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Limaki, Hamidreza Khalatbari (2014) In-vivo and in-vitro study of mechanism of action of 4 hydroxyisoleucine as an amino acid derived from fenugreek seed with anti-diabetic and properties. Doctoral thesis, London Metropolitan University.
Diabetes is a progressive multi-factorial metabolic syndrome with serious short and long term complications affecting many of organs with high increasing prevalence in the world. Using herbs and their derivatives for treating diabetes has a long history in many traditional cultures across the world. Molecules and compounds were isolated from herbs are the basis of many therapeutics which we are using in medicine for treating a variety of health conditions. The seeds of fenugreek, Trigonella foenum graecum, commonly used as a spice in Middle Eastern countries and widely used in South Asia and Europe, are known to have anti-diabetic properties. In 1979, Hardman identified an unusual amino acid (2S, 3R, 4S) 4-hydroxyisoleucine (4HO-Ile) in a fenugreek seed extract as an active compound in fenugreek seed. It was so far found only in fenugreek seed, which is responsible for its anti-diabetic properties. Studies on 4-hydroxyisoleucine effects on type 2 diabetes and insulin resistant animal models revealed that it has anti-diabetic properties of enhancing insulin secretion under hyperglycaemic conditions, and increasing insulin sensitivity. Unfortunately, the available published researches for 4-hydroxyisoleucine are limited and its mechanism of actions is not clear. Here we describe for the first time the anti-diabetic activity of 4-hydroxyisoleucine in a model of type 1 diabetes as all the previous works focused on 4-hydroxyisoleucine activity in type 2 diabetes and insulin resistant condition. Treatment of streptozotocin-treated type 1 diabetes rats, where levels of insulin are much reduced, by 65%, compared to normal animals, with daily doses of 4-hydroxyisoleucine at 50 mg/kg/day for four weeks could reduce plasma glucose in the diabetic group. Moreover the high levels of lipids (cholesterol, HDL, III LDL and triglyceride) and uric acid in the diabetic rats, could be restored to levels found in non-diabetic controls by the treatment with 4-hydroxyisoleucine. These results demonstrate that 4-hydroxyisoleucine has significant anti-diabetic activities in type 1 diabetes as well as previously studied type 2 diabetes and insulin resistance model that are independent of insulin. The findings suggest the potential of 4HO-Ile as an adjunct to diabetes treatment and for type 1 as well as type 2 diabetes.
To investigate the insulin-independent effects of 4-hydroxyisoleucine further, the cell based experiments were designed to assess the effect of 4-hydroxyisoleucine on cellular glucose uptake and ATP content after one day incubation. Isoleucine was added to the experiment as a positive control because firstly it has some level of anti-diabetic properties according to previously published studies and secondly it has similar molecular backbone as 4-hydroxyisoleucine. BRIN-BD 11, a functional and glucose responsive pancreatic beta cell, was selected as a cell model which is not insulin-responsive and dependent on the insulin signalling pathway for glucose uptake. Use of the model provides the opportunity to study the mechanisms of action of both 4-hydroxyisoleucine and isoleucine independently. We adopted a unique approach using inhibitors to target suggested pathway and molecules within the cell which may be involved in both 4-hydroxyisoleucine and isoleucine mechanism of actions. The results revealed that 4-
hydroxyisoleucine and isoleucine could increase glucose uptake in BRIN-BD 11 cells, but as previously suggested, 4-hydroxyisoleucine activity is in direct correlation with glucose concentration. 4-hydroxyisoleucine has higher activity in higher concentrations of glucose. 4-hydroxyisoleucine increased the glucose uptake much greater than isoleucine at 11mM and 22mM concentration of glucose in IV cell culture medium. Endpoint measurements of ATP content of the BRIN-BD11 cells did not show any significant changes between 4-hydroxyisoleucine and isoleucine groups and control as well as an insulin level measurement in culture medium after 24 hours. The results showed that there are substantial differences between isoleucine and 4-hydroxyisoleucine mechanisms of action unlike their similar glucose uptake stimulatory effect which is greater in 4-hydroxyisoleucine. 4-hydroxyisoleucine activity strongly dependent on new protein synthesis and GLUT 1 activity. GLUT 1 is widely available in most of the cells and it controls the basal glucose uptake independent of insulin. The connection between GLUT 1 and 4-hydroxyisoleucine effect in cellular level, supports the idea that 4-hydroxyisoleucine utilises the glucose basal consumption and uptake of cells. Mitochondrial calcium channel signalling inhibition affects 4-hydroxyisoleucine and isoleucine functionality as well as inhibition of mitochondria pyruvate carrier. Real time monitoring of cell metabolism by Seahorse XF-24 autoanalyser after 24 hours incubation with 4-hydroxyisoleucine and isoleucine revealed that both 4-hydroxyisoleucine and isoleucine activities are strongly dependent on mitochondrial respiration but 4-hydroxyisoleucine significantly up-regulates glycolysis which is not affected by isoleucine. The connection between mitochondria calcium signalling and contradictory behaviour of 4-hydroxyisoleucine and isoleucine support the very important role of mitochondria in their mechanisms of action.
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