Ciclopirox enhances pancreatic islet health by modulating the unfolded protein response in diabetes

Abstract

Pancreatic dysfunction during diabetes is linked to the induction of endoplasmic reticulum (ER) stress on pancreatic beta (β) cells. Our laboratory recently discovered that p21 protects from diabetes by modifying the outcome of ER stress response. In the present study, we explored the antidiabetic activity of ciclopirox (CPX), an iron chelator and recently described activator of p21 expression. The effects of CPX in beta cell survival and function were assessed in cultured islets in vitro as well as in diabetic mice in vivo. The consequences of CPX in high glucose-induced insulin release and reactive oxygen species (ROS) production were also evaluated. Islet survival assays confirmed the significance of p21 in the regulation of glucotoxicity and suggested that CPX counteracts glucotoxicity in a manner that depends on p21. In vivo, administration of CPX in wild-type (WT) diabetic mice restored glucose homeostasis. In WT-cultured islets, CPX suppressed the expression of ER stress markers BiP, GRP94, and CHOP and reduced the levels of ROS during culture at high glucose. This reduction of ER stress may be associated with the ability of CPX to inhibit insulin release. Iron citrate stimulated insulin release, which was inhibited by CPX that functions as an iron chelator. It is conceivable that inhibition of insulin production constrains ER stress in islets promoting their survival and thus protecting from diabetes in vivo. This unfolded protein response (UPR)-antagonizing activity of CPX suggests application for the management not only of diabetes but also of other conditions related to ER stress.

Keywords: Beta cells; Chaperone; Glucose; Glucotoxicity; Unfolded protein response.

Figure 1. Islet survival in relation to genotype at increasing glucose concentrations

a. Survival of cultured pancreatic islets of each genotype at increasing amounts of glucose for 24h and 48h as indicated in the figure. *, P<0.05; **, P<0.005 Student’s T test. b. Relative survival of islets of each genotype in high glucose for 48h expressed as % vs. survival at normal glucose. All values are expressed as average +/− SD.

Figure 2. Assessment of pancreatic islet survival in the presence of CPX

a. Experimental design of how islet survival was assessed in the presence of CPX at 20μΜ or vehicle (VEH) for 24h under different increasing glucose amounts in the culture media, then they were let to recover for 48h in normal glucose-containing media. A pool of several islets has been used as described in the methods. b. Survival of islets of different genotypes in relation to glucose concentration in the presence or absence of CPX. *, P<0.05 vs. untreated with CPX. #, P<0.05 vs. islets cultured in normal glucose, Student’s T test. All values are expressed as average +/− SD.

Figure 3. CPX restores glucose homeostasis in diabetic mice

a. p21 expression in the pancreas of wild type mice non-diabetic (upper panel) or diabetic with glucose at 180 mg/dl (lower panel) that received CPX. Mice received orally CPX at 0–25 mg/kg daily for 5 days and the levels of p21 were assessed. Actin levels were used as positive control. b. Fasting glucose levels of wild type mice fed with HFD and sucrose after CPX at 25mg/kg daily administration. Administration of CPX was initiated about 50 days after initiation of the diabetes-inducing diet when glucose levels exceeded 180 mg/dl (arrows) and continued daily thereafter. c. Glucose tolerance and insulin sensitivity (d) of diabetic mice that have been treated with saline or CPX. For this experiment mice with similar levels of fasting glucose were selected between experimental groups. e. Ferritin levels in the sera of non-diabetic and diabetic mice that received CPX. All values are expressed as average +/− SD.

Figure 4. Fasting blood glucose levels of non-diabetic wt mice non affected by CPX

Fasting glucose for non-diabetic wt mice treated with CPX (a) glucose tolerance (b) and insulin sensitivity (c) of wt mice receiving CPX. All values are expressed as average +/− SD.

Figure 5. Islet cell morphology after Ciclopirox treatment in HFD-induced diabetic mice and control mice

a. Pancreatic islet morphology (H&E staining) of wt non-diabetic (upper panel) or diabetic (lower panel) mice receiving CPX or saline. Yellow arrows show islets. White asterisks indicate areas in the exocrine panceas with cells exhibiting intense eosinophilic granular cytoplasm. b. Quantification of total islets expressed as the % vs. controls of the product between islet numbers in cross-sections and the weight of each pancreas. All values are expressed as average +/− SD.

Figure 6. CPX decreases insulin release and attenuates the consequences of glucose-induced ER stress in islets

a. Insulin release in wt islets following glucose stimulation at 17mM or 25mM, or 40 mM KCl and treated with CPX at 20μΜ for 30min and 60min respectively. *, P<0.01 vs. untreated. b. Expression of insulin, CHOP, GRP94 and BiP in wt islets treated with high glucose alone or in combination with CPX (in KRB). Actin levels were assessed as loading control. High glucose experiment was performed in triplicates. All values are expressed as average +/− SD.

Figure 7. Iron citrate elevates ER-stress markers, whereas Ciclopirox restores ER-stress homeostasis

a. The effects of iron citrate and ciclopirox at the indicated concentrations in insulin, BiP, GRP94 and CHOP levels in islets cultured at 5.5mM glucose. Actin levels were assessed as loading control. The high glucose experiment was performed in triplicates. b. Effect of CPX at 20μM (in KRB) for 24h in ROS production (in arbitrary units, AU) that was triggered by high (25mM) or moderate (17mM) glucose (Glu) or iron citrate (Fe) (in 5.5mM glucose). TBHP was used as positive control for ROS production. *. P<0.05, Student’s t-test. All values are expressed as average +/− SD.

Figure 8

Diagrammatic depiction of the presumed role of CPX in regulating islets’ survival by modulating p21 expression, insulin release and ER stress induction.