Genetic ablation or chemical inhibition of phosphatidylcholine transfer protein attenuates diet-induced hepatic glucose production

Abstract

Phosphatidylcholine transfer protein (PC-TP, synonym StARD2) is a highly specific intracellular lipid binding protein that is enriched in liver. Coding region polymorphisms in both humans and mice appear to confer protection against measures of insulin resistance. The current study was designed to test the hypotheses that Pctp-/- mice are protected against diet-induced increases in hepatic glucose production and that small molecule inhibition of PC-TP recapitulates this phenotype. Pctp-/- and wildtype mice were subjected to high-fat feeding and rates of hepatic glucose production and glucose clearance were quantified by hyperinsulinemic euglycemic clamp studies and pyruvate tolerance tests. These studies revealed that high-fat diet-induced increases in hepatic glucose production were markedly attenuated in Pctp-/- mice. Small molecule inhibitors of PC-TP were synthesized and their potencies, as well as mechanism of inhibition, were characterized in vitro. An optimized inhibitor was administered to high-fat-fed mice and used to explore effects on insulin signaling in cell culture systems. Small molecule inhibitors bound PC-TP, displaced phosphatidylcholines from the lipid binding site, and increased the thermal stability of the protein. Administration of the optimized inhibitor to wildtype mice attenuated hepatic glucose production associated with high-fat feeding, but had no activity in Pctp-/- mice. Indicative of a mechanism for reducing glucose intolerance that is distinct from commonly utilized insulin-sensitizing agents, the inhibitor promoted insulin-independent phosphorylation of key insulin signaling molecules.

Conclusion: These findings suggest PC-TP inhibition as a novel therapeutic strategy in the management of hepatic insulin resistance.

Figure 1. Pctp^−/− mice are resistant to diet-induced glucose intolerance

(A) Reduced fasting plasma glucose concentrations in mice lacking PC-TP. Wild type (n = 6 – 10) and Pctp^−/− (n = 8 – 10) mice were fed a high fat (60% kcal) diet for up to 18 w. Values for chow fed wild type and Pctp^−/− mice of similar age to mice fed high fat for 8 w are replotted from reference (6). (B) Decreased hepatic glucose production in Pctp^−/− mice. Values determined by hyperinsulinemic euglycemic clamp studies for wild type mice (n = 6) and Pctp^−/− mice (n = 8) fed the high fat diet for 18 w. (C) Pyruvate tolerance tests reflect changes in hepatic glucose production. Wild type (n = 4) and Pctp^−/− (n = 5) mice fed high fat diet for 12 w were subjected to pyruvate tolerance tests. The barplot gives mean AUC values. *P < 0.05 versus wild type mice.

Figure 2. Structure-activity relationships of PC-TP inhibitors

Structures and inhibitory characteristics of small molecules derived from compounds A1 and B1. “NA” denotes that a compound exhibited no activity, and “-” denotes that no measurement was performed. Syntheses of small molecular inhibitors are illustrated in Supporting Information.

Figure 3. Compound A1 binds PC-TP, displaces phosphatidylcholine and increases thermal stability

(A) Binding of compound A1 to PC-TP as assessed by surface plasmon resonance. Upper panel: Compound A1 (50 – 100 µM) was injected for 150 s to allow binding and then dissociation from PC-TP bound to a CM5 chip. Representative sensograms are shown overlaid and zeroed on the y-axis to the average baseline before injection. The injection start time for each sample was set to zero on the x-axis. Lower panel: Selected inhibitors were analyzed by surface plasmon resonance. Rate constants of association (k_a) and dissociation (k_d) were determined by non-linear least squares analysis of the sensogram data (SigmaPlot, Systat Software, San Jose, CA) and used to calculate equilibrium dissociation constants, K_D = k_d/k_a. (B) Displacement of phosphatidylcholine from PC-TP by compound A1. Upper panel: Emission scans of Pyr-PC in the absence of PC–TP or with PC–TP plus compound A1. Lower panel: Relative affinity constants, K_rel, were determined according to the equation (33) ΔF(392 nM) = −1/K_rel·([X]/[Pyr– PC])·ΔF(392 nM) + ΔF_max for X = phosphatidylcholine or compound A1 by linear regression of ΔF plotted against the product of ΔF(392 nM) and the molar ratio [X]/[Pyr-PC]. The inset bar plot displays values of K_rel (n = 3 – 4 determinations) for phosphatidylcholine and compound A1. *P = 0.0001. (C) Stabilization of PC-TP by compound A1 using a thermal shift (ThermoFluor) assay. Fluorescence intensity (upper panel) and first derivative, dF/dT of the melting curves (lower panel) for PC–TP, PC–TP plus phosphatidylcholine and PC–TP with compound A1. Values of T_m were determined using LightCycler Protein Melting software (Roche), which identified T_m as minimum values of −dF/dT when plotted as functions of temperature. The inset bar plot shows the comparison of the T_m values (n = 3 determinations) of PC–TP plus phosphatidylcholine, and PC–TP plus compound A1. *P = 0.02.

Figure 4. Compound A1 improves glucose homeostasis in high fat fed wild type but not Pctp^−/− mice

Improved glucose and pyruvate tolerance tests in wild type mice treated with compound A1. High fat fed wild type mice and Pctp^−/− mice were injected i.p. 5 d per w with 3 mg/kg of compound A1 for 12 w. Mice were subjected to glucose tolerance tests (A: wild type - vehicle n = 4, compound A1 n = 8; B: Pctp^−/− - vehicle n = 3, compound A1 n = 4) and pyruvate tolerance tests (C: wild type - vehicle n = 4, compound A1 n = 7; D: Pctp^−/− - vehicle n = 3, compound A1 n = 3). The inset bar plots provide AUC values for wild type mice and Pctp^−/− mice. *P < 0.05 versus vehicle treatment.

Figure 5. Compound A1 enhances insulin signaling

(A) Compound A1 increases phosphorylation of selected mediators of insulin signaling in cultured cells. Human hepatocytes (left panel) and HEK 293E cells (right panel) were serum starved overnight, then treated with compound A1 for 1 h and harvested for immunoblot analyses. Results are representative of 2 experiments for human hepatocytes and 3 experiments for HEK 293E cells. (B) Compound A1 increases phosphorylation of Akt and S6K in livers of wild type mice. Homogenates of liver samples from mice fasted overnight were subjected to immunoblotting and bands (Supporting Fig. 5B) were quantified by densitometry. *P < 0.05 versus vehicle treatment.