Genomic knockout ofalms1in zebrafish recapitulates Alström syndrome and provides insight into metabolic phenotypes

Abstract

SCIENTIFIC ABSTRACTAlström 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 alms1 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 SUMMARYThese 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.

Consensus clinical management guidelines for Alström syndrome

“…The knockdown of Alms1 resulted in excess secreted insulin and cellular transport, especially in insulin-related pathways, prior to the onset of adipogenesis. These findings support a role for Alms1 in regulating secretion and membrane depolarization in β-cells [ 58 ]. In addition, a role for ALMS1 in glucose homeostasis and GLUT4 translocation in adipose tissue of ALMS1 GT/GT mouse model was shown [ 54 ].…”

“…In the ALMS1 GT/GT and foz/foz, mouse models, hyperinsulinemia develops early and pancreatic islets show beta cell proliferation, thus suggesting that both IR and increased insulin secretion might contribute to glucose intolerance [ 10 , 56 ]. Using zebrafish models, loss of Alms1 , resulted in a significant decrease in β-cell production and under prolonged exposure to high glucose conditions, β-cells were unable to continually expand as a result of decreased proliferation and increased cell death [ 57 , 58 ]. The knockdown of Alms1 resulted in excess secreted insulin and cellular transport, especially in insulin-related pathways, prior to the onset of adipogenesis.…”

alms1mutant zebrafish do not show hair cell phenotypes seen in other cilia mutants

“…It is also possible that alms1 might not be playing a role in zebrafish hair cells due to a lack of conservation to the mammalian homolog as much of the N-terminal half of the protein is specific to mammals (Hearn 2019). However, as the zebrafish mutant shows multiple expected phenotypes due to defects in other tissues (Nesmith et al 2019) if any of these issues were responsible for the lack of phenotype we see, it would have to be relatively hair cell specific. We can also not rule out more subtle defects, particularly in inner ear hair cells, that were not observed by the methods we used.…”

“…Animals: Experiments used either 5 days post-fertilization (dpf) Danio rerio (zebrafish) larvae or adult zebrafish over three months of age. We used the previously described alms1 umd2 mutant line, which causes a premature stop codon (Nesmith et al 2019). Mutant larvae were generated by either crossing two heterozygous animals together ,or one heterozygous animal to a homozygous mutant, and comparing homozygous mutants to either wild-type or heterozygous siblings born at the same time.…”

“…Similar to mouse mutants, zebrafish alms1 mutants share many phenotypes with Alström Syndrome patients, including retinal degeneration, kidney and cardiac defects, increased fat disposition in the liver, a propensity for obesity, hyperinsulinemia, and glucose response defects (Nesmith et al 2019). However, hair cells and the auditory and vestibular systems of these fish have not been examined.…”