Fig 2: IGF-1 neutralizing antibody or IGF-1 targeting siRNAs reduced IGF-1Rß and Akt phosphorylation and diminish neurite outgrowth. DRG neurons from control rats were cultured, treated with different doses of IGF-1 neutralizing antibody and underwent A, B Western blotting for Akt phosphorylation and IGF-1Rß or C, D neurite outgrowth measurement. In A, B, total protein bands were used for normalization. In C, D, normal goat IgG was used as a control antibody. In E–G, DRG neurons from control rats were cultured, treated with different doses of two IGF-1 targeting siRNAs and underwent E real-time PCR assay for IGF-1 or F, G neurite outgrowth measurement. In E, four transcript variants (a pair of primers designed for the detection of two variants 1 and 2 or 3 and 4) of IGF-1 that produce protein were analyzed. In F, G, exogenous hIGF-1 was added alone or along with IGF-1 knock-down as control groups. Data are mean ± SEM of N = 4; *p < 0.05 or **p < 0.01 or ****p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s post hoc test

Fig 3: NFAT1 and CEBPß transcription factors activated IGF-1 gene expression and were less enriched at the Igf1 promoter in DRG tissue derived from diabetic rats. About A 1.2 kb of IGF-1 gene promoter region was chosen to design ChIP assay. Five regions were used for amplification and each pair of primers was designed to include one transcription factor-binding site. In B, DRG tissues derived from adult control and STZ-diabetic rats underwent ChIP assay using NFAT1 and CEBPß antibodies for pull down followed by IGF-1 promoter region (five regions in total stats for three are shown) amplification using ChIP-qRT-PCR analysis. In C, diagram of the mutated transcription factor (NFAT1 and CEBPß) binding sites on promoter region of Igf1 gene is given. A total of 4 binding sites (3a for NFAT1-binding site 3, 3b for CEBPß-binding site 3, 5 for NFAT1- and CEBPß-binding site 5) from (A) were mutated. In D, luciferase activity of the mutated and wild type IGF-1 promoter was measured in HEK293 cells. In E, DRG neurons from STZ-diabetic (Db) rats were transfected with NFAT1, CEBPß or both, and different transcript variants (Tv1,2,3,4) of IGF-1 were measured using qRT-PCR. Data are mean ± SEM of N = 4–5 animals or N = 4–6 culture groups; *p < 0.05 or **p < 0.01 or ****p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Tukey’s post hoc test

Fig 4: IGF-1-overexpressing plasmid enhanced glycolysis, mitochondrial respiration, and neurite outgrowth. DRG neurons from control rats were cultured, transfected with different doses of hIGF-1 (transcript variant 4)-overexpressing plasmid and 200 ng control GFP plasmid (ctrl). In A, B, mitochondrial OCR was measured in live neurons after 6 h. In C–F, glycolysis parameters and total neurite outgrowth were calculated. Total protein in mg was used to normalize OCR and ECAR data. Data are mean ± SEM of N = 4–6; *p < 0.05 or **p < 0.01 or ***p < 0.001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s post hoc test

Fig 5: The level of endogenous IGF-1 was reduced in DRG tissue from type 1 and type 2 diabetic rodents, in part, via hyperglycemia-activated polyol pathway activity and restored by exogenous hIGF-1. In A–D DRG tissues from control (Ctrl), hIGF-1-treated (STZ-Db + hIGF-1), untreated STZ-diabetic (STZ-Db), Zucker diabetic fatty (ZDF) rats and db/db mice were homogenized and underwent A qRT-PCR or B–D ELISA for IGF-1 detection. In E, DRG neurons derived from Ctrl and STZ-Db rats were cultured and media was collected after 2 days to measure secreted IGF-1 protein. DRG neurons derived from control (F, G, H) or diabetic (I) rats were cultured in the presence of 25 mM glucose with/without insulin or hIGF-1 treatment or in the presence of 5 mM glucose. RNA was extracted and utilized for real-time PCR assay. In G, 25 mM mannitol was used to control for osmotic pressure compared with 25 mM D-glucose. In H, sorbinil (10 and 100 µM), an aldose reductase inhibitor (ARI), was applied. Data are mean ± SEM of N = 3–6; *p < 0.05 or **p < 0.01 or ****p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s or Tukey’s post hoc test