Clinical parameters. Demographic information was self-reported by participants and is provided in Supplemental Table 1 (supplemental material available online with this article; https://doi.org/10.1172/JCI180157DS1). Participants were a subset from a prior study (5) for whom pristine residual samples were available for analysis. These included 13 individuals with PBH (11 female, 2 male; mean age 48 ± 11, BMI 30.6 ± 4.9, 3.1 ± 1.6 years post-RYGB), 10 individuals post-RYGB without symptomatic hypoglycemia (Asx) (8 female, 2 male; age 48 ± 8, BMI 29.9 ± 4.4, 2.6 ± 1.0 years post-RYGB), and 8 overweight/obese individuals without T2D and without history of gastrointestinal surgery (Ow/Ob) (5 female, 2 male; age 48 ± 9, BMI 41.8 ± 10.8). Hemoglobin A1c did not differ between groups. Fasting glucose, insulin, triglycerides, homeostatic model assessment for insulin resistance (HOMA-IR), and Matsuda index were significantly lower, and HDL significantly higher, in both surgical groups as compared with nonsurgical Ow/Ob individuals, consistent with improved glucose metabolism and insulin sensitivity postoperatively, but did not differ between PBH and Asx.

During mixed meal testing, nonsurgical participants displayed the expected meal-related rise in glucose with return to baseline by 120 minutes. However, patterns were distinct in the asymptomatic post-surgical patients, with greater glycemic excursions versus controls; participants with PBH had lower glucose levels at 30 minutes versus asymptomatic (128 ± 32.3 mg/dL vs. 162 ± 38.3 mg/dL at 30 minutes, P = 0.019; Supplemental Figure 1A). At 2 hours, PBH and Asx glucose levels were similar, but significantly lower than in nonsurgical individuals (PBH 68.7 ± 11.3 mg/dL; Asx 70.3 ± 8.6 mg/dL; Ow/Ob 98.3 ± 11.4 mg/dL; P < 0.001 for both PBH vs. Ow/Ob and Asx vs. Ow/Ob).

Fasting insulin and C-peptide were reduced in PBH and Asx groups compared with Ow/Ob, but did not differ between PBH and Asx. After meal ingestion, insulin and C-peptide levels increased markedly in PBH, returning to baseline by 2 hours (Supplemental Figure 1, B and C). Plasma GLP-1 was significantly increased in both surgical groups, with even higher levels in PBH at 30 minutes (209.3 vs. 125.8 pmol/L for Asx, P < 0.001; Supplemental Figure 1D). Gastric inhibitory polypeptide (GIP) levels did not differ between PBH and Asx, but GIP levels in Asx were higher than those in Ow/Ob at 30 minutes (P < 0.01) (Supplemental Figure 1E). Glucagon levels did not differ in the fasting state but were higher after meals in both surgical groups (Supplemental Figure 1F). Plasma cortisol did not differ between groups (Supplemental Figure 1G).

Metabolite patterns differ in PBH. Unsupervised hierarchical clustering of the 189 metabolites detected at all 3 time points and with significant FDR less than 0.05 (F test) is presented in the heatmap in Figure 1A, with a full list of cluster components provided in Supplemental Table 2 and full statistical analysis in Supplemental Table 3. As expected, ingestion of the mixed meal in nonsurgical controls resulted in decreased abundance of multiple lipid species and increased abundance of AAs at 30 minutes, with return to baseline at 120 minutes.

Metabolite abundance in individuals with PBH, Asx, and Ow/Ob in the fasting state and at 30 and 120 minutes after mixed meal ingestion. (A) Heatmap of z-scored log2-transformed metabolite abundance for the top 190 most altered metabolites between groups, with unsupervised hierarchical clustering of rows. Cluster annotations include lipid species (clusters 1–6), amino acids (AA; cluster 7), tryptophan metabolites (cluster 8), carnitines (CAR; cluster 9), and tricarboxylic acid (TCA) cycle metabolites (cluster 10). (B–D) Pathway enrichment analysis for PBH versus Asx individuals in the fasting state and 30 and 120 minutes after meal ingestion, respectively. Downregulated pathways are colored blue, while upregulated pathways are colored red. The –log10P value for enrichment is represented on the x axis; the vertical line indicates nominal P less than 0.05.

Exploratory analysis of these 189 metabolites revealed that 158 metabolites were altered in Asx individuals versus nonsurgical controls, reflecting the impact of surgery (FDR < 0.25). Sixty-two metabolites were altered in PBH versus Asx post-surgical individuals, and 178 metabolites were differentially abundant in both comparisons. Interestingly, 19 of these 178 had opposite directionality between PBH and Asx, including adenine, oxalate, and hydroxyphenylpyruvate (Supplemental Figure 2, A–C). These findings support the concept that PBH is not simply an “extreme” example of post-surgical metabolic perturbations.

Pathway analysis was performed using the Fry function of the rotation gene set test (ROAST) in the limma R package. Multiple pathways were enriched (FDR < 0.25) in the comparison of PBH and Asx post-surgical groups (Figure 1, B–D). In the fasting state, pathways downregulated in PBH included spermidine/spermine biosynthesis, tryptophan and methionine metabolism, phospholipid synthesis, and glycolysis (Figure 1B). After meals, both spermidine/spermine and methionine metabolism pathways remained downregulated at 30 minutes (Figure 1C). By 2 hours, enriched pathways were upregulated in PBH, including starch/sucrose and pyruvate metabolism, gluconeogenesis, and citric acid cycle pathways (Figure 1D). Additional comparisons are presented in Supplemental Figure 3 (PBH vs. Ow/Ob) and Supplemental Figure 4 (Asx vs. Ow/Ob).

Correlation of metabolites with glucose. As expected, both AAs and lipids were highly correlated with glucose levels. The top metabolites that correlated with glucose in the fasting state (FDR < 0.05) (Supplemental Table 4) were AAs (Phe, Tyr, Met, Leu, Trp, His, Ala, and Asn), with similar patterns at 30 minutes. Additional metabolites correlating with glucose in the fasting state included NMMA (N-methylmalonamic acid), carnitine, UDP (uridine-5′-diphosphate), and oxalate. At 30 minutes, metabolites correlating with glucose included AAs (Leu, Met, Ile, Phe, Tyr, Gln, Pro, Arg, Trp, Gly, Ser, His, Val, Lys, Thr), citrulline, choline, NMMA, and ornithine, and the triglycerides C56:1, C48:0, and C50:0 (FDR < 0.05). There were no metabolites significantly correlating (FDR < 0.05) with glucose at 120 minutes (Supplemental Table 4).

Given that hypoglycemia typically develops 2–3 hours after meals, we asked whether metabolites present in the fasting state correlated with post-meal glucose. While relationships were not as robust as for the matched-time analyses, fasting levels of 17 metabolites were positively correlated with glucose at 120 minutes, including lactate, AAs (Gln, Pro, Trp, Tyr, betaine), carnitine, and multiple lipids (lysophosphatidylcholine [LPC] 18:0, 18:2, 20:3, and 22:6; lysophosphatidylethanolamine [LPE] 18:1 and 22:6) (FDR < 0.25) (Supplemental Figure 5).

Plasma levels of TCA cycle intermediates are increased in PBH. Hypoglycemia ultimately may reflect interaction between excessive insulin secretion, inadequate counterregulatory hormone levels and/or action, reduced glycogen stores, and inadequate substrate or activity within gluconeogenic pathways. As noted above, multiple AAs were reduced in PBH, potentially contributing to reduced formation of intermediates such as pyruvate (alanine), acetyl-CoA (tryptophan, tyrosine, leucine, isoleucine, and phenylalanine), and succinyl-CoA (methionine, valine, and threonine). We interrogated metabolite patterns in central glucose metabolic pathways, projecting differences in PBH onto a pathway map at each time point (Figure 2).

Model demonstrating the metabolites of the glycolysis, gluconeogenesis, and TCA cycle pathways during mixed meal tolerance test. Individuals with PBH and Asx are compared in the fasting state (A) and at 30 minutes (B) and 120 minutes (C) after mixed meal ingestion. Metabolites are colored blue if decreased (nominal P < 0.05), red if increased (nominal P < 0.05), and white if unchanged in PBH versus Asx.

In the fasting state (Figure 2A), PBH was characterized not only by decreased levels of glucose (20% lower, P = 0.001, FDR = 0.04) but also by decreases in gluconeogenesis intermediates such as glucose-6-phosphate (40% reduction, P = 0.02, FDR = 0.18) and pyruvate (80% lower, P < 0.0001, FDR = 0.003). Conversely, TCA cycle intermediates such as aconitate, isocitrate, and succinate were increased by 30% in PBH versus Asx in the fasting state (P < 0.05 for all, FDR < 0.25). Moreover, the ketone β-hydroxybutyrate was increased by 80%, as noted above (fold change = 1.8, P = 0.04, FDR < 0.25).

At 30 minutes after meals, only glucose and pyruvate remained lower (20% and 50% lower, P < 0.01, FDR < 0.1), and isocitrate remained higher (30% higher, P = 0.009, FDR = 0.14), in PBH versus Asx (Figure 2B). At 2 hours, 3-phosphoglycerate was 2-fold higher (P = 0.002, FDR = 0.002), while several TCA intermediates were again higher in PBH, including 30%–40% increases in aconitate, isocitrate, and malate (all P < 0.01, FDR < 0.01) (Figure 2C).

Lipid, ketone, and bile acid metabolism is perturbed in PBH. Multiple lipid species were decreased in both surgical groups at 30 minutes, consistent with postprandial rises in insulin secretion. Additional lipid species differentially abundant in PBH versus Asx included LPE 20:4 and C22:6, LPC C22:6, and triacylglycerol (TAG) C50:5, C54:5, and C56:7, all of which were decreased by 30%–50% in PBH versus Asx (P < 0.05, FDR < 0.25) (Supplemental Figure 6, A–F). By contrast, C56:1 TAG was markedly increased in PBH versus Asx at 30 and 120 minutes (29- and 59-fold higher, P = 0.01, FDR < 0.25 for both; Supplemental Figure 6G). In agreement, abundance of C56:1 TAG was inversely correlated with glucose levels at both 30 (r = –0.67, P = 0.01) and 120 minutes (r = –0.65, P = 0.02) in PBH, but not in the fasting state.

Ketones, produced in the liver from 2-carbon products of β-oxidation, can be utilized by some tissues as an alternative fuel when glucose availability is limited, as with prolonged fasting. Interestingly, the ketone β-hydroxybutyrate was increased in the fasting state by 80% in PBH versus Asx (P = 0.04, FDR < 0.25) and more than 3-fold in comparison with Ow/Ob (P < 0.001, FDR = 0.01). These differences remained at 120 minutes (Supplemental Figure 6H).

Bile acids have been proposed as mediators of glycemic improvement after RYGB (4), potentially via effects to increase both incretin hormones and FGF-19 (4, 10). Fasting levels of the conjugated bile acid taurocholate were 2.5-fold higher in PBH versus Asx (P = 0.03, FDR = 0.22) (Supplemental Figure 6I). However, there were no significant differences in fasting or postprandial levels of other measured bile acids in PBH versus asymptomatic post-surgical participants (4, 9, 11).

Biomarkers of insulin resistance and T2D are decreased in PBH. Previous studies have identified metabolite biomarkers of insulin resistance and risk for T2D (12, 13). These include AAs (branched-chain AAs, Phe, and Tyr), and triglyceride species characterized by lower carbon number and double bond content (14). Indeed, post-surgical individuals had reductions in these biomarker AAs and lipids, consistent with improved metabolism after RYGB, with further reductions in PBH. Moreover, α-hydroxybutyrate, an organic acid derived from α-ketobutyrate (produced by AA catabolism) and associated with insulin resistance, was 30% lower in PBH at both fasting (P = 0.03, FDR = 0.21) and 30 minutes after meals (P = 0.03, FDR = 0.22), as compared with Asx (Supplemental Figure 7A). Likewise, 2-aminoadipate, a product of lysine degradation associated with insulin resistance (13, 15), was also 6-fold lower in PBH versus Asx (P = 0.001, FDR = 0.04) in the fasting state (Supplemental Figure 7B).

Levels of B-complex vitamins are reduced in PBH. B-complex vitamins play important roles as cofactors for key reactions in cellular metabolism. Several water-soluble B vitamins, including niacinamide (B3) and pantothenate (B5), were decreased 2-fold in the fasting state in PBH versus Asx (P < 0.05 for all, FDR < 0.25). Pantothenate was also 2-fold lower at 30 and 120 minutes (P < 0.01 for both, FDR < 0.05) (Supplemental Figure 7, C and D). Moreover, fasting glucose was positively correlated with levels of both vitamins B3 (fasting: r = 0.48, P = 0.02, FDR 0.11) and B5 (fasting: r = 0.52, P = 0.009, FDR = 0.07; 30 minutes: r = 0.49, P = 0.017, FDR = 0.14).

AA levels are reduced in PBH in the fasting state. In the fasted state, 10 AAs were significantly reduced in PBH as compared with both asymptomatic and nonsurgical groups. Leucine, isoleucine, valine, alanine, tryptophan, phenylalanine, threonine, tyrosine, methionine, and asparagine were reduced by 17%–29% (P < 0.05, FDR < 0.25) in PBH versus Asx and Ow/Ob groups (Figure 3, A–J). After meal ingestion, AA levels increased markedly in both surgical groups, but only leucine, tryptophan, and phenylalanine remained lower in PBH as compared with Asx. AA levels for each participant are presented in Supplemental Figure 8. Notably, the reductions in AAs in PBH included both essential and non-essential AAs, and both gluconeogenic (e.g., Ala) and ketogenic (e.g., Leu) AAs. Arginine abundance was increased in both PBH and Asx compared with Ow/Ob (Figure 3K), while glutamine did not differ between groups (Figure 3L). The sum of all AAs was strongly positively correlated with glucose in the fasting state (r = 0.78, P = 0.001) and at 30 minutes (r = 0.59, P = 0.033); these correlations did not remain at 120 minutes (r = 0.07, P = 0.82) (Figure 3, M–O). The top-ranking specific AAs correlated with glucose levels both in the fasting state and at 30 minutes are provided in Supplemental Figure 9.

Abundance of 12 plasma AAs in PBH, Asx, and Ow/Ob individuals in the fasting state and at 30 and 120 minutes after mixed meal ingestion. (A–L) Abundance of leucine (A), isoleucine (B), valine (C), alanine (D), tryptophan (E), phenylalanine (F), threonine (G), tyrosine (H), methionine (I), asparagine (J), arginine (K), and glutamine (L). (M–O) Correlation between glucose levels and sum of AA abundance in the fasting state (red, M) and at 30 minutes (red, N) and 120 minutes (red, O) after meal. Data are mean ± SD. *,#,$P < 0.05; **,##P < 0.01; ###P < 0.001. Two-way ANOVA with Tukey’s multiple-comparison test was performed.

Serotonin levels are uniquely increased in the postprandial state in individuals with PBH. Both pathway and individual metabolite analysis revealed differential abundance of tryptophan pathway intermediates in PBH, including tryptophan itself and its downstream metabolites kynurenic acid, xanthurenate, and serotonin (Figure 4). For example, tryptophan levels were 40% lower in the fasting state in PBH versus Asx (P = 0.003, FDR = 0.06), and 20%–30% lower at 30 and 120 minutes (P = 0.028 and 0.019, respectively, FDR < 0.25) (Figure 4A). Similarly, kynurenic acid was reduced by 70% at all 3 time points (P < 0.01 for all, FDR < 0.25), while xanthurenate was 8-fold lower in the fasting state (P = 0.003, FDR = 0.06) and 6-fold lower at 2 hours (P = 0.008, FDR < 0.01) (Figure 4, B and C).

Abundance of tryptophan, tryptophan-derived metabolites, and serotonin in PBH, Asx, and Ow/Ob individuals in the fasting state and after mixed meal ingestion. Center: Schematic of tryptophan metabolism and downstream metabolites. (A–E) Abundance of tryptophan (A), kynurenic acid (B), xanthurenate (C), serotonin (D), and serotonin by ELISA (E). (F–H) Serotonin ELISA of individual participants in PBH (red), Asx (green), and Ow/Ob (blue) groups. Data are mean ± SD. *,#P < 0.05; **,##P < 0.01; ###P < 0.001;****P < 0.0001 for PBH vs. Asx; ####P < 0.0001 for PBH vs. Ow/Ob. Two-way ANOVA with Tukey’s multiple-comparison test was performed. Blue color of circles in the schematic indicates metabolites with decreased abundance, while white indicates unchanged abundance.

Serotonin levels were 10-fold lower in the fasting state in PBH, as compared with Asx (P = 0.019, FDR = 0.16) (Figure 4D). Interestingly, serotonin patterns in response to meal ingestion were distinct in PBH, increasing 3.2- and 4.7-fold at 30 and 120 minutes, respectively; by comparison, serotonin levels decreased after meal ingestion in Asx and nonsurgical groups.

To confirm alterations in serotonin dynamics in PBH, we analyzed serotonin using ELISA on samples collected in the fasting state and at 30 and 120 minutes after a meal originating from a newly recruited cohort described in Supplemental Table 5. Consistent with the metabolomics data, serotonin levels in individuals with PBH increased 1.9-fold after a meal (P < 0.001), whereas Asx and Ow/Ob patients had a 1.4- to 1.6-fold (P < 0.001 for both) decrease in serotonin levels over the same time course (Figure 4E). Serotonin levels for each participant are presented in Figure 4, F–H, demonstrating the distinct responses in all individuals with PBH.

Correlation analysis between metabolites and glucose levels during mixed meal tolerance test showed that tryptophan was correlated with glucose in the fasting state (r = 0.67, P < 0.001) and at 30 (r = 0.65, P < 0.001) and 120 minutes (r = 0.47, P = 0.023) (Supplemental Table 4). While there was no correlation between serotonin (measured by metabolomics assay) and glucose (Supplemental Table 4), plasma serotonin, measured by ELISA, was inversely correlated with glucose at 120 minutes after the meal in PBH (r = –0.78, P = 0.007) (Supplemental Figure 10).

To exclude the possibility that high plasma insulin or low glucose levels could contribute to observed increases in serotonin in the postprandial state, we analyzed serotonin levels in response to hyperinsulinemic-hypoglycemic clamps in PBH, Asx, and Ow/Ob participants (Supplemental Figure 16A). Consistent with the design of the clamp, plasma glucose levels at 100 minutes were similarly reduced in all groups (PBH: 51 ± 7 mg/dL; Asx: 51 ± 7 mg/dL; Ow/Ob: 53 ± 5 mg/dL; Supplemental Figure 16B). However, serotonin levels did not differ between groups, either in the fasting state (PBH: 103 ± 57 ng/mL; Asx: 152 ± 73 ng/mL; Ow/Ob: 174 ± 81 ng/mL) or during experimental hypoglycemia (PBH: 142 ± 72 ng/mL; Asx: 119 ± 89 ng/mL; Ow/Ob: 187 ± 53 ng/mL) (Supplemental Figure 16C). Similarly, serotonin levels did not change after insulin injection in mice (2 U/kg, i.p.) (Supplemental Figure 16, D and E) as compared with equal-volume saline at either 15 minutes (insulin: 430 ± 166 ng/mL; vs. saline: 344 ± 56 ng/mL) or 30 minutes (insulin: 344 ± 87 ng/mL; vs. saline: 351 ± 175 ng/mL) (Supplemental Figure 16F). Together, these results in both humans and mice suggest that hyperinsulinemia or hypoglycemia per se is not likely to mediate increases in postprandial serotonin in PBH.

Serotonin induces hypoglycemia in mice. Given that serotonin levels were uniquely increased in the postprandial state in individuals with PBH (4.7-fold at 120 minutes), and not influenced by experimental hypoglycemia or hyperinsulinemia, we sought to determine whether serotonin could modulate glucose levels in vivo in healthy C57BL/6J mice. We injected wild-type mice with serotonin (20 mg/kg, i.p.) or an equal volume of saline (Figure 5A). In serotonin-injected mice, serotonin levels increased rapidly to a peak of 539 ng/mL at 30 minutes (Figure 5B). To determine the impact of serotonin on glucose metabolism, we measured blood glucose at baseline and at 15, 30, and 60 minutes. Glucose levels were significantly reduced in serotonin-injected male mice at 15 (–39%, P < 0.001), 30 (–43%, P < 0.002), and 60 minutes (–44%, P < 0.018) (Figure 5C), with reductions of similar magnitude in females (–29% at 15 minutes, P < 0.001; –48% at 30 minutes, P < 0.001; and –48% at 60 minutes, P = 0.005) (Supplemental Figure 11). We next assessed whether serotonin-induced reduction in glucose was mediated by an insulin-dependent mechanism; plasma insulin increased 5- and 2.5-fold at 30 and 60 minutes after serotonin injection versus saline (P = 0.004 and P = 0.002) (Figure 5D). Interestingly, serotonin administration also significantly increased plasma GLP-1 levels 1.6- to 2.1-fold at 15, 30, and 60 minutes after injection (P = 0.011, P < 0.001, and P < 0.001, respectively) (Figure 5E).

Serotonin reduces glucose and increases insulin and GLP-1 secretion. (A) Experimental protocol showing exogenous administration of serotonin (20 mg/kg) or saline in C57BL/6J mice. (B) Serotonin levels achieved by serotonin injection. (C) Glucose levels after serotonin or saline injection. (D and E) Insulin levels (D) and GLP-1 levels (E) after serotonin or saline injection. In all panels, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Tukey’s multiple-comparison test.

To mimic the postprandial glucose dynamics and exaggerated insulin secretion characteristic of PBH in mice, we developed an insulin-augmented mixed meal tolerance test. After a 4-hour fast, mice were gavaged with a liquid mixed meal (200 μL Ensure Compact, Abbott Laboratories) and injected with insulin (2 U/kg, i.p.; Humulin R, Lilly), before injection with serotonin or saline (Figure 6A). Consistent with prior experiments, serotonin (20 mg/kg) also reduced glucose by 38% versus saline in this setting, achieving levels below fasting level (serotonin, 80 mg/dL, vs. saline, 128 mg/dL, at 60 minutes; P = 0.021; Figure 6B).

Serotonin induces hypoglycemia during meal tolerance test; hypoglycemia is blocked by cyprophetadine and more specifically by ketanserin. Reductions in glucose are blocked by the serotonin antagonist cyproheptadine and the specific serotonin receptor 2 antagonist ketanserin. (A) Scheme showing administration of cyproheptadine (50 mg/kg) or saline and, after 30 minutes, oral gavage with 200 μL of Ensure, insulin injection (2 U/kg), and administration of serotonin (20 mg/kg) or saline in C57BL/6J mice. MMTT, mixed meal tolerance test. (B) Glucose levels during experiment. (C) Scheme showing administration of ketanserin (5 mg/kg) and exogenous administration of serotonin (20 mg/kg) or saline in C57BL/6J mice. (D) Glucose levels after serotonin or saline injection. (E and F) Insulin levels (E) and GLP-1 levels (F) after serotonin or saline injection. In all panels, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Tukey’s multiple-comparison test.

Given that serotonin increased GLP-1 levels, we asked whether GLP-1 receptor blockade would attenuate serotonin affects. We first confirmed that the GLP-1 receptor antagonist avexitide (30 mmol/kg) blocked semaglutide-induced increases in insulin secretion (Supplemental Figure 13B) and, in parallel, attenuated the hypoglycemic effects of semaglutide (Supplemental Figure 13C) and reduced GLP-1 levels (Supplemental Figure 13D). Next, we tested whether avexitide could block serotonin-induced hypoglycemia (Supplemental Figure 14A). Treatment of mice with avexitide rapidly increased glucose levels in comparison with saline-injected mice (P = 0.012 at 15 minutes). However, avexitide was unable to block serotonin-induced hypoglycemia (Supplemental Figure 14B). Likewise, avexitide did not affect serotonin-induced increases in insulin and GLP-1 (Supplemental Figure 14, C and D). Thus, these data suggest that GLP-1 receptor–mediated signaling is not likely to be a primary mechanism mediating serotonin-induced hypoglycemia.

Serotonin-induced hypoglycemia in mice is blocked by serotonin receptor antagonism. First, we sought to investigate pathways by which serotonin induced hypoglycemia. Serotonin has pleiotropic effects on many aspects of metabolism, mediated via multiple receptor subtypes (16).

To test whether the effects of serotonin on glucose levels could be blocked by broad antagonism of serotonin receptor subtypes, the nonspecific antagonist cyproheptadine or an equal volume of saline was injected intraperitoneally 30 minutes before the mixed meal (Figure 6A). Cyproheptadine completely blocked the hypoglycemia induced by serotonin administration, with glucose of 170 mg/dL at 60 minutes versus 80 mg/dL with serotonin alone (P = 0.005) (Figure 6B). Interestingly, cyproheptadine pretreatment also blunted the increase in plasma serotonin as compared with saline at 15 (75%, P < 0.001), 30 (72%, P = 0.008), and 60 minutes (74%, P = 0.034) (Supplemental Figure 12A). Likewise, serotonin-induced increase in GLP-1 (1.7- to 2.1-fold, P < 0.001; Supplemental Figure 12B) was abolished by pretreatment with cyproheptadine (–1.9- to –2.1-fold, P < 0.001 for all; Supplemental Figure 12B). These results suggest that increased plasma serotonin may contribute to regulation of postprandial glucose metabolism, potentially via effects to increase both GLP-1 and insulin secretion.

We next investigated whether inhibition of specific serotonin receptor subtypes could block serotonin-induced hypoglycemia, selecting inhibitors of 5-HTR3 and 5-HTR2, given their demonstrated role in metabolism and insulin secretion (17–19). The 5-HTR3 antagonist ondansetron (3 mg/kg) was administered 30 minutes before injection of serotonin (20 mg/kg) or an equal volume of saline (Supplemental Figure 15A). Ondansetron did not impact serotonin-induced reductions in glucose (Supplemental Figure 15B), nor was it able to block serotonin-stimulated increases in insulin (Supplemental Figure 15C) or GLP-1 (Supplemental Figure 15D).

We next tested the impact of the 5-HTR2 antagonist ketanserin, injecting male mice with ketanserin (5 mg/kg) 30 minutes before serotonin administration (20 mg/kg) (Figure 6C). Ketanserin abolished the impact of serotonin, increasing blood glucose by 86% (P < 0.05) (Figure 6D). Likewise, ketanserin reduced serotonin-stimulated insulin levels by 44% (P < 0.05) (Figure 6E) and reduced serotonin-stimulated GLP-1 levels by 33% (P < 0.05) (Figure 6F). These data suggest that 5-HTR2 may mediate glycemic effects of serotonin and that inhibition could be tested as a potential strategy for PBH.