High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Otis, Jessica P.; Farber, Steven A.

doi:10.3791/54735

Cited by 17 publications

(19 citation statements)

References 23 publications

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“…When MTP is mutated or absent, B-lp production is reduced or absent and TG accumulates in cytoplasmic lipid droplets (LDs) [ 54 – 56 ]. We have previously shown that accumulation of LDs in intestinal enterocytes of zebrafish larvae fed a high-fat meal causes the gut to be opaque [ 57 ] ( S4 Fig ), most likely due to the lipid droplets’ ability to scatter light [ 58 , 59 ]. Therefore, we hypothesized that the yolk opacity in the mttp mutant embryos is due to aberrant accumulation of LDs in the cytoplasm of the YSL.…”

Section: Resultsmentioning

confidence: 99%

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein

et al. 2020

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Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttp stl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.

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Section: Resultsmentioning

confidence: 99%

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein

et al. 2020

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“…From 4 dpf until the last day of treatment renewal—at 9 dpf—larvae were fed daily with a SD, 10% of fat (Tetramin, Tetra, Blacksburg, VA, USA), or with a HFD made of chicken egg yolk, ~53% of fat (Sigma-Aldrich, St. Louis, MO, USA), for 1 h before medium renewal. Both diets were also previously used in zebrafish [ 27 , 100 , 101 ]. At 5 dpf, larvae were exposed with 43 mM ethanol directly added to the incubation medium and/or by 25 nM B[a]P in dimethyl sulfoxide (DMSO)—DMSO final proportion: 0.001% v/v —or by this vehicle only until 12-dpf.…”

Section: Methodsmentioning

confidence: 99%

Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae

et al. 2018

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The rise in prevalence of non-alcoholic fatty liver disease (NAFLD) constitutes an important public health concern worldwide. Including obesity, numerous risk factors of NAFLD such as benzo[a]pyrene (B[a]P) and ethanol have been identified as modifying the physicochemical properties of the plasma membrane in vitro thus causing membrane remodeling—changes in membrane fluidity and lipid-raft characteristics. In this study, the possible involvement of membrane remodeling in the in vivo progression of steatosis to a steatohepatitis-like state upon co-exposure to B[a]P and ethanol was tested in obese zebrafish larvae. Larvae bearing steatosis as the result of a high-fat diet were exposed to ethanol and/or B[a]P for seven days at low concentrations coherent with human exposure in order to elicit hepatotoxicity. In this condition, the toxicant co-exposure raised global membrane order with higher lipid-raft clustering in the plasma membrane of liver cells, as evaluated by staining with the fluoroprobe di-4-ANEPPDHQ. Involvement of this membrane’s remodeling was finally explored by using the lipid-raft disruptor pravastatin that counteracted the effects of toxicant co-exposure both on membrane remodeling and toxicity. Overall, it can be concluded that B[a]P/ethanol co-exposure can induce in vivo hepatotoxicity via membrane remodeling which could be considered as a good target mechanism for developing combination therapy to deal with steatohepatitis.

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“…Embryonic feeding assays were performed as previously published (41). Briefly, equal amounts of BODIPY FL C12 (Life Technologies) egg yolk were provided to 7 days post fertilization (dpf) larvae for a period of 4 hours at 28.5 °C.…”

Section: Methodsmentioning

confidence: 99%

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

Nesmith

Hostelley

Leitch

et al. 2018

Preprint

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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.

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High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Cited by 17 publications

References 23 publications

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein

Membrane Remodeling as a Key Player of the Hepatotoxicity Induced by Co-Exposure to Benzo[a]pyrene and Ethanol of Obese Zebrafish Larvae

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

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