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The carefully orchestrated events that result in a protective immune response are coordinated to a large extent by cytokines produced by Th1 and Th2 cell subsets. Th1 cells preferentially produce IL-2 and IFN-γ, resulting in a cellular response that helps to eliminate infected cells. In contrast, Th2 cells produce IL-4, IL-5, IL-6, and IL-10, stimulating an Ab response that attacks extracellular pathogens, thereby preventing the cells from becoming infected. To elucidate the mechanisms of differential regulation of cytokine genes by these two different subsets of T cells, we established an in vitro differentiation model of freshly isolated human peripheral blood T cells in which IFN-γ was used as an index gene to study the transcriptional regulation. The data presented here demonstrate that the IFN-γ promoter undergoes differential methylation during in vitro differentiation: the promoter becomes hypermethylated in Th2 cells, whereas it is hypomethylated in Th1 cells. Hypermethylation in Th2 cells results in chromatin condensation and exclusion of CREB proteins from the IFN-γ promoter. Treatment with 5-azacytidine, a demethylating agent, causes Th2 cells to reverse histone condensation and enables CREB recruitment to the hypomethylated promoter. This results in the increased production of IFN-γ. These data indicate the importance of promoter methylation in the regulation of the IFN-γ gene during differentiation.

The number of patients with diabetes mellitus, who exhibit insulin resistance, is increasing recently all over the world. 1) The major causes have been suggested to be functional disorders in insulin secreting capacity and in carbohydrate metabolism deterioration with aging. 2,3) In the type 2 diabetic patients without obesity, who have been frequently found in Japanese, Chinese and Korean, the reduction in insulin secretion seems to be more important than the increase in insulin resistance. 4) These defects may be caused by long term hypersecretion of insulin in response to the intake of high calorie food owing to westernized food habits. The hypersecretion of insulin leads to depletion of the insulin stock and hence to the decline in insulin secretion from the pancreatic beta cells.Several kinds of knock-out mice have been recently developed as animal models for diabetes. Although these models express several important molecular pathological features of diabetes, they carry some disadvantages in that they do not exactly express some of the symptoms of type 2 diabetes. Moreover, maintenance of these genetically-modified mice requires intensive managements in large-scaled facilities so that it needs so much cost. In order to overwhelm these disadvantages of genetically-modified model mice, we attempted to establish a type 2 diabetic model with non-obese features mouse by a non-genetic way.Streptozotocin (STZ) is a widely used chemical inducer for type 1 diabetes. 5) STZ has been shown to produce free radicals in the body which specifically cut DNA chains in the pancreatic beta cells, resulting in disorder of the function of pancreatic beta cell and, at a later phase, destruction of the beta cells by necrosis. 6) These processes evoke activation of the poly ADP ribose synthase to repair the damaged DNA, 7) and a large amount of nicotinamide dinucleotide 8) is consumed for this restoration. 9) Here, consumption of NAD is effectively supplemented by intake of nicotinamide (NA). Meanwhile, in some clinical practice, a high dose of NA is occasionally administered to diabetic patients to prevent the progress of insulin-dependent diabetes (IDDM). 10) Type 2 diabetic model in rats with combined application of STZ and NA has been already reported. 11) Thus it was considered that the similar procedures would be applicable for the prevention from STZ-induced excessive destruction of beta cells in mice. We report here the establishment of a novel method for producing non-obese type 2 diabetic mouse model with a non-genetic way, as well as its pathophysiological characterization. We also show that the present mouse model is useful for searching drugs that are beneficial to treatment of the non-obese type 2 diabetes. MATERIALS AND METHODS Animals and DrugsAll animal experiments were carried out according to the "Principles of Laboratory Animal Care" (NIH publication number 85-23, revised 1985) and the Guidelines of the Animal Investigation Committee, Chiba University, Japan.Male C57BL/6J mice of 5-6 week old age were purchas...

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