Impact of heavy drinking on plasma acetate. On a typical drinking day, the heavy drinkers consumed 8 ± 1 drinks (n = 7), and the light drinkers consumed 1 or 0 (i.e., some drank very rarely). For the heavy drinkers, the last drink was 3 ± 1 days earlier (n = 5, 2 did not answer that question), while for the light drinkers, one had a drink 7 days before, and the others consumed no alcohol for at least 20 days and in several cases over 2 months. Heavy drinkers tested sober, with breath alcohol of 0 on the test days, having drunk no fewer than 2 days before the study, and in one case one week before the study. The averaged longest interval without drinking for heavy drinkers was 5 ± 1 days. Heavy drinkers had significantly greater levels of plasma acetate before infusion (0.20 ± 0.13 mM, range: 0.09–0.48 mM) compared with those of light drinkers (0.09 ± 0.01 mM, range: 0.08–0.11 mM) (P = 0.05) (Figure 1A). Differences in acetate before infusion between the groups may reflect slower acetate clearance rates in the heavy drinkers. Plasma glucose showed no significant difference after overnight fasting, with levels before infusion of 5.0 ± 0.5 mM for heavy drinkers and 4.8 ± 0.4 mM for light drinkers (P = 0.6). During the acetate infusion, subjects showed no significant plasma glucose changes (overall group effect, P = 0.61), with variations of less than 2% (Figure 1B). Plasma lactate and β-hydroxybutyrate enter the brain by the same monocarboxylic acid transporter as acetate, and so differences could potentially affect acetate entry to the brain by competitive inhibition. β-Hydroxybutyrate differed insignificantly between the 2 groups at baseline (0.17 ± 0.01 mM and 0.21 ± 0.11 mM for light and heavy drinkers, respectively, P = 0.13) and after 120 minutes (0.09 ± 0.03 mM and 0.61 ± 0.42 mM, respectively, P = 0.27). The rise in β-hydroxybutyrate during the infusion was significant in the heavy drinkers (P = 0.05), if no Bonferroni correction is applied to account for the multiple measures in plasma, but not in the light drinkers (P = 0.28). However, plasma lactate showed no significant differences between groups before and during the acetate infusion, with lactate levels before infusion of 0.95 ± 0.13 mM and 0.83 ± 0.10 mM for light and heavy drinkers, respectively (P = 0.46), rising slightly to 1.03 ± 0.12 mM and 0.84 ± 0.12 mM, respectively, by the end of the infusion (P = 0.30). The rise in lactate was also insignificant for each group (P = 0.93 and P = 0.56 for heavy and light drinkers, respectively) or for all data together (P = 0.63). Infusion of [2-13C]acetate increased the plasma acetate from baseline (~0.1 mM) to approximately 1 to 2 mM within 5 minutes (Figure 1C) among both groups. The steady-state plasma acetate concentrations between heavy drinkers and light drinkers were comparable (1.2 ± 0.3 mM for heavy drinkers and 1.3 ± 0.2 mM for light drinkers, P = 0.78; Figure 1C). Plasma 13C-acetate enrichments followed a similar pattern as plasma acetate concentrations, rising from 0% to 70%~80% within 5 minutes. No significant differences were seen between groups at steady state (72% ± 5% for heavy drinkers and 77% ± 5% for light drinkers, P = 0.5; Figure 1D).

Figure 1 Plasma acetate and glucose concentrations during [2-13C]acetate infusion. (A) Plasma acetate concentrations before infusion. Symbols represent individual concentrations; horizontal bars indicate the mean. (B) Averaged plasma glucose concentrations during acetate infusion. (C) Averaged plasma acetate concentrations during [2-13C]acetate infusion. (D) Averaged plasma acetate 13C enrichments during the acetate infusion. Gray diamonds represent heavy drinkers (HD), and white diamonds represent light drinkers (LD). Values with error bars represent mean ± SEM.

Impact of heavy drinking on 13C labeling in the brain. Figure 2 shows representative 13C-MRS spectra during the steady-state portion of the 13C-acetate infusion in a heavy drinkers and light drinkers, normalized to the subjects’ own natural 13C abundance of N-acetylaspartate (NAA) C3 and C6 resonances. The 1H MRS measurements showed no significant differences between heavy drinkers and light drinkers with respect to any of the metabolites, respectively, including the ratio of NAA to water (P = 0.10), that of glutamate to water (P = 0.49), that of glutamine to water (P = 0.62), and that of GABA to water (P = 0.4). The most abundant 13C labeling occurred in the glutamate and glutamine C4 positions. Heavy drinkers had higher 13C labeling incorporation across the time course in glutamate C4 (P = 0.01) and glutamine C4 (P = 0.021) relative to that of light drinkers, including at the end point (P = 0.0013 and 0.012, respectively) (Figure 3, A and B). Glutamate and glutamine C3 are also labeled, which occurs as 13C is processed through multiple turns of the TCA cycle.

Figure 2 Steady-state 13C-spectra of a heavy drinker (top spectrum) and a light drinker (lower spectrum). The heavy drinker had markedly greater 13C labeling than the light drinker. Acquisition parameters were as follows: 765 complex points; 5,000 Hz bandwidth; repetition time, 2.5 seconds. A total of 640 acquisitions were grouped to make each spectrum, each representing the final 27 minutes observed while the glutamate and glutamine C4 labeling were at a steady state. The spectra were zero filled to 16,384 complex points, windowed with –2 Hz Lorentzian and 6 Hz Gaussian broadening, and Fourier transformed.

Figure 3 Averaged time courses of the percentage of 13C enrichment of Glu4 and Gln4 for (A) heavy drinkers and (B) light drinkers. Heavy drinkers showed higher 13C labeling in both Glu4 and Gln4, consistent with greater utilization of acetate. Gln4 is represented by black diamonds and Glu4 is represented by black squares in heavy drinkers. Gln4 is represented by white diamonds and Glu4 is represented by white squares in light drinkers. Values with error bars represent group mean ± SEM.

The time courses of 13C enrichments of glutamate and glutamine C4 and the steady-state 13C enrichments of glutamate and glutamine C3 were analyzed with a mathematical model of brain acetate metabolism in CWave software (33) to calculate the metabolic fluxes (Table 1 and Figure 4). Metabolic modeling of the individual time courses showed that heavy drinkers had a greater c erebral m etabolic r ate of ac etate (CMR ac ) than light drinkers, with values of 0.069 ± 0.008 mmol/min/kg and 0.048 ± 0.006 mmol/min/kg, respectively (P = 0.02) (Figure 5A). 13C resonances of GABA, acetate, and other metabolites were small due to their low overall concentrations in the brain. 13C-GABA was undetectable in light drinkers but was detected at a level of 0.09 ± 0.03 mmol/kg in heavy drinkers, indicating that 13C-GABA labeling in heavy drinkers is also greater than that in light drinkers (Figure 2). In light drinkers, 13C-GABA was necessarily much lower than in heavy drinkers. Some idea of the limit can be drawn from the standard deviation of the spectral noise, ± 0.029 mmol/kg. To detect the presence of labeled GABA across the group, signal-to-noise ratios of at least 1.5 would be needed, which have a detection limit of 0.044 mmol/kg. The fraction of glutamate that resides in astroglia (FracGluA) was set to 0.10, calculated from data in ref. 34 for a similar voxel, but was tested also for a much lower value (0.01), which was found to have a negligible impact on the results. The rate of glutamate-glutamine cycling (V cycle ) was calculated relative to the neuronal TCA cycle (V tcaN ), using the steady-state enrichments of glutamate and glutamine C4, as stated in Table 1. The value of V cycle was 0.18 ± 0.03 mmol/kg/min and 0.28 ± 0.02 mmol/kg/min in light and heavy drinkers, respectively (P = 0.008). Two other parameters, the astroglial rate of pyruvate dehydrogenase (V pdhA ) and the rate of glutamine synthesis (V gln ), were defined from other rates, as defined in Table 1. V pdhA was estimated to be 0.08 ± 0.02 mmol/kg/min and 0.04 ± 0.02 mmol/kg/min in light and heavy drinkers (P = 0.13), and the estimated value of V gln was 0.22 ± 0.03 mmol/kg/min and 0.32 ± 0.02 mmol/kg/min in light and heavy drinkers (P = 0.008). It is important to note that lactate from the blood exchanges with lactate that is generated in the brain, and its effect is combined with that of glucose in the rate of flow through pyruvate dehydrogenase.

Figure 4 Metabolic pathways showing brain uptake of [2-13C]acetate and transfer of the 13C labeling to glutamine and glutamate in astroglia and neurons. Astroglia consume acetate, while neurons and astroglia both consume glucose (Glc). The acetate labeled at the methyl group, whose carbon is designated 2 (Ac 2 ), enters the astroglia and the TCA cycle to form C2-labeled acetyl CoA (Ac 2 CoA). In the first turn of the Krebs cycle, it labels the C4 of astroglial α-ketoglutarate (α-KG A4 ), which exchanges to form glutamate C4 (Glu A4 ). Astroglia convert glutamate to glutamine, forming glutamine C4 (Gln 4 ), which is transferred to neurons, converted, and mixed with the large neuronal pool of glutamate (Glu N4 ). Some of the glutamate is released as part of glutamate-glutamine cycling, and some exchanges to form neuronal α-ketoglutarate (α-KG N4 ). In both compartments, the carbon continues through the cycle and labels oxaloacetate (OAA) and labels glutamate and glutamine at C3 (data not shown) but does not return to the C4 of glutamate and glutamine. Meanwhile, the vast majority of glucose remains unlabeled and dilutes the pool of acetyl CoA and the Krebs cycle intermediates in neurons and astroglia. AcCoA, acetyl-CoA; Lac, lactate; Pyr, pyruvate.

Figure 5 Metabolic rates calculated based on individual 13C time courses of Glu4 and Gln4 and the steady state of Glu3 and Gln3. The value of V xA , which is the rate of exchange between astroglial α-ketoglutarate and glutamate, has not been determined. The kinetics was therefore tested over a range of values of V xA . Its minimum possible value is equal to the rate of the TCA cycle V tcaA , and, for values above 10×V tcaA , there is negligible difference in the kinetic impact compared with infinity (70), so V xA = 10×V tcaA was selected as the maximum of the range. (A) CMR ac was calculated assuming V xA = 10×V tcaA . CMR ac was significantly greater in the heavy drinking group (P = 0.02). (B) Astroglial TCA cycle (V tcaA ) rates did not differ (P = 0.58) when V xA = 10×V tcaA . (C) CMR ac was calculated assuming V xA = V tcaA , showing significant differences between heavy drinkers and light drinkers (P = 0.01). (D) V tcaA showed no difference between heavy drinker and light drinker groups when V xA = V tcaA (P = 0.99). Values with error bars represent group mean ± SEM. Symbols represent individual concentrations; horizontal bars indicate the mean.

Table 1 Equations used in CWave software to calculate metabolic rates

The metabolic fitting required that the rate of exchange between astrocytic α-ketoglutarate and glutamate (V xA ) have an assumed value (Table 1). We fitted the data for Figure 5, A and B, with a value of the exchange rate that was 10-fold greater than the astrocytic TCA cycle rate (V tcaA ). To test the sensitivity of the model to the value of V xA , fitting was repeated with V xA = V tcaA . The sensitivity test of V xA showed that for the minimum value of V xA , which is equal to V tcaA , CMR ac was 0.103 ± 0.013 mmol/kg/min and 0.061 ± 0.008 mmol/kg/min for heavy drinkers and light drinkers, respectively, while for V xA = 10 × V tcaA , CMR ac was 0.069 ± 0.008 mmol/kg/min for heavy drinkers and 0.048 ± 0.006 mmol/kg/min for light drinkers. In neither case did the value of V tcaA , which was 0.08–0.13 mmol/kg/min (Figure 5, B and D), differ significantly between heavy drinkers and light drinkers, although in both cases heavy drinkers showed significantly higher CMR ac than that of light drinkers (P = 0.02) (Figure 5, A and C).

Impact of heavy drinking on brain acetate concentrations. Brain 13C-acetate concentrations during the steady-state portion of the infusion of [2-13C]acetate were 80% higher in heavy drinkers (0.071 ± 0.014 mmol/kg) than in light drinkers (0.039 ± 0.007 mmol/kg) (P = 0.06; Figure 6A). The ratios of brain/blood 13C-acetate concentrations in heavy drinkers were 84% greater (0.049 ± 0.007) than those in light drinkers (0.026 ± 0.006) (P = 0.02; Figure 6B). Because brain acetate levels and brain/blood ratios were higher, while at the same time the brain was consuming more acetate, heavy drinkers must have had elevated blood-brain transport of acetate compared with that of light drinkers.

Figure 6 Brain acetate concentrations during steady-state [2-13C]acetate infusion, showing heavy drinkers have increased [2-13C]acetate in brain. (A) Steady-state brain [2-13C] acetate concentrations during the [2-13C]acetate infusion. (B) Ratios of brain/blood acetate concentrations at steady-state [2-13C]acetate infusion. Values with error bars represent group average ± SEM. Symbols represent individual concentrations; horizontal bars indicate the mean.

Relationship of acetate uptake to recent drinking history. Steady-state glutamate C4 13C enrichments were significantly correlated with the number of drinks consumed in the past 30 days (P = 0.0005, r2 = 0.8; Figure 7A) and past 60 days (P = 0.0006, r2 = 0.8). Steady-state glutamine C4 13C enrichments were correlated with the number of drinking days in the past month (P = 0.03, r2 = 0.6; Figure 7B), although the latter comparison did not survive a Bonferroni correction. The relationship suggests that the increase in the metabolite labeling is an adaptation of the brain to obtain more energy from acetate during chronic heavy drinking.