Human IGF1 extends lifespan and enhances resistance to Plasmodium falciparum infection in the malaria vector Anopheles stephensi

Abstract

The highly conserved insulin/insulin-like growth factor (IGF) signaling (IIS) pathway regulates metabolism, development, lifespan and immunity across a wide range of organisms. Previous studies have shown that human insulin ingested in the blood meal can activate mosquito IIS, resulting in attenuated lifespan and increased malaria parasite infection. Because human IGF1 is present at higher concentrations in blood than insulin and is functionally linked with lifespan and immune processes, we predicted that human IGF1 ingested in a blood meal would affect lifespan and malaria parasite infection in the mosquito Anopheles stephensi. Here we demonstrate that physiological levels of ingested IGF1, like insulin, can persist intact in the blood-filled midgut for up to 30 h and disseminate into the mosquito body, and that both peptides activate IIS in mosquito cells and midgut. At these same levels, ingested IGF1 alone extended average mosquito lifespan by 23% compared with controls and, more significantly, when ingested in infected blood meals, reduced the prevalence of Plasmodium falciparum-infected mosquitoes by >20% and parasite load by 35-50% compared with controls. Thus, the effects of ingested IGF1 on mosquito lifespan and immunity are opposite to those of ingested insulin. These results offer the first evidence that insect cells can functionally discriminate between mammalian insulin and IGF1. Further, in light of previous success in genetically targeting IIS to alter mosquito lifespan and malaria parasite transmission, this study indicates that a more complete understanding of the IIS-activating ligands in blood can be used to optimize transgenic strategies for malaria control.

Fig. 1.

Stimulation of immortalized Anopheles stephensi embryo-derived (ASE) cells with human insulin-like growth factor 1 (IGF1) induces phosphorylation of ERK, Akt and FOXO. ASE cells were treated with diluent buffer (0.1% BSA/PBS) as a treatment control or with 0.013 μmol l⁻¹ IGF1 (0.1 μg ml⁻¹) or 0.133 μmol l⁻¹ IGF1 (1.0 μg ml⁻¹) for 5 min. (A) Representative western blot showing phosphorylation of signaling proteins. (B–D) Mean ± s.e.m. fold induction of protein levels normalized first to the GAPDH loading control and then to the treatment control: (B) pAkt, (C) pFOXO and (D) pERK. Data were analyzed by ANOVA for overall significance followed by Bonferroni post-test for pairwise comparisons (α=0.05). Experiments were replicated five to seven times and P-values are noted on the graphs.

Fig. 2.

Human insulin and IGF1 persist intact in the midgut during blood digestion and disperse into the body of A. stephensi. (A,C) Representative autoradiographs of abdomen (A, 4 day exposure; C, 0.75 day exposure) and head/thorax extract samples (A, 28 day exposure; C, 5 day exposure) processed 0 to 48 h post blood meal (PBM) containing radiolabeled insulin (A, 5.9×10⁻⁴ μmol l⁻¹) or IGF1 (C, 0.133 μmol l⁻¹). Three body part equivalents were loaded per lane. Insulin is the 3.8 kDa molecular weight marker (MW) in the Kaleidoscope Polypeptide Standards (BioRad) loaded in an adjacent lane. (B,D) Mean (±s.e.m.) amount of radiolabeled insulin (B) or IGF1 (D) per A. stephensi body part (abdomen or head/thorax) from three cohorts of experimental females.

Fig. 3.

Blood ingestion induces rapid phosphorylation of the insulin receptor in the A. stephensi midgut. Mosquitoes were fed washed red blood cells (RBCs) alone or supplemented with 1.7×10⁻⁴ μmol l⁻¹ insulin or IGF1 at 0.013 or 0.133 μmol l⁻¹. Midguts were dissected from non-RBC-fed females (NF; shown with the 0.5 h groups) and RBC-fed females at 0.5, 1 and 3 h post blood meal (PBM) and processed for electrophoresis and western blotting. (A) Representative blots showing the phosphorylated mosquito insulin receptor (pMIR, arrows) in midgut samples from females treated as above. pMIR was detected as a single ~500 kDa immunoreactive protein. (B) Mean fold increase in pMIR in midgut samples for each treatment group at each time point relative to the non-RBC-fed group. Data were analyzed by ANOVA followed by Bonferroni multiple comparison tests for pairwise comparisons of means (α=0.05). Experiments were replicated five times with separate cohorts of mosquitoes.

Fig. 4.

Provision of human IGF1 in the blood meal induces phosphorylation of FOXO and p70S6K and inhibits phosphorylation of ERK in the A. stephensi midgut. Mosquitoes were fed blood meals containing 0.013 or 0.133 mol l⁻¹ IGF1 or an equivalent volume of diluent buffer (0.1% BSA/PBS). Midguts were dissected at 0.5–1 h after the initiation of blood feeding. Data are represented as the mean ± s.e.m. fold induction of phospho-protein levels for p-FOXO (A), p-p70S6K (B) and p-ERK (C) quantified by densitometry and normalized first to the GAPDH loading control and then to the buffer control. Normally distributed data were analyzed by ANOVA for overall significance followed by Bonferroni multiple comparison tests for pairwise comparison of means (ERK; α=0.05). Non-normally distributed data were analyzed by Friedman's test for overall significance followed by Dunn's multiple comparison tests for pairwise comparison of means (FOXO and p70S6K; α=0.05). Experiments were replicated five times and P-values are displayed on the graphs.

Fig. 5.

Survivorship curves from Experiment 3 showing the effects of IGF1 on A. stephensi lifespan. Low levels of human IGF1 (0.013 μmol l⁻¹ IGF1) extend A. stephensi lifespan, while survivorship of mosquitoes treated with higher levels of IGF1 (0.133 μmol l⁻¹ IGF1) was not different from that of buffer-fed (0.1% BSA/PBS) controls. For these studies, 3- to 5-day-old A. stephensi females were fed weekly blood meals supplemented with physiologically low (0.013 μmol l⁻¹) or high (0.133 μmol l⁻¹) IGF1 or the IGF1 diluent buffer (0.1% BSA/PBS). A total of 300 mosquitoes were used per treatment and experiments were replicated three times with separate cohorts of mosquitoes.

Fig. 6.

Provision of human IGF1 in an infected blood meal reduces Plasmodium falciparum development in A. stephensi. Mosquitoes were fed blood meals containing malaria parasites and diluent buffer (0.1% BSA/PBS) as a control treatment, or an identical blood meal with 1.33 nmol l⁻¹ to 0.133 μmol l⁻¹ IGF1. Midguts were dissected and oocysts were counted at 10 days following infection. (A) IGF1 treatment decreased the number of oocysts per midgut relative to controls. This experiment was replicated four times with separate cohorts of mosquitoes and analyzed by ANOVA to determine whether the oocyst intensity in the controls differed among replicates. No differences were found, so the data were pooled across replicates and analyzed by Kruskal–Wallis to test for overall significance and Dunn's post-test for pairwise comparisons of means. (B) IGF1 treatment decreased the prevalence of malaria infection, i.e. the percent of midguts with at least one oocyst out of the total number of midguts dissected. Differences between groups were analyzed using Fisher's exact test and P-values are noted on the graph. (C) Human IGF1 did not affect growth of asexual-stage P. falciparum. Replicate cultures of P. falciparum NF54 were incubated with increasing concentrations of human IGF1. Relative growth is compared with the diluent buffer control, which is set at 100%. Data from three independent experiments were analyzed by ANOVA and by Bonferroni multiple comparison tests for pairwise comparison of means (α=0.05). No significant differences among treatment groups and controls were observed.