L-valine is a powerful stimulator of GLP-1 secretion in rodents and stimulates secretion through ATP-sensitive potassium channels and voltage-gated calcium channels

GLP-1 is a key regulator of glucose homeostasis and energy intake, and pharmacologic GLP-1 receptor agonists effectively treat type 2 diabetes and obesity. Enhancing endogenous GLP-1 secretion via nutrition could engage local sensory pathways and co-secreted gut hormones (e.g., PYY, oxyntomodulin) and may reduce adverse effects. However, mechanisms by which protein digestion products stimulate GLP-1 remain incompletely defined due to the complexity of peptides and amino acids generated during digestion. Prior perfusion studies identified L-valine as a particularly potent luminal stimulator of GLP-1 in the proximal rat small intestine, prompting investigation of the molecular basis. The study tested whether L-valine’s GLP-1 stimulation depends on (1) Na+-coupled uptake and associated depolarization, (2) intracellular oxidation leading to closure of KATP channels, and/or (3) activation of voltage-gated Ca2+ channels, and assessed in vivo efficacy and distal gut effects.

Previous work showed protein hydrolysates stimulate GLP-1 in perfused rat intestine via absorptive processes, implicating intracellular sensing (Modvig et al., 2019). Among amino acids, L-valine was distinguished as the strongest luminal GLP-1 secretagogue in rat small intestine (Modvig et al., 2021). Glucose-stimulated GLP-1 secretion often depends on SGLT1-mediated Na+ co-transport and activation of voltage-gated Ca2+ channels (Kuhre et al., 2015), and metabolism with KATP channel closure has been implicated in carbohydrate sensing in L-cells (Reimann et al., 2008). Amino acid transport across the intestine involves apical B0 transporters (high affinity for L-valine) and proton-coupled systems (PAT1), though L-valine is not a PAT1 substrate. Human data on amino acid-driven GLP-1 release are variable (e.g., glutamine and arginine can increase GLP-1), and one study reported no clear glucoregulatory or appetite effects of intraduodenal L-valine in healthy men (Elovaris et al., 2019), with GLP-1 not measured.

Study design combined in vivo mouse experiments, ex vivo isolated perfused rat small intestine and colon, and in vitro GLUTag L-cell assays. In vivo: Male C57BL/6JRj mice (12 weeks; n=8/group for L-valine and D-glucose; n=6 control) were fasted 5 h. At −30 min, oral neprilysin inhibitor sacubitril (0.3 mg/kg) and DPP-4 inhibitor sitagliptin (10 mg/kg) were given to preserve active GLP-1. At 0 min, mice received oral L-valine (1 g/kg; 20 µL/g), D-glucose (2 g/kg; 20 µL/g), or water, mixed with acetaminophen (100 mg/kg) to assess gastric emptying. Tail blood was sampled at 0, 5, 10, and 30 min (plus glucose at 60 min) for glucose, active GLP-1 (ELISA), and total amino acids. Ex vivo perfusion: Male Wistar rats (~250 g) underwent isolation and single-pass vascular perfusion (small intestine ~37 cm; flow 7.5 mL/min; colon ~10 cm; flow 3 mL/min) with oxygenated modified Krebs-Ringer buffer (37 °C) containing 3.5 mM glucose, 0.1% BSA, 5% dextran T-70, 5 mM fumarate/pyruvate/glutamate, and 10 µM IBMX. Luminal saline flow was 0.25 mL/min (small intestine) or 0.15 mL/min (colon). Protocols included two luminal L-valine stimulations (50 mM; minutes 11–25 and 46–60) with 20-min washout; positive control bombesin (BBS; 10 nM) was administered intravascularly at the end. For Na+ depletion, luminal NaCl was replaced with isosmotic KCl (0.11%) during minutes 26–60. For Ca2+ channel involvement, nifedipine (10 µM) was infused intravascularly from minute 36–60. For KATP testing, diazoxide (250 µM) was infused intravascularly from minute 36–60. Venous effluent was collected each minute for GLP-1 (and in colon experiments, PYY) by RIA; total L-amino acids in effluent assessed absorption. In vitro GLUTag assays: Cells (passages 31–34) were cultured in DMEM (5.6 mM glucose, 10% FBS, pen/strep, Glutamax). For secretion assays, cells in KRH buffer (no glucose/albumin) were exposed to L-valine (50 µM–50 mM) vs baseline KRH with 5.6 mM glucose; supernatants were collected for GLP-1 measurement (mid-region antibody). For calcium imaging, cells loaded with Fluo-4 AM were stimulated with L-valine (e.g., 10 and 50 mM), with inhibitors applied in some wells: nifedipine (2 µM), diazoxide (100 µM), EDTA (1 mM). Fluorescence was recorded for 90 s. Statistics: Perfusion data presented as mean ± SEM; 10-min baseline and last 10 min of stimulation used to compute total and baseline-subtracted responses. Paired/unpaired t-tests or ANOVA with appropriate post hoc tests were used. In vivo time-course by two-way ANOVA with Šídák multiple comparisons; AUCs by one-way ANOVA with Tukey. Significance at P<0.05.

- Oral L-valine (1 g/kg) in male mice increased plasma active GLP-1 to levels comparable to oral D-glucose (2 g/kg); GLP-1 tAUC0–60 min did not differ between L-valine and D-glucose (P>0.05). Peak active GLP-1: 5 min for L-valine (21.84 ± 5.9 pM) vs 10 min for glucose (17.76 ± 3.4 pM). Plasma total amino acids rose rapidly at 5 min after L-valine (P<0.0001), indicating rapid absorption. Gastric emptying (acetaminophen AUC) did not differ among groups (P>0.05). - Perfused rat small intestine: Luminal L-valine (50 mM) robustly stimulated GLP-1 (P<0.0001). Baseline-subtracted second L-valine response averaged 6.1 ± 3.9 pM (vs first 11.2 ± 4.9 pM; P<0.002). BBS consistently confirmed tissue responsiveness. - Voltage-gated Ca2+ channels: Nifedipine (10 µM) markedly reduced L-valine–stimulated GLP-1 during the second stimulation vs control second stimulation (1.2 ± 1.9 pM vs 6.1 ± 3.9 pM; P<0.01; n=7–8). In GLUTag cells, L-valine increased intracellular Ca2+ at 10 and 50 mM (P<0.05). - Sodium independence: Replacing luminal NaCl with KCl did not alter L-valine–induced GLP-1 (baseline-subtracted: 6.0 ± 2.1 pM vs 6.1 ± 3.9 pM; P>0.05; n=6–8), arguing against Na+-coupled uptake as the depolarizing trigger. - KATP channels: The KATP opener diazoxide (250 µM) strongly inhibited L-valine–induced GLP-1 (baseline-subtracted: 0.9 ± 0.8 pM vs 6.1 ± 3.9 pM; P<0.01; n=6–8). In GLUTag cells, diazoxide, nifedipine, and EDTA each reduced the L-valine–induced Ca2+ response. - Perfused colon: Luminal L-valine (50 mM) tended to increase GLP-1 (53.7 ± 20.8 pM baseline vs 81.4 ± 31.2 pM; P=0.0575) and PYY (21.2 ± 3.3 pM vs 39.3 ± 11.8 pM; P>0.05). L-valine was absorbed (total amino acids increased from 388.5 ± 55.8 µM to 603.7 ± 104.4 µM; P<0.05), but less than in proximal small intestine in prior work, aligning with weaker hormone responses.

The work addresses how L-valine stimulates GLP-1 secretion, demonstrating that oral L-valine is as effective as glucose in elevating active GLP-1 in mice and that luminal L-valine potently triggers GLP-1 release in perfused proximal small intestine. Mechanistically, L-valine-induced secretion depends on membrane depolarization leading to activation of voltage-gated Ca2+ channels, as nifedipine strongly attenuated the response and L-valine elevated intracellular Ca2+ in GLUTag cells. Depolarization does not require Na+-coupled uptake, given preserved secretion under luminal Na+ depletion, contrasting with glucose’s SGLT1-dependent mechanism. Instead, findings support a metabolism-dependent pathway in L-cells whereby L-valine metabolism increases ATP/ADP, closes KATP channels, reduces K+ efflux, depolarizes the membrane, and opens Ca2+ channels to drive exocytosis. In the colon, modest absorption and correspondingly weaker GLP-1 and PYY responses suggest proximal absorption is important for L-valine’s efficacy, though microbial metabolites in the distal gut may contribute to hormone secretion under physiologic conditions. These insights refine understanding of protein-derived nutrient sensing by L-cells and identify L-valine as a robust amino acid stimulus engaging KATP and Ca2+ channel signaling, informing strategies to nutritionally amplify endogenous GLP-1 and co-secreted hormones.

L-valine is a potent stimulator of GLP-1 secretion in rodents in vivo and ex vivo. The data support a mechanism wherein intracellular metabolism of L-valine leads to closure of KATP channels, membrane depolarization, and activation of voltage-gated Ca2+ channels, independent of Na+-coupled luminal uptake. L-valine also tends to enhance GLP-1 and PYY secretion in the colon, albeit less robustly, consistent with lower distal absorption. Future studies should evaluate L-valine’s capacity to augment postprandial GLP-1 and glycemic control in humans (e.g., as a preload), delineate contributions of alternative uptake pathways (e.g., H+-coupled transport), explore sex and strain differences, and define dose-response relationships within physiologic concentration ranges.

- In vitro GLUTag cells are non-polarized, potentially altering amino acid uptake routes relative to native apical/basolateral transporters. - In perfused small intestine, the second L-valine response was consistently smaller than the first, likely reflecting incomplete washout or desensitization with the 20-min interval. - The luminal L-valine concentration (50 mM) exceeds typical postprandial concentrations for a single amino acid, limiting direct physiological extrapolation. - Only male Wistar rats and male C57BL/6JRj mice were studied; sex and strain differences were not assessed. - Human efficacy was not tested; translational relevance to human GLP-1 secretion and glycemic outcomes remains to be established. - Colon experiments showed modest absorption and non-significant hormone increases, limiting conclusions about distal gut effects.