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Background: Fructose 2,6 bisphosphate the potent stimulator of phosphofructosekinase-1, is involved in the control of glycolysis, gluconeogenesis. Experimental and clinical studies have demonstrated that levels of fructose 2,6 bisphosphate increased in peripheral blood mononuclear cells in diabetics suggest that disturbance in immune cells play a major role in increased susceptibility to infection. These altered metabolic products and oxydated stress may play a role in the development of diabetic co mplication.
Objective:The present research is designed to study the effect of fasting blood sugar and HbA1c on fructose 2,6 bisphosphate in diabetics and comparing and correlating the same parameter with control subjects.
Material and Methods: Two hundred patients with type-II diabetes and fifty control subjects were selected for this study. Fasting blood sugar and glycated hemoglobin were estimated. Fructose 2,6 bisphosphate was estimated and correlated from peripheral blood mononuclear cells.
Result:Results of this study showed highly significant fructose 2,6 bisphosphate levels in immune cells in diabetic group. A significantly positive correlation of fructose 2,6 bisphosphate with FBS and HbA1c was seen in diabetic group.
Conclusion: Higher levels of FBS and HbA1c are associated with increased levels of fructose 2,6 bisphosphate of peripheral blood mononuclear cells in diabetes reduces immunity.
Fructose 2, 6 bisphosphate is detected in all mammalian tissue as well as in fungi and plants.1 Fructose 2,6 bisphosphate is potent positive allosteric effector of phosphofructokinase-1 which is rate limiting enzyme for glycolysis.2 Fructose 2,6 bisphosphate is formed by phosphofructokinase-2. Phosphofructokinase-2 is a bifunctional protein that has both, the kinase activity that produces fructose 2,6 bisphosphate and phosphatase activity that converts fructose 2,6 bisphosphate back to fructose 6 phosphate and inorganic phosphate (Pi).3 High fructose 2,6-bisphosphate levels mediate enhanced glycolysis and bifunctional enzyme phosphofructokinase-2/fructose 2,6-bisphosphatase catalyses the formation and degradation of fructose 2,6-bisphosphate.4 Diabetes causes substantial changes in the fructose 2,6-bisphosphate system. In hepatocyte, diabetes mellitus enhances phosphorylation of fructose 2,6-bisphosphate leading to a decrease in the activity of the enzyme causing hyperglycemia. In peripheral blood lymphocytes fructose 2,6-bisphosphate system is slightly different from that of hepatocyte.5 Hyperglycemia increases intracellular fructose 2,6-bisphosphate in immune cells.6 These findings may help to clarify the impaired functions in immune cell, in patients with diabetes.7 As diabetes progress patients are at increased risk of developing infections. Complication due to depressed immunity patient with diabetes mellitus has infections more often then those with diabetes mellitus. The course of the infection is also more complicated in these patients.8 Good metabolic control is a major factor in limiting the development and spread of infection and most importantly, the development of diabetes complication predispose to infection.9 These factors include genetic susceptibility to infection, altered cellular humoral immune defense mechanism and local factor including poor blood supply and nerve damage and alterations in metabolism associated with diabetes mellitus.10 Haemoglobin A1c (HbA1c) is widely used to determine levels of long-term blood glucose, judge the adequacy of diabetes management, and adjust therapies.11 Consistently high HbA1c levels increase the risk for long-term disabling and potentially life-threatening complications, including cardiovascular disease, kidney disease, eye damage and nerve damage.6 A significant relation ship has been seen in some studies between intracellular fructose 2,6- bisphosphate levels and long term glycemic control as assessed by HbA1c.12
In present study, emphasis has been given to estimate the level of fructose 2, 6 bisphosphate, which impaired immune systems in lymphocyte, has an implication for outcome of the infection and correlate with HbA1c to reduce life-threatening complications in diabetes.
A total of two hundred and fifty subjects were included in this study. Two hundred subjects were diagnosed type-II diabetics and fifty individuals selected as control were completely asymptomatic. In all cases age, sex, weight, height and duration of illness were recorded. Diagnosed type-II diabetic patients were taken from diabetic clinics JPMC Karachi. About 8ml of blood was taken after an over night fast. Peripheral mononuclear cells (PBMC) were separated by ficoll gradients from 6ml of venous blood. PBMC were homogenized in 50mM NaOH and incubated at 80â-¦C for 10mins. After centrifugation the supernatants were used to assay for fructose 2,6 bisphosphate by Van Schaftigen method.13 The serum glucose was estimated by kit method supplied by Spinreact SA spain. HbA1c was estimated by fast ion exchange resins separation method using the kit supplied by Human Germany. Statistically analysis was performed with SPSS 15.0 software.
Two hundred and fifty subjects studied and distributed as fifty healthy normal subjects as control group and two hundred diagnosed type-II diabetes mellitus. Table-1 shows mean values with standard error of mean (mean±s.e.m). Age, height, weight and body mass index of control compared with diabetic subjects. Height, weight and body mass index of diabetics were significantly higher when compared to control. No statistically significant difference observed in age. Table-2 shows the intergroup comparison of fasting blood glucose, glycated hemoglobin and fructose 2,6 bisphosphate. The mean values of fasting blood glucose of diabetics were significantly higher (P<0.001) when compared to control. Similar results were found with HbA1c and fructose 2, 6 bisphosphate of diabetic group was significantly higher (P<0.001) when compared to control group. Table-3 depicts the comparison of relationship of fructose 2, 6 bisphosphate and serum fasting blood glucose and Co-efficient of correlation "r" of fructose 2, 6 bisphosphate with HbA1c showed significantly positively correlated (P<0.001) fructose 2, 6 bisphosphate compared with fasting blood glucose. However, it also shows non-significant relationship between diabetic and normal subjects, while fructose 2, 6 bisphosphate showed significantly positively correlation (p<0.001) with HbA1c. Figure-3 and 4 represents correlation coefficient of fructose 2, 6 bisphosphate versus fasting blood sugar (FBS) and glycated hemoglobin (HbA1c).
Comparison of age, height, weight and body mass index of control with the diabetic subjects.
The values are expressed as Mean± S.E. The number of observation and units are given in parenthesis.
44.90 ± 1.36
61.23 ± 1.54
63.87* ± 1.43
1.68* ± 1.33
27.63* ± 0.31
*P<0.001 significant when compared to control.
Comparison of fasting blood glucose, HbA1c and Fructose 2, 6 bisphosphate of control and diabetic subjects.
The values are expressed as Mean± S.E.M.
The number of observation and units are given in parenthesis.
86.54 ± 1.95
3.15 ± 0.11
137.70* ± 1.61
8.45* ± 0.11
6.91* ± 0.11
*P<0.001 significant when compared to control.
Co-efficient of correlation ( r ) of Fructose 2, 6 bisphosphate (pmol) vs FBS and HbA1c
r = -0.23
r = 0.15
r = 0.665*
r = 0.28
r = -0.05
r = 0.534*
* Correlation is significant at the p<0.001 level
Figure # 1 Figure # 2
Correlation ( r ) of Fructose 2, 6 Correlation( r ) of Fructose2,6
bisphosphate (pmol) vs FBS bisphosphate(pmol) vs HbA1c
Diabetes is a chronic disorder that occurs when the pancreas does not produce enough insulin or body can not effectively use the insulin it produce.14 Infection tends to occur with greater frequency and severity in diabetic patients than in non-diabetics. A specific defect in innate and adaptive immune function was identified by many in vitro studies.15 Immune cells require glucose uptake and metabolism for normal function. High glucose levels inhibit proliferation of peripheral mononuclear cells. Activated T cells have dramatically increased metabolic requirement, glucose metabolism and aerobic glycolysis due to fuel this demand.16
It was proposed decades ago that fasting glucose value is higher in diabetics.17 In our study we measured fasting blood sugar to determine the control of diabetes and assess the metabolic condition and were in agreement of the above statement. Also in our study diabetic patients had higher fasting blood sugar values (137.70 ± 1.61mg/dl) than control subjects (86.54 ± 1.95mg/dl) (p<0.001) as expected. Measurement of HbA1c provides a retrospective index of glycemic control over the 4 to 8 weeks before its determination helps diabetes management and adjustment of therapy. In one study,15 it was found that high glycosylated Hb levels are most likely to exhibit close association with particular infection and hyporesponsiveness. In another study,18 it was found significantly higher HbA1c in a group of 1480 subjects with diabetic nephropathy. Atsume et al.,12 conducted an investigation to see the effect of long term hyperglycemia on intracellular fructose 2,6 bisphosphate in immune cells, suggested that subjects with higher HbA1c values have an increase intracellular fructose 2,6 bisphosphate in immune cells. Results of our study also showed significant difference in HbA1c (p<0.001) in diabetics as compared to controls actual values being 8.45 ± 0.10 and 4.33 ± 0.11 per cent respectively. Immune cells activation requires an increase in glucose uptake and anaerobic glycolysis. Fructose 2,6 bisphosphate is a powerful activator of rate limiting enzyme of glycolysis, defects causes decrease in immune function in diabetic patients. Infection occur with increased frequency and severity in diabetes. Atsumi et al., (2007)12 showed intracellular fructose 2,6 bisphosphate levels in peripheral blood mononuclear cells in diabetics and observed significantly higher than age matched control subjects, our results also showed positively significant relationship between fructose 2,6 bisphosphate levels and HbA1c (r â• 0.451, p<0.001). Results of Vander et al.,(2009)6 indicated that activated thymocytes from diabetic rats showed two fold more fructose 2,6 bisphosphate than cells from normal rats, our study also in agreement of the above mentioned studies with significantly high (p<0.001) levels of fructose 2, 6 bisphosphate in immune cells of diabetics when compared with normal subjects. We also observed a significant positive correlation between intracellular fructose 2,6 bisphosphate levels and long term glycemic control as assessed by HbA1c. These finding may help to clarify the impaired function in immune cells in patients with diabetes.
It is concluded that in diabetes fructose 2,6 bisphosphate is increased in immune cells causes decreased in numbers of immune cells which may induce chance of infection.