Fuel Metabolism Following 3 Days on a Carbohydrate-Free Diet vs. 3 Days of Fasting in Men with Type 2 Diabetes: A Randomized Controlled Crossover Trial

Citation: Nuttall FQ, Almokayyad RM, Gannon MC (2018) Fuel Metabolism Following 3 Days on a CarbohydrateFree Diet vs. 3 Days of Fasting in Men with Type 2 Diabetes: A Randomized Controlled Crossover Trial. J Nutr Health Sci 5(2): 204 Volume 5 | Issue 2 Journal of Nutrition and Health Sciences


Introduction
Our laboratory is interested in the effect of variations in the diet on human physiology and metabolism in subjects with and without type 2 diabetes (T2DM). We are particularly interested in determining several factors related to fuel metabolism.

Materials and Methods
This is a randomized, crossover study design with a four-week washout period. On one occasion seven male subjects received a calorie-sufficient, CHO-free diet for 72 h consisting of <3% carbohydrate, 15% protein, ~85% fat (Table 1). On another occasion, all subjects starved for 72 h. This was preceded by a standard diet consisting of 55% carbohydrate, 15% protein, 30% fat. Recruitment began in June of 2009; follow-up was completed in June of 2010.
Recently we have used two different approaches to maximally eliminate carbohydrate from the diet: a carbohydrate-free (CHOfree) diet and fasting. The metabolic effects of both have been compared in subjects with T2DM. Our overall objective has been to compare the results when the metabolic fuel being oxidized is largely fat, without a significant loss of lean body mass. Seventy-two hours of fasting is a time period over which a loss in non-water body mass is minimal [1]. Data from a 72-h fast were compared to 72-h macronutrient-sufficient, no carbohydrate, high fat diet in men with T2DM.
In the present report, weight changes, resting metabolic rate (RMR), plasma urea nitrogen, thyroid hormones, fuel oxidation, urine sodium, potassium, calcium, creatinine and urea nitrogen data are provided and discussed. The 24-hr glucose, insulin and glucagon responses [2] as well as the ghrelin and leptin responses [3] from these same subjects have been published.

Day 1 Breakfast
Scrambled eggs (with butter, heavy cream, cheese), Fried salt pork Ingestion of water was encouraged. Black coffee, tea without sugar or cream, and calorie-free beverages were allowed. Activity was limited to quiet diversions such as reading or watching TV. Blood samples were obtained during the first and last 24 h of the 72-h intervention. Urine also was collected during the first and last 24 h as well. The subjects were under supervision the entire time.
Weight and blood pressure was determined daily in the AM, as was indirect calorimetry.

Assays
Weight was determined on a digital scale (Scalitronix, White Plains, NY) in pajamas; blood pressure was determined using an automatic Dinemap Instrument (Critikon/Mediq, Pennsauken, NJ); indirect calorimetry (RMR and non-protein respiratory quotient (nRQ) was determined using a Medgraphics CPX Express apparatus (Medical Graphics Corp, St. Paul, MN), at 0800 h and over a 30-minute period.

Calculations
The quantification of the carbohydrate (glucose) being oxidized was calculated based on the indirect calorimetry data. In the post-absorptive state, i.e., 14 h overnight without food, it was assumed that the glucose utilized came from endogenous sources (glycogen metabolism and gluconeogenesis) [5]. Two quantification methods were used. In the first, the fraction of energy utilization due to carbohydrate was determined using the nRQ data. In the second, quantitative excretion of potassium was used. During glycogenolysis in the liver, release of an amount of intracellular water containing 0.45 mmoles of potassium/g of glycogen has been reported [6,7]. This potassium enters the circulation, and in an equilibrium state, would be excreted in the urine. Thus, we have used the quantity of potassium in the urine as a surrogate for net glycogen/glucose utilization as fuel.
The caloric value of glycogen is 4.1 Kcal/g compared to glucose which is 3.7 Kcal/g [8]. Thus, the amount of glucose contributed from glycogen is increased by 11%.
Serum/plasma/urine data: creatinine, TSH, T 4 , T 3 , sodium, potassium, calcium, urea nitrogen and glucose were determined by an automated method on an Abbott Architect ci 8200 analyzer (Abbott Park, IL).
Plasma was analyzed for urea nitrogen over two 24-h periods (from 0800 on day 1 and 0800 on day 4). Some blood samples were not available during the overnight h for one subject while ingesting the CHO-free diet. Therefore, that complete data set for the plasma urea nitrogen 24-h profiles is presented for 6 subjects.
Dietary content of protein, fat and carbohydrate was estimated using diet analysis modules "Nutritionist Pro" (Axxya Systems LLC, Stafford, TX,) and "VistA" (Veterans Health Information Systems and Architecture; Department of Veterans Affairs, Veterans Health Administration, US Government). Both are based on United States Department of Agriculture (USDA) databases.
Non-protein fuel oxidation was determined from published tables based on nRQ [9]. Protein fuel oxidation was calculated based on the 24-h urinary urea nitrogen excretion [10].
Protein metabolized generally is calculated by quantifying the total daily nitrogen lost. Urine urea nitrogen excretion makes up the great bulk, but small amounts are lost in the feces and from skin desquamation. In our study only the urinary urea nitrogen excretion was quantified and used to calculate protein metabolism (1 g N=6.25 g protein) [8].

Area Determination
The net and total integrated 24-h area responses were calculated with a computer program based on the trapezoid rule [11].

Statistics
Statistics were determined with Prism 4 software by Graphpad (LaJolla, CA) using paired Student's t-test. A p-value less than 0.05 was the criterion for significance. Data are presented as the mean±SEM.

Body Weight
The mean initial body weight was 97 kg and 96 kg at the beginning of the CHO-free and fasting arms, respectively, indicating stability of the body weight over ~5 weeks. The mean body weight decreased from 97 to 95 kg after ingesting a carbohydrate-free diet for 72 h [2]. The weight loss was essentially linear.

Results
During fasting, the mean body weight decreased from 96 to 93 kg 72 h later. In contrast to the linear weight loss with the CHO-free diet, the majority of the weight loss occurred within the first 24 h of fasting (Table 2). That the data were obtained at 0800 hr. and thus are representative of the caloric intake from the previous day.

Blood Pressure
The CHO-free diet, as well as fasting, resulted in a small decrease in blood pressure. However, only the decrease in diastolic pressure when the subjects had fasted was significant ( Table 2).

Calculated Food Energy Intake
The mean calculated food energy intake when the subjects ingested the standard diet on day one was 2464 Kcal/24 h for both arms of the study. When ingesting the CHO-free diet it was 2436 Kcal/24 h (

Non-Protein Respiratory Quotient (nRQ)
The initial mean nRQs were 0.84 on the first day of the CHO-free diet and 0.85 when fasting. The non-protein fuel mix being oxidized based on published tables was 47% carbohydrate, 53% fat for the CHO-free diet and 51% carbohydrate, 49% fat with fasting [9]. The nRQ subsequently decreased to 0.79 at the end of the 72-hr CHO-free diet and 0.78 with fasting. At this time the calculated non-protein fuel mix being oxidized was 30% carbohydrate, 70% fat with the CHO-free diet and 26% carbohydrate, 74% fat with fasting. Thus, the nRQ data were similar whether the subjects fasted or ingested the CHO-free diet ( Table 2). Urine Urea Nitrogen ( Table 3): The initial 24 h quantitative urine urea nitrogen excretions also were similar. They decreased only modestly with ingestion of the CHO-free diet or with fasting. The decrease was identical in both arms of the study (1.6 g), however, significant only with fasting. Values are means±SEM *P≤0.05 Standard vs Treatment by Student's t test^ P=0.02 by Wilcoxon signed rank test **The average of the 2 "Pre" data sets for volume and glucose, and individual post data set for volume and glucose were published previously [2]. 24 Hour collection for Standard (i.e. Control) was from 0800 day 1 to 0800 day 2 24 Hour collection for CHO-Free and Fasting was from 0800 day 4 to 0800 day 5 The creatinine excretion was extraordinarily low for the control diet before fasting, for an unexplained technical reason Table 3: 24-Hour Urine Data (n=7) Urine Creatinine ( Table 3): The initial quantitative urine creatinine was lower at the beginning of the fasting arm of the study compared to that at the beginning of the CHO-free diet arm. This was a uniform finding, and likely was an assay error. Over the last 24 h the creatinine had only increased by 69 mg with the CHO-free diet but by 204 mg with fasting. Had the initial creatinine excretions been similar, the slight increase at the end of the study would have been similar. (Table 3): The mean urine volumes as well as the sodium, potassium and calcium content were similar when the subjects ingested the standard diet on the two different occasions. Both the CHO-free diet and fasting resulted in a decrease in the urine volume, sodium, potassium, and calcium excretion compared to the standard diet. In general the decrease was greatest when the subjects fasted. During the fast, all of the decreases compared to the standard diet were statistically significant. The potassium decreases were similar and statistically significant in both arms. Plasma/Serum Data ( Table 4): The circulating creatinine, TSH, and T 4 concentrations remained unchanged. The T 3 decreased in both arms of the study. The HbA1c and glucose data have been published previously and are included for the possible interest of the reader [2].

Meals
In people without diabetes, the weight loss of short-term fasting (~1 kg/day) was reportedly reproduced by a CHO-free diet [1,12]. It was associated in both metabolic states by a similar sodium-induced diuresis. Addition of dietary CHO resulted in prompt sodium retention. Diuresis-associated weight loss was greatest during the first 24 h, and generally lasted only 3-4 days. It was

Discussion
The RMR, as well as body weight, at the initiation of each arm of the study was similar when the subjects ingested a mixed diet, although the data were obtained weeks apart. At the end of the study the RMR had decreased by 11% with the CHO-free diet and 16% with fasting, a statistically non-significant difference. We are not aware of comparative RMR data in people with T2DM between a short-term fast and a low or CHO-free diet. Thus, whether the current data are typical of the subjects with T2DM in general, remains to be determined. In contrast to the modest decrease noted in these men with T2DM, in normal young men and women, an increase in RMR has been reported with short-term fasting or no change after fasting for 7 days [14][15][16][17][18]. With a CHO-free diet, Bergstrom et al reported a decrease in RMR in fit young men [19]. However, others reported no change in normal subjects [20][21][22].
In subjects without diabetes, many authors have reported that a CHO-free or a very low CHO diet, or fasting, induced a decrease in plasma T 3 [22][23][24][25][26][27]. As reported by the current Senior Author, the decrease in T 3 can be observed within 8 h when CHO-free meals are ingested [28]. In our subjects, a decrease in total T 3 was present and was similar when ingesting the low-CHO diet or fasting.
To our knowledge, there are only limited nRQ data obtained in normal subjects ingesting a CHO-free diet. In 7 males the calculated fuel mixture being oxidized was 12% CHO, 88% fat after 4 days [20]; similar data were reported in 6 males [19]. Thus, when CHO was not available, fuel consumption was essentially from fat. With fasting, a low CHO: high fat oxidation ratio (~10% CHO: 90% fat) has been reported frequently [14][15][16][17]25]. Our data indicate CHO still provided a significant component to the oxidized fuel and was similar in each arm. For unknown reasons, these subjects with T2DM had not completely adapted to a typical fat-derived fuel mixture. Circulating glucose concentrations also were higher than in normals [2]. Whether this could result in a greater glucose production rate is a consideration.
In normal subjects, the gluconeogenesis rate reportedly remains unchanged with a short-term CHO-free diet or with fasting [29,30]. The predominant change in glucose production is due to a change in the net rate of glycogen oxidation. In the present study, the net glucose utilization (glycogen-dependent glucose oxidized) was estimated by two different methods. The first was based on the RMR and nRQ data, the second on the 24-h urinary potassium excretion. Results were similar. Traditionally oxidation of liver glycogen as fuel is considered to occur during the first 1-2 days of fasting. If glycogen is depleted, the only source of CHO fuel is through recycling of glucose via the Cori cycle, amino acids from protein, plus a small amount derived from fat. In normal people, needle biopsy data indicated that liver glycogen was indeed rapidly (24 h) reduced during short-term fasting or ingestion of a CHO-free diet [31]. However, it was still present and became stable, but at a low level, and was not re-accumulated until CHO was ingested [31]. Similar data were obtained by others later using NMR spectroscopy to quantify liver glycogen. This was compared to the overall glucose production rate [32]. There was essentially no net glycogenolysis from 46-64 h of fasting, and 96% of glucose production was due to gluconeogenesis. Owen et al also reported that oxidation of CHO from glycogen was undetectable after 2 days of fasting in obese subjects, some with T2DM [27]. However, other indirect data suggest that considerable glycogen is still present in subjects with T2DM after 64-72 h of fasting and can be a source of fuel, if gluconeogenesis is impaired [32,33]. Glycogen may actually be increased after a 72-h fast [34]. In addition, inhibition of gluconeogenesis by ethanol administration did not result in hypoglycemia [35], which would be expected if glycogen was not present and mobilizable as fuel [33]. The amount of glucose released by glucagon injection also was reported to be much greater in subjects with T2DM after a 3 day fast [34]. Our data based on RMR and nRQ calculations, also are compatible with liver glycogen being increased and mobilizable as fuel in these subjects. The source of excessive glycogen stores and the mechanism by which a relatively high CHO utilization occurs when fasting or not ingesting CHO remains unexplained. Our data indicate it cannot be due to an accelerated metabolism of protein.
Circulating urea nitrogen concentrations remained unchanged following mixed meals, CHO-free meals, or with fasting. Also, the concentration did not vary throughout 24 h, indicating lack of a meal-related effect of ingested protein, and lack of a circadian rhythm. With fasting it remained stable even though the urinary excretion decreased, suggesting a strong intrinsic system for regulating the circulating urea and presumably total body protein turnover. We are not aware of comparative data. In normal subjects with short-term fasting, urea nitrogen excretion reportedly remains the same as when ingesting mixed meals or increases modestly [36,37]. However, other authors reported a decrease [32,38,39]. After 72 h on a CHO-free diet, urine nitrogen excretion reportedly doubled in normal subjects [40]. Also, an increase was reported in normal subjects ingesting a CHO-free diet over 10 days [41]. The present results in subjects with T2DM are different. Urea nitrogen excretion was similar following mixed meals on 2 different occasions or with CHO-free meals, and was only modestly lower with fasting. Thus, the amount of protein used for fuel was similar regardless of the dietary changes. Creatinine excretion also was similar, compatible with a stable utilization of creatine by muscle, and maintenance of skeletal mass. If so, the excreted urea was the result of oxidation of non-structural proteins in the associated with little change in potassium excretion, indicating little loss of non-fat body mass [12]. Later, this was documented by determination of total body potassium [13]. In contrast, in the present study, when the CHO-free diet was ingested, weight loss was linear and only 0.5 kg/day. This was associated with little change in urine volume or sodium excretion. A much greater weight loss occurred with fasting (1 kg/day). The majority occurred during the first 24 h, likely associated with a major initial sodium diuresis. Urine volume decreased 25% and sodium excretion decreased 65% by the last 24 h. Thus, these results were similar to those obtained previously by others [12]. Initially a major but similar glucosuria was present. It essentially disappeared by the last 24 h [2], which could complicate interpretation of the data.
so-called labile protein pool [25]. Maintenance of skeletal muscle mass with short-term fasting also has been observed in normal subjects [27].
We suggest that glucose sensing provides a mechanism for assuring an adequate type and amount of fuel for the brain and other organs under all possible variations in macronutrient ingestion, as well as when exogenous fuel is lacking. In the present study, in one case the fuel supply was exogenous while in the other it was endogenous, indicating the ability of the body to similarly regulate the mixture of fuels being oxidized regardless of source.

Conclusion
The study strengths are: a well-controlled observational study with 2 different methods of inducing a dietary carbohydrate deficiency, the metabolic stability of the subjects, and a separate control diet for each arm of the study. The study limitations are: the relatively small number of subjects and males only; thus the results may or may not be representative of subjects with T2DM, in general.
We were not able to find contemporary data in subjects without diabetes ingesting a carbohydrate-free diet to compare to our study. It is known that certain indigenous peoples (Greenland Eskimos, Central Plains Indians), explorers in the early 20th century, etc. were able to exist for long periods of time on a carbohydrate-free diet [42][43][44]. These were presumably normal healthy adults. However, detailed quantitative measurements of fuel metabolism are not available for these subjects.
While not directly related to the present study, there is a literature regarding the effect of high-or low-carbohydrate diets in normal subjects interested in improving athletic performance.
Since the 1960s, when techniques for muscle biopsies were popularized by Bergstrom et al, high muscle glycogen concentration has been associated with enhanced physical performance [19,45]. High carbohydrate diets, and/or carbohydrate loading are techniques used to increase muscle glycogen concentration.
An observation by Pernow and Saltin suggested that some "heavy exercise" was possible following reduction of muscle glycogen, provided that an adequate supply of non-esterified fatty acids (NEFAs) was available to the muscle [46]. Subsequently Phinney et al. reported that chronic ketosis, induced by dietary carbohydrate restriction, was not deleterious to physical performance [22,47]. Current literature exists in support of enhanced exercise performance with both high-carbohydrate and ketogenic diets (for example, [48][49][50][51]).
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