Effect of Chronic Ethanol Consumption on Exogenous Glucose Metabolism in Rats Using [1-¹³C], [2-¹³C], and [3-¹³C]glucose Breath Tests

Abstract

The C3 carbon of glucose molecules becomes the C1 carbon of pyruvate molecules during glycolysis, and the C1 and C2 carbons of glucose molecules are metabolized in the tricarboxylic acid (TCA) cycle. Utilizing this position-dependent metabolism of C atoms in glucose molecules, [1-¹³C], [2-¹³C], and [3-¹³C]glucose breath tests are used to evaluate glucose metabolism. However, the effects of chronic ethanol consumption remain incompletely understood. Therefore, we evaluated glucose metabolism in ethanol-fed rats using [1-¹³C], [2-¹³C], and [3-¹³C]glucose breath tests. Ethanol-fed (ERs) and control rats (CRs) (n = 8 each) were used in this study, and ERs were prepared by replacing drinking water with a 16% ethanol solution. We administered 100 mg/kg of [1-¹³C], [2-¹³C], or [3-¹³C]glucose to rats and collected expired air (at 10-min intervals for 180 min). We compared the ¹³CO₂ levels (Δ¹³CO₂, ‰) of breath measured by IR isotope ratio spectrometry and area under the curve (AUC) values of the ¹³CO₂ levels-time curve between ERs and CRs. ¹³CO₂ levels and AUCs after administration of [1-¹³C]glucose and [2-¹³C]glucose were lower in ERs than in CRs. Conversely, the AUC for the [3-¹³C]glucose breath test showed no significant differences between ERs and CRs, although ¹³CO₂ levels during the 110–120 min interval were significantly high in ERs. These findings indicate that chronic ethanol consumption diminishes glucose oxidation without concomitantly reducing glycolysis. Our study demonstrates the utility of ¹³C-labeled glucose breath tests as noninvasive and repeatable methods for evaluating glucose metabolism in various subjects, including those with alcoholism or diabetes.

INTRODUCTION

Chronic ethanol intake has various effects on glucose kinetics, including decreased insulin-induced glucose uptake in the skeletal muscles,¹⁾ inhibition of gluconeogenesis,²⁾ reduced absorption of glucose,³⁾ and decreased resistance to insulin.⁴⁾ Chronic ethanol consumption suppresses metabolism via the tricarboxylic acid (TCA) cycle (aerobic metabolism) and enhances glycolysis (anaerobic metabolism) even in aerobic environments due to elevated ratios of the reduced/oxidized form of nicotinamide adenine dinucleotide as a result of ethanol metabolism.^5,6) Different carbon positions in glucose molecules have different metabolic pathways. C3 carbon in a glucose molecule becomes C1 carbon in the pyruvate molecule, which produces CO₂ via pyruvate decarboxylation, primarily reflecting glucose metabolism in glycolysis. The C1 carbon in the glucose molecule is the C2 carbon in the acetyl-CoA molecule, which produces CO₂ at the third turn of the TCA cycle. C2 carbon in the glucose molecule is C1 carbon in acetyl-CoA, which produces CO₂ at the second turn of the TCA cycle.⁷⁾ Our previous studies on breath tests using ¹³C-labeled glucose in diabetic patients and Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a commonly used animal model for type 2 diabetes mellitus, have confirmed that the metabolism of carbon atoms in glucose molecules depends on their positions and reported the differences in glucose breath test (GBT) results between diabetic humans/rats and healthy controls.^8,9) However, changes in exogenous glucose metabolism due to chronic ethanol consumption have not been sufficiently evaluated despite the well-known effects of ethanol on glucose metabolism, as described above.^1–7) Thus, we aimed to evaluate the metabolic changes of glycolysis and the TCA cycle in ethanol-fed rats using [1-¹³C], [2-¹³C], and [3-¹³C]glucose breath tests.

MATERIALS AND METHODS

Animal Model

The experimental protocols were approved by the Animal Care Committee of Toho University (Permit Number: 21-54-390). Sixteen female F344/DuCrj rats aged 4 weeks were purchased from CLEA Japan Inc. (Tokyo, Japan). After 1 week of acclimatization, all the rats were housed in separate cages in a quiet, temperature- and humidity-controlled room (22–24 °C and 50–60%, respectively) under a 12-/12-h light/dark cycle. All the rats were allowed free access to a powdered diet (CE-7; CLEA Japan Inc.) and fresh tap water, replaced between 9:00 and 10:00 daily. To create a rat model of long-term consumption of Japanese sake, the most commonly consumed Japanese alcoholic beverage, we replaced fresh tap water with 16% ethanol solution (Japanese Sake, Ozeki Co., Ltd., Nishinomiya, Japan) as the drinking fluid in eight female ethanol-fed rats (ERs) from 5 weeks of age, based on a previous study.¹⁰⁾ [1-¹³C], [2-¹³C], and [3-¹³C]glucose breath tests were performed on rats between 12 and 14 weeks of age. As control rats (CRs), eight female rats were pair-fed an isocaloric mash food containing sucrose as a caloric substitute for ethanol. The body weight of all rats and the amount of ingested ethanol by the ERs were measured daily and recorded.

Body Weights and the Amount of Ingested Ethanol by the ERs

The body weights of the ERs during the GBTs (12–14 weeks of age) were 152–172 g. The daily measured amount of ingested 16% ethanol solution in each ER was 9.22–10.43 mL/d. Hence, the net ethanol intake was 1.47–1.67 g/d. As the ERs ingested the solution for 7–9 weeks, the cumulative total of ingested amounts of ethanol reached 72–105 g per rat prior to the GBTs (12–14 weeks of age).

¹³C-GBT

Three replicates of GBT with [1-¹³C], [2-¹³C], or [3-¹³C]glucose as the labeling substance were performed on 16 female rats, including eight ERs and eight control rats aged 12 − 14 weeks, using ¹³C-labeled glucose (99.5 atom% ¹³C) purchased from Cambridge Isotope Laboratories (Tewksbury, MA, U.S.A.). All rats were made to fast for 24 h. Expired air from the rats was obtained by a collecting system comprising an animal chamber and a pump invented by Uchida et al.¹¹⁾ (Fig. 1). Initially, all the rats were placed in chambers of the collecting system to collect 1.2 L of baseline breath gas over 10 min. After collecting the baseline gas, rats were removed from the chambers to administer ¹³C-glucose. Next, 100 mg/kg of ¹³C-glucose was rapidly administered by inserting a thin metal tube through the mouth into the stomachs of rats. The rats were placed in the chambers of the collecting system immediately after the administration of 100 mg/kg of ¹³C-glucose once again to collect exhaled gas. The exhaled gas was collected 18 times (every 10 min for a total collection time of 180 min). The ¹³C level was estimated as the ¹³CO₂/¹²CO₂ isotope ratio and is expressed as delta over baseline per mil (Δ¹³CO₂, ‰) using non-dispersive IR isotope ratio spectrometry (POC-one; Otsuka Electrics Co., Ltd., Hirakata, Japan). The ¹³C values were used to construct the breath ¹³CO₂ expiration–time curve. The area under the curve (AUC) of the breath ¹³CO₂ expiration–time curve for up to 180 min (AUC₁₈₀) was used as a marker of glucose metabolism.

Fig. 1. Exhaled Gas Collection System

Exhaled breath gas is continuously aspirated by the rotating pump and collected in a sample bag. After collection, examiners connect the sample bag containing collected breath to an infrared isotope ratio spectrometer for analysis (POC-one).

Statistical Analysis

The results are presented as means, unless otherwise indicated. A student’s t-test was utilized to assess inter-group differences in expired ¹³CO₂ levels at each time point and the AUC₁₈₀ using the Statistical Package (JMP v. 6.0, Japanese edition). Statistical significance was set at p < 0.05.

RESULTS

The ¹³CO₂ concentration (¹³CO₂/¹²CO₂ isotope ratio and delta [Δ‰] to baseline) in exhaled gas after [1-¹³C]glucose administration peaked at 70 min for ERs, whereas it peaked at 110 min for CRs, and was lower for ERs than CRs at all time points, except 30–70 min (Table 1, Fig. 2). The ¹³CO₂ levels in the expired breath gas after the administration of [2-¹³C]glucose were also lower for ERs than CRs at all time points except at 60–120 min (Table 2, Fig. 3). The breath ¹³CO₂ expiration–time curve of CRs after the administration of [2-¹³C]glucose was bimodal, with a high peak at 130 min (Table 2, Fig. 3). In both GBTs with [1-¹³C]glucose and [2-¹³C]glucose, the peaks of the expiration–time curve of CRs were delayed in the CRs when compared to the ERs (Figs. 2, 3). In CRs, the peak was earlier in GBT with [1-¹³C]glucose than in GBT with [2-¹³C]glucose (110 vs. 130 min, Table 1, Fig. 2, Table 2, Fig. 3). Unlike the results of GBTs with [1-¹³C]glucose and [2-¹³C]glucose, the breath ¹³CO₂ expiration–time curves of [3-¹³C]glucose displayed a similar convex shape in both the ERs and CRs; the ¹³CO₂ levels were significantly higher in ERs only at 100–110 min (Table 3, Fig. 4). The excretion of ¹³CO₂ in the ERs was delayed when compared to the CRs, and the ¹³CO₂ level in the ERs remained higher than in the CRs throughout the latter half of the GBTs with [3-¹³C]glucose. The AUC₁₈₀ for [1-¹³C] and [2-¹³C]glucose breath tests were larger in CRs than in ERs (p < 0.01). However, the AUC₁₈₀ for the [3-¹³C]glucose breath test did not significantly differ between ERs and CRs (p = 0.39; Fig. 5).

Table 1. Comparison of ¹³CO₂ Levels in the [1-¹³C]glucose Breath Test

Time point	13CO2 levels (‰) in ER (n = 8)	13CO2 levels (‰) in CR (n = 8)	p-Value
10 min	32.01 (± 14.10)	73.00 (± 31.00)	0.0043*
20 min	74.86 (± 34.47)	113.09 (± 27.31)	0.0276*
30 min	114.56 (± 41.42)	125.64 (± 24.01)	0.5200
40 min	143.28 (± 43.51)	160.91 (± 32.56)	0.3700
50 min	165.05 (± 43.70)	192.56 (± 23.82)	0.1400
60 min	180.00 (± 43.44)	206.50 (± 24.82)	0.1500
70 min	188.79 (± 38.18)	208.10 (± 24.78)	0.2500
80 min	188.59 (± 29.71)	219.45 (± 18.73)	0.0260*
90 min	187.54 (± 22.50)	221.04 (± 21.77)	0.0091*
100 min	182.09 (± 15.50)	227.11 (± 37.18)	0.0069*
110 min	178.65 (± 10.93)	235.00 (± 38.99)	0.0015*
120 min	170.98 (± 7.21)	223.78 (± 26.53)	0.0001*
130 min	162.89 (± 8.18)	228.82 (± 22.70)	0.0001*
140 min	153.85 (± 11.53)	216.40 (± 49.29)	0.0036*
150 min	142.66 (± 13.30)	170.48 (± 14.02)	0.0011*
160 min	133.06 (± 16.84)	161.10 (± 13.78)	0.0027*
170 min	124.69 (± 18.56)	152.40 (± 12.39)	0.0034*
180 min	116.04 (± 18.09)	144.85 (± 12.79)	0.0025*
AUC	344.75 (± 46.76)	429.38 (± 28.70)	0.0003*

Notes: * p-Value <0.05. AUC, area under the curve; CR, control rat; ER, ethanol-fed rat.

Fig. 2. Comparison of Mean ¹³CO₂ Levels in the Expired Breath Using the [1-¹³C]glucose Breath Test

The ¹³CO₂ levels in the expired breath gas after the administration of [1-¹³C]glucose peak at 70 min for ER (green), at 110 min for CR (red), and are lower for ER than CR at all time points except 30–70 min. The tinted areas around each line indicate the standard deviation. CR, control rat; ER, ethanol-fed rat.

Table 2. Comparison of ¹³CO₂ Levels in the [2-¹³C]glucose Breath Test

Time point	13CO2 levels (‰) in ER (n = 8)	13CO2 levels (‰) in CR (n = 8)	p-Value
10 min	20.90 (± 4.25)	39.64 (± 23.14)	0.0566
20 min	54.60 (± 12.07)	72.79 (± 17.29)	0.0305*
30 min	89.54 (± 15.03)	111.79 (± 19.24)	0.0230*
40 min	118.09 (± 18.95)	142.39 (± 25.45)	0.0496*
50 min	142.57 (± 19.46)	176.84 (± 29.74)	0.0183*
60 min	161.61 (± 18.98)	185.85 (± 38.89)	0.1437
70 min	173.16 (± 21.45)	192.99 (± 19.90)	0.0761
80 min	178.06 (± 22.77)	186.44 (± 18.80)	0.4363
90 min	181.79 (± 23.16)	187.91 (± 17.88)	0.5638
100 min	180.89 (± 22.54)	185.95 (± 16.39)	0.6162
110 min	182.68 (± 21.49)	186.99 (± 14.76)	0.6480
120 min	177.11 (± 20.22)	178.84 (± 17.55)	0.8581
130 min	168.16 (± 19.13)	206.55 (± 32.28)	0.0142*
140 min	160.18 (± 17.14)	198.90 (± 45.80)	0.0521
150 min	151.18 (± 17.81)	201.80 (± 39.84)	0.0086*
160 min	143.01 (± 16.21)	189.75 (± 40.84)	0.0145*
170 min	132.50 (± 14.91)	167.51 (± 40.08)	0.0461*
180 min	123.85 (± 13.92)	158.46 (± 36.98)	0.0353*
AUC	324.25 (± 35.54)	369.62 (± 21.65)	0.0099*

Notes: * p-Value <0.05. AUC, area under the curve; CR, control rat; ER, ethanol-fed rat.

Fig. 3. Comparison of Mean ¹³CO₂ Levels in Expired Breath Using the [2-¹³C]glucose Breath Test

The ¹³CO₂ levels in expired breath after the administration of [2-¹³C]glucose are significantly lower in the ER (green) than in the CR (red) at all time points, except at 60–120 min. The CR curve is bimodal, with a high peak at 130 min. The tinted areas around each line indicate the standard deviation. CR, control rat; ER, ethanol-fed rat.

Table 3. Comparison of ¹³CO₂ Levels in the [3-¹³C]glucose Breath Test

Time point	13CO2 levels (‰) in ER (n = 8)	13CO2 levels (‰) in CR (n = 8)	p-Value
10 min	62.09 (± 28.40)	51.76 (± 24.25)	0.4473
20 min	127.40 (± 51.92)	131.69 (± 47.41)	0.8655
30 min	180.25 (± 61.54)	184.51 (± 56.21)	0.8870
40 min	210.65 (± 58.56)	218.22 (± 59.18)	0.8007
50 min	231.59 (± 47.92)	234.49 (± 55.72)	0.9127
60 min	240.89 (± 39.32)	240.45 (± 47.34)	0.9840
70 min	244.52 (± 28.64)	238.50 (± 35.37)	0.7137
80 min	243.16 (± 20.82)	232.04 (± 24.91)	0.3489
90 min	238.65 (± 17.01)	221.95 (± 16.92)	0.0691
100 min	229.65 (± 15.05)	213.14 (± 14.36)	0.0415*
110 min	224.51 (± 17.27)	206.16 (± 16.88)	0.0496*
120 min	211.59 (± 18.49)	192.79 (± 21.70)	0.0832
130 min	200.69 (± 20.78)	180.72 (± 24.10)	0.0977
140 min	189.10 (± 18.67)	167.24 (± 26.11)	0.0746
150 min	173.59 (± 20.62)	156.04 (± 25.84)	0.1554
160 min	160.26 (± 20.87)	146.57 (± 23.23)	0.2355
170 min	147.12 (± 20.72)	137.32 (± 22.97)	0.3853
180 min	135.49 (± 21.34)	126.21 (± 22.55)	0.4123
AUC	462.00 (± 57.14)	438.12 (± 50.09)	0.3890

Notes: * p-Value <0.05. AUC, area under the curve; CR, control rat; ER, ethanol-fed rat.

Fig. 4. Comparison of Mean ¹³CO₂ Levels in Expired Breath on the [3-¹³C]glucose Breath Test

¹³CO₂ levels in the expired breath after the administration of [3-¹³C]glucose are significantly higher in the ER (green) than in the CR (red) only at 100–110 min. The tinted areas around each line indicate the standard deviation. CR, control rat; ER, ethanol-fed rat.

Fig. 5. Comparison of AUC₁₈₀ in GBTs Using [1-¹³C]glucose (A), [2-¹³C]glucose (B), and [3-¹³C]glucose (C)

The AUC₁₈₀ of the [1-¹³C]glucose breath test was smaller in ERs (blue) than in CRs (orange, p = 0.0003). Similarly, the AUC₁₈₀ of the [2-¹³C]glucose breath test was smaller in ERs (blue) than in CRs (orange, p = 0.0040). However, the AUC₁₈₀ of the [3-¹³C]glucose breath test showed no significant difference between ERs (blue) and CRs (orange, p = 0.3900). Error bars indicate the standard deviation. AUC, area under the curve; CR, control rat; ER, ethanol-fed rat; GBT, glucose breath test.

DISCUSSION

Our study evaluated the changes in glucose metabolism induced by chronic alcohol ingestion in rats using GBT. It showed attenuated [1-¹³C]glucose and [2-¹³C]glucose metabolism in ERs. It also showed the delayed peak of ¹³CO₂ levels on the [1-¹³C]glucose breath test and the bimodal characteristics with the surge at 130 min on the [2-¹³C]glucose breath test in CRs.

Previous studies on the metabolic effects of chronic ethanol consumption reported the inhibition of glucose oxidation in adipose tissue, reducing both the pentose phosphate shunt and TCA cycle activity.¹²⁾ As shown in Fig. 4, the trends of ¹³CO₂ levels in the ER and CR groups were similar during the earlier phase after [3-¹³C]glucose administration, whereas a tendency of higher ¹³CO₂ levels in the ER group was observed during the latter half. Considering that C3 carbon in glucose molecules becomes C1 carbon in pyruvate molecules in glycolysis and that C1 carbon and C2 carbon in glucose molecules are metabolized in the TCA cycle, our results suggest that chronic ethanol consumption suppresses metabolism via the TCA cycle, which is consistent with previous studies.¹²⁾ Considering that C2 and C1 carbon of glucose produce CO₂ at the second and third turns of the TCA cycle, respectively, the earlier peak of GBTs with [1-¹³C]glucose when compared to that of [2-¹³C]glucose in CR is inconsistent with the order of metabolism of carbon atoms of glucose in the TCA cycle. Further studies are needed to clarify the reasons for the inconsistency.

Our previous studies in OLETF rats⁸⁾ and in early-stage diabetic patients⁹⁾ with GBT showed the suppression of [1-¹³C]glucose and [2-¹³C]glucose metabolism, as observed in this study using ERs. The effect of chronic alcohol consumption on glucose metabolism may be similar to that in early-stage diabetes through the suppression of [1-¹³C]glucose and [2-¹³C]glucose oxidation in both glycolysis and the TCA cycle. Further research is warranted to validate this hypothesis.

The present study has several limitations. First, 24-h fasting as a preparation for the experiment may have affected glucose metabolism. Second, we did not evaluate sex differences in this study. We used female rats because previous studies, including our study with breath tests, showed that females are more susceptible to ethanol exposure than males.^10,13,14) However, the lack of analysis using male rats may be a limitation of this study. Finally, we need to consider the methodological limitations of GBT. As the assumption in the 75 g oral glucose tolerance test (OGTT), a commonly used test for diagnosis of diabetes-related conditions, we assumed that all glucose molecules of the 100 mg/kg of exogenously provided glucose in this experiment were metabolized immediately and in an anterograde manner in the liver. However, we could not exclude irregular metabolism during the experiment, including metabolism by intestinal bacteria or retrograde metabolism. Although we could not measure plasma glucose levels, simultaneously measuring plasma glucose levels might provide more information, which may help to evaluate the utility of the glucose breath test for monitoring temporal changes in glucose metabolism.

In summary, our study suggested that chronic ethanol consumption reduces glucose oxidation while glycolysis remains unaffected. Our study proposes the efficacy of ¹³C-labeled GBTs as a noninvasive and repeatable tool for evaluating specific biochemical reactions in glucose metabolism.

Acknowledgments

We thank Dr. Tsunehiko Imai for providing excellent experimental instructions.

Conflict of Interest

The authors declare no conflict of interest.

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