Loss of functional beta cell mass by beta cell apoptosis and inadequate insulin production is a hallmark of type 1 and type 2 diabetes. During the last award period, we have identified thioredoxin-interacting protein (TXNIP) as a critical factor controlling beta cell apoptosis and showed that beta cell TXNIP was upregulated in diabetes, whereas TXNIP deficiency protected against diabetes by promoting beta cell survival. Now we discovered that TXNIP regulates beta cell microRNA expression and particularly induces beta cell expression of a specific microRNA, miR-204. While miR-204 has not been previously implicated in beta cell biology, our preliminary findings suggest that this microRNA is upregulated in diabetes, blocks insulin production and serum levels correlate with the decline of functional beta cell mass associated with diabetes. Our hypothesis is therefore that that TXNIP induces miR-204 resulting in a cascade of events that inhibit insulin production and promote loss of functional beta cell mass. We propose 4 Specific Aims to test this hypothesis. 1. To study the in vivo effects of TXNIP and miR-204 inhibition on functional beta cell mass under normal and diabetic conditions, we will take advantage of our whole body (HcB-19) and beta cell-specific TXNIP-deficient (bTKO) mouse models as well as our double mutant obese and insulin resistant leptin- and TXNIP-deficient mouse (ob/hcb) and will assess the effects of TXNIP deficiency on islet miR-204 expression and beta cell insulin production as compared to control mice. We will also use a novel miR-204 knockout mouse (204KO) to determine the specific role of miR-204 in the control of insulin production and functional beta cell mass under normal conditions and in response to diet- induced obesity and diabetes. 2. To analyze the cellular effects of altered beta cell TXNIP and miR-204 expression, we will overexpress and inhibit TXNIP and miR-204 in INS-1 cells and human islets and use isolated bTKO and 204KO islets and assess changes in insulin expression and secretion. 3. To determine the molecular mechanisms by which miR-204 and TXNIP control beta cell function, we will follow-up on our initial studies suggesting that miR-204 acts via MafA downregulation and will assess how miR-204 and TXNIP affect expression level, insulin promoter occupancy and 3'UTR function of MafA and human MafB. To do so, we will again use our INS-1 cell lines, isolated bTKO and 204KO islets and primary human islets. 4. To evaluate miR- 204 as a potential novel serum marker for functional beta cell mass, we will induce controlled beta cell destruction using multiple low-dose streptozotocin injections as well as analyze spontaneous non-obese diabetic (NOD) mice as a second model of diabetes and will measure serum miR-204 in parallel with whole pancreas insulin content and beta cell mass at different stages of beta cell loss. The results of these studies will provide an in depth understanding of the newly discovered TXNIP/miR- 204/MafA signaling pathway controlling insulin synthesis and may help reveal novel markers for the measurement of functional beta cell mass.
The results of the proposed studies will shed new light on a thus far underappreciated control mechanism governing pancreatic beta cell function and insulin production and will elucidate the molecular mechanisms involved. By revealing potential novel microRNA markers and therapeutic targets to measure and enhance endogenous insulin production, they will also set the stage for the development of better treatment approaches for patients with diabetes.
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