Research ArticleMetabolism

The short-chain fatty acid propionate increases glucagon and FABP4 production, impairing insulin action in mice and humans

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Science Translational Medicine  24 Apr 2019:
Vol. 11, Issue 489, eaav0120
DOI: 10.1126/scitranslmed.aav0120
  • Fig. 1 Propionate induces hyperglycemia in mice.

    (A) Time course of the change in blood glucose was analyzed in wild-type mice after intraperitoneal injection of pyruvate or propionate (at three different doses) (n = 4 mice per group, N = 2). (B and C) Propionate and pyruvate (15 mmol/kg each) were injected intraperitoneally (B) or administered orally (C) to female mice, and blood glucose was measured over 2 hours (n = 5 mice per group or n = 4 mice per group, respectively, N = 2; delivered dose was 15 mmol/kg). (D) Propionate was measured using gas chromatography–mass spectrometry (GC-MS) in the systemic circulation of mice at 0 and 20 min after oral administration of propionate and in the portal circulation 20 min after oral administration of propionate. Propionate was orally administered at a dose of 15 mmol/kg, 5 hours after food withdrawal (n = 5 mice per time point; experiment was performed once). (E and F) After a propionate bolus (1 g/kg), administered orally or rectaly to male mice, blood glucose and the appearance of radiolabeled propionate were measured in blood (n = 3 mice per group; experiment was performed once). (G) Glycemic response to intraperitoneal injection of propionate (15 mmol/kg) after 18 hours of fasting (n = 6 mice per group; experiment was done once). (H) Biochemical analysis of liver glycogen 15 min after intraperitoneal injection of propionate or pyruvate (15 mmol/kg) (n = 8 mice per group; administration was after a 5-hour fasting, N = 2). (I) Glycogen-loaded primary rat hepatocytes were treated with propionate (1 mM) or glucagon (100 nM) in substrate-free Dulbecco’s modified Eagle’s medium (DMEM) for 3 hours, and glucose appearing in the medium was measured using a glucose oxidase–based assay (n = 4 wells per treatment, N = 3). All results are reported as means ± SEM. Statistical differences between two groups were determined using unpaired two-tailed Student’s t test; differences among three or more groups were compared using one-way analysis of variance (ANOVA) and Tukey post hoc analysis. Responses to glucose tolerance tests between mice groups were compared using two-way ANOVA with Bonferroni post hoc analysis. *P < 0.05; **P < 0.005.

  • Fig. 2 Glucagon and FABP4 are required for propionate-induced hyperglycemia.

    (A and B) Plasma concentrations of glucagon and FABP4 were measured 30 min after propionate or pyruvate administration given 5 hours after food withdrawal (n = 8 male mice per group). All experiments were performed using either sodium propionate or sodium pyruvate (15 mmol/kg body weight) (N = 2). (C and D) Genetic deletion of the glucagon receptor in the liver (Gcgrfl/fl ΔLiver) or total genetic ablation of FABP4 protected mice from propionate-induced hyperglycemia as shown by blood glucose measurements (n = 4 male mice per group, n = 5 male mice per group, N = 2). (E) Tail vein injection of polyclonal antibody against FABP4 (0.2 mg/kg) resulted in an attenuated response to propionate as shown by blood glucose measurements (n = 4 male mice per group, N = 2). IgG, immunoglobulin G. (F) Reconstitution of circulating FABP4 using recombinant FABP4 (50 μg/kg) abolished the protective effect of Fabp4 genetic deficiency as shown by blood glucose measurements (n = 8 male mice per group). All results are reported as means ± SEM. Statistical differences between two groups were determined using unpaired two-tailed Student’s t test; responses to glucose tolerance tests between mice groups were compared using two-way ANOVA with Bonferroni post hoc analysis. *P < 0.05; **P < 0.005.

  • Fig. 3 The hormonal and metabolic effects of propionate are mediated by activation of the sympathetic nervous system.

    (A) Glucagon secretion was measured in isolated pancreatic islets from three wild-type C57/B6 mice. Glucagon was measured in the culture medium at low- and high-glucose concentrations in the presence or absence of 1 mM propionate (n = 15 islets for glucagon production assay). (B) Western blot (IB) showing the amount of FABP4 secreted from mouse adipose tissue explants at the indicated concentrations of propionate. Forskolin was used as a positive control (representative blot of n = 3). (C) Differentiated Neuro2A cells were treated with propionate or KCl (10 mM final), and the change in plasma membrane potential was measured (n = 8 wells per treatment; repeated with three consecutive passages of cells). ns, not significant. (D) Norepinephrine measurements were taken 15 min after intraperitoneal injection of propionate (15 mmol/kg) or PBS. Hexamethonium (Hex; 20 mg/kg) or PBS were intraperitoneally injected 7 min before the injection of either propionate or PBS (n = 5 mice per group, N = 2). (E) Blockade of sympathetic nervous system activity with phentolamine (Phent; 1 mg/kg), hexamethonium (20 mg/kg), or both (Hex + Phent) before intraperitoneal injection of propionate (15 mmol/kg), inhibited propionate-induced hyperglycemia as shown by blood glucose measurements. (F) Glucagon and (G) FABP4 plasma concentrations were collected at 30 min after intraperitoneal injection of propionate or PBS as described in (E) (n = 5 mice per group; experiment was done once). All results are reported as means ± SEM. Islet glucagon secretion was compared using a two-way ANOVA statistical test, and there was no statistical difference between treatment groups. Statistical differences between three or more groups were compared using one-way ANOVA and Tukey post hoc analysis. Responses to a glucose tolerance test between mouse groups were compared using two-way ANOVA with Bonferroni post hoc analysis. *P < 0.05; **P < 0.005.

  • Fig. 4 The metabolic and hormonal effects of propionate in human participants.

    (A) At 30 and 60 min after consumption of a meal supplemented with either placebo or 1 g of calcium propionate, plasma propionate was measured in 14 healthy participants by GC-MS. (B) At 30 min after meal consumption, plasma norepinephrine was assayed. Time course of the change in plasma glucagon (C) and serum FABP4 concentrations (D) from baseline were analyzed at 30-min intervals after the mixed-meal challenge. (E) The Matsuda insulin sensitivity index (ISI-M) was calculated using blood glucose and serum insulin during the mixed-meal test. (F) Time course of the change in serum insulin at 30-min intervals and serum C-peptide (G) at 30 min in response to the mixed meal. (H) Plasma glucose concentrations during the 240-min postprandial time course. The meal was consumed after an 8-hour fasting. All results are reported as means ± SEM. Statistical differences between two groups at indicated time points were compared using paired t test. Hormonal and glucose response to placebo or propionate throughout the entire sampling was compared using repeated-measures two-way ANOVA with Bonferroni post hoc analysis. *P < 0.05.

  • Fig. 5 Chronic propionate treatment induces FABP4-dependent weight gain and impairs glucose homeostasis in mice.

    (A) Weekly body weight measurements were taken during chronic treatment of mice with low-dose sodium propionate (150 μg/ml) or sodium chloride (91.2 μg/ml, to provide a molar equivalent of sodium as the experimental group) that was added to the drinking water (n = 7 mice per group, N = 2). (B) Whole-body fat mass of the two groups was determined by dual-energy x-ray absorptiometry at the end of the experiment. (C) Blood glucose measurements were taken in conscious animals 6 hours after food withdrawal at week 18 of treatment with either sodium propionate or sodium chloride. (D) Plasma glucagon measurements were taken in conscious animals 6 hours after food withdrawal at week 18 of treatment with either sodium propionate or sodium chloride. (E) Plasma FABP4 measurements were taken in conscious animals 6 hours after food withdrawal at week 18 of treatment with either sodium propionate or sodium chloride. (F) Plasma insulin measurements were taken in conscious animals 6 hours after food withdrawal at week 18 of treatment with either sodium propionate or sodium chloride. (G) Hyperinsulemic-euglycemic clamp studies were performed in conscious mice at the end of the intervention with either sodium propionate or sodium chloride (n = 8 animals per group). (H) Average glucose infusion rate in the clamp experiment during steady state. (I) Calculated endogenous glucose production rate in the clamp experiment during steady state. (J) Fabp4−/− mice were placed on chronic sodium propionate or sodium chloride treatment, and body weight was monitored weekly. (K) An insulin tolerance test (0.75 U/kg) was performed on Fabp4−/− mice (red lines) and wild-type littermate controls (black lines) after chronic propionate treatment (solid lines) or sodium chloride treatment as a control (dashed lines) (n = 8 animals per group). All results are reported as means ± SEM. Statistical differences between two groups were determined using unpaired two-tailed Student’s t test. Response to insulin tests between mouse groups and weight gain over time were compared using two-way ANOVA with Bonferroni post hoc analysis. *P < 0.05.

  • Fig. 6 Correlation of plasma propionate with insulin resistance in overweight and obese human participants.

    Plasma propionate was measured in 160 overweight or obese participants in the DIRECT study, at baseline and at 6 months after a dietary intervention. The study population was divided into tertiles (T1, T2, and T3) based on the change in circulating propionate from baseline to 6 months (red bars). The change in insulin resistance was assessed for each group using the HOMA-IR calculation (black bars). Correlations between plasma propionate concentrations and metabolic outcomes were performed by general linear models after adjustment for age, sex, dietary group, and baseline value of the respective outcome.

  • Table 1 Characteristics of participants in the clinical trial.

    Demographic, anthropometric, and biochemical characteristics of the 14 healthy participants randomized to a double-blind, placebo-controlled, crossover study assessing the effects of the food preservative propionate on postprandial metabolism after an 8-hour fasting. Baseline measurements were taken at the screening visit. WBC, white blood cells; TSH, thyroid-stimulating hormone.

    n14
    Mean age (years)41 ± 14
    Male, n (%)9 (64%)
    Race, n (%)
      Caucasian9 (64%)
      African-American2 (14%)
      Other3 (22%)
    Body weight (kg)74 ± 10.3
    BMI (kg/m2)23.7 ± 2.3
    Blood pressure (mmHg)
      Systolic119 ± 14
      Diastolic75 ± 10
    Fasting glucose (mg/dl)89 ± 8
    HbA1c (%)5.5 ± 0.3
    Hemoglobin (g/dl)13.6 ± 1.2
    WBC (cells/mm3)5.6 ± 1.2
    Creatinine (mg/dl)0.9 ± 0.1
    TSH (mIU/liter)1.9 ± 0.9

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/489/eaav0120/DC1

    Fig. S1. Propionate induces hyperglycemia and hyperinsulinemia in mice.

    Fig. S2. Fabp4 deficiency does not affect glucagon secretion in response to propionate administration.

    Fig. S3. The effect of sympathetic blockade on blood glucose.

    Fig. S4. Results of hyperinsulinemic-euglycemic clamp studies.

    Fig. S5. Proposed model for data presented in this study.

    Data file S1. Source data for Figs. 1 to 6 and figs. S1 to S4.

  • The PDF file includes:

    • Fig. S1. Propionate induces hyperglycemia and hyperinsulinemia in mice.
    • Fig. S2. Fabp4 deficiency does not affect glucagon secretion in response to propionate administration.
    • Fig. S3. The effect of sympathetic blockade on blood glucose.
    • Fig. S4. Results of hyperinsulinemic-euglycemic clamp studies.
    • Fig. S5. Proposed model for data presented in this study.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Source data for Figs. 1 to 6 and figs. S1 to S4.

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