Identification of the fasting hepatic steatosis mutant rmn. (A) Whole-mount ORO staining of larvae. The yolk lipid (arrowhead) is exhausted 5 dpf. Vascular lipid staining (arrows) ceases by the end of 5 dpf. The surfactant-lined swim bladder stained with ORO (asterisk). (B) Whole-mount BODIPY 493/503 staining of 6-dpf larvae. This dye also stained the vitreous humor of the eyes (arrowhead) and the ventricles of the CNS (arrow). (C) Confocal stacks of livers fixed and stained as in B. A single slice of a rmn mutant liver is shown in the inset. (D) Transmission electron microscopy of liver sections showing that the rmn mutant hepatocytes have cytoplasmic lipid droplets (ld). The nuclear (n) and mitochondrial (m) morphology appears normal in rmn mutants. Higher-magnification micrographs also show multilamellar structures (asterisks) suggestive of multivesicular bodies and elongated tubular (t) precursors of these structures in both wild-type (WT) and the rmn mutant livers. Similarly, autophagosomal structures (arrowheads) were observed in both wild-type and rmn mutant livers. Bar, 1 μm.

Increased neutral lipids in rmn mutants. (A) β-Hydroxybutyrate (n = 10 larvae for each genotype), cholesterol (n = 15), cholesteryl esters (n = 50), free fatty acids (n = 40), and triacylglycerol (n = 50) measured in whole-body extracts of larvae. (*) P < 0.01 for the age-matched comparator. (B) Neutral lipid levels in fed and fasted adult livers. (*) P < 0.01 for the nutritional status comparator (n = 4). (C) Adult liver protein mass in fed and fasted animals (n = 4). (D) Blood glucose and triacylglycerol from fed and fasted animals. (*) P < 0.01 for the age-matched comparator (n = 4). In all panels, wild-type is shown in open bars, and rmn mutant is solid bars.

Positional cloning of the rmn locus. (A) The rmn locus was narrowed to a 100-kb critical interval on chromosome 12. Genetic distance of markers is reported on the radiation hybrid map in centiRays (cR) and on the MGH meiotic map in centiMorgans (cM) above the individual bacterial artificial chromosome and fosmid clones used to span this region of the genome. The recombination events (reported as recombinants/meioses) at the labeled polymorphic markers (poly-CA or “CA repeat”) are shown below individual clones. Clone acronym definitions and the names of the clones between zK29B5 and zC29A20 are in the Supplemental Material. (B) Fine mapping narrowed the critical interval to the space between two CA repeats within introns of slc16a6a and calcoco2 on the clone zK106L8 (white region flanked by gray and red portions of the chromosome). Coding exons are filled red boxes. The 5′ and 3′ untranslated regions (UTRs) are unfilled boxes. (C) Sanger traces of slc16a6a cDNAs prepared with primers in the 5′ and 3′ UTRs indicating that exon 2 is skipped in rmn mutants. The position of leucine codon 86 and methionine codon 97 are shown. (D) Cartoons of slc16a6a cDNAs prepared as in C. (E) Sanger traces of genomic DNA spanning exon 2 of slc16a6a reveal retrotransposon sequences in rmn mutants. An in-frame stop codon is shown. (F) Cartoon of the insertion mutation destroying exon 2 of slc16a6a. (G, left) The sequenced products shown in E differ in size: Amplification of exon 2 and flanking intronic sequences of slc16a6a from homozygous wild-type animals (+/+), heterozygous rmn carriers (+/rmn), and homozygous rmn mutant animals (rmn/rmn) reveals a 450-bp increase in the rmn allele product. (Left panel) Heterozygous carriers proved difficult to score using this primer pair because the mutant allele amplified less robustly in the presence of the wild-type allele (primers A and B). (Right panel) Thus, we resorted to a three-primer strategy to amplify either the sequences deleted by the insertion or a small portion of the insertion. The latter approach led to confident scoring of heterozygous carriers. The molecular mass standards are shown in kilobase pairs.

Slc16a6a is a β-hydroxybutyrate transporter. (A) RT–PCR of mature slc16a6a and rpp0 transcripts at the indicated ages. (B) Whole-mount in situ hybridization with slc16a6a riboprobes. (C) The topology of the predicted wild-type and hypothetical rmn Slc16a6a proteins. (D) Ponceau S staining of nitrocellulose membranes following transfer of proteins resolved by sodium dodecyl sulfate polyacrylamide electrophoresis. (E) Immunoblotting with custom anti-Slc16a6a IgGs of the membrane in D. The membrane was deliberately overexposed in order to increase the chance of detecting a smaller protein band in the rmn mutant. (F) Current tracings of Xenopus oocytes expressing Slc16a6a (or control-injected oocytes, in black) that were perfused with monocarboxylic acids (5 mM) for the indicated time period. Oocytes were voltage-clamped to a holding potential of −70 mV. (G) Current–voltage relation of a Slc16a6a-expressing oocyte perfused with β-hydroxybutyrate.

(A) A model of the role of Slc16a6a in hepatic ketone body metabolism. Dietary and stored reduced carbon atoms in the form of fatty acids (intestine and adipose, respectively) can be delivered to the liver, where, after partial β-oxidation, they can be repackaged as ketone bodies for feeding the peripheral organs during fasting. Triacylglycerol can also be packaged into VLDL particles (liver) or chylomicrons (intestine) for delivery to the periphery. The intestine and skeletal muscle also provide ketogenic amino acids, the most abundant of which is leucine. In birds, reptiles, and mammals, the rate-limiting enzyme of ketogenesis is mitochondrial HMG-CoA synthase 2 (gray reactions), which arose in birds through gene duplication of the cytosolic HMG-CoA synthase 1 gene (). Amphibians, fish, and other lower vertebrates have only the cytosolic, cholesterol-synthetic HMG-CoA synthase 1 and rely on increased flux through deamination and oxidation of leucine (which also occurs in higher vertebrates) to make mitochondrial HMG-CoA for ketogenesis (; ). Slc16a6a is a β-hydroxybutyrate-transporting MCT that is expressed in the liver. In rmn mutants, this transporter is inactivated, and the carbon atoms that would otherwise be secreted as fuel during fasting are diverted back to de novo lipogenesis and storage in lipid droplets. The peripheral ketone body transporters are not known (“?”), but Slc16a1, Slc16a7, and Slc16a3 are expressed in brain endothelial cells, neurons, and astrocytes, respectively (). In zebrafish, there is evidence that Slc5a family members are expressed in muscle and can transport sodium hydroxybutyrate (). (CoA) Coenzyme A; (Cpt1) Carnitine palmitoyl transferase 1; (Bckdh) Branched chain ketoacid dehydrogenase; (Chyl) chylomicron particle; (FAD) flavine adenine dinucleotide; (FADH₂) reduced FAD; (NAD⁺) nicotinamide adenine dinucleotide; (NADH₂) reduced NAD; (VLDL) very low-density lipoprotein particle. (B) Larvae were incubated with L-[¹⁴C(U)]-leucine, and then lipids were analyzed as described in the Materials and Methods to calculate the ¹⁴C incorporation ratio. Values greater than unity reflect increased relative incorporation of ketogenic precursor carbon atoms into neutral lipids in rmn mutants.

Genetic and nutritional rescue of rmn mutants. (A) Single-cell, homozygous rmn mutant embryo larvae were injected with wild-type zebrafish slc16a6a or human SLC16A6 cDNAs under the control of either bactin2 (broadly expressed) or fabp10a (liver-limited) promoter elements. Larvae were then stained with ORO on 6 dpf. (B) Whole-mount ORO staining of larvae fed from 5 to 12 dpf. The intestinal lumen contents were also stained with ORO, reflecting the lipid content of the larval diet. (C) Whole-mount ORO staining of larvae fed from 5 to 12 dpf and then removed from food. (D) In previously fed rmn mutants, hepatic steatosis appears after 3 d of fasting. (E) Survival curve for animals fed from 5 to 12 dpf and then removed from food (n = 50 larvae of each genotype). The median age at death was 20 ± 2 dpf for wild-type larvae and 18 ± 1.7 dpf for rmn mutant larvae (log rank test: χ² = 62.3 on one degree of freedom; P = 2.9 × 10⁻¹⁶).