Functional characterization and immunolocalization of the transporter encoded by the life-extending gene Indy

Abstract

Caloric restriction extends life span in a variety of species, highlighting the importance of energy balance in aging. A new longevity gene, Indy (for I'm not dead yet), which doubles the average life span of flies without a loss of fertility or physical activity, was postulated to extend life by affecting intermediary metabolism. We report that functional studies in Xenopus oocytes show INDY is a metabolite transporter that mediates the high-affinity, disulfonic stilbene-sensitive flux of dicarboxylates and citrate across the plasma membrane by a mechanism that is not coupled to Na(+), K(+), or Cl(-). Immunocytochemical studies localize INDY to the plasma membrane with most prominent expression in adult fat body, oenocytes, and the basolateral region of midgut cells and show that life-extending mutations in Indy reduce INDY expression. We conclude that INDY functions as a novel sodium-independent mechanism for transporting Krebs and citric acid cycle intermediates through the epithelium of the gut and across the plasma membranes of organs involved in intermediary metabolism and storage. The life-extending effect of mutations in Indy is likely caused by an alteration in energy balance caused by a decrease in INDY transport function.

Fig 1.

INDY is a transporter. A >50-fold increase in [¹⁴C]succinate transport was seen in Xenopus oocytes injected with Indy cRNA, as compared with water-injected controls.

Fig 2.

INDY's transport selectivity is similar to mammalian sodium-dicarboxylate cotransporters. Effects of 10 mM of each indicated anion (as sodium salts) on INDY-mediated [¹⁴C]succinate uptake were assayed by iso-osmotic replacement of NaCl in the media. [¹⁴C]Succinate transport was strongly inhibited by succinate (as expected for a saturable transport process), citrate, α-ketoglutarate, glutarate, and fumarate but not by pyruvate, lactate, PAH, sulfate, or phosphate.

Fig 3.

INDY is a relatively high-affinity succinate transporter. Lineweaver–Burk plot indicates relatively high affinity (K_m = 39 μM) for succinate transport mediated by INDY.

Fig 4.

INDY is not a cotransporter. INDY-mediated transport of succinate is not coupled to Na⁺, K⁺, or H⁺ or driven by an exchange of Cl⁻. The replacement of NaCl by KCl, choline Cl, LiCl, KCl, or Na gluconate or imposition of an outside-acid pH gradient (pH 6.0) had no effect on the uptake of succinate by the INDY-expressing oocytes.

Fig 5.

INDY-mediated succinate transport is disulfonic stilbene-sensitive. Transport was almost completely inhibited by 0.1 mM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), was modestly reduced by 1 mM phloretin or furosemide, and was unaffected by 1 mM probenecid.

Fig 6.

INDY mediates citrate transport. Compared with water-injected control, uptake of [¹⁴C]citrate was stimulated >50-fold into oocytes injected with Indy cRNA. Citrate uptake was not reduced by replacement of NaCl by KCl but was stimulated by imposition of an outside-acid pH gradient (pH 6.0). Citrate uptake was virtually abolished by 0.1 mM DIDS.

Fig 7.

Immunolocalization of INDY in control and mutant adult flies. Immunostaining of 5 μm paraffin sections of gut (A and B), fat body (C and D), and oenocytes (E and F) of control (A, C, E) and Indy mutant adult flies (B, D, F) by using rabbit anti-INDY primary antibody (1:5,000) and goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:200) and developed as described in Vectastain Elite ABC kit. Independent antibodies were raised to two different peptides of INDY. Both anti-peptide antibodies showed the same specificity. The preimmune rabbit antibody from each showed no specific staining on control tissue sections at 1:500–1:5,000. (Bars = 10 μm.)