Fig. 8: Effects of astrocyte-specific Cre-mediated Pdk2 gene ablation on diabetes-induced hypothalamic inflammation and changes in feeding behavior.
To determine the role of hypothalamic PDK2 in the regulation of neuroinflammation and feeding behavior, we administered AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP (4.3 × 109 virus particles) into the mediobasal hypothalamus of Pdk2 floxed mice, bilaterally, using a stereotaxic device 1 w before STZ injection. The schematic diagram presents the experiment timeline and route of administration (a). The relative expression of Tnf-α (#p = 0.0141, AAV5-GFAP-mCherry-Cre+STZ), IL-1β (#p = 0.0316, AAV5-GFAP-mCherry-Cre+STZ), and IL-6 (##p = 0.0016, AAV5-GFAP-mCherry-Cre+STZ) mRNA in the hypothalamic tissues isolated from AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP and STZ-injected mice was evaluated by real-time RT-PCR (b). GFAP (##p = 0.0020, AAV5-GFAP-mCherry-Cre + STZ) and Iba-1 (##p = 0.0053, AAV5-GFAP-mCherry-Cre+STZ) immunofluorescence staining revealed their increased immunoreactivity in the hypothalamus of diabetes Pdk2 floxed mice, but Pdk2-deficiency in hypothalamic astrocytes attenuated such an increase in the immunoreactivities at 3 w post-STZ injection (c). Microscope data were gathered using five randomly selected fields captured at the same magnification. Scale bar indicates 200 µm. Lactate concentration (##p = 0.0030, AAV5-GFAP-mCherry-Cre+STZ) in hypothalamic tissues collected from AAV5-GFAP-mCherry-Cre- or AAV5-GFAP-eYFP- and STZ-injected mice was measured with an HPLC analyzer (d). Quantification of the lactate concentration from HPLC graphs is shown. Food intake (*p = 0.0393, AAV5-GFAP-mCherry-Cre+STZ at 4 w) was assessed following AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP and STZ administration (e). Arrows indicate the time points of adeno-associated virus (AAV), and STZ administration. The relative expression of Npy (#p = 0.0140, AAV5-GFAP-mCherry-Cre+STZ), Agrp (#p = 0.0380, AAV5-GFAP-mCherry-Cre+STZ) and Pomc (##p = 0.0090, AAV5-GFAP-mCherry-Cre + STZ) mRNA in hypothalamic tissues isolated from AAV5-GFAP-mCherry-Cre- or AAV5-GFAP-eYFP- and STZ-injected mice just after completion of behavioral assessments was evaluated by real-time RT-PCR (f). mRNA expression results are displayed as the fold increase of mRNA expression normalized to Gapdh. *p < 0.05, **p < 0.01, or ***p < 0.001 versus the vehicle-treated control animals (b–d, f) or STZ/AAV5-GFAP-eYFP+STZ-treated animals (e); #p < 0.05, ##p < 0.01, or ###p < 0.001 versus indicated groups. One-way ANOVA (b–d, f) and two-way ANOVA (e) with Tukey’s post hoc test, n = 3 (b, d, f), n = 5 (c), and n = 6 (e); mean ± SEM. Value of “n” indicates the number of animals. Source data are provided as a Source data file. w week(s); IR immunoreactivity.
