Tequila Regulates Insulin-Like Signaling and Extends Life Span in Drosophila melanogaster

Abstract

The aging process is a universal phenomenon shared by all living organisms. The identification of longevity genes is important in that the study of these genes is likely to yield significant insights into human senescence. In this study, we have identified Tequila as a novel candidate gene involved in the regulation of longevity in Drosophila melanogaster. We have found that a hypomorphic mutation of Tequila (Teq ^f01792), as well as cell-specific downregulation of Tequila in insulin-producing neurons of the fly, significantly extends life span. Tequila deficiency–induced life-span extension is likely to be associated with reduced insulin-like signaling, because Tequila mutant flies display several common phenotypes of insulin dysregulation, including reduced circulating Drosophila insulin-like peptide 2 (Dilp2), reduced Akt phosphorylation, reduced body size, and altered glucose homeostasis. These observations suggest that Tequila may confer life-span extension by acting as a modulator of Drosophila insulin-like signaling.

Metabolic processes are considered as central components of life-span regulation. Nutritional manipulation, such as dietary restriction, has been shown to prolong healthy life span across species, from yeast to flies to mammals (1–3). Insulin/IGF-like signaling is the major pathway responding to the energy and metabolic status of the body. In flies, there are eight genes encoding the Drosophila insulin-like peptides, Dilp1-8, which were originally identified based on a sequence similarity search using four conserved cysteine residues within the insulin A chain as bait (4–6). The sequence comparison reveals a 20%–35% similarity of the various Dilps to the mature human insulin peptide. Dilps are predicted to resemble preproinsulin at the structural level and require peptidase processing for their final activation. Four Dilps (1, 2, 3 and 5) are produced by median neurosecretory cells of the pars intercerebralis, also called insulin-producing neurons, which appear to function as pancreatic β cells in mammals (7).

Dilps signal through the insulin receptor and the insulin receptor substrate (chico, in flies), leading to activation of phosphoinositide-3-kinase and protein kinase B/Akt. This kinase cascade eventually phosphorylates the forkhead transcription factor (dFOXO), causing dFOXO retention in the cytoplasm via binding to 14-3-3 proteins. Despite the fact that mutations resulting in reduced activity of insulin/IGF-like signaling have been shown to increase life span in several evolutionarily distant species, the role of insulin/IGF-like signaling in dietary restriction–induced life-span extension remains controversial (8–12).

Tequila was originally identified as a multiple-domain serine protease that is transcriptionally upregulated following fungal or bacterial infection (13). More recently, it has been recognized as a neurotrypsin ortholog that is involved in long-term memory (LTM) formation (14,15). Intriguingly, Tequila may regulate memory formation in a nutrient-dependent manner, because Tequila mutant flies display LTM deficiency when fed a normal diet but have normal LTM on a starvation diet (15,16). Tequila is highly expressed in the brain and fat body of adult Drosophila melanogaster, further suggesting that it may have a functional relationship with metabolic regulation. In this study, we report novel functions of Tequila that may modulate insulin-like signaling and extend life span in D melanogaster.

Materials and Methods

Flies and Life-Span Assays

w ¹¹¹⁸, Tequila ^f01792, Elav-Gal4, PPL-Gal4, Dilp2-Gal4, c739-Gal4, Elav-gene switch (GS), MB-GS driver, UAS-GFP, and UAS-Tequila RNAi (VDRC #45232) fly stocks were raised on standard sucrose/yeast/cornmeal food. UAS-Tequila was generated from a full-length Tequila D isoform cDNA and subcloned into the pUAST vector. We used the Tequila D isoform for overexpression experiments due to its higher expression level in adult flies compared with the other isoforms (13). The effectiveness of the UAS-Tequila RNAi and UAS-Tequila constructs used for Tequila knockdown and overexpression, respectively, was verified by quantitative polymerase chain reaction (qPCR, Figure 1 and Supplementary Figure 1). For life-span assays, all flies were backcrossed into the w ¹¹¹⁸ background for at least five generations, as described previously (17). Flies that had eclosed within 48 hours (approximately 100 males and 100 females) were transferred to a 1-L population cage and maintained in a humidified, temperature-controlled incubator with 12-hour on/off light cycle at 25°C (18). Fresh food was provided every other day, and the number and sex of dead flies were scored. Fly food contained 5% dextrose, 5% yeast, 2% agar, and 0.23% Tegosept (Apex). For the Gene Switch experiment, 200 µM RU486 (mifepristone) in ethanol, or ethanol alone as control, was added to the food.

Figure 1.

Tequila mutation induces life-span extension. (A) Tequila mRNA is significantly downregulated in Teq ^f01792 homozygous mutant flies (blue) compared with control flies (w ¹¹¹⁸, red). Experiments were done in triplicate, and each replicate contained more than 30 flies for each group. (B and C) Extended life spans of male (B) and female (C) Teq ^f01792 homozygous mutant flies (blue) compared with control flies (w ¹¹¹⁸, red). Statistical analysis of life spans is shown in Table 1. (D and E) Teq ^f01792 homozygous mutant flies show smaller body size (D) and reduced body weight (E). (F and G) Food intake and female fecundity are not altered in Teq ^f01792 homozygous mutant flies (blue) compared with control flies (w ¹¹¹⁸, red). Data were collected from 8–10 replicates for each group. Each replicate contained 10 flies for the body weight (E) and feeding assays (F), and three mating pairs for the female fecundity measurements (G).

Quantification of mRNA

Total RNA was prepared from at least 30 flies using the NucleoSpin RNA Kit (Macherey-Nagel). The RNA was converted to cDNA using oligo-d(T)₁₅ (Invitrogen) and SuperScript III reverse transcriptase (Invitrogen) as described previously (19). qPCR was carried out using a StepOnePlus Real-Time PCR System (Applied Biosystems), SYBR Green Master Mix (Fermentas), and gene-specific primers 5′-CATTGGTGTGGAGCTGTGGTAAT-3′ and 5′-CTCG GCTATGTTTGCGTAGTGAT-3′ for Tequila and 5′-AATGGGTGT CGCTGAAGAAGTC-3′ and 5′-GACGAAATCAAGGCTAAGGT CG-3′ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A two-step PCR reaction was carried out with denaturation at 95°C for 15 seconds, annealing and extension combined at 60°C for 1 minute in a total of 40 cycles. The mRNA expression level of each target gene compared with GAPDH was quantified by subtraction: Ct (specific gene) − Ct (GAPDH) = ΔCt. A difference of one PCR cycle equates to a twofold change in mRNA expression level. The uniqueness of amplicons was confirmed using dissociation curves.

Immunostaining

Fly brains or Drosophila Schneider (S2) cells (Invitrogen) were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes at room temperature, treated with PBS containing 0.25% Triton X-100 (PBST) for 1 hour, blocked with PBST and 10% normal goat serum for 1 hour, and incubated with primary antibodies diluted in blocking solution for 1 day at 4°C. After being washed in PBST three times, samples were incubated with secondary antibody for 1 day at 4°C. Samples were washed and incubated with 1:250 Alexa Fluor 635 streptavidin (Molecular Probes). Primary antibodies used were rabbit anti-hemagglutinin (1:250, Bethyl) and rabbit anti-Tequila (1:50, a kind gift from Dr. T. Preat). Secondary antibody was biotinylated anti-rabbit (1:200 Molecular Probes). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (The Jackson Laboratory). Brain samples were mounted in FocusClear (CelExplorer Labs), and images were captured using confocal microscopy (Zeiss LSM 700).

Cell Culture, Plasmid Transfection, and Western Blot Analysis

Drosophila S2 cells were cultured in M3 medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and penicillin/streptomycin (Gibco) at 25°C. For transfection, 5×10⁶ S2 cells per well were subcultured in a 6-well culture plate, and plasmids were introduced into cells by Effectene transfection reagent (Qiagen) according to the manufacturer’s instructions. Full-length Dilp2 and Tequila (D isoform) cDNAs were subcloned into pENTR-D/TOPO (Invitrogen) and recombined into the destination vectors, pAWH (DGRC) and pUWR (DGRC), respectively, using LR Clonase (Invitrogen). Cell lysates were harvested 48 hours after transfection. For Western blot analysis, fly tissues or S2 cells were lysed in radioimmunoprecipitation assay buffer (Thermo); proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes using standard procedures (20). The antibodies used were rabbit anti-phospho-Akt (Ser505, 1:1000, Abcam), mouse anti-tubulin (1:1000, Developmental Studies Hybridoma Bank), and rabbit anti-hemagglutinin (HA) (1:4000, Bethyl). Protein signals were visualized with horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence reagent (Thermo).

Body Weight, Feeding, and Fecundity Measurements

For body weight measurements, 10 male or female 5-day-old flies were anesthetized (CO₂) and weighed immediately, using a microbalance (Sartorius). In feeding assays, 10-day-old flies maintained on high calorie food were transferred to fresh vials of the same food, with addition of 0.5% FD&C no. 1 blue food dye. After 6 hours, 10 flies were homogenized in a single tube containing PBS, and the amount of ingested dye was determined by spectrophotometer for dye absorbance at 620nm. Female fecundity was determined by daily counting of eggs produced by three mating pairs. Flies were passed daily to new vials containing appropriate food, and the number of eggs laid was counted and recorded for the first 20 days of adult life.

Circulating Dilp2 and Oral Glucose Tolerance Test

Hemolymph Dilp2 level was quantified following our recently developed enzyme immunoassay (EIA) (21). Briefly, about 0.5 µL of hemolymph was collected from 15 decapitated female flies (5-day-old). Hemolymph was diluted in PBS and incubated overnight in a 96-well EIA plate (Corning Incorporated). After the incubation with anti-DILP2 antibody (1:2500) and a horseradish peroxidase–conjugated secondary antibody (1:2500), the plate was developed by adding TMB solution (3,39,5,59-teramethylbenzidine; American Qualex antibodies) and incubated for 15 minutes at room temperature. The reaction was stopped by addition of 1M phosphoric acid, and the absorbance was recorded at 450nm using a plate reader. For the glucose tolerance test, female flies were fasted for 16 hours on 2% agar before being transferred to vials containing 10% dextrose-soaked filters for 1 hour. Flies were then transferred to vials containing water-soaked filters for 30 and 60 minutes, respectively. A total of 30 flies were collected in a microcentrifuge tube and centrifuged at 5000g for 10 minutes to obtain the hemolymph. The amount of circulating glucose was measured using a glucose assay kit (Sigma).

Starvation Challenge and Paraquat-Induced Oxidative Stress

Flies that were 10-day-old were transferred to vials containing either 2% agar (starvation) or filter paper soaked in a solution of 20mM paraquat and 6% dextrose. The number of dead flies was counted every 3–4 hours.

Statistics

All data are expressed as mean ± SEM. Survival curves were analyzed by the Kaplan–Meier procedure and log-rank test. Data for all other assays were analyzed using Student’s t test.

Results

Tequila Mutants Are Long-Lived Flies

To test whether any alteration in Tequila gene expression affects life span in flies, we obtained a mutant fly line carrying a hypomorphic mutation of Tequila (Teq ^f01792) (15). The Teq ^f01792 flies were backcross to w ¹¹¹⁸ flies for at least five generations for genetic background isogenization, and we further confirmed that Teq ^f01792 homozygous mutant flies showed reduced Tequila mRNA expression (Figure 1A). The physical appearance of Teq ^f01792 flies was smaller, with reduced body weight in both male and female flies (Figure 1D and E). The life span of the mutant flies was examined in a 1-L demographic cage, and we found that both male and female homozygous Teq ^f01792 flies displayed significant life-span extension compared with genetically matched w ¹¹¹⁸ control flies (Figure 1B and C and Table 1). The homozygous Teq ^f01792 mutation did not show noticeable trade-offs for longer life span that are commonly seen in other long-lived mutant flies, such as decreased female fecundity and feeding behavior (Figure 1F and G).

Table 1.

The Effect of Tequila on Drosophila Life Span (LS)

Gender	Strain	Number	Mean LS (day/hr)	Extension (%)
Male	w 1118	424	35.7
	Teq f01792	199	47.4	32.8*
Female	w 1118	430	33.3
	Teq f01792	399	43.1	29.1*
Male	Elav-Gal4>+	149	47.5
	+>UAS-Teq RNAi	216	52
	Elav-Gal4>UAS-Teq RNAi	197	60.7	21.7*/14.3*
Female	Elav-Gal4>+	152	45.2
	+>UAS-Teq RNAi	217	47.3
	Elav-Gal4>UAS-Teq RNAi	215	55.1	18.0*/14.2*
Male	Elav-GS> UAS-Teq RNAi (RU−)	239	43.9
	Elav-GS >UAS-Teq RNAi (RU+)	208	57.9	31.9*
Female	Elav-GS> UAS-Teq RNAi (RU−)	165	40.5
	Elav-GS >UAS-Teq RNAi (RU+)	215	58.4	44.2*
Male	PPL-Gal4>+	288	44.2
	+>UAS-Teq RNAi	315	45.4
	PPL-Gal4>UAS-Teq RNAi	403	36.8	−20.1*/−23.4*
Female	PPL-Gal4>+	332	46.8
	+>UAS-Teq RNAi	415	44.9
	PPL-Gal4>UAS-Teq RNAi	394	32	−46.3*/−40.3*
Male	Dilp2-Gal4>+	195	42.4
	+>UAS-Teq RNAi	215	45.8
	Dilp2-Gal4>UAS-Teq RNAi	208	59.3	28.5*/22.8*
Female	Dilp2-Gal4>+	169	40.2
	+>UAS-Teq RNAi	216	37.5
	Dilp2-Gal4>UAS-Teq RNAi	215	59.3	32.2*/36.8*
Male	c739-Gal4>+	218	50.3
	+>UAS-Teq RNAi	317	50.4
	c739-Gal4>UAS-Teq RNAi	220	49.8	−1.0/−1.2
Female	c739-Gal4>+	224	48.7
	+>UAS-Teq RNAi	218	47.4
	c739-Gal4>UAS-Teq RNAi	220	48.4	−0.6/−2.1
Male	MB-GS> UAS-Teq RNAi (RU-)	211	55.3
	MB-GS >UAS-Teq RNAi (RU+)	208	55.9	1.1
Female	MB-GS> UAS-Teq RNAi (RU−)	157	68.5
	MB-GS >UAS-Teq RNAi (RU+)	213	70.1	2.3
Male	Dilp2>+	184	54.7
	Dilp2>Teq	190	55.2	0.9
Female	Dilp2>+	171	56
	Dilp2>Teq	213	51.4	−8.2*
Starvation
Male	w 1118	99	41.6 hr
	Teq f01792	98	54.6 hr	31.3*
Female	w 1118	100	71.1 hr
	Teq f01792	100	78.5 hr	10.4*
Oxidative stress
Male	w 1118	49	20.9 hr
	Teq f01792	50	27.9 hr	33.5*
Female	w 1118	49	22.5 hr
	Teq f01792	50	24.5 hr	8.9

*p Value <0.01 compared with genetic matched control by log-rank test.

Long-lived mutants are generally more stress resistant than their wild-type counterparts (22–24). In agreement with this, we found that both male and female Teq ^f01792 homozygous mutant flies were more resistant to starvation-induced mortality compared with w ¹¹¹⁸ flies (Figure 2A and B and Table 1). Male Teq ^f01792 homozygous mutants were also resistant to paraquat-induced oxidative stress, but female mutant flies showed only a trend of increased survival that did not reach statistical significance (Figure 2C and D and Table 1).

Figure 2.

Tequila mutation induces stress resistance. (A and B) Enhanced survival of male and female Teq ^f01792 homozygous mutant flies (blue) on starvation challenge compared with control flies (w ¹¹¹⁸, red). (C) Enhanced survival of male Teq ^f01792 homozygous mutant flies (blue) on paraquat-induced oxidative stress challenge compared with control flies (w ¹¹¹⁸, red). (D) No difference in survival is observed between female control (w ¹¹¹⁸, red) and Teq ^f01792 homozygous mutant flies (blue) on paraquat-induced oxidative stress. Statistical analysis is shown in Table 1.

Tequila-Induced Life-Span Extension Is Associated With Insulin-Producing Neurons

Tequila is known to be expressed in Drosophila brain and fat body, which are involved in cognitive and immune regulation, respectively (13,15). In order to identify which tissue might mediate the Tequila deficiency–induced life-span extension, we employed the Gal4/UAS binary system to specifically knockdown Tequila mRNA expression in either brain or fat body. Flies carrying a normal allele (+) and either a constitutive pan-neuronal driver (Elav-Gal4), an inducible pan-neuronal driver (Elav-GS), or a constitutive fat body–specific driver (PPL-Gal4), were crossed to UAS-Tequila RNAi (TeqRNAi) flies, and life spans of the progeny were analyzed. We found that constitutive Tequila inactivation in neurons extended the life span of both male and female flies (Figure 3A and B and Table 1). Life-span extensions were also observed in both male and female mutant flies carrying Elav-GS and UAS-Tequila RNAi when food was supplemented with RU486 after eclosion (Figure 3C and D and Table 1). Thus, adult neuronal inactivation of Tequila is essential for the observed increased longevity in flies. Constitutive Tequila inactivation in fat body, on the other hand, shortened the life span of both male and female flies (Figure 3E and F and Table 1).

Figure 3.

Neuron-specific inactivation of Tequila induces life-span extension. (A and B) Extended life spans in male (A) and female (B) mutant flies with constitutive, neuron-specific knockdown of Tequila (Elav-Gal4>TeqRNAi, blue) compared with control flies (+>TeqRNAi, green; Elav-Gal4>+, red). (C and D) Extended life spans of male (C) and female (D) mutant flies carrying the RU486-inducible driver for neuron-specific knockdown of Tequila (Elav-GS>TeqRNAi; RU+, blue) compared with uninduced control flies (RU-, red). RU486 was fed to adult flies throughout the life span. (E and F) Shortened life spans in male (E) and female (F) mutant flies with constitutive, fat body knockdown of Tequila (PPL-Gal4>TeqRNAi, blue) compared with control flies (+>TeqRNAi, green; PPL-Gal4>+, red). Statistical analysis of life spans is shown in Table 1.

We further investigated which neuronal cell type might be involved in Tequila-induced life-span extension. We first used constitutive (c739-Gal4) and inducible (MB-GS) mushroom body drivers to reduce Tequila expression, because Tequila mRNA and protein have been shown to be expressed in the mushroom body following LTM conditioning (15). However, we found no effect on the life span of mutant flies carrying Tequila knockdown in LTM-related mushroom body (Figure 4A–D and Table 1). Because manipulation of insulin-producing neurons in Drosophila brain affects the life span of flies (25,26), we examined the expression pattern of Tequila in the insulin-producing neurons of Drosophila brain. Using immunohistochemistry, we found that anti-Tequila immnuoreactivity was indeed associated with insulin-producing neurons (Figure 4E–H). We also found that knockdown of Tequila mRNA expression specifically in the insulin-producing neurons (Dilp2>TeqRNAi) induced significant life-span extension in both male and female flies (Figure 4I and J and Table 1). These data suggest that Tequila-induced life-span extension is associated with insulin-producing neurons.

Figure 4.

Insulin-producing neurons, but not mushroom body, mediate Tequila-induced life-span extension. (A and B) No life-span extension is seen in male (A) and female (B) mutant flies carrying constitutive, mushroom body knockdown of Tequila (c739-Gal4>TeqRNAi, blue) compared with control flies (+>TeqRNAi, green; c739-Gal4>+, red). (C and D) No life-span extension is seen in male (C) and female (D) mutant flies carrying the RU486-inducible driver for mushroom body knockdown of Tequila (MB-GS>TeqRNAi; RU+, blue) compared with uninduced control flies (RU-, red). RU486 was fed to adult flies throughout the life span. (E–H) Confocal images showing that Tequila immunoreactivity (anti-Teq, red) colocalizes with the insulin-producing neurons (green). The insulin-producing neurons are labeled by green fluorescence (Dilp2>GFP). Cell nuclei are labeled with 4′,6-diamidino-2-phenylindole (blue). Scale bars = 5 µm. (I and J) Extended life spans of male (I) and female (J) mutant flies carrying constitutive, insulin-producing neuron knockdown of Tequila (Dilp2>TeqRNAi; blue) compared with control flies (+>TeqRNAi, green; Dilp2>+, red). Statistical analysis of life spans is shown in Table 1.

Tequila Regulates Insulin-Like Signaling

To investigate the functional relationship of Tequila and insulin-like peptides, Drosophila S2 cells were transfected with pAWH-Dilp2-HA and/or pUWR-Tequila-red fluorescent protein constructs (Figure 5A). Successfully transfected cells were identified by colocalization of red fluorescent protein and/or anti-hemagglutinin immunoreactivity (Figure 5B–E). We subsequently lysed and harvested the cells and found that there were elevated levels of smaller Dilp2 fragments in cells that had been cotransfected with both pAWH-Dilp2-HA and pUWR-Tequila-red fluorescent protein constructs in Western blot analysis, implying that Dilp2 might act as substrate for the peptidase activity of Tequila in transfected Drosophila S2 cells (Figure 5F). We postulated that this putative peptidase digestion process might be essential for activation of the Dilps, because both circulating Dilp2 and Akt phosphorylation, a downstream molecular signature of insulin signaling, are reduced in Teq ^f01792 homozygous mutant flies (Figure 5G–I). Moreover, we also tested the glucose homeostasis of flies in an oral glucose tolerance test. In control (w ¹¹¹⁸) flies, typical glucose clearance kinetics were observed when the flies were subjected to fasting and subsequent addition of glucose solutions to food (Figure 5J). Teq ^f01792 homozygous mutant flies showed slower glucose clearance kinetics, indicated by much higher circulating glucose levels even at 60 minutes after glucose injection (Figure 5J).

Figure 5.

Tequila regulates insulin-like signaling. (A–E) Drosophila S2 cells transfected with pAWH-Dilp2-HA (green) and/or pUWR-Tequila-red fluorescent protein (red) constructs can be observed under a fluorescence microscope. Cell nuclei are labeled with 4′,6-diamidino-2-phenylindole (blue). Scale bars = 5 µm. (F) Increased smaller molecular weight Dilp2 fragments were detected in cell lysates of Tequila and Dilp2 cotransfected S2 cells using an anti-hemagglutinin antibody in Western blot analysis. Anti-α-tubulin (Tub) is shown as an internal control. (G) Reduced circulating Dilp2 were detected in the hemolymph of Teq ^f01792 homozygous mutant flies (blue) compared with control flies (w ¹¹¹⁸, red). Experiments were done in eight replicates, and each replicate contained 15 flies for each group. (H and I) Akt phosphorylation (anti-p-Akt) is decreased in Teq ^f01792 homozygous mutant flies (blue) compared with control flies (w ¹¹¹⁸, red) in Western blot analysis. Experiments were done in triplicate, and each replicate contained 15 flies for each group. (J) An oral glucose tolerance test shows that Teq ^f01792 homozygous mutant flies have abnormal kinetics for glucose clearance. Experiments were done in triplicate, and each replicate contained 30 flies for each group. (K and L) Akt phosphorylation (anti-p-Akt) is increased in mutant flies carrying constitutive, insulin-producing neuron overexpression of Tequila (Dilp2>Teq, blue) compared with control flies (+>Teq or Dilp2>+, red) in Western blot analysis. Experiments were done in triplicate, and each replicate contained 15 flies for each group. (M and N) Normal life span in male (M) and slightly shortened life span in female (N) mutant flies carrying constitutive, insulin-producing neuron overexpression of Tequila (Dilp2>Teq, blue) compared with control flies (Dilp2>+, red). Statistical analysis of life spans is shown in Table 1.

If Tequila is required for Dilp2 maturation, we would expect that Tequila overexpression might increase insulin-like signaling. We overexpressed Tequila in insulin-producing neurons (Dilp2>Teq) and found that Akt phosphorylation was significantly increased in these mutant flies compared with control flies (Figure 5K and L). However, Tequila overexpression in insulin-producing neurons (Dilp2>Teq) only slightly reduced life span in female flies and had no effect on male flies (Figure 5M and N). It is possible that both the control and mutant flies in our experimental setting already sustained a high level of insulin-like signaling, in which case, enhanced Tequila expression would not necessarily have a large influence on Drosophila life span.

Discussion

This study was undertaken with the goal of identifying novel longevity genes, and as one of these genes, Tequila was evaluated for its potential to regulate longevity. Despite its action in LTM formation, and possibly in the immune response (13–15), relatively little is known about the biological function of Tequila in aging. The observation that Tequila inactivation induced life-span extension was verified by a number of different genetic approaches. We showed that a hypomorphic mutation of Tequila (Teq ^f01792) was capable of inducing life-span extension. Moreover, cell-specific RNAi expression confirmed that reduction of Tequila expression in insulin-producing neurons could function to extend life span. Thus, the insulin/IGF-signaling pathway appears to be critical for Tequila-mediated life-span extension.

In mammalian cells, maturation of insulin first requires conversion of preproinsulin to proinsulin by removal of an N-terminal signal peptide. Proinsulin then undergoes proteolytic processing during protein secretion to become the bioactive insulin hormone (27). Although the maturation process of Dilps remains unknown, analysis of their amino acid sequence predicts several likely cleavage sites (4). Because Tequila is recognized as a neurotrypsin ortholog, we examined the possibility that Dilps might be a substrate for the putative protease activity of Tequila. Our data provide some evidence supporting this possibility, demonstrated by (i) an increase in smaller Dilp2 fragments in transfected Drosophila S2 cells, (ii) reduced circulating Dilp2, (iii) altered Akt phosphorylation, and (iv) disrupted glucose homeostasis in Tequila mutant flies. Although we do not currently have evidence of the specific cleavage sites or for bioactivity of the processed Dilp2, our data are suggestive that Tequila could be involved in activation of Dilp2-mediated signaling.

In contrast to the action of Tequila in the insulin-producing neurons, fat body–specific inactivation of Tequila resulted in reduced life span in flies. One possible explanation for this is that Tequila inactivation in fat body may cause altered Dilp6-mediated signaling, and Dilp6 has been shown to regulate life span by repressing Dilp2 expression and secretion from the insulin-producing neurons of Drosophila brain (21). Our future studies will include further examination of Dilp6 expression or secretion from fat body in Tequila mutant flies. The proposed function of Tequila may also be involved in immune response (13), and chronic fat body–specific upregulation of immune response has been shown to induce enhanced pathogen resistance but reduced life span (28). Although it is unclear at what level Tequila may be involved in immune regulation in flies, the amino acid sequence and functional analysis of Sp22D protein, a Tequila ortholog in the mosquito Anopheles, suggest that it may serve as a signaling molecule upon pathogen intrusion or wounding in insects (29). These observations support the hypothesis that Tequila may have multiple functions in different biological contexts.

It is well established that metabolic trade-offs can modulate the life history evolution of growth, reproduction, and somatic maintenance. Analysis of mutant flies having reduced insulin/IGF-like signaling has identified several common phenotypes that include growth retardation, reduced fecundity, altered metabolism, increased resistance to starvation and oxidative stress, and extended life span (4,7,25). Tequila mutant flies display several of these characteristic phenotypes, but we did not find any reduction in female fecundity. Thus, we are not able to demonstrate a strong connection linking Tequila between insulin/IGF-like signaling and reproductive regulation. Testing under multiple food conditions will be needed in order to establish the contribution of Tequila to fly reproductive regulation, because fly fecundity is known to fluctuate significantly in order to permit Drosophila to adapt to varying nutritional status.

In summary, our studies have identified Tequila as a novel longevity-regulating gene in Drosophila. We have shown that Tequila has the potential to act as a modulator of fly insulin/IGF signaling, possibly through proteolytic processing of Dilps, and that it can regulate life span.

Supplementary Material

Supplementary figure can be found at: http://biomedgerontology.oxfordjournals.org/

Funding

This study was supported by grants from the Ministry of Science and Technology, Taiwan (102-2311-B-007-008-MY3 to Dr. H.-D.W.), (100-2311-B-002-017-MY3 and 103-2321-B-002-093 to Dr. P.-Y.W.), and the National Taiwan University Hospital (UN102-029 and UN103-055) to Drs. M.-S.W. and P.-Y.W.