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, 7 (4), e14005

Short-chain Fatty Acid Delivery: Assessing Exogenous Administration of the Microbiome Metabolite Acetate in Mice

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Short-chain Fatty Acid Delivery: Assessing Exogenous Administration of the Microbiome Metabolite Acetate in Mice

Tyler B Shubitowski et al. Physiol Rep.

Abstract

Short-chain fatty acids (SCFAs) are fermentation by-products of gut microbes which have been linked to positive effects on host physiology; the most abundant SCFA is acetate. Exogenous administration of acetate alters host metabolism, immune function, and blood pressure, making it a biologic of interest. The effects of acetate have been attributed to activation of G-protein-coupled receptors and other proteins (i.e., HDACs), often occurring at locations distant from the gut such as the pancreas or the kidney. However, due to technical difficulties and costs, studies have often delivered exogenous acetate without determining if systemic plasma acetate levels are altered. Thus, it is unclear to what extent each method of acetate delivery may alter systemic plasma acetate levels. In this study, we aimed to determine if acetate is elevated after exogenous administration by drinking water (DW), oral gavage (OG), or intraperitoneal (IP) injection, and if so, over what timecourse, to best inform future studies. Using a commercially available kit, we demonstrated that sodium acetate delivered over 21 days in DW does not elicit a measurable change in systemic acetate over baseline. However, when acetate is delivered by OG or IP injection, there are rapid, reproducible, and dose-dependent changes in plasma acetate. These studies report, for the first time, the timecourse of changes in plasma acetate following acetate administration by three common methods, and thus inform the best practices for exogenous acetate delivery.

Keywords: Drinking water; i.p; injection; oral gavage; plasma acetate.

Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Figure 1
Figure 1
Acetate Colorimetric Kit demonstrates specificity for acetate. Adding exogenous acetate directly to plasma results in an increase in absorbance (A). This robust increase in absorbance is only seen when acetate is added to plasma (B: “plasma” indicates plasma alone, all other bars are plasma + indicated compound). Data shown as mean ± SEM of triplicate wells. Acetate: all pairwise comparisons are significant, < 0.001 (indicated by **); Propionate: 1 mmol/L dose significantly different from all other doses (< 0.001), 0 mmol/L significantly different from 0.15 and 0.3 mmol/L (= 0.003) (indicated by *). Formate: < 0.04 for 0 mmol/L versus 1 mmol/L (indicated by #). L‐lactate: significant decrease in absorbance with increasing dose (< 0.04 for 0.15 mmol/L versus 0.3 mmol/L; indicated by #).
Figure 2
Figure 2
Plasma acetate does not change following sodium acetate DW treatment. Plasma was collected from nine mice (= 3 females, six males) at four timepoints over 28 days; mice were treated with 200 mmol/L sodium acetate DW from Day 0–21 and plasma acetate was measured using the Acetate Colorimetric Kit (A). In a separate experiment, plasma was collected from eight mice (= 4 females, open symbols; = 4 males, closed symbols) after 21 days of treatment with either control water or acetate water. Plasma acetate was then measured by EIGC/MS (B). Finally, plasma was collected from eight mice (= 4 females and four males) prior to and immediately following a 24 h water deprivation, along with at nine timepoints over 24 h while treated with 200 mmol/L sodium acetate DW (C); acetate was measured using the Acetate Colorimetric Kit. For C, all timepoints are = 8 with the exception of 5 h (= 7) and 8 h (= 6), due to technical difficulties. There were no significant differences present at any of the time points, between males or females, in any of the treatment conditions. DW, drinking water.
Figure 3
Figure 3
Plasma acetate increases following acetate oral gavage. Sodium acetate was delivered by gavage at 2 g/kg (A; = 4 male, = 4 female) and 1 g/kg (B; = 4 male, = 4 female) following a baseline collection. This method of delivery significantly elevated plasma acetate at both doses and in both sexes. Baseline is indicated by a dotted line; #denotes significant change from baseline in male acetate value, $denotes significant change from baseline in female acetate value, ^denotes significant difference between males and females.
Figure 4
Figure 4
Plasma acetate increases following acetate IP injection. Sodium acetate was delivered IP at 1 g/kg (A; = 5 male, = 4 female) and 0.5 g/kg (B; = 4 male, = 4 female) following a baseline collection. This method of delivery significantly elevated plasma acetate at both doses and in both sexes. Baseline is indicated by a dotted line; #denotes significant change from baseline in male acetate value, $denotes significant change from baseline in female acetate value, ^denotes significant difference between males and females.
Figure 5
Figure 5
Urine acetate elevates following acetate IP injection. Sodium acetate was delivered at 1 g/kg, IP to male (A) and female (B) mice (= 4 and 4) following a baseline collection. Urine was collected, when available, at 15 min intervals after injection. Each mouse is individually plotted. In all cases, the first urine sample collected post‐injection showed an increase in acetate, indicating renal clearance of acetate from plasma. The collective fold changes in males and females demonstrate a statistically significant increase (one‐way ANOVA) in acetate at the “peak” timepoint (C). ***< 0.001
Figure 6
Figure 6
MAP decreases following acetate IP injection. Sodium acetate, or a volume‐matched saline control, was delivered at 1 g/kg IP to male mice following a baseline pressure reading. MAP was significantly lower in the acetate group at minutes 19, 21, 28, and 29 (= 3, t‐test, saline vs. acetate, < 0.05 using Bonferroni correction for multiple comparisons). MAP, mean arterial pressure.

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