Fig. 7: The role of PDK2 and lactic acid in regulating the AMPK signaling and food intake-related neuropeptide circuitry in diabetic mouse hypothalamus.
p-AMPK (#p = 0.0157, KO + STZ) and AMPK protein levels in the hypothalamic tissues isolated from WT and Pdk2 KO mice at 3 w post-STZ/vehicle injection were assessed by Western blot analysis (a). Co-immunofluorescence staining of mouse brain tissues showed p-AMPK and βIII-tubulin-positive neurons (###p = 1.4E-7, not co-localized with p-AMPK; and (###p = 3.7E-7, co-localized with p-AMPK) in the ARC at 3 w post-STZ/vehicle injection (b). The percentage of p-AMPK-positive neurons was calculated from the total number of cells present per mm2 using six randomly selected fields. Scale bar indicates 100 µm. The expression of p-AMPK (**p = 0.0051, lactic acid) and AMPK proteins (c), as well as Agrp (d) in mHypoE-N41 cells, and Pomc mRNA in mHypoE-N43/5 cells (e) after treating with lactic acid (7.1 mM) for 72 h was assessed by Western blot and real-time RT-PCR, respectively. The effect of Compound C (16 µM) and metformin (100 µM) on lactic acid-induced increased expression of Agrp mRNA (##p = 0.0011, lactic acid+ComC) in mHypoE-N41 cells was observed (d). Results for mRNA expression are displayed as the fold increase of gene expression normalized to Gapdh. The diagram shows the proposed lactic acid-AMPK axis in the regulation of the AgRP (f). Representative traces of INMDA induced by NMDA (100 μM) in the absence or presence of l-lactate (7.1 mM) in NPY/AgRP-expressing neurons (left) and POMC-expressing neurons (right) (g). l-lactate-induced changes of the NMDA-induced currents in NPY and POMC neurons (##p = 0.0006, POMC) (h). Representative traces of sEPSCNMDA in the absence (left) or presence (right) of l-lactate in NPY neurons (i). Cumulative probability distribution for inter-event interval (left) and amplitude (right) of sEPSCNMDA with or without l-lactate (j). The plot includes 195 events for control (thin lines) and 143 events for l-lactate (thick lines). Representative traces before, during, and after the application of NMDA in the absence (left) or presence (right) of l-lactate in NPY neurons in the current-clamp condition (k). NMDA-induced changes of action potential frequency in the absence or presence of l-lactate in NPY neurons (*p = 0.0220, NMDA + l-lactate) (l). Open and closed circles represent the value from individual neurons and their average respectively. Insets represent the l-lactate-induced changes in the frequency (left) and amplitude (right) of sEPSCNMDA. Dotted lines represent the relative control of basal frequency and amplitude of sEPSCNMDA. *p < 0.05, **p < 0.01, or ***p < 0.001 versus the vehicle-treated control animals/cells. Two-way ANOVA (a, b), one-way ANOVA with Tukey’s post hoc test (d, h), two-tailed Student’s t-test (c, e), two-tailed paired t-test (h, j, l), unpaired t-test, n = 9 individual neurons (h), n = 195 events for control and 143 for l-lactate (j), n = 6 individual neurons (l) n = 3 animals per group (a) or sister wells (c–e), and n = 5 animals per group (b); mean ± SEM. Source data are provided as a Source data file. ComC Compound C; Met metformin; L-lac l-lactate; Amp Amplitude; Freq Frequency; NC negative control; ns not significant.
