A Genetic Model to Study Increased Hexosamine Biosynthetic Flux

Abstract

Recently, we identified harvest moon (hmn), a fully penetrant and expressive recessive zebrafish mutant with hepatic steatosis. Larvae showed increased triacylglycerol in the absence of other obvious defects. When we attempted to raise these otherwise normal-appearing mutants to adulthood, we observed a developmental arrest and death in the early juvenile period. In this study, we report the positional cloning of the hmn locus and characterization of the defects caused by the mutation. Using bulk segregant analysis and fine mapping, we find that hmn mutants harbor a point mutation in an invariant residue within the sugar isomerase 1 domain of the gene encoding the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP) glutamine-fructose-6-phosphate transamidase (Gfpt1). The mutated protein shows increased abundance. The HBP generates β-N-acetyl-glucosamine (GlcNAc) as a spillover pathway from glucose. GlcNAc can be O-linked to seryl and threonyl residues of diverse cellular proteins (O-GlcNAc modification). Although some of these O-GlcNAc modifications serve an essential structural role, many others are dynamically generated on signaling molecules, including several impacting insulin signaling. We find that gfpt1 mutants show global increase in O-GlcNAc modification, and, surprisingly, lower fasting blood glucose in males. Taken together with our previously reported work, the gfpt1 mutant we isolated demonstrates that global increase in O-GlcNAc modification causes some severe insulin resistance phenotypes (hepatic steatosis and runting) but does not cause hyperglycemia. This animal model will provide a platform for dissecting how O-GlcNAc modification alters insulin responsiveness in multiple tissues.

Figure 1.

Hepatic steatosis in hmn mutants is not modified by nutritional status. (a) Diffuse accumulation of neutral lipid droplets in never-fed 7-dpf livers stained with eosin following osmium impregnation is seen in hmn mutant livers. (b) Never-fed larvae were live-sorted at 7 dpf with Nile Red staining. They were fixed and stained with ORO at the indicated ages and scored for hepatic steatosis. (c) Larvae were live-sorted at 7 dpf and fed until fixation and staining with ORO at the indicated ages.

Figure 2.

Positional cloning of the hmn mutant reveals a substitution mutation in an invariant residue of Gfpt1 protein within the first sugar isomerase domain. (a) A map cross of heterozygous hmn carriers to the polymorphic WIK line was performed. A single pair of heterozygous progeny was bred to generate all the individuals used to positionally clone the locus. First, larvae were fixed and stained with ORO and then sorted into two pools. DNA from 21 WT and 21 hmn mutant larvae were examined by bulk segregant analysis using a standard set of simple sequence-length polymorphism microsatellite markers (SSLP, z markers) on a meiotic map (6). This allowed us to assign the mutated gene to the south subtelomeric region of chromosome 8. (b) Examination of 51 additional progeny from this single pair allowed us to narrow the locus further to a single contig. cDNAs were cloned and sequenced for all protein-coding genes within the critical interval. (c) At position 987 of the gfpt1 coding sequence, a G-to-A transition mutation was identified in hmn mutants (and was confirmed to have been induced in the mutagenized male through sequencing genomic DNA). (d) The mutation changed codon 329 from an E to a K residue. (e) Comparison among multiple species from yeast to humans revealed that E329 (zebrafish numbering, shaded in red) is an invariant residue. Many neighboring residues are also invariant (yellow) or highly conserved among the species examined. (f) Inspection of the crystal structure of human GFPT1 (a homodimer, with both chains shown in different colors) shows that the invariant E239 residue is in close proximity (9.6 Å) to the fructose-6-phosphate substrate. (g) The HBP is shown, with the rate-limiting enzyme GFPT, catalyzing the conversion of fructose-6-phosphate to glucosamine-6-phosphate via a transamidation-requiring glutamine.

Figure 3.

Gfpt1^E329K mutants have higher Gfpt1 protein abundance and increased O-GlcNAc modification, but lower fasting blood glucose. (a) Livers from three adult WT and gfpt1^E329K mutants were dissected following an overnight fast. The livers were pooled and homogenized in protein extraction buffer. Proteins (15 μg per sample) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (molecular weight standards shown in kDa) and transferred to polyvinylidene fluoride membranes, and immunoblot analyses were performed with anti-GFPT1, anti–O-GlcNAc modification, and anti-Tubb IgGs. As expected, the anti-GFPT1 IgG detected a single band of nearly 80 kDa; multiple O-GlcNAc modified proteins were detected in both groups. (b) Animals were fasted overnight prior to measurement of blood glucose.