Intestinal serine protease inhibition increases FGF21 and improves metabolism in obese mice

Abstract

Trypsin is the major serine protease responsible for intestinal protein digestion. An inhibitor, camostat (CS), reduced weight gain, hyperglycemia, and dyslipidemia in obese rats; however, the mechanisms for these are largely unknown. We reasoned that CS creates an apparent dietary protein restriction, which is known to increase hepatic fibroblast growth factor 21 (FGF21). Therefore, metabolic responses to CS and a gut-restricted CS metabolite, FOY-251, were measured in mice. Food intake, body weight, blood glucose, branched-chain amino acids (LC/MS), hormone levels (ELISA), liver pathology (histology), and transcriptional changes (qRT-PCR) were measured in ob/ob, lean and diet-induced obese (DIO) C57BL/6 mice. In ob/ob mice, CS in chow (9-69 mg/kg) or FOY-251 (46 mg/kg) reduced food intake and body weight gain to a similar extent as pair-fed mice. CS decreased blood glucose, liver weight, and lipidosis and increased FGF21 gene transcription and plasma levels. In lean mice, CS increased liver FGF21 mRNA and plasma levels. Relative to pair feeding, FOY-251 also increased plasma FGF21 and induced liver FGF21 and integrated stress response (ISR) transcription. In DIO mice, FOY-251 (100 mg/kg po) did not alter peak glucose levels but reduced the AUC of the glucose excursion in response to an oral glucose challenge. FOY-251 increased plasma FGF21 levels. In addition to previously reported satiety-dependent (cholecystokinin-mediated) actions, intestinal trypsin inhibition engages non-satiety-related pathways in both leptin-deficient and DIO mice. This novel mechanism improves metabolism by a liver-integrated stress response and increased FGF21 expression levels in mice. NEW & NOTEWORTHY Trypsin inhibitors, including plant-based consumer products, have long been associated with metabolic improvements. Studies in the 1980s and 1990s suggested this was due to satiety hormones and caloric wasting by loss of protein and fatty acids in feces. This work suggests an entirely new mechanism based on the lower amounts of digested protein available in the gut. This apparent protein reduction may cause beneficial metabolic adaptation by the intestinal-liver axis to perceived nutrient stress.

Keywords: camostat; gluconeogenesis; integrated stress response; liver; obesity; protein dilution; small intestine; triglycerides; type 2 diabetes.

Fig. 1.

Metabolic changes in ob/ob mice given a range of doses of camostat (CS) in chow (0.08, 0.25, and 0.8 mg/g) for 7 days. A: compound intake resulted in average daily doses of 9.2, 25.5, and 69.0 mg/kg CS. B: food intake was reduced and maintained at a lower level through day 7 in CS-treated (69 and 26 mg·kg⁻¹·day⁻¹) mice; pair-fed mice (chow containing vehicle) were matched for the highest CS dose. C: %decreased body weight was similar in both CS and pair-fed mice compared with vehicle-treated mice. D: at termination, fasted blood glucose was reduced in 69 mg/kg group compared with pair-fed, but similar in vehicle, pair-fed and the lower CS-dosed groups. Terminal plasma insulin levels (E) and total glucagon-like peptide-1 (GLP1; F) were similar across the groups. G: fecal protein levels were increased by all CS doses relative to pair-fed mice. ○, Vehicle; ■, CS (9, 26, and 69 mg/kg); ◇ and dashed line, Pair-Fed; n = 8/group. Data are illustrated as box-and-whisker plots with individual plots in this and all subsequent figures. Bars indicate significance level by ANOVA with Tukey’s or Dunnett’s posttest. Percent weight loss correlated with fasted blood glucose (H) and plasma triglyceride (TG) levels (G).

Fig. 2.

Liver changes in ob/ob mice after 7 days of camostat (CS) in chow. A: liver weight (as %body weight) was reduced by the highest dose of CS compared with vehicle and pair-fed groups, where pair-fed was also lower than vehicle weight. P value by ANOVA and Tukey’s posttest comparisons. B: liver weight was correlated with 5-h-fasted blood glucose levels in vehicle- and CS-treated groups. C: CS-treated mice had lower lipidosis grade compared with pair-fed in hematoxylin-eosin-stained livers. P value by Kruskal-Wallis ANOVA and Dunn’s posttest comparison. D: representative digital images illustrate decreased size of livers in CS-treated mice (69 mg/kg) compared with vehicle-treated mice. Widespread vacuolization and diffuse lipid droplet accumulation noted in vehicle-treated is reduced in CS-treated examples, also shown in insets at higher-power magnification. Scale bars, 2.5 mm at low-power magnification and 250 µm in insets.

Fig. 3.

Metabolic changes in ob/ob mice are recapitulated after 24-h treatment with camostat (CS; 69 mg/kg in feed) with tissue transcriptional responses. Reduced food intake (A), decreased body weight gain (B), decreased fed blood glucose (C), and increased fecal protein (D) compared with vehicle-treated. P value by Student’s t-test, n = 8/group. In a separate repeat experiment (n = 6/group), tissue was obtained for transcriptional level of metabolic genes. E: liver had relatively reduced gluconeogenic mRNA [glucose-6-phosphatase catalytic subunit (G6PC)] and increased fibroblast growth factor 21 (FGF21) mRNA. F: pancreas had increased expression of trypsinogens [protease, serine 1 and 3 (PRSS1, PRSS3)], serine peptidase inhibitor Kazal type (SPINK), and FGF21 mRNA. G: plasma FGF21 levels were increased by CS. H: ileum mucosa had increased transcription of multiple metabolic target genes. I: duodenal mucosa transcriptional responses were unaltered using the same panel as for ileum, except for a small increase in peptide transporter 1 (SLC15A1). P < 0.05 by unpaired Student’s t-test. ACLY, ATP citrate lyase; PC, phosphocreatine; PCK1, phosphoenolpyruvate carboxykinase-1; SCT, secretin; GIP, gastric inhibitory polypeptide; SST, somatostatin; GAST, gastrin; PGC1α, peroxisome proliferator-activated receptor-γ coactivator 1α; LCT, long-chain triglyceride; ACLY, ATP citrate lyase; ISG15, interferon-stimulated gene 15; AHR, aryl hydrocarbon receptor; FXR, farsenoid X receptor.

Fig. 4.

In lean C57BL/6 mice, 24-h camostat (CS) treatment (69 mg/kg) compared with vehicle reduced food intake (A), decreased body weight (B), and increased liver fibroblast growth factor 21 (FGF21) mRNA fivefold (C) with increased integrated stress response (ISR) target gene transcription, such as asparagine synthetase (ASNS). ATP citrate lyase (ACLY) was the only gluconeogenic gene decreased. D: plasma FGF21 was detected in all CS-treated mice, whereas FGF21 was beneath quantification threshold of 98.8 pg/ml in vehicle-treated (shown as ½ lower limit of quantification). E: plasma branched-chain amino acids (BCAA) valine, isoleucine, and leucine were unchanged by CS in lean mice. Bar indicates P < 0.05 by unpaired Student’s t-test, n = 6/group. MIST1, muscle, intestine, and stomach expression 1; ATF4, activation transcription factor 4; GADD45, growth arrest and DNA damage inducible-45α; G6PC, glucose-6-phosphatase catalytic subunit; NUPR1, nuclear protein-1.

Fig. 5.

Metabolic changes in ob/ob mice administered gut-restricted camostat (CS) metabolite 4-(4-guanidinobenzoyloxy phenyl) acetic acid (FOY-251) in feed for 24 h compared with vehicle-treated mice. FOY-251 reduced food intake but did not increase kaolin intake (A), decreased body weight (B), increased fecal protein concentration (C), decreased fed blood glucose (D), and increased plasma fibroblast growth factor 21 (FGF21; E) (n = 7–8/group). F: in a separate group of mice given the same treatment, plasma branched-chain amino acids (BCAA) were reduced (n = 6/group). ○, vehicle; ■, FOY-251; Bar indicates P value by unpaired Student’s t-test.

Fig. 6.

Metabolic effects in ob/ob mice after camostat (CS) metabolite (CSM) 4-(4-guanidinobenzoyloxy phenyl) acetic acid (FOY-251) in feed for 7 days compared with vehicle or pair-fed groups (n = 5–6/group). A: food intake was reduced in FOY-251-treated mice; pair-fed mice were matched to this. B: pair-fed and FOY-251 treatment reduced %body weight (BW) to the same extent. C: FOY-251 decreased terminal fed blood glucose (C) but terminal plasma insulin levels were not different between groups (D). E: FOY-251 treatment reduced liver weight compared with pair-fed mice. F: plasma fibroblast growth factor 21 (FGF21) levels were increased by FOY-251 compared with vehicle and pair-fed groups. G: plasma branched-chain amino acids (BCAA) were decreased by both FOY-251 and pair feeding relative to vehicle. ○, Vehicle; ■, FOY-251; □, Pair-Fed. Bar indicates P value; *P < 0.05 vs. pair-fed and FOY-251-treated groups, by one-way ANOVA and Tukey’s post hoc test, n = 6/group.

Fig. 7.

Transcriptional changes in various metabolic tissues in ob/ob mice from the same experiment as Fig. 6. A: liver ATP citrate lyase (ACLY) was decreased by camostat (CS) metabolite 4-(4-guanidinobenzoyloxy phenyl) acetic acid (FOY-251), whereas glucose-6-phosphatase catalytic subunit (G6PC) and phosphoenolpyruvate carboxykinase-1 (PCK1) were increased in the pair-fed group. B: in pancreas, fibroblast growth factor 21 (FGF21) mRNA was not significantly increased, whereas insulin (INS1) and glucagon (GCG) were strongly suppressed by FOY-251 treatment. C: liver FGF21 transcription was increased 15-fold, accompanied by induction of all integrated stress response target genes in FOY-251-treated mice. D: ileum mucosa of hormones (gastrin was removed because of widely variable fold increases due to very low control levels), gluconeogenic enzymes, and transcription factors and UCP1 in white adipose tissue (WAT) were not altered. F: in dorsal vagal complex (DVC), satiety hormone and vagal signaling peptide receptor mRNA was not altered by FOY-251 treatment, whereas G protein-coupled receptor 65 (GPR65) was decreased in the pair-fed group. Bar indicates P < 0.05 by unpaired Student’s t-test. PC, phosphocreatine; MIST1, muscle, intestine, and stomach expression 1; ATF4, activation transcription factor 4; NUPR1, nuclear protein-1; GADD45α, growth arrest and DNA damage inducible-α; ASNS, asparagine synthetase; SCT, ; SST, ; GIP, gastric inhibitory polypeptide; FXR, farsenoid X receptor; UCP1, uncoupling protein-1; NYP2R, neuropeptide Y2 receptor; CNR1, cannabinoid receptor 1; HCRTR1, hypocretin receptor 1; CCKRR, cholecystokinin A receptor; CCKRB, cholecystokinin receptor B; PAR2, protease-activated receptor 2; GLP1R, glucagon-like peptide-1 receptor.

Fig. 8.

Metabolic changes in diet-induced obese mice after camostat (CS) metabolite 4-(4-guanidinobenzoyloxy phenyl) acetic acid (FOY-251) given by oral gavage (100 mg/kg) for 4 days (A–F, n = 8/group) and in response to oral glucose challenge after a single dose (G–I, n = 10/group). In FOY-251-treated DIO mice, there was decreased food intake (A) and body weight gain (B) with increased fecal protein (C) compared with vehicle-treated mice. Plasma samples from bleeds taken 15 and 60 min after the last dose of FOY-251 had modestly reduced insulin at 60 min time point (D) and no change in total GLP1, glucagon-like peptide-1 (tGLP1; E) but markedly increased fibroblast growth factor 21 (FGF21) levels (F) at 15 and 60 min. Acute single dose of FOY-251 given before oral glucose challenge had (G) no effect on peak glucose levels (H) but modestly reduced the area under the curve (AUC) of the glucose excursion with increased plasma insulin levels (I) at 15 min compared with baseline.

Fig. 9.

Working model of the potential metabolic benefits of reducing protein digestion through a trypsin-fibroblast growth factor 21 (FGF21) axis. First, partially hydrolyzed protein in the presence of trypsin inhibition reduces formation of peptones, mimicking a state of intestinal protein dilution. This adds to trypsin inhibitor-evoked cholecystokinin (CCK) secretion to reduce food intake. Second, apparent protein dilution is detected by the liver, decreasing lipogenesis [(ACLY)] and substrate utilization and inducing integrated stress response (ISR) target genes to increase FGF21. In the pancreas, FGF21 mRNA may be locally induced in an insulin- and glucagon-independent fashion with no change in circulating insulin levels. Overall, branched-chain amino acid (BCAA) levels in obese mice are reduced to the same extent as in pair-fed and lean mice. These mechanisms could all contribute to reduced liver weight, liver lipidosis, and hyperglycemia. Third, circulating FGF21 primarily acts on central nervous system βklothoR1c to reduce food intake and would also be expected to increase energy expenditure, warranting further evaluation in future studies. Fourth, in large intestine, increased protein may alter bacterial nitrogen utilization and nutrient signaling, especially bioactives related to putrefaction. ACLY, ATP citrate lyase; G6PC, glucose-6-phosphatase catalytic subunit.