Feng Y et al. (2013), FGT-1 is the major glucose transporter in C. el...

XB-ART-48428

Biochem J 2013 Dec 01;4562:219-29. doi: 10.1042/BJ20131101.

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FGT-1 is the major glucose transporter in C. elegans and is central to aging pathways.

Feng Y , Williams BG , Koumanov F , Wolstenholme AJ , Holman GD .

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Caenorhabditis elegans is widely used as a model for investigation of the relationships between aging, nutrient restriction and signalling via the DAF-2 (abnormal dauer formation 2) receptor for insulin-like peptides and AGE-1 [ageing alteration 1; orthologue of PI3K (phosphoinositide 3-kinase)], but the identity of the glucose transporters that may link these processes is unknown. We unexpectedly find that of the eight putative GLUT (glucose transporter)-like genes only the two splice variants of one gene have a glucose transport function in an oocyte expression system. We have named this gene fgt-1 (facilitated glucose transporter, isoform 1). We show that knockdown of fgt-1 RNA leads to loss of glucose transport and reduced glucose metabolism in wild-type worms. The FGT-1 glucose transporters of C. elegans thus play a key role in glucose energy supply to C. elegans. Importantly, knockdown of fgt-1 leads to an extension of lifespan equivalent, but not additive, to that observed in daf-2 and age-1 mutant worms. The results of the present study are consistent with DAF-2 and AGE-1 signalling stimulating glucose transport in C. elegans and this process being associated with the longevity phenotype in daf-2 and age-1 mutant worms. We propose that fgt-1 constitutes a common axis for the lifespan extending effects of nutrient restriction and reduced insulin-like peptide signalling.

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Species referenced: Xenopus
Genes referenced: gnl3 ins pik3ca slc2a1

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	Figure 1. Comparison of FGT-1A and -1B with GLUT1 and GLUT4Alignment allows comparison of invariant and non-charged residues, particularly in the C-terminal half of the protein. Boxes with a dotted-fill highlight charged residues in the TM regions. Highly conserved signature residues (boxed residues), particularly in the TM7 region, are thought to be important in substrate recognition (see the Supplementary Online Data at http://www.biochemj.org/bj/456/bj4560219add.htm for more information).
	Figure 2. Glucose transport activity of C. elegans GLUT-like transporters expressed in Xenopus oocytes(A) The levels of GLUT-like transporters at the cell surface as determined using exofacial HA epitopes. cRNAs for transporters HA-tagged in the first exofacial loop were injected into oocytes. The HA tag at the cell surface was detected with an anti-HA antibody and a β-galactosidase-conjugated secondary antibody. Results are means±S.E.M. for three separate experiments. P<0.05 compared with the water-injected control oocytes. (B) Glucose-transport activity associated with the indicated putative GLUT-like transporters (without HA tags). Results are means±S.E.M. for three separate experiments. ns, not significantly different compared with the water-injected control oocytes. (C) Comparison of glucose-transport activities, in the presence and absence of the HA tags, that are associated with the expression of hGLUT1 and C. elegans FGT-1A and FGT-1B. Results are means±S.E.M. for three separate experiments. P<0.05 compared with the water-injected control oocytes. (D) Uptake of 50 μM D-glucose via FGT-1A expressed in oocytes. Time courses were found to be linear. Results are means±S.E.M. for three separate experiments. (E) Rates of maximal transport for glucose in Xenopus oocytes expressing 50 ng of cRNA for FGT-1A and FGT-1B. Oocytes were incubated with the indicated concentrations of D-glucose (S). The rates of [14C]glucose uptake (v, pmol glucose/oocyte per min) were used in least-square fits to the Michaelis–Menten equation. Results are means±S.E.M. for three separate experiments.
	Figure 3. Knockdown of fgt-1a and fgt-1b in wild-type and signalling mutant C. elegans strains(A) C. elegans wild-type strain (N2) L1 larvae were seeded on to NGM plates seeded with fgt-1a or fgt-1b RNAi bacteria (with or without 1 mM IPTG to induce knockdown). The young adult worms were transferred on to NGM plates (50 μM FUDR, with or without 1 mM IPTG) seeded with fgt-1a or fgt-1b RNAi bacteria. The worms were maintained on the plates without exhaustion of bacteria food source for 10 days. Total RNAs of each sample were extracted and the extent of knockdown of the total fgt-1 mRNA was determined by qPCR using primers to a region present in both transcripts. Results are means±S.E.M. for three separate experiments. (B) C. elegans L1 larvae of wild-type strain (N2) or strains mutated in the DAF-2 receptor [daf-2 (e1370)], or the PI3K [age-1 (hx546)] were seeded on to NGM plates seeded with fgt-1a and fgt-1b RNAi bacteria mixture (1:1 ratio) (with or without 1 mM IPTG to induce knockdown). The young adult worms were transferred to NGM plates (50 μM FUDR, with or without 1 mM IPTG) seeded with fgt-1a and fgt-1b RNAi bacteria mixture (1:1 ratio). The worms were maintained on the plates without exhaustion of bacteria food source for 10 days. Total RNA of each sample were extracted and the extent of knockdown of the total fgt-1 mRNA was determined by qPCR using primers to a region present in both transcripts. Results are means±S.E.M. for three separate experiments. *P<0.05 compared with the uninduced control. mRNA levels in mutant strains daf-2 and age-1 were not significantly different (n.s.) compared with the wild-type strain (P=0.072 and 0.075 respectively).
	Figure 4. Knockdown of fgt-1 influences whole-body glucose uptake and metabolism in C. elegans(A) Whole-body 2DG uptake in the indicated C. elegans strains was determined. P<0.05 compared with the control uninduced RNAi. ΨP<0.05 compared with the wild-type C. elegans strain fed with control uninduced RNAi bacteria. (B) Whole-body glucose oxidation in the indicated C. elegans strains was determined from the conversion of medium 500 μM [14C]glucose into 14CO2 which was trapped in KOH. P<0.05 compared with the control RNAi. ΨP<0.05 compared with the wild-type C. elegans strain fed with control uninduced RNAi bacteria. (C) Whole-body glucose into fat conversion in the indicated C. elegans strains was determined from the 14C fat content extracted from worm lysates with heptane. *P<0.05 compared with the control RNAi. ΨP<0.05 compared with the wild-type C. elegans strain fed with control RNAi bacteria. Results are means±S.E.M. for three separate experiments.
	Figure 5. fgt-1 knockdown extends lifespan in C. elegans(A) Determination of lifespan for wild-type (N2) worms exposed to fgt-1a RNAi (▲) and fgt-1b RNAi (●) with high (20 mM) glucose concentration medium. ∆ and ○, control uninduced RNAi. (B) Determination of lifespan for wild-type (N2) worms exposed to fgt-1a RNAi (▲) and fgt-1b RNAi (●) together with NGM. ∆ and ○, control uninduced RNAi. (C) Determination of lifespan for wild-type (N2) worms exposed to a fgt-1a and fgt-1b RNAi mixture (1:1 ratio) together with NGM. ▲, fgt-1 RNAi; ∆, control uninduced RNAi.
	Figure 6. The lifespan-extending effects of fgt-1 knockdown are reduced on the daf-2 background, whereas there is no significant lifespan extension on the age-1 background(A) Determination of lifespan for daf-2 (e1370) mutant worms exposed to fgt-1a RNAi (▲) and fgt-1b RNAi (●) with NGM. ∆ and ○, control uninduced RNAi. (B) Determination of lifespan for daf-2 (e1370) mutant worms exposed to a fgt-1a and fgt-1b RNAi mixture (1:1 ratio) together with NGM. ▲, fgt-1 RNAi; ∆, control RNAi. (C) Determination of lifespan for age-1 (hx546) mutant worms exposed to fgt-1a RNAi (▲) and fgt-1b RNAi (●) together with NGM. ∆ and ○, control uninduced RNAi. (D) Determination of lifespan for age-1 (hx546) mutant worms exposed to a fgt-1a and fgt-1b RNAi mixture (1:1 ratio) together with NGM. ▲, fgt-1 RNAi; ∆, control uninduced RNAi.

References [+] :

Al-Hasani, Roles of the N- and C-termini of GLUT4 in endocytosis. 2002, Pubmed