Full size table Experimental design A To investigate the effect of DDW on metabolic changes occur in diabetes, diabetic and control rats were divided into two main groups: half of the animals were given DDW 25 ppm Dand the other half received normal tap water ppm Dboth provided ad libitum. To determine whether DDW exerts its effects, at least partially, by modifying the action of insulin, diabetic rats were further divided into subgroups according to insulin treatment.
The control rats did not receive insulin treatment. We did not want to achieve euglycaemia by insulin treatment, but to prevent severe acute complications only. Therefore, two different doses of insulin Huminsulin Lilly Normal I. The insulin was administered subcutaneously in equal portions twice daily 8.
Treatments started at the 2nd week after STZ injection and lasted for an additional 8 weeks.
Treatments started at the 2nd week after STZ injection and lasted for 4 weeks. Food intake, water consumption, and body weight were measured daily. Blood samples from the tail vein and 24 h urine samples were collected once a week. The plasma samples were obtained by centrifugation at ×g for 15 min at 4 °C.
After measuring the volume, urine was centrifuged at × g for 10 min and the supernatant was used for analysis. Plasma glucose Plasma Glucose was determined spectrophotometrically using reagent kits from Reanal Finechemical Co. Budapest, Hungary.
The plates were analyzed by a Biorad microplate reader. Plasma fructosamine concentration Plasma fructosamine concentration was determined spectrophotometrically using the micro method developed by Oppel et al. In brief, fructosamine reagent was prepared by dissolving 50 mg nitroblue tetrazolium NBT, from Sigma, Budapest, Hungary in Standard was prepared from bovine serum albumin as described previously in detail [ 23 ]. Twenty µl of plasma or an adequate volume of standard solution was pipetted into the wells of a well plate in three parallels, respectively.
After the addition of µl reagent into each well, the plates were profoundly shaken and then incubated at 37 °C for 10 min.
The initial absorbance was read at nm A1. Following a subsequent incubation for 10 min, the absorbance was read again at nm A2. Fructosamine concentration Csample was calculated from Eq. In brief, 30 µl of blood samples collected without anticoagulants were hemolysated in µL deionised water. After further washing of the columns with washing buffer 10 mLµL of the hemolysate were transferred to the top of the columns and allowed to soak in.
Unbound hemoglobins were eluted by passing 8 mL of washing buffer through the column. The unbound fraction, containing most of the hemoglobin was diluted to 15 mL with washing buffer.
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The absorbance of each fraction was measured at nm and the amount of hemoglobin bound glycosylated was calculated as a percentage of the total. Isolation of the membrane fraction of soleus muscle Isolation of the membrane fraction of soleus muscle was performed according to Villanueva-Peñacarrillo ML et al.
Briefly, soleus muscles from each rat hind limb were removed and trimmed of connective tissue, fat and nerves. The homogenate was centrifuged at ×g for 10 min at 4 °C, and the pellet was discarded.
The supernatant was then centrifuged at ×g for 60 min in a Beckman SW55 rotor. The pellet was resuspended in 0. The pellet was finally resuspended and homogenized in washing buffer, and the total membrane protein content was measured by the Bradford method. Western blot analyses The samples were prepared in 2 × Laemmli buffer containing mmol dithiothreitol and boiled in a water bath for 15 min.
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The ODs of bands were determined by densitometry. All chemicals not mentioned otherwise were purchased from Sigma Budapest, Hungary. Statistical analyses The results are presented as the mean ± SEM of n observations.
Results DDW as drinking water reduced plasma glucose concentration and improved the metabolic parameters in diabetic rats To evaluate the effect of DDW on the glucose metabolism in streptozotocin STZ -induced diabetic rat model, first we aimed to test the 25 diabetes mellitus gyógyszer D-concentration, which was the lowest D-concentration of DDW available.
In animals without any insulin treatment, DDW alone did not affect blood glucose concentration.
These results indicate that deuterium depletion, in the presence of insulin, can affect blood glucose levels in diabetic animals. Open and closed circles represent the data obtained from animals receiving either DDW 25 ppm or tap water, respectively.
Numbers in brackets show the number of animals in each group Full size image To evaluate the changes of metabolic parameters in non-diabetic rats receiving drinking water with ppm or 25 ppm D content for four weeks were individually placed in metabolic cages for 24 h and the amount of fluid and food consumed was measured and the volume of urine excreted and its glucose content were determined. To evaluate the changes of metabolic parameters in STZ-treated diabetic rats receiving drinking water with ppm or 25 ppm D content for four weeks were individually placed in metabolic cages for 12 h, without insulin administration, and the amount of fluid and food consumption, the volume of urine excretion and its glucose content were measured, respectively.
In conclusion, the D content of the drinking water did not significantly affect the parameters studied in either non-diabetic or STZ-treated animals without insulin administration.
Next, to determine the most effective D-concentration, DDW with 25, 75,and ppm Exercise induced hypoglycemia in non diabetics was tested along with the application of a low insulin dose only. Surprisingly, we found that the ppm value, which was closer to the natural D level, was the most effective D-concentration in reducing blood glucose levels in diabetic animals. The inset shows the values for the symbols. The amount of fluid and food consumed was measured, and bullosis diabeticorum treatment volume of urine excreted and its glucose content were determined.
Table 3. Figures 3 — 5 show the summary of three experiments in which the following D-concentrations were applied: 25, 75,ppm and ppm in the control group.
Low dose of insulin was used along with all D-concentrations. The inset graphs show the magnified part of the curve between the deuterium content of ppm and ppm, respectively.
Animals receiving drinking water-containing ppm deuterium had the lowest fructosamine levels among the DDW-treated animals. The inset graphs show the magnified part exercise induced hypoglycemia in non diabetics the curve in the range of the deuterium content between and ppm.
Furthermore, symbols indicate the significance level of data compared to animals receiving 25 ppm deuterium-containing drinking water. DDW did not influence the half-life of insulin in blood The first experiment revealed that the presence of insulin is essential for DDW to be able to reduce blood glucose concentration.
To exclude the possibility that deuterium depletion may stabilize the insulin in the plasma which results in lower blood glucose levels, the insulin concentration of the blood plasma was determined after the administration of DDW. The insulin concentration was the highest two hours after administration and there was a gradual decrease for 10 h until the next administration, but the data did not support the hypothesis that DDW influences insulin stability Fig. The essential role of insulin in the mechanism influenced by the lowering D-concentration was also confirmed by following the correlation between insulin and glucose concentration after every two hours of administration.
The data show that the blood glucose concentration was the lowest two hours after the administration, when the insulin level was the highest. DDW dose-dependently potentiates the effect of insulin, in part, due to the increased GLUT4 protein translocation from the cytoplasm to the membrane As glucose is cleared from the bloodstream by a family of facilitative transporters GLUTs and the GLUT4 isoform is the major insulin-responsive transporter, we planned to measure the amount of GLUT4 in the membrane fraction of the soleus muscle.
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Figure 8 shows that in non-diabetic rats the GLUT4 level was high and remained the same independently of D-concentration in the drinking water 25 ppm or ppm. Inset shows a representative immunoblot of insulin-promoted GLUT4 translocation to the membrane fraction. Bars indicate the mean values of the densitometry evaluation of the GLUT4 protein corresponding spots on the immunoblots and expressed as a percentage of the value of the tap water given to control group non-diabetic animals.
In diabetic rats, we found a strong correlation between the amount of GLUT4 in the membrane fraction and the blood glucose concentration. As the blood glucose concentration was the highest in the control group, the lowest GLUT4 level was detected in the membrane, but at the same time the ppm DDW resulted in the lowest glucose concentration.
This can be explained with the highest GLUT4 level being in the membrane. Discussion There has been increasing evidence that naturally occurring deuterium has a central role in living organisms since the first paper was published [ 9 ].
In this study, we proved that the changes in D-concentrations in water may potentiate the insulin-regulated membrane trafficking by recruiting membrane vesicles exercise induced hypoglycemia in non diabetics the GLUT4 glucose transporters from the interior of cells to the cell surface.
One of the most striking results was that it was not the lowest D-concentration 25 ppm that exerted the most significant stimulus on the insulin signal transduction system, but a narrow subnatural concentration range between and ppm.
We also suggest that the D-concentration varies in the different nutrients depending on the place of cultivation, country of origin, the biochemical pathways for photosynthetic CO2-fixation in plants [ 3637 ], and the ratio of the main organic compounds carbohydrates, proteins, lipids also influences cellular processes and has a major impact on metabolism.
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Based on our experimental data, deuterium-depleted water can offer clinical benefits in the treatment of patients with metabolic syndrome by increasing insulin sensitivity. The results presented here serve as novel evidence that the naturally occurring deuterium has an important role in living organisms. Further research is needed to explore whether a similar mechanism could be responsible for the beneficial effect of DDW on the parameters of glucose metabolism in human subjects, however this is beyond the scope of the current study.
Nevertheless, deuterium depletion can potentially offer an effective and innocuous tool for treating not only malignant but also metabolic diseases.
Data availability All data generated or analysed during this study are included in this published article. References 1. Jancsó G Isotope effects. Van Nostrand Reinhold, New York, pp —