A Caenorhabditis elegans model of insulin resistance: altered macronutrient storage and dauer formation in an OGT-1 knockout

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is an evolutionarily conserved modification of nuclear pore proteins, signaling kinases, and transcription factors. The O-GlcNAc transferase (OGT) catalyzing O-GlcNAc addition is essential in mammals and mediates the last step in a nutrient-sensing "hexosamine-signaling pathway." This pathway may be deregulated in diabetes and neurodegenerative disease. To examine the function of O-GlcNAc in a genetically amenable organism, we describe a putative null allele of OGT in Caenorhabditis elegans that is viable and fertile. We demonstrate that, whereas nuclear pore proteins of the homozygous deletion strain are devoid of O-GlcNAc, nuclear transport of transcription factors appears normal. However, the OGT mutant exhibits striking metabolic changes manifested in a approximately 3-fold elevation in trehalose levels and glycogen stores with a concomitant approximately 3-fold decrease in triglycerides levels. In nematodes, a highly conserved insulin-like signaling cascade regulates macronutrient storage, longevity, and dauer formation. The OGT knockout suppresses dauer larvae formation induced by a temperature-sensitive allele of the insulin-like receptor gene daf-2. Our findings demonstrate that OGT modulates macronutrient storage and dauer formation in C. elegans, providing a unique genetic model for examining the role of O-GlcNAc in cellular signaling and insulin resistance.

Fig. 1.

A deletion in the ogt-1 gene of C. elegans results in altered nuclear pore glycosylation. (A) Position of the deletion in ogt-1(ok430) is shown. The deletion removes the central portion of the protein producing a product lacking catalytic activity. (B) Levels of GlcNAcitol released by β elimination were measured. Nucleoporin-specific Abs directed against either O-GlcNAc (RL-2) (C) or protein determinants (RL-1) (D) were used to examine nuclear pore glycosylation in N2 and ogt-1(ok430) strains as indicated. (E) Another anti-O-GlcNAc Ab (CTD110) (27) was used for immunobloting. (C–E Lower) Corresponding Ponceau stains for each blot.

Fig. 2.

O-GlcNAc is absent in ogt-1(ok430). Embryos were fixed and processed as described in Materials and Methods and stained with either RL-2 (anti-O-GlcNAc) (A) or RL-1 (protein epitope) (B) mAbs to nucleoporins. DNA was stained with DAPI as indicated. No O-GlcNAc was detectable on nuclear pores in ogt-1(ok430) homozygous mutants.

Fig. 3.

Nuclear transport is unaffected in ogt-1(ok430). The nuclear transport of transcription factors SKN-1 (A) and HLH-1 (B) was examined as described in Materials and Methods. (A) SKN-1 nuclear localization is shown (Upper) and overlaid onto a corresponding Nomarski image (Lower). (B) HLH-1 localization is shown (Upper) and overlaid upon a DAPI nuclear staining (Lower). Neither the kinetics of transport nor the nuclear localization of either transcription factor was noticeably altered in the ogt-1(ok430) mutant animals. Three other transcription factors, HLH-2, ELT-2, and LIN-26 showed similar results (data not shown).

Fig. 4.

Macronutrient storage in ogt-1ok(430). N2 and ogt-1(ok430) strains were assayed for their storage of glycogen, trehalose, and glucose as described in Materials and Methods. (A Left) Levels of each of the stored carbohydrates is shown. A 2.5- to 3-fold elevation in sugar storage was observed; this representative experiment was repeated nine times with nearly identical results. (A Right)(Lower) Carminic acid was detected by fluorescence microscopy and was predominantly found in the intestine of the animal. The carminic acid-positive structures are a distinct subset of granules in the intestinal cells in both N2 and ogt-1(ok430). (Upper) A corresponding Nomarski image is shown. (B Left) Storage of neutral lipids in N2 and ogt-1(ok430) was quantified in three replicate experiments as described in Materials and Methods. The reduction in neutral lipid storage was ≈70%. (B Right) (Lower) Enlargement of the anterior intestinal cells of the ogt-1(ok430) strain that was allowed to take up Nile red to label lipid droplets. (Upper) A corresponding Nomarski image is shown.

Fig. 5.

Loss of OGT activity leads to alteration of dauer formation in C. elegans. (A) Insulin-like-signaling C. elegans. Key components of the insulin-like-signaling pathway leading to dauer formation are shown. Without DAF-2 signaling, DAF-16 is not repressed, and the dauer pathway is initiated. Mammalian homologs of the C. elegans proteins exist for each case. (B) Loss of OGT activity suppresses dauer formation. To test the role of ogt-1 in the insulin-signaling pathway, we used an allele (e1370) of the daf-2 gene that encodes an insulin-like receptor. At the restrictive temperature (25°C), daf-2(e1370) is a constitutive dauer with 100% of animals entering diapause. However, by culturing animals at slightly lower temperatures, the percentage of animals entering dauer can be manipulated. We tested temperatures at 0.5°C increments between 22°C and 24°C by incubating cultures in an Echotherm incubator (Torrey Pines Scientific, San Marcus, CA) with the temperature monitored by using a Barnant thermocouple thermometer (Barnant, Barrington, IL). We found that 22.5°C was the optimal temperature leading to sensitized daf-2(e1370) mutants for these assays. Adults of each strain tested were allowed to lay embryos overnight at 15°C. Cultures were shifted to 22.5°C and scored 3 d later. Animals scored as dauers were arrested and displayed canonical visible phenotypes; all other animals were scored as nondauers. At 22.5°C, no dauers were observed in cultures of N2 (n = 222) or ogt-1(ok430) mutants (n = 432), whereas daf-2(e1370) mutants had 93% dauers (n = 579). In contrast, ogt-1(ok430) daf-2(e1370) double mutant cultures had only 27% dauers (n = 332) at 22.5°C, demonstrating the loss of OGT-1 activity suppressed daf-2(e1370) dauer formation at this temperature.