Diabetic rats and mice are resistant to porcine and human insulin: flawed experimental models for testing islet xenografts.

BACKGROUND: Islet transplantation is potentially a promising therapy for the restoration of carbohydrate control to diabetic patients. However, the global application of islet transplantation requires a ubiquitous source of beta cells. The xenotransplantation of porcine islets would provide such a source. Success in porcine islet xenografting has been achieved in diabetic primates. However, there are few reports of reversal of diabetes with porcine islet xenografts in rodent models of diabetes, relative to the number of successful rodent experiments performed as allografts. Here we report for the first time the inability of porcine (and human) insulin to control blood glucose levels in diabetic rodents determined by a series of dose escalating studies. METHODS: Insulin was administered intravenously to streptozotocin induced diabetic Lewis rats, Balb/c and athymic Balb/c mice (n = 5 per group) at the following doses: Group I "physiological dose" (pd) of 0.16 U/kg for a total dose of 40 mU to a 250 g rat. Group II received 0.64 U/kg (4xpd), group III 1.6 U/kg (10xpd) and group IV 6.4 U/kg (40xpd). Blood glucose levels were monitored in each animal at seven time points: 0 (pre-injection), 10 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h and 3 h post-injection. Serum insulin levels were also determined. RESULTS: Diabetic Lewis rats achieved a maximum reduction in blood glucose from 22.1 +/- 1.8mmol/l to 8.0 +/- 3.1 mmol/l (a 63.7% reduction), 90 minutes post-injection of 6.4 U/kg dose of porcine insulin (40xpd). Human insulin was less effective at reducing blood glucose levels in rats than porcine insulin (P < 0.001). Porcine insulin reduced blood glucose levels in Balb/c mice from a mean of 18.2 +/- 2.1 mmol/l to a hypoglycemic minimum of 1.26 +/- 0.18 mmol/l a reduction of 93.0%, 60 min post-injection of the maximum dose of 6.4 U/kg. Balb/c mice were significantly more responsive to porcine insulin than Lewis rats at doses of 0.64 U/kg (P < 0.001), 1.6 U/kg (P < 0.05) and 6.4 U/kg (P < 0.001). Athymic Balb/c nude mice reached a maximum reduction in blood glucose from 21.6 +/- 1.8 mmol/l to 3.6 +/- 0.9 mmol/l (a 83.4% reduction) 120 min post-injection at a dose of 6.4 U/kg. Overall, athymic Balb/c nude mice were more resistant to porcine insulin than immunocompetent Balb/c mice at doses of 0.64 U/kg (P < 0.001), 1.6 U/kg (P < 0.001) and 6.4 U/kg (P < 0.05). Insulin diluent alone marginally increased blood glucose levels in all animals tested. CONCLUSIONS: Our results suggest that restoration of normoglycemia in diabetic rodents is not ideal for testing porcine islets xenografts since the reversals of diabetes in these species requires 20 to 40 times the dose of porcine insulin used in humans.

The intrinsically disordered TC-1 interacts with Chibby via regions with high helical propensity.

Thyroid cancer 1 (TC-1) is a 106-residue naturally disordered protein that has been found to associate with thyroid, gastric, and breast cancers. Recent studies showed that the protein functions as a positive regulator in the Wnt/beta-catenin signaling pathway, a pathway that is known to play essential roles in developmental processes and causes tumor formation when misregulated. By competing with beta-catenin for binding to Chibby (Cby), a conserved nuclear protein that antagonizes the beta-catenin-mediated transcriptions, TC-1 up-regulates a number of beta-catenin target genes that are known to be involved in the aggressive behavior of cancers. In order to gain a molecular understanding of the role TC-1 plays in regulating the Wnt/beta-catenin signaling pathway, detailed structural studies of the protein and its interaction with Cby are essential. In this work, we used nuclear magnetic resonance (NMR) spectroscopy to elucidate the structure of TC-1 and its interaction with Cby. Our results indicate that even though TC-1 is naturally disordered, the protein adopts fairly compact conformations under nondenaturing conditions. Chemical shift analysis and relaxation measurements show that three regions (D44-R53, K58-A64, and D73-T88) with high-helical propensity are present in the C-terminal portion of TC-1. Upon addition of Cby, significant broadening of resonance signals derived from these helical regions of TC-1 was observed. The result indicates that the intrinsically disordered TC-1 interacts with Cby via its transient helical structure.