Pancreatic β-cells are a critical cell type in the pathology of diabetes. Models of genetic syndromes featuring diabetes can provide novel mechanistic insights into regulation of β-cells in the context of disease. We previously examined β-cell mass in models of two ciliopathies, Alström Syndrome (AS) and Bardet-Biedl Syndrome (BBS), which are similar in the presence of metabolic phenotypes, including obesity, but exhibit strikingly different rates of diabetes. Zebrafish models of these disorders show deficient β-cells with diabetes in AS models and an increased β-cells absent diabetes in BBS models, indicating β-cell generation or maintenance that correlates with disease prevalence. Using transcriptome analyses, differential expression of several exocrine pancreas proteases with directionality that was consistent with β-cell numbers were identified. Based on these lines of evidence, we hypothesized that pancreatic proteases directly impact β-cells. In the present study, we examined this possibility and found that pancreatic protease genes contribute to proper maintenance of normal β-cell numbers, proliferation in larval zebrafish, and regulation of AS and BBS β-cell phenotypes. Our data suggest that these proteins can be taken up directly by cultured β-cells and ex vivo murine islets, inducing proliferation in both. Endogenous uptake of pancreatic proteases by β-cells was confirmed in vivo using transgenic zebrafish and in intact murine pancreata. Taken together, these findings support a novel proliferative signaling role for exocrine pancreas proteases through interaction with endocrine β-cells.

Genome analysis of diverse human populations has contributed to the identification of novel genomic loci for diseases of major clinical and public health impact. Here, we report a genome-wide analysis of type 2 diabetes (T2D) in sub-Saharan Africans, an understudied ancestral group. We analyze ~18 million autosomal SNPs in 5,231 individuals from Nigeria, Ghana and Kenya. We identify a previously-unreported genome-wide significant locus: ZRANB3 (Zinc Finger RANBP2-Type Containing 3, lead SNP p = 2.831 × 10 −9 ). Knockdown or genomic knockout of the zebrafish ortholog results in reduction in pancreatic β-cell number which we demonstrate to be due to increased apoptosis in islets. siRNA transfection of murine Zranb3 in MIN6 β-cells results in impaired insulin secretion in response to high glucose, implicating Zranb3 in β-cell functional response to high glucose conditions. We also show transferability in our study of 32 established T2D loci. Our findings advance understanding of the genetics of T2D in non-European ancestry populations.

Alström syndrome is an autosomal recessive obesity ciliopathy caused by loss-of-function mutations in the ALMS1 gene. In addition to multi-organ dysfunction, such as cardiomyopathy, retinal degeneration, and renal dysfunction, the disorder is characterized by high rates of obesity, insulin resistance and early onset type 2 diabetes mellitus (T2DM). To investigate mechanisms linking disease phenotypes we generated a loss-of-function deletion of a lms1 in the zebrafish using CRISPR/Cas9. We demonstrate conserved phenotypic effects including cardiac defects, retinal degeneration, and metabolic deficits that included propensity for obesity and fatty livers in addition to hyperinsulinemia and glucose response defects. Gene expression changes in β-cells isolated from alms1−/− mutants revealed changes consistent with insulin hyper-secretion and glucose sensing failure, which were also identified in cultured murine β-cells lacking Alms1 . These data present a zebrafish model to assess etiology and new secretory pathway defects underlying Alström syndrome-associated metabolic phenotypes. Given the hyperinsulinemia and reduced glucose sensitivity in these animals we also propose the alms1 loss-of-function mutant as a monogenic model for studying T2DM phenotypes. AUTHOR SUMMARY These data comprise a thorough characterization of a zebrafish model of Alström syndrome, a human obesity syndrome caused by loss-of-function deletions in a single gene, ALMS1 . The high rates of obesity and insulin resistance found in these patients suggest this disorder as a single-gene model for Type 2 Diabetes Mellitus (T2DM), a disorder caused by a variety of environmental and genetic factors in the general population. We identify a propensity for obesity, excess lipid storage, loss of β-cells in islets, and hyperinsulinemia in larval and adult stages of zebrafish alms1 mutants. We isolated β-cells from the alms1 mutants and compared the gene expression profiles from RNASeq datasets to identify molecular pathways that may contribute to the loss of β-cells and hyperinsulinemia. The increase in genes implicated in generalized pancreatic secretion, insulin secretion, and glucose transport suggest potential β-cell exhaustion as a source of β-cell loss and excess larval insulin. We propose this mutant as a new genetic tool for understanding the metabolic failures found in Type 2 Diabetes Mellitus.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.