Effect of fat on inflammation and insulin resistance in human mononuclear cells and myeloid cell insulin receptor knockout mice
Deopurkar, Rupali Anil
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Obesity and diabetes are major risk factors for cardiovascular disease. Both have been shown to be associated with oxidative stress and inflammation. These processes are also involved in the pathogenesis of atherosclerosis. It is possible that chronically increased macronutrient intake results in a proinflammatory state in obesity. We have previously shown that glucose intake results in oxidative stress and inflammatory changes in the circulating peripheral blood mononuclear cells (MNC). But little is known about the acute effects of lipids on these cells. In this work, we studied the effects of intake of lipid in the MNC. We hypothesized that intake of lipids produces inflammation in the MNC acutely which results in chronic subclinical inflammatory state which may lead to insulin resistance. It has been shown previously that insulin inhibits various mediators of inflammation. It is possible that insulin may be antiatherogenic. In this context, we studied the effects of high fat diet in mice lacking insulin receptor in the myelogenous leukocytes (monocytes and granulocytes). Since insulin has an antiinflammatory effect, mice lacking insulin receptor on these cells will be more susceptible to inflammation and atherosclerosis, when fed high fat diet. Since inflammation is also linked to insulin resistance and type 2 diabetes mellitus, we also studied the effect of lack of insulin receptor in myelogenous leukocytes on glucose tolerance. Twelve fasting human subjects were given 33 g of dairy cream. Blood samples were collected before and 1, 2, 3 hour after intake of cream. MNC were isolated and plasma and serum was collected. RNA was isolated from the circulating MNC and expression of various genes involved in inflammation was assessed by RT-PCR. Western blotting and EMSA was used to determine the quantity and binding activity respectively of proteins and transcription factors of interest in the context of inflammation and oxidative stress. Thin layer chromatography was done to assess the accumulation of different types of lipids in the MNC. The concentration of circulating inflammatory mediators was measured in plasma and serum. Similar experiments were done on ten normal healthy lean subjects who ingested water instead of cream. A knock out mouse lacking insulin receptor in monocytes and granulocytes was created and characterized. High fat diet was given to these mice and glucose tolerance test and insulin tolerance tests were done. The expression of inflammatory markers at the level of mRNA was assessed in the lymphocytes, liver, muscle, fat and brain. We also attempted to study the insulin signaling in the liver and the skeletal muscle. Our findings show that following cream intake there is an increase in the intranuclear NFκB binding, a decrease in IκB protein level and an increase in IKK kinase activity in the MNC, consistent with a proinflammatory effect. There is an increase in the mRNA expression of proinflammatory genes, IL-1β, c-fos and Egr-1 and the procoagulatory and antifibrinolytic, PAI-1. There was also increase in gene expression of SOCS3, a protein that interferes with insulin signaling. The plasma concentration of inflammatory mediators like IL-18 and MIF increased following lipid intake. There was also significant increase in the serum concentration of PAI-1; adhesion molecule, sICAM-1 and MMP-9, a protein involved in plaque rupture. Water challenge served as the control and did not induce any change in the above mentioned parameters. Our work in the animal model demonstrated that female mice lacking insulin receptor in the myeloid cell line have impaired glucose tolerance. We also attempted to study inflammation and insulin resistance in tissues, including the liver, skeletal muscle, fat and brain. Thus, our work demonstrates that intake of dairy cream induces acute proinflammatory changes in the MNC and plasma, which are potentially relevant to atherogenesis and to the development of inflammation and insulin resistance in obesity, a state of chronic over nutrition. The work in the animal model underlines the importance of the role of insulin in the myelogenous leukocytes in the development of global insulin resistance. This research will help us understand the pathogenesis of atherosclerosis and complications associated with chronically increased macronutrient intake such as in obesity. This better understanding may help in the search of new interventions to prevent atherosclerosis and also may lead to modification in our eating habits to promote a healthier lifestyle.