Expression of metabolic hormone signalling genes in the brain of a triprolyl-human amylin transgenic mouse model of type 2 diabetes

There is a need for greater understanding of the molecular mechanism underlying the development of insulin resistance in type 2 diabetes, and new models in which to study this. The hormone amylin is postulated to be involved in the development of this disease, as human amylin (hA) forms amyloid in the pancreases of diabetic patients, and wild-type hA oligomers have been shown to be cytotoxic to β-cells. By contrast, rodent amylin is non-amyloidogenic, so mice expressing wild-type hA have been developed. However, amylin-evoked β-cell loss in these models limits the secretion of amylin and insulin. We have developed transgenic mice that overexpress [25, 28, 29triprolyl]human amylin, a non-amyloidogenic variant of amylin, designated the Line 44 (L44) model. These mice develop obesity and hyperglycaemia. We examined the expression of 43 genes involved in amylin, insulin and leptin signalling in the brain of this model, at different disease stages, using the NanoString nCounter (NanoString Technologies, Seattle, WA, USA). After onset of diabetes, mice remained hyperglycaemic for a period of time but blood glucose levels eventually returned to normoglycaemia. The incidence of diabetes (defined as having blood glucose measurements > 11 mM over 3 consecutive weeks) was gene-dose dependent; 81% of homozygous mice and 59% of hemizygous mice developed diabetes over 400 days. A proportion of nontransgenic mice (33%) also developed diabetes. As these were bred from hemizygous pairs this may be the result of epigenetic or environmental factors. Experimental L44 mice were bred from hemizygous x hemizygous pairs. We analysed both the hemizygous and homoygous genotypes, with age-matched nontransgenic mice as controls (littermates if possible). Blood glucose and body weight were measured weekly from weaning. At predetermined time points mice were euthanised and brain samples taken. These time points were T1: 100 days (before the onset of diabetes), T2: diabetes oneset (3 blood glucose measurements > 11 mM) and T3: 400 days (post-diabetic). The brain was split into 4 regions: the hindbrain (including the cerebellum), left cortex, right cortex and midbrain (the remaining part of the brain, including the forebrain). RNA was extracted from each sample and analysed using the Nanostring nCounter platform to examine the expression of genes involved in the signalling pathways of amylin, insulin and leptin. Due to their large number, samples were pooled together for analysis. Two samples were pooled only if they were of the same genotype and collection time. The same quantity of RNA from each sample was mixed, thus results represent the mean of the two samples. E.g. sample T1 HB HEM 1/2 consists of RNA extracted from the hindbrain of mouse HEM 1 and HEM 2 at T1.