Beta-Hydroxybutyrate Suppresses Hepatic Production of the Ghrelin Receptor Antagonist LEAP2

Abstract

Introduction: Liver-expressed antimicrobial peptide-2 (LEAP2) is an endogenous ghrelin receptor antagonist, which is upregulated in the fed state and downregulated during fasting. We hypothesized that the ketone body beta-hydroxybutyrate (BHB) is involved in the downregulation of LEAP2 during conditions with high circulating levels of BHB.

Methods: Hepatic and intestinal Leap2 expression were determined in 3 groups of mice with increasing circulating levels of BHB: prolonged fasting, prolonged ketogenic diet, and oral BHB treatment. LEAP2 levels were measured in lean and obese individuals, in human individuals following endurance exercise, and in mice after BHB treatment. Lastly, we investigated Leap2 expression in isolated murine hepatocytes challenged with BHB.

Results: We confirmed increased circulating LEAP2 levels in individuals with obesity compared to lean individuals. The recovery period after endurance exercise was associated with increased plasma levels of BHB levels and decreased LEAP2 levels in humans. Leap2 expression was selectively decreased in the liver after fasting and after exposure to a ketogenic diet for 3 weeks. Importantly, we found that oral administration of BHB increased circulating levels of BHB in mice and decreased Leap2 expression levels and circulating LEAP2 plasma levels, as did Leap2 expression after direct exposure to BHB in isolated murine hepatocytes.

Conclusion: From our data, we suggest that LEAP2 is downregulated during different states of energy deprivation in both humans and rodents. Furthermore, we here provide evidence that the ketone body, BHB, which is highly upregulated during fasting metabolism, directly downregulates LEAP2 levels. This may be relevant in ghrelin receptor-induced hunger signaling during energy deprivation.

Keywords: LEAP2; energy deprivation; exercise; ketogenic diet; ketone bodies.

Figure 1.

LEAP2 levels decrease by fasting and increase with obesity. (A) Relative Leap2 mRNA expression levels from liver, duodenum, and jejunum samples in 2 treatment groups; ad libitum–fed (ad lib.) mice and mice fasted for 24 hours; n = 9 in ad libitum–fed and n = 10 fasted mice (B) Relative Leap2 mRNA expression levels from liver in 2 groups; lean and HFD-fed DIO mice; n = 6 in lean mice and 9 in HFD-fed DIO mice (C) Mean BW of lean vs obese human individuals; lean human individuals n = 10 (averaged from 2 separate days), obese human individuals n = 17 (D) LEAP2 plasma levels in lean vs obese human individuals; lean human individuals n = 10 (averaged from 2 separate days), obese human individuals n = 17. Data were normalized to reference gene Ywhaz and subsequently normalized to corresponding control (A-B). Data were analyzed by unpaired t test. One outlier was identified and excluded due to technical error and exclusion of outlier did not diverge from the results of the main analysis and result (A [fasted mice in duodenum and jejunum] , in both lean and HFD-fed DIO mice (1 in each) and D in humans with obesity. ** P < 0.01; *** P < 0.001, **** P < 0.0001, ns = not significant. Abbreviations: DIO; diet-induced obesity, HFD; high-fat diet, LEAP2; liver-expressed antimicrobial peptide 2.

Figure 2.

Circulating LEAP2 decrease in response to exercise while BHB levels increase. (A) Experimental protocol for human endurance exercise study, (B) Time course of BHB plasma levels on the following time points; before endurance exercise, and 30, 60, 120, and 180 minutes after exercise (C) Time course of LEAP2 plasma levels on the following timepoints; before endurance exercise, and 30, 60, 120, and 180 minutes after exercise. Data were analyzed by one-way ANOVA repeated measures followed by Tukey post hoc test (B-C) n = 9. * P < 0.05; ** P < 0.01. Abbreviations: BHB; beta-hydroxybutyrate, EIA; enzyme immunoassay, LEAP2; liver-expressed antimicrobial peptide 2.

Figure 3.

Ketogenic diet downregulates Leap2 expression in mouse liver. (A) Treatment groups, (B) BW in chow-cohort, (C) Blood glucose levels in chow-cohort, (D) BHB blood levels in chow-cohort, (E) Relative Leap2 expression levels in liver in chow-cohort, (F) Relative Leap2 expression levels in duodenum in chow-cohort (G) Relative Mct1 expression levels in liver in chow-cohort, (H) Relative Mct1 expression levels in duodenum in chow-cohort, (I) Relative Hcar2 expression levels in liver in chow-cohort, (J) BW in HFD-cohort, (K) Blood glucose levels in HFD-cohort, (L) BHB blood levels in HFD-cohort, (M) Relative Leap2 expression levels in liver in HFD-cohort, (N) Relative Leap2 expression levels in duodenum in HFD-cohort, (O) Relative Mct1 expression levels in liver in HFD-cohort, (P) Relative Mct1 expression levels in in duodenum in HFD-cohort, (Q) Relative Hcar2 expression levels in in liver in HFD-cohort. Data were normalized to reference gene Ywhaz and subsequently normalized to either chow or HFD control (E-I and M-Q, respectively). Data were analyzed by unpaired t test (B-I), and one-way ANOVA followed by Tukey post hoc test (J-Q). Group^Chow, n = 7; Group^Chow-KD, n = 7; Group^HFD, n = 10; Group^HFD-KD, n = 9; Group^HFD-Chow n = 9. Outliers were identified and exclusion of outlier did not diverge from the results of the main analysis and result (E, I, M, N, P). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Abbreviations: BHB; beta-hydroxybutyrate, HFD; high-fat diet, Leap2; Liver-expressed antimicrobial peptide 2.

Figure 4.

In vivo administration of exogenous BHB downregulates circulating LEAP2 levels and Leap2 expression in mouse liver and duodenum. (A) Plasma levels of BHB in 2 treatment groups; vehicle and BHB (100 mg/kg BW), 2 hours after oral administration, (B) Blood glucose levels in 2 treatment groups; vehicle and BHB (100 mg/kg BW), 2 hours after oral administration, (C-E) Relative Leap2 expression levels in 2 treatment groups; vehicle and BHB (100 mg/kg BW), in liver, duodenal, and jejunal, respectively, 2 hours after oral administration, (F) Systemic LEAP2 plasma levels from vehicle and BHB (100 mg/kg BW), 2 hours after oral administration, (G-I) Relative Mct1 expression levels in 2 treatment groups; vehicle and BHB (100 mg/kg BW), in liver, duodenal, and jejunal, respectively, 2 hours after oral administration, (J-L) Relative Hcar2 expression levels in 2 treatment groups; vehicle and BHB (100 mg/kg BW), in liver, duodenum, and jejunum, 2 hours after oral administration. Data were normalized to reference gene Ywhaz and subsequently normalized to vehicle (C-E + G-L). All data were analyzed by unpaired t test. n = 10 in each group. Outliers were identified and exclusion of outlier did not diverge from the results of the main analysis and result (C, G, H, K, L). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, ns = not significant. Abbreviations: BHB; beta-hydroxybutyrate, Hcar2; hydroxycarboxylic acid receptor 2, LEAP2/Leap2; Liver-expressed antimicrobial peptide 2, Mct1; monocarboxylate transporter 1.

Figure 5.

Leap2 expression is downregulated by BHB in isolated mouse hepatocytes. (A) Relative Leap2 expression levels after 4 hours incubation with 1 and 10 mM BHB in mouse hepatocytes, (B) Relative Hcar2 expression levels after 4 hours incubation with 1 and 10 mM BHB in mouse hepatocytes, (C) Relative Leap2 expression levels after 4 hours incubation with 10 nM, 100 nM, and 1 uM AR277 in mouse hepatocytes, (D) Relative Mct1 expression levels after 4 hours incubation with 1 and 10 mM BHB in mouse hepatocytes, (E) Relative Leap2 expression levels after 4 hours incubation with 10 nM, 100 nM TSA in mouse hepatocytes, (F) Relative Leap2 expression levels after 4 hours incubation with 0.1 uM, 1 uM, and 10 uM TMP269 in mouse hepatocytes. Data from each mouse were normalized to reference gene Ywhaz and subsequently normalized to control and collected in the same analysis. Data were analyzed by one-way ANOVA followed by Tukey post hoc tests (A-B). All data were presented as means ± SEM. n = 10-12 mice (A and B), 6 (C), 7 (D), 5 (E) and 3 (F). n is the number of biologically independent samples for which data were averaged from duplicate, triplicate, or quadruplicate measurements. * P < 0.05. Abbreviations: BHB; beta-hydroxybutyrate, Hcar2; hydroxycarboxylic acid receptor 2, Leap2; Liver-expressed antimicrobial peptide 2, Mct1; monocarboxylate transporter 1, TSA; trichostatin a.

Figure 6.

Graphical illustration of BHB-induced LEAP2 regulation. Graphical illustration demonstrating that LEAP2 is reduced during energy-deprived conditions and that BHB directly downregulates LEAP2 expression and secretion in hepatocytes. Abbreviations: BHB; beta-hydroxybutyrate, LEAP2; Liver-expressed antimicrobial peptide 2.