Diazepam Binding Inhibitor Control of Eu- and Hypoglycemic Patterns of Ventromedial Hypothalamic Nucleus Glucose-Regulatory Signaling

Figures & data

This is a visual representation of the abstract.

Figure 1 High-performance Capillary Electrophoresis-Electrospray Ionization-Quadrupole-Linear Ion Trap Mass Spectrometry (QTRAP CESI-MS/MS) Quantification of Diazepam Binding Inhibitor (Dbi) and Octadecaneuropeptide (ODN) in Ventromedial Hypothalamic Nucleus (VMN) Dorsomedial (VMNdm) versus Ventrolateral (VMNvl) Divisions. Groups of Dbi or scramble (SCR) siRNA-pretreated male rats (n = 3/group) were sacrificed by microwave fixation 1 h after subcutaneous (sc) injection of vehicle (V) or neutral protamine Hagedorn insulin (INS; 10.0 U/kg bw). VMNvl and VMNdm tissues were each bilaterally micropunch-dissected from 100 micron-thick fresh frozen sections cut from −1.80 to −2.30 mm posterior to bregma, using a hollow 0.50 mm-diameter punch tool, for CESI-QTRAP MS/MS analysis (see the Materials and Methods section). Data depict mean Dbi mRNA [Figures 1A (VMNdm) and 1D (VMNvl)], Dbi protein [Figures 1B (VMNdm) and 1E (VMNvl), or ODN protein [Figures 1C (VMNdm) and 1F (VMNvl)] measures ± S.E.M. following treatment groups: SCR siRNA/V (solid white bars, n = 3), Dbi siRNA/V (diagonal-striped white bars, n = 3), SCR siRNA/INS (solid gray bars, n = 3), and Dbi siRNA/INS (diagonal-striped gray bars, n = 3). Results of statistical analyses are as follows: VMNdm Dbi mRNA: F_(3,8): 45.65, p < 0.001; Knockdown main effect: F_(1,8): 34.62, p < 0.001; INS main effect: F_(1,8): 62.05, p < 0.001; Knockdown/INS interaction: F_(1,8): 40.27; p < 0.001; VMNvl Dbi mRNA: F_(3,8): 69.86, p < 0.001; Knockdown main effect: F_(1,8): 177.75, p < 0.001; INS main effect: F_(1,8): 18.45, p = 0.003; Knockdown/INS interaction: F_(1,8): 13.38; p = 0.006]; F_(3,8): 45.65, p < 0.001, Knockdown main effect: F_(1,8): 34.62, p < 0.001; INS main effect: F_(1,8): 62.05, p < 0.001; Knockdown/INS interaction: F_(1,8): 40.27; p < 0.001]; VMNdm Dbi protein: F_(3,8): 4.90, p = 0.032; Knockdown main effect: F_(1,8): 0.702, p = 0.426; INS main effect: F_(1,8):1.01, p = 0.344; Knockdown/INS interaction: F_(1,8): 12.98; p = 0.007]; VMNdm ODN protein: F_(3,8): 14.07, p = 0.001, Knockdown main effect: F_(1,8): 1.97, p = 0.198; INS main effect: F_(1,8): 9.45, p = 0.015; Knockdown/INS interaction: F_(1,8): 30.78, p < 0.001; VMNvl Dbi protein: F_(3,8): 13.15, p = 0.002, Knockdown main effect: F_(1,8): 1.97, p = 0.198; INS main effect: F_(1,8): 6.83, p = 0.031; Knockdown/INS interaction: F_(1,8): 30.66, p < 0.001; VMNvl ODN protein: F_(3,8): 37.87, p < 0.001; Knockdown main effect: F_(1,8): 22.29, p = 0.002; INS main effect: F_(1,8): 91.27, p < 001; Knockdown/INS interaction: F_(1,8): 0.06, p = 0.816]. Statistical differences between discrete pairs of treatment groups are denoted as follows: *p < 0.05; **p < 0.01; and ***p < 0.001.

Figure 2 Effects of Dbi Gene Knockdown on Eu- or Hypoglycemic Neurotransmitter and Metabolic Sensor Marker Gene Expression Patterns in VMNvl γ-Aminobutyric Acid (GABAergic) Neurons. Groups of male rats (n = 4/group) were pretreated by bilateral administration of Dbi or SCR siRNA to the VMN 7 days prior to sc V or INS injection. Individual VMNvl glutamate decarboxylase (Gad)_65/67-immunopositive neurons were laser-catapult-microdissected from 10 micron-thick fresh frozen sections for multiplex single-cell qPCR analyses. mRNA data were normalized to the housekeeping gene GAPDH by the 2^−ΔΔCt method. Data depict mean normalized Gad1/Gad₆₇ (Figure 2A), Gad2/Gad₆₅ (Figure 2B), glucokinase (Gck; Figure 2C), glucokinase-regulatory peptide (Gkrp; Figure 2D), 5’-AMP-activated protein kinase-alpha1 (AMPKα1/Prkaa1; Figure 2E), or AMPK-alpha2 (AMPKα2/Prkaa2; Figure 2F) mRNA measures ± S.E.M. for the following treatment groups: SCR siRNA/V (solid white bars, n = 8), Dbi siRNA/V (diagonal-striped white bars, n = 8), SCR siRNA/INS (solid gray bars, n = 8), and Dbi siRNA/INS (diagonal-striped gray bars, n = 8). Outcomes of statistical analyses are as follows: Gad1: F_(3,28): 115.48, p < 0.001; Knockdown main effect: F_(1,28): 58.82, p < 0.0001; INS main effect: F_(1,28): 155.34, p < 0.001; Knockdown/INS interaction: F_(1,28): 132.28, p < 0.001; Gad2: F_(3,28): 193.99, p < 0.001; Knockdown main effect: F_(1,28): 98.60, p < 0.0001; INS main effect: F_(1,28): 214.88, p < 0.001; Knockdown/INS interaction: F_(1,28): 268.50, p < 0.001; Gck: F_(3,28): 1425.23, p < 0.001; Knockdown main effect: F_(1,28): 497.17, p < 0.0001; INS main effect: F_(12,8): 2786.75, p < 0.001; Knockdown/INS interaction: F_(12,8): 991.77, p < 0.001; Gkrp: F_(3,28): 248.72, p < 0.001; Knockdown main effect: F_(1,28): 182.30, p < 0.0001; INS main effect: F_(1,28): 336.03, p < 0.0001; Knockdown/INS interaction: F_(1,28): 227.83, p < 0.001]; AMPKα1/Prkaa1: F_(3,28): 358.40, p < 0.001; Knockdown main effect: F_(1,28): 401.23, p < 0.0001; INS main effect: F_(1,28): 316.17, p < 0.001; Knockdown/INS interaction: F_(1,28): 357.81, p < 0.001; AMPKα2/Prkaa2: F_(3,28): 164.77, p < 0.001, Knockdown main effect: F_(1,28): 55.19, p < 0.0001; INS main effect: F_(1,28): 6.39, p = 0.017; Knockdown/INS interaction: F_(1,28): 432.73, p < 0.001]. Statistical differences between discrete pairs of treatment groups are denoted as follows: *p < 0.05; **p < 0.01; and ***p < 0.001.

Figure 3 VMNdm GABAergic Nerve Cell Neurotransmitter and Metabolic Sensor Marker Gene Expression during Eu- versus Hypoglycemia; Effects of Dbi Gene Knockdown. Experimental design, treatment group designations, target gene profiles, analytical and statistical methods, and data presentation format are identical to those described in Figure 2 above, except that GAD-immunopositive neurons were laser-catapult-microdissected from the VMNdm. Outcomes of statistical analyses are as follows: Gad1: F_(3,28): 35.44, p < 0.001; Knockdown main effect: F_(1,28): 21.68, p < 0.0001; INS main effect: F_(1,28): 26.88, p < 0.0001; Knockdown/INS interaction: F_(1,28): 57.77; p < 0.001]; Gad2: F_(32,8): 28.17, p < 0.001, Knockdown main effect: F_(1,28): 22.09, p < 0.0001; INS main effect: F_(1,28): 56.50, p < 0.0001; Knockdown/INS interaction: F_(1,28): 5.91, p = 0.022]; Gck: F_(3,28): 45.97, p < 0.001; Knockdown main effect: F_(1,8): 56.53, p < 0.0001; INS main effect: F_(1,28): 40.52, p < 0.001; Knockdown/INS interaction: F_(1,28): 40.84, p < 0.001]; Gkrp: F_(3,28): 11.19, p < 0.001; Knockdown main effect: F_(1,28): 13.58, p = 0.001; INS main effect: F_(1,28): 13.78, p = 0.001; Knockdown/INS interaction: F_(1,28): 5.99, p = 0.021]; AMPKα1/Prkaa1: F_(3,28): 145.46, p < 0.001, Knockdown main effect: F_(1,28): 123.46, p < 0.0001; INS main effect: F_(1,28): 143.73, p < 0.00; Knockdown/INS interaction: F_(1,28): 169.21, p < 0.001]; AMPKα2/Prkaa2: F_(3,28): 84.67, p < 0.001; Knockdown main effect: F_(1,28): 71.92; p < 0.0001; INS main effect: F_(1,28): 32.30; p < 0.001; Knockdown/INS interaction: F_(1,28): 149.78; p < 0.001]. *p < 0.05; **p < 0.01; and ***p < 0.001.

Figure 4 Effects of Dbi Gene Silencing on Eu- or Hypoglycemic Neurotransmitter and Metabolic Sensor Marker Gene Expression Patterns in VMNvl Nitrergic Neurons. Groups (n = 4/group) of Dbi or SCR siRNA-pretreated, V- or INS-injected male rats were sacrificed one hour after sc injection. Individual VMNdm neuronal nitric oxide synthase (nNOS/NOS1)-immunopositive neurons were laser-catapult-microdissected from 10 micron-thick fresh frozen sections for multiplex single-cell qPCR analyses. Data depict mean normalized nNOS/NOS1 (Figure 4A), Gck (Figure 4B), Gkrp (Figure 4C), AMPKα1/Prkaa1 (Figure 4D), or AMPKα2/Prkaa2 (Figure 4E) mRNA measures ± S.E.M. for SCR siRNA/V (n = 8), Dbi siRNA/V (n = 8), SCR siRNA/INS (n = 8), Dbi siRNA/INS (n = 8) treatment groups. Outcomes of statistical analyses are as follows: nNOS: F_(3,28): 365.90, p < 0.001; Knockdown main effect: F_(1,28): 337.27, p < 0.0001; INS main effect: F_(1,28): 483.78; p < 0.001; Knockdown/INS interaction: F_(1,28): 276.65; p < 0.001; Gck: F_(3,28): 328.81, p < 0.001; Knockdown main effect: F_(1,28): 822.64, p < 0.0001; INS main effect: F_(1,28): 7.36, p = 0.011; Knockdown/INS interaction: F_(1,28): 156.42, p < 0.001; Gkrp: F_(3,28): 93.08, p < 0.001; Knockdown main effect: F_(1,28): 173.19, p < 0.001; INS main effect: F_(1,28): 92.90, p < 0.001; Knockdown/INS interaction: F_(1,28): 13.14, p = 0.001; AMPKα1/Prkaa1: F_(3,28): 122.73, p < 0.001; Knockdown main effect: F_(1,28): 24.09, p < 0.0001; INS main effect: F_(1,28): 218.50, p < 0.001; Knockdown/INS interaction: F_(1,28): 125.60, p < 0.001; AMPKα2/Prkaa2: F_(3,28): 160.07, p < 0.001, Knockdown main effect: F_(1,28): 168.68, p < 0.0001; INS main effect: F_(1,28): 121.08, p < 0.001; Knockdown/INS interaction: F_(1,28): 190.44, p < 0.001. Statistical differences between discrete pairs of treatment groups are denoted as follows: *p < 0.05; **p < 0.01; and ***p < 0.001.

Figure 5 Eu- versus Hypoglycemic Neurotransmitter and Metabolic Sensor Marker Gene Expression Patterns in VMNdm Nitrergic Neurons: Effects of Dbi Gene Knockdown. Experimental design, treatment group designations, target gene profiles, analytical and statistical methods, and data presentation format are identical to that described in Figure 2 above, except that nNOS-immunopositive neurons were laser-catapult-microdissected from the VMNdm. Outcomes of statistical analyses are as follows: nNOS: F_(3,28): 78.52, p < 0.001; Knockdown main effect: F_(1,28): 65.83, p < 0.0001; INS main effect: F_(1,28): 526.22, p < 0,001; Knockdown/INS interaction: F_(1,28): 113.52, p < 0.001; Gck: F_(3,28): 859.64, p < 0.001; Knockdown main effect: F_(1,28): 531.39, p < 0.0001; INS main effect: F_(1,28): 1197.25, p < 0.001; Knockdown/INS interaction: F_(1,28): 850.27, p < 0.001; Gkrp: F_(3,28): 14.82, p < 0.001; Knockdown main effect: F_(1,28): 1.04, p = 0.317; INS main effect: F_(1,28):4.44, p = 0.044; Knockdown/INS interaction: F_(1,28): 38.99, p < 0.001; AMPKα1/Prkaa1: F_(3,28): 39.14, p < 0.001; Knockdown main effect: F_(1,28): 14.03, p = 0.001; INS main effect: F_(1,28): 20.78, p < 0,001; Knockdown/INS interaction: F_(1,28): 82.60, p < 0.001; AMPKα2/Prkaa2: F_(3,28): 88.86, p < 0.001; Knockdown main effect: F_(1,28): 46.27, p < 0.0001; INS main effect: F_(1,28): 183.48, p < 0.001; Knockdown/INS interaction: F_(1,28): 36.81, p < 0.001. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6 Effects of VMN Dbi Gene Silencing on Plasma Glucose and CounterRegulatory Hormone Profiles in Eu- or Hypoglycemic Male Rats. Plasma samples were obtained from groups of Dbi or SCR siRNA-pretreated male rats one hour after sc injection of V or INS, and analyzed for glucose (Figure 6A), glucagon (Figure 6B), or corticosterone (Figure 6C) concentrations. In each panel, individual treatment group data depict mean concentrations ± S.E.M. for n = 4 samples. Data were analyzed by two-way ANOVA and Student–Newman–Keuls post-hoc test, using GraphPad Prism, Vol. 8 software. Results of statistical analyses are as follows: glucose: F_(3,12): 170.27, p < 0.001; Knockdown main effect: F_(1,8): 1.18, p = 0.299; INS main effect: F_(1,12): 485.76, p < 0.001; Knockdown/INS interaction: F_(1,12): 23.87, p < 0.001; glucagon: F_(3,12): 34.66, p < 0.001; Knockdown main effect: F_(1,8): 0.51, p = 0.489; INS main effect: F_(1,12): 102.85, p < 0.001; Knockdown/INS interaction: F_(1,12): 0.61, p = 0.450; corticosterone: F_(3,12): 147.23, p < 0.001; Knockdown main effect: F_(1,12): 0.19, p = 0.671; INS main effect: F_(1,12): 359.18, p < 0.001; Knockdown/INS interaction: F_(1,12): 82.33, p < 0.001. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7 Summary of Outcomes of CESI-QTRAP MS/MS and Single Cell Multiplex qPCR Analysis Analyses of VMN tissue or Laser-Catapult-Microdissected Glucose-Regulatory Neurons. The left side of the cartoon illustrates the effects of Dbi gene knockdown on VMNdm versus the VMNvl ODN tissue content during insulin-induced hypoglycemia (HYPO). The right side depicts the effects of Dbi/ODN signaling on euglycemic (EU) versus HYPO patterns of transmitter marker gene expression in GABAergic or nitrergic neurons laser-microdissected from the VMNdm or VMNvl.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.