Circulating levels of IL-1B+IL-6 cause ER stress and dysfunction in islets from prediabetic male mice.

Elevated levels of circulating proinflammatory cytokines are associated with obesity and increased risk of type 2 diabetes, but the mechanism is unknown. We tested whether proinflammatory cytokines IL-1B+IL-6 at low picogram per milliliter concentrations (consistent with serum levels) could directly trigger pancreatic islet dysfunction. Overnight exposure to IL-1B+IL-6 in islets isolated from normal mice and humans disrupted glucose-stimulated intracellular calcium responses; cytokine-induced effects were more severe among islets from prediabetic db/db mice that otherwise showed no signs of dysfunction. IL-1B+IL-6 exposure reduced endoplasmic reticulum (ER) calcium storage, activated ER stress responses (Nos2, Bip, Atf4, and Ddit3 [CHOP]), impaired glucose-stimulated insulin secretion, and increased cell death only in islets from prediabetic db/db mice. Furthermore, we found increased serum levels of IL-1B and IL-6 in diabetes-prone mice at an age before hyperglycemia was exhibited, suggesting that low-grade systemic inflammation develops early in the disease process. In addition, we implanted normal outbred and inbred mice with subcutaneous osmotic mini-pumps containing IL-1B+IL-6 to mimic the serum increases found in prediabetic db/db mice. Both IL-1B and IL-6 were elevated in serum from cytokine-pump mice, but glucose tolerance and blood glucose levels did not differ from controls. However, when compared with controls, isolated islets from cytokine-pump mice showed deficiencies in calcium handling and insulin secretion that were similar to observations with islets exposed to cytokines in vitro. These findings provide proof of principle that low-grade systemic inflammation is present early in the development of type 2 diabetes and can trigger ER stress-mediated islet dysfunction that can lead to islet failure.

Evidence that low-grade systemic inflammation can induce islet dysfunction as measured by impaired calcium handling.

In obesity and the early stages of type 2 diabetes (T2D), proinflammatory cytokines are mildly elevated in the systemic circulation. This low-grade systemic inflammation exposes pancreatic islets to these circulating cytokines at much lower levels than seen within the islet during insulitis. These low-dose effects have not been well described. We examined mouse islets treated overnight with a low-dose cytokine combination commonly associated with inflammation (TNF-alpha, IL-1 beta, and IFN-gamma). We then examined islet function primarily using intracellular calcium ([Ca(2+)](i)), a key component of insulin secretion and cytokine signaling. Cytokine-treated islets demonstrated several features that suggested dysfunction including excess [Ca(2+)](i) in low physiological glucose (3mM), reduced responses to glucose stimulation, and disrupted [Ca(2+)](i) oscillations. Interestingly, islets taken from young db/db mice showed similar disruptions in [Ca(2+)](i) dynamics as cytokine-treated islets. Additional studies of control islets showed that the cytokine-induced elevation in basal [Ca(2+)](i) was due to both greater calcium influx through L-type-calcium-channels and reduced endoplasmic reticulum (ER) calcium storage. Many of these cytokine-induced disruptions could be reproduced by SERCA blockade. Our data suggest that chronic low-grade inflammation produces circulating cytokine levels that are sufficient to induce beta-cell dysfunction and may play a contributing role in beta-cell failure in early T2D.

A practical guide to rodent islet isolation and assessment.

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.

Glucose metabolism, islet architecture, and genetic homogeneity in imprinting of [Ca2+](i) and insulin rhythms in mouse islets.

We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca2+](i)) that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed "islet imprinting." We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J) as well as outbred mouse strains (Swiss-Webster; CD1). Second, imprinting was observed in NAD(P)H oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca2+](i) oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a K(ATP)-channel opener that blocks [Ca2+](i) influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca2+](i) oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca2+](i) oscillations. Lastly, to test whether the imprinted [Ca2+](i) patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca2+](i) oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.