systems may become adapted during the development of hypertrophy for augmenting Ca2+-efflux from the hypertrophied myocardial cell and this may prevent the occurrence of intracellular Ca2+ overload in a stable form of cardiac hypertrophy.

To evaluate changes in Ca2+ transport activities in the cardiac sarcoplasmic reticulum (microsomes) and mitochondria, cardiac hypertrophy was induced in rabbits by constricting the abdominal aorta. The animals showed a stable non-failing left heart hypertrophy between 16-22 weeks after the operation. ATP-dependent Ca2+ uptake and Ca2+ binding activities were depressed in microsomes from hypertrophied rabbits in comparison with sham-operated controls (P less than 0.05). These changes were seen at different concentrations of free Ca2+ (10(-7) to 10(-4)M) and were accompanied by alterations in the phospholipid content of the microsomal fraction. Mitochondrial Ca2+ transport activities and phospholipid content remained unchanged in the hypertrophied heart. The results of this study identify a specific lesion in the sarcotubular membrane and suggest that the depressed Ca2+ transport activity in the microsomal fraction from the hypertrophied myocardium may be due to changes in its phospholipid composition.

Irrespective of the type and nature of stressful stimulus, elevated levels of circulating catecholamines are commonly observed. A small amount of catecholamines released in acute and mild stress is considered to increase heart function beneficially by binding to the β-adrenergic receptors, activating the adenylate cyclase system and raising the intracellular concentration of calcium (1,2). In this regard it should be mentioned that β-adrenergic receptors are coupled to adenylate cyclase by guanine nucleotide binding protein, and their activation by catecholamines is associated with formation of cyclic AMP, phosphorylation of Ca2+-channels through protein kinase and subsequent increase in the entry of Ca2+ into the myocardial cell. On the other hand, an excessive amount of catecholamine released in chronic and severe stress is believed to produce cardiotoxic effects which are associated with the occurrence of an intracellular Ca2+ overload and abnormalities in the excitation-contraction coupling process (3,4,5). Although participation of the β-adrenergic receptor-adenylate cyclase mechanism in raising the intracellular concentration of calcium is obvious at initial stages, its role in later stages with prolonged exposure of the myocardium to high doses of catecholamines is not clear at present. This view is based on the fact that prolonged exposure of tissue to catecholamines has been shown to produce desensitization of adrenergic receptors (6,7,8).

Circulating levels of catecholamines are increased dramatically under stressful conditions and these hormones are generally considered responsible for the development of stress-associated cardiomyopathy. In fact varying degrees of ultrastructural abnormalities as well as cellular necrosis have been shown to occur in the myocardium upon injecting animals with large doses of catecholamines including isoproterenol (1,2,3). Although earlier studies have demonstrated the occurrence of intracellular Ca2+ overload during the development of catecholamine-induced necrosis (4,5,6), the exact mechanisms for its pathogenesis are poorly understood. Since catecholamines are known to activate the β-adrenergic receptor-adenylate cyclase system and thereby increase calcium influx, it is generally assumed that the occurrence of intracellular Ca2+ overload due to high levels of circulating catecholamine is a consequence of massive Ca2+ entry through calcium channels in heart sarcolemma. Previous studies have indicated that other membrane systems such as sarcoplasmic reticulum and mitochondria, which are also known to regulate the cytoplasmic concentration of Ca2+, also are affected during the occurrence of catecholamine-induced cardiomyopathy (3,7,8).