A metabolic dysregulator in hiding Propionate is a short-chain fatty acid that is a commonly used food preservative. Tirosh et al. now show that propionate induces postprandial hyperglycemia in mice, resulting in a counter-regulatory hormonal response. Propionate increased norepinephrine release by the sympathetic nervous system, leading to an increase in circulating glucagon and the adipokine fatty acid–binding protein 4 (Fabp4), which jointly induced liver glycogenolysis and compensatory hyperinsulinemia. Long-term exposure of mice to a daily low dose of propionate resulted in a gradual weight gain and insulin resistance. Studies in humans highlight the potential contribution of propionate in the diet to the development of insulin resistance and obesity.

Abstract The short-chain fatty acid propionate is a potent inhibitor of molds that is widely used as a food preservative and endogenously produced by gut microbiota. Although generally recognized as safe by the U.S. Food and Drug Administration, the metabolic effects of propionate consumption in humans are unclear. Here, we report that propionate stimulates glycogenolysis and hyperglycemia in mice by increasing plasma concentrations of glucagon and fatty acid–binding protein 4 (FABP4). Fabp4-deficient mice and mice lacking liver glucagon receptor were protected from the effects of propionate. Although propionate did not directly promote glucagon or FABP4 secretion in ex vivo rodent pancreatic islets and adipose tissue models, respectively, it activated the sympathetic nervous system in mice, leading to secretion of these hormones in vivo. This effect could be blocked by the pharmacological inhibition of norepinephrine, which prevented propionate-induced hyperglycemia in mice. In a randomized, double-blind, placebo-controlled study in humans, consumption of a propionate-containing mixed meal resulted in a postprandial increase in plasma glucagon, FABP4, and norepinephrine, leading to insulin resistance and compensatory hyperinsulinemia. Chronic exposure of mice to a propionate dose equivalent to that used for food preservation resulted in gradual weight gain. In humans, plasma propionate decreased with weight loss in the Dietary Intervention Randomized Controlled Trial (DIRECT) and served as an independent predictor of improved insulin sensitivity. Thus, propionate may activate a catecholamine-mediated increase in insulin counter-regulatory signals, leading to insulin resistance and hyperinsulinemia, which, over time, may promote adiposity and metabolic abnormalities. Further evaluation of the metabolic consequences of propionate consumption is warranted.

INTRODUCTION According to the International Diabetes Federation, about 415 million people worldwide suffer from diabetes (1). Despite extensive research efforts, medical and surgical treatments, prevention programs, and public health policies designed to curb this trend, the rate of diabetes incidence is projected to further increase by more than 50% by 2040, becoming one of the greatest threats to global health (1). The dramatic increase in the incidence of obesity and diabetes over the past 50 years cannot be attributed solely to genetics and thus must involve contributing environmental and dietary factors. Among these, one factor that warrants attention is the extensive use of chemicals in the processing, preservation, and packaging of foods. It was recently suggested that the lack of evidence linking the wide use of chemicals and food additives to metabolic health is due to the absence of detailed studies evaluating these possibilities (2). Propionate (propionic acid), a naturally occurring short-chain fatty acid (SCFA), is a potent mold inhibitor that is widely used as a food preservative in cheeses and baked goods, as well as in animal feeds and artificial flavorings (3, 4). The metabolic actions of propionate were first described in 1912 by Ringer, who demonstrated a significant increase in glucose production after administration of propionate to dogs and concluded that this three-carbon molecule is converted to glucose through gluconeogenesis (5), although he also recognized that more glucose was produced than could be theoretically explained by stoichiometric conversion of propionate to glucose (5). Subsequently, propionate was shown to strongly stimulate endogenous glucose production in other mammals (6–8). Given the unique property of propionate to increase glucose production, it is widely used as an energy source for dairy cows and sheep to increase the concentration of glucose in milk (9). Recently, elegant studies using labeled propionate demonstrated that the direct conversion to glucose could explain no more than 5% of the increase in endogenous glucose production observed in propionate-infused rats, and the remainder was attributed to increased pyruvate carboxylase activity through an, as of now, unclear mechanism (7). In a small study in healthy human volunteers, propionate added to bread as a food preservative resulted in higher postprandial insulin compared to placebo-supplemented bread, suggesting a potential induction of postmeal insulin resistance (10). The hyperglycemia and hyperinsulinemia observed after exposure to exogenous propionate are somewhat in contrast to the beneficial metabolic effects attributed to endogenously produced propionate and other SCFAs. In the colon, these molecules are produced primarily by fermentation of undigested carbohydrates (11, 12). Several health-related benefits of dietary fibers such as increased postmeal satiety and decreased body weight and fat mass have been attributed to the production of SCFAs from fermentation (13, 14). However, the direct biological and metabolic effects of SCFAs, in general, and propionate, in particular, are not defined, and their potential link to human obesity and metabolic alterations remains to be established. For example, one study in humans showed that fecal concentrations of propionate, which reflect that in the portal and systemic circulation (12), were found to directly correlate with body mass index (BMI) (15). In an independent study, higher amounts of propionate and its intestinal transporter were found in overweight and obese participants compared to lean volunteers (16). This observation also has been reported recently in obese children (17). Moreover, small interventional trials conducted in humans failed to demonstrate any beneficial effects of propionate administration (18, 19). Although one study reported reduced postprandial glucose concentrations after propionate treatment, this was found to be secondary to impaired digestion of the meals and not because of improved glucose tolerance (20). Despite decreased absorption, propionate administration resulted in increased triglycerides and decreased high-density lipoprotein cholesterol, a common lipid abnormality observed in insulin-resistant states (21). Here, we report that propionate induced an increase in hepatic glucose production in mice, mediated by activation of the sympathetic nervous system, and a subsequent surge in circulating concentrations of fasting counter-regulatory hormones: norepinephrine, glucagon, and fatty acid–binding protein 4 (FABP4 or aP2). Mice lacking either Fabp4 or the liver glucagon receptor were resistant to the effects of propionate and did not show an increase in endogenous glucose production. To assess the potential translational impact of our observations, we conducted a double-blinded, placebo-controlled crossover study in healthy human volunteers. The addition of propionate to a mixed meal at a concentration similar to that used as a food preservative resulted in an increase in postprandial glucagon, FABP4, and norepinephrine concentrations compared to a placebo-supplemented meal, leading to an increase in blood glucose and compensatory hyperinsulinemia. Chronic treatment of mice with a low dose of propionate, equivalent to the amount used in food preservation, resulted in increased adiposity and insulin resistance.

DISCUSSION In this study, we report that exposure to the SCFA propionate, a food preservative, led to a rapid activation of the sympathetic nervous system and concomitantly an increase in the fasting hormones, glucagon and FABP4, in the postprandial state in mice. This increase resulted in enhanced endogenous glucose production, primarily most likely due to hepatic glycogenolysis, leading to hyperglycemia and compensatory hyperinsulinemia. Human consumption of propionate at a dose used to extend shelf life and preserve food was sufficient to reproduce the hormonal response to acute propionate exposure observed in mice. Furthermore, chronic exposure of mice to an equivalent daily propionate dose resulted in an increase in plasma concentrations of the insulin counter-regulatory hormones, glucagon and FABP4, and the development of insulin resistance, hyperinsulinemia, and gradual weight gain. The relevance of propionate to insulin resistance and obesity in humans was also suggested in a large, long-term, dietary interventional study (DIRECT), in which the reduction in plasma propionate in response to a weight-loss diet was independently associated with improved insulin sensitivity. The unfavorable metabolic effects that we observed upon both acute and chronic administration of propionate contrast with some reports of metabolic benefits attributed to propionate. Some studies have demonstrated that propionate suppresses food intake (30–32), inhibits lipolysis, and reduces plasma fatty acid content (18, 33, 34). However, these effects were reported when high concentrations of propionate (>4% of food) were used, more than 10-fold higher than the concentrations used for preserving foods. In studies where lower concentrations of propionate [0.5 to 2% (w/w) of food] were used, no beneficial effects could be demonstrated (32). In addition, despite the suggested favorable metabolic effects associated with gut microbiota–derived SCFAs, mice lacking one of the potential propionate receptors (the G protein–coupled receptor 43) were protected against the effects of a high-fat diet with a lower body fat mass and improved glucose control and insulin sensitivity (35). Reciprocally, a recently published study in Toll-like receptor 5–deficient mice demonstrated that the metabolic pathologies previously reported in this model are mediated by increased portal delivery of gut microbiota–derived propionate and other SCFAs to the liver (36). Furthermore, in line with our findings, Perry et al. (7), directly measured pyruvate cycling relative to mitochondrial pyruvate metabolism in rats and demonstrated that propionate administration increased this ratio by ~30-fold, with a concomitant increase in the rate of endogenous glucose production up to 100%. Thus, a combination of multiple mechanisms may account for the increase in endogenous glucose production observed after propionate administration to rodents. These include activation of the sympathetic nervous system leading to glucagon and FABP4 release and glycogenolysis, as well as direct activation of gluconeogenesis by increased pyruvate cycling. Thus, the sensitivity of the endogenous glucose production machinery to propionate not only makes propionate an unsuitable tracer to assess hepatic glucose metabolism (as suggested by Perry et al.) (7) but also indicates that propionate may act as a “metabolic disruptor” in the postprandial state when added to human foods. Data in humans also indirectly support a detrimental rather than a beneficial metabolic effect of propionate. Association studies in humans have indicated a relationship between propionate and an increase in BMI (15–17). Consistent with our findings (Fig. 4), the interventional study showed a higher postprandial insulin concentration with similar blood glucose concentration in healthy participants consuming propionate-containing bread (10). In addition, impaired postprandial suppression of SCFAs was also observed after eating propionate-containing bread, suggesting inefficient suppression of adipose tissue lipolysis, a condition that is known to result in augmented adipocyte release of FABP4 (24). Neither catecholamines nor glucagon, which stimulate both lipolysis and FABP4 release (22, 24), were measured in these studies (10). Together, the current literature suggests that orally delivered propionate does not mimic the beneficial metabolic effects attributed to SCFAs derived from bacteria in the colon and may result in adverse metabolic effects, including insulin resistance and glucose intolerance. These differential effects of oral propionate versus colonic propionate were also apparent in our tracer experiments (Fig. 1, E and F). This apparent discrepancy may be explained by different doses and routes of administration and local effects of propionate on proximal enterocytes versus distal colonocytes, as has been recently suggested for acetate (37). This apparent discrepancy may also be explained by the interactions of propionate and the colonic mucosa with other SCFAs and metabolites produced by the gut microbiota. The observation of a potential increase in endogenous glucose production leading to postprandial hyperinsulinemia is of concern, especially given the addition of propionate to processed foods and the compelling evidence that chronic hyperinsulinemia can drive obesity and metabolic abnormalities (38–40). Additional studies in larger human populations, with longer exposure to various doses of propionate, are needed to better elucidate the various metabolic effects of propionate in humans. An additional conclusion arising from our work is that there is an apparent interaction between the biological activities of both FABP4 and glucagon. The inability of propionate to induce hyperglycemia when Fabp4 was either genetically deleted or pharmacologically neutralized, despite an intact increase in plasma glucagon, is intriguing. This finding suggests that FABP4 may be required for glucagon-induced glycogen breakdown and may serve as a bona fide counter-regulatory endocrine signal. Glucagon bioactivity has not been evaluated in the Fabp4−/− mouse, but this may provide more insight into the type 2 diabetes-resistant phenotype of these mice under high-fat diet conditions. Unraveling the potential biological and mechanistic interactions of FABP4 and glucagon represents an important area of future investigation. Our study has several limitations. First, the acute metabolic effects of propionate observed in the human study and the association between insulin resistance and propionate concentration in the DIRECT study do not demonstrate a direct causal relationship between oral propionate consumption and the global epidemics of obesity and diabetes. In addition, we also did not examine the metabolic impact of chronic dietary propionate exposure in humans. Larger interventional studies targeting a reduction in environmental propionate and assessing metabolic and anthropometric outcomes are necessary to fully translate our findings in mice to humans. In addition, blood propionate concentrations reflect not only the systemic absorption of exogenous propionate (environmental exposure and production by gut bacteria) but also endogenously produced propionate. Propionate is the degradation product of some amino acids and of odd-chain fatty acids. Thus, blood propionate is a composite of various metabolic pathways and we cannot estimate the differential contribution of propionate-based preserved foods on systemic propionate concentrations. We were able to demonstrate that even trace amounts of propionate added to a single mixed meal were sufficient to rapidly increase blood propionate concentrations. Last, participants in the acute study were healthy, nonobese, and normoglycemic. Thus, they were able to mount a hyperinsulinemic response to overcome (at least in part) the postprandial insulin resistance induced by propionate. Therefore, the potential hyperglycemic effects of propionate-containing foods on prediabetic and overtly diabetic participants cannot be concluded from this study. Our findings may have implications for the current practice of food preservation. Given that the U.S. Food and Drug Administration has declared propionate to be generally recognized as safe with no known adverse effects, there is currently no limitation on its utilization other than as required by good manufacturing practice (3). Here, we report that exogenous propionate leads to a rapid activation of the sympathetic nervous system, resulting in an increase in both glucagon and FABP4. The increase of both of these fasting hormones in the postprandial state drives enhanced endogenous glucose production, likely due to glycogenolysis, leading to hyperglycemia and compensatory hyperinsulinemia. The hormonal responses to high-dose propionate observed in mice were also observed in humans given a much lower dose that was equivalent to that in processed foods, although the hyperglycemic response was milder. Nonetheless, repeated daily exposure to propionate for prolonged periods, as evident in our chronic propionate treatment of mice, may have important implications for public health and should stimulate a renewed interest in examining the potential actions and underlying mechanisms associated with food components such as propionate in humans. There are alternatives that could be used for food preservation [for review, see (41)], and if those molecules prove to be neutral in their metabolic activities, then simple alterations in manufacturing practices may yield public health benefits.

MATERIALS AND METHODS Study design The objectives of the study were to assess whether the commonly used food preservative propionate affects postprandial glucose metabolism, both in mice and in humans, and to identify its mechanism of action. Experiments conducted in mice included glucose tolerance tests in response to propionate or pyruvate and blood biochemistry measurements after test compound administration. The Harvard Medical Area Standing Committee on Animals approved all in vivo studies. Sample size for mouse groups was chosen on the basis of pilot experiments that ensured a power of 90% and a significance of 5%. All samples were included in the analysis unless they fell more than 2 SDs from the mean. No randomization was used for animal studies. Investigators analyzing data were blinded to mouse genotype and treatment groups. Individual level data for the mouse experiments are included in data file S1. We conducted a double-blind, randomized, placebo-controlled, crossover clinical study (#NCT 01889446) to test whether propionate, given as a food supplement to humans, resulted in altered postprandial metabolism. The rationale was to use an amount of propionate that was similar to that used for food preservation. Thus, we supplemented a 500-kcal propionate-free meal with 1000 mg of calcium propionate or placebo as detailed below. Inclusion criteria for the study were age of 18 to 65 years, good health as evidenced by history and physical exam, and BMI of 20 to 29.9 kg/m2. Exclusion criteria were fasting plasma glucose > 110 mg/dl, HbA1c > 6.0%, current illness other than treated hypothyroidism, blood pressure > 135/85 or systolic blood pressure < 90 mmHg, hepatic disease (transaminase more than three times normal), renal impairment (creatinine clearance < 60 ml/min), history of drug or alcohol abuse, participation in any other concurrent clinical trial, pregnant women, and participants with a history of food allergies. Baseline characteristics were collected at the screening visit. Fourteen healthy volunteers were randomized into two groups, provided with a mixed meal after 8 hours of fasting that did or did not contain 1000 mg of calcium propionate (also known as E282) provided to us by Niacet Corporation. After a 1-week washout, participants were crossed over to the other arm and provided with the other mixed meal. Blood samples were collected at time 0, after which the mixed meal supplemented with placebo or propionate was consumed within 15 min, and blood samples were serially collected every 30 min thereafter for 4 hours. All assays of blood, including those for C-peptide, glucagon, and catecholamine, were analyzed at the LabCorp laboratories using validated assays that are used for clinical care of patients. Determination of FABP4 was performed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). The ISI-M, which was found to correlate with the rate of whole-body glucose disappearance during a euglycemic insulin clamp study (42), was calculated using glucose and insulin values obtained during the mixed-meal test. ISI-M = 10,000/(G 0 × I 0 × G mean × I mean )1/2, where G and I represent plasma glucose (mg/dl) and insulin (μU/ml) concentrations, respectively, and “0” and “mean” indicate the fasting value and mean value during administration of the mixed-meal test, respectively. The Brigham and Women’s Hospital (BWH) Institutional Review Board (IRB) approved this study, which was conducted at the outpatient facility of the BWH Clinical and Translational Science Center. Informed consent was obtained from all participants. An additional patient cohort from DIRECT was used to study the effects of weight loss interventions on plasma propionate concentrations and metabolic characteristics. The DIRECT study was a workplace dietary intervention trial that took place in Dimona, Israel (29). Briefly, 322 overweight or obese participants (age, 52 ± 7 years; women, 14%; BMI, 31 ± 4) were randomized to one of the following weight-loss diets: a low-fat, restricted-calorie diet; a Mediterranean restricted-calorie diet; and a low-carbohydrate nonrestricted-calorie diet. Here, we subjected plasma samples from the DIRECT study for metabolomics analysis. These plasma samples were obtained from 160 participants at the baseline visit and after 6 months of the dietary intervention (the weight loss nadir). The samples were analyzed by the Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany using electrospray tandem mass spectrometry (43). Correlation between propionate and metabolic outcomes was performed by general linear models after adjustment for age, sex, dietary group, and baseline value of the respective outcome. This study was approved by the IRB of Sheba Medical Center, Tel-Hashomer, Israel. Animals Male littermates in the C57BL/6J background at 6 weeks of age were obtained from the Jackson laboratory (Bar Harbor, ME, stock no. 000664). All mice were maintained on a 12-hour light/dark cycle in a pathogen-free barrier facility with free access to water and food. The Harvard Medical Area Standing Committee on Animals approved all in vivo studies. Sample size was chosen on the basis of pilot experiments that ensured a power of 90% and a significance of 5%. All samples were included in analysis unless they fell more than 2 SDs from the mean. No randomization was used for animal studies. Investigators analyzing data were blinded to mouse genotype and treatment groups. Wild-type or Fabp4-deficient (Fabp4−/−) mice (44) on C57BL/6J background were used to evaluate the effects of propionate in the presence or absence of the adipokine FABP4. The liver-specific glucagon receptor–deficient mice were generated by crossing the floxed G-coupled glucagon receptor mice (GCGRfl/fl, provided to us by D. Drucker, University of Toronto) (23) with albumin gene promoter–driven Cre recombinase mice (Alb_Cre, B6.Cg-Speer6-ps1Tg(Alb-cre)21Mgn/J; the Jackson laboratory, Bar Harbor, ME, stock no. 003574). The characterization of this mouse model was previously described in detail (23). For studies assessing the metabolic consequences of chronic, low-dose exposure to propionate, we conducted three independent experiments in which either C57BL6 mice or wild-type and Fabp4−/− mice (6 weeks old) were exposed to propionate (~15 mg/kg) (the food preservative sodium propionate; Sigma-Aldrich, St. Louis, MO) added to the drinking water. On the basis of measurements of daily food and water intake (28), we estimate this concentration to correspond to supplementation of food by 0.15 to 0.3% (w/w) of propionate, equivalent to the propionate content in a processed food–based diet (3). The same molar ratio of sodium chloride was added to the drinking water of the control group. Weekly body weight and blood glucose were measured. Blood was collected at 6 weeks of intervention for assessment of glucagon, insulin, and FABP4 concentrations. Insulin tolerance test (0.75 units/kg body weight) was performed after 6-hour food withdrawal. Hyperinsulinemic-euglycemic clamp studies were performed as previously described (22). Briefly, 4 days before experiments, mice were anesthetized, and the right jugular vein was catheterized with a PE10 polyethylene tube (inside and outside diameters, 0.28 and 0.61 mm, respectively; Becton Dickinson, Franklin Lakes, NJ) filled with heparin solution (100 USP U/ml). The distal end of the catheter was tunneled under the skin, exteriorized in the intrascapular area, heat-sealed, and then placed in a pocket under the skin until the day of the experiment, when it was re-exteriorized. During hyperinsulinemic-euglycemic clamp, steady-state tracer analysis was used for calculations, and glucose-specific activity was verified for steady state. Propionate and pyruvate tolerance tests C57BL/6J mice aged 10 to 14 weeks were injected intraperitoneally with propionate at concentrations of 0.5 to 2.0 g/kg body weight (5 to 20 mmol/kg) or equal molar ratios of sodium pyruvate (both from Sigma-Aldrich, St. Louis, MO). All studies were performed after 5 hours of food withdrawal, unless stated otherwise in the figure legend. Similar experiments were performed using an oral gavage instead of intraperitoneal injections. PBS was used as a vehicle. All treatments were administered after adjustment of the pH to 7.4. Blood glucose was measured at baseline and at 15- to 30-min intervals (as indicated in the figures) using a Breeze2 glucometer (Bayer, Leverkusen, Germany). Hexamethonium (20 mg/kg) and/or phentolamine (1 mg/kg) or PBS were injected 7 min before the intraperitoneal injection of propionate, and both blood glucose and hormones were measured as indicated in the figure legends. A polyclonal anti-mouse FABP4 antibody (Santa Cruz Biotechnology, Dallas, TX) or an immunoglobulin G control were intravenously injected to the tail vein of wild-type mice 1 week before the propionate tolerance test. A concentration of 50 μg/kg body weight of a recombinant FABP4 protein produced in Escherichia coli (or PBS as a vehicle control) was injected with propionate into Fabp4−/− mice. Rectal propionate administration Anesthetized animals were either orally gavaged or rectally administered 1-14C–labeled propionate (0.1 mCi/kg) (MP Biomedicals, Santa Ana, CA) mixed with sodium propionate (1 g/kg) and trace amounts of dextran blue (for visual confirmation of administration). Rectal administration was performed via 3-cm PE50 tubing attached to a blunt 23G needle. After administration, animals were sampled via tail vein bleeding for blood collection. At the end of the experiment, animals were euthanized via cervical dislocation, and digestive tract was removed for visual inspection of dextran blue. At all instances, orally administered bolus was confined to stomach and rectally administered bolus was confined between duodenum and distal colon. Radioactivity was measured by decolorizing 5 μl of blood with hydrogen peroxide and mixing it with Ecoscint H (National Diagnostics, Atlanta, GA) at 1:10 (v/v) ratio and reading on a MicroBeta2 instrument (PerkinElmer, Waltham, MA). Hormonal response to propionate or pyruvate treatment Mouse serum concentration of FABP4 was determined using a commercially available ELISA system (BioVendor, Czech Republic). Noradrenaline was measured using an ELISA obtained from Eagle Biosciences Inc. (Nashua, NH). Plasma for glucagon determination was collected in EDTA-containing tubes supplemented with aprotinin and was assayed using a Glucagon Quantikine ELISA system (R&D Systems, Minneapolis, MN). Plasma concentration of insulin was measured using the Mercodia Mouse Insulin ELISA (Uppsala, Sweden). Liver glycogen determination Liver glycogen content was determined using a phenol-sulfuric acid–based colorimetric method after perchloric acid extraction (45). Briefly, about 100 mg of liver tissue was snap-frozen in 2-ml round bottom Eppendorf tubes in liquid nitrogen until sample processing. On the day of sample processing, liver was homogenized in 10% perchloric acid using 1:3 (v/v) ratio of tissue to 1.0-mm zirconium oxide beads (#ZrOB10; Next Advance, Troy, NY). Homogenized samples were aliquoted into three equal parts after centrifugation. One aliquot was saved for analysis of free sugars (S 0 ). The other homogenate was mixed with concentrated sulfuric acid (1:10, v/v) for hydrolysis of sugars (S h ). Although vortexing, 5% phenol was added (1:2, v/v) to the sample for color development. After 30-min incubation, 200 μl of the sample was transferred to a microplate for absorbance measurement at 490 nm. Glycogen from bovine liver (#G0885, Sigma-Aldrich) was used to generate the standard curve for glycogen, and dextrose was used to generate standard curve for free sugars. Glycogen content was calculated by subtracting free sugars from final glycogen hydrolyzed homogenate (S h − S 0 ). The last aliquot was used to measure protein concentration. Curve fitting and linear regression analysis was done using SoftMax Pro version 5.1. Membrane polarization in Neuro2a cells Neuro2a cells were obtained from the American Type Culture Collection (#CCL-131; Manassas, VA) at passage 27 and maintained in DMEM with 10% fetal bovine serum (FBS) supplementation. Before the experiments, the cells were seeded onto a 96-well black, clear-bottom plates, and differentiation was induced with serum deprivation. After 2 days, when neurite growth was observed, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS; #59331C, Sigma-Aldrich) and incubated with Screen Quest Membrane Potential Assay Kit reagents (#36005; AAT Bioquest, Sunnyvale, CA) as per the manufacturer’s instructions. Measurements were performed on a Spectramax Paradigm instrument (Molecular Devices, San Jose, CA). FABP4 secretion from adipose tissue Perigonadal adipose depots were removed for preparation of explants. Adipose tissue samples were washed in PBS and serum-free DMEM consecutively and minced into roughly 2-mm-sized pieces with scissors. Explants were washed with DMEM and incubated for 1 hour in the same medium for recovery. After recovery, fresh DMEM was added and secreted FABP4 in culture medium was measured every 15 min in the presence or absence of increasing concentrations of propionate or forskolin (20 μM). Samples were subjected to Western blot analysis using an anti-FABP4 antibody (Santa Cruz Biotechnology). Glucagon secretion from isolated pancreatic islets The methods for isolating islets from mice were described previously (46). Briefly, the pancreatic duct was perfused with 2.5 ml of Liberase RI (3 mg/ml) (Roche, Penzberg, Germany), after which it was excised and disaggregated by shaking for 24 min at 37°C. The islets were partially isolated by sedimentation and then hand-picked from the acinar tissue debris under a dissecting microscope. To stimulate glucagon secretion, islets were transferred from high glucose (22.4 mM) to low glucose (2.8 mM) medium. Conditioned medium was assayed for glucagon production using a commercial glucagon ELISA system (R&D Systems). Glucose production in primary hepatocytes Primary rat hepatocytes plated onto 24-well collagen-coated plates were acquired from the Massachusetts General Hospital Cell Resource Core (Boston, MA). All cells were changed to low serum medium (199 medium supplemented with 0.1% FBS, penicillin/streptomycin, and 1 μM dexamethasone) overnight upon arrival. For glycogenolysis experiments, cells were washed with DPBS (Thermo Fisher Scientific, Waltham, MA) and changed to glycogen loading medium [phenol red–free DMEM supplemented with 10 mM glucose, 15 mM fructose, and Humulin (100 mU/ml) (Eli-Lilly, Indianapolis, IN)] for 2 hours. At the end of glycogen loading, cells were washed three times with DPBS and changed to substrate-free DMEM and incubated in the presence or absence of stimulants for 3 hours. For gluconeogenesis experiments, cells were washed three times with DPBS and incubated with substrate-free DMEM and 1 μM dexamethasone for 3 hours. At the end of the glycogen depletion period, cells were washed again and fresh substrate-free DMEM with indicated substrates was added to the cells. Medium was harvested at the end of 2 hours. At the completion of each experiment, cells were lysed with 0.5 N NaOH and neutralized with 0.5 N HCl before protein concentration determination using commercial Bradford assay. Glucose secreted into the medium was assayed using Amplex Red–based glucose oxidase assay (Thermo Fisher Scientific, #A22189). Statistics All values are presented as means ± SEM unless stated otherwise in the figure legends. Paired t tests were used to compare changes in hormone concentrations at 30 min versus the baseline within groups of mice or human participants. Paired t tests were also used to compare the 30-min concentrations of norepinephrine and C-peptide between treatment groups in the crossover human study. Unpaired t tests were used to compare FABP4, glucagon, and norepinephrine amounts between propionate and pyruvate treatment groups in mice. For the propionate and pyruvate tolerance tests in mice and for comparing glucagon and FABP4 responses in the human study, we used a two-way ANOVA with Bonferroni post hoc analysis. One-way ANOVA and Tukey post hoc analysis were used to compare area under the curve between multiple groups. A two-tailed P value <0.05 was considered statistically significant. All statistical analyses were done using GraphPad Prism 7.0.

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.

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Acknowledgments: We thank the Union Chemique Belge (UCB) for providing recombinant FABP4 used in some replicate experiments and D. Drucker (Toronto, Canada) for providing the GCGRfl/fl mouse model through a material transfer agreement. Funding: This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (NIH) under award number K08 DK097145 (to A.T.). G.S.H. is supported by JDRF and NIH (AI116901) for work on FABP4. Additional support for this work (to A.T.) was received from the Nutrition Obesity Research Center at Harvard (P30-DK040561), the Cardiovascular, Diabetes and Metabolic Disorder Research Center of the Brigham Research Institute (CVDM-BRI), and the Israeli Ministry of Health Research and Fellowship Fund on food and nutrition with implications on public health. E.S.C. was partially supported by the Genes and the Environment Training Program (#5T32ES016645). Author contributions: A.T., E.S.C., G.T., K.C.C., and G.S.H. designed the experiments. A.T., E.S.C., G.T., K.E.I., M.A., M.R., K.S.H., I.R., and R.L. performed the propionate tolerance test studies, and A.T., E.S.C., and G.T. analyzed the data. A.T., E.S.C., G.T., and K.E.I. designed, performed, and analyzed the euglycemic-clamp studies. E.S.C. and G.T. designed, performed, and analyzed the propionate tracer experiment. E.S.C. designed, performed, and analyzed the in vitro primary hepatocyte and Neuro2a cell line studies. A.T. and R.G. designed and performed the mixed-meal study in humans. Y.H., L.Q., and I.S. performed and analyzed the results obtained from the DIRECT trial. K.E. performed and analyzed ex vivo pancreatic islet experiments. A.T., E.S.C., G.T., K.C.C., and G.S.H. wrote the manuscript with input from all authors. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials.