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Gluconeogenesis during endurance exercise in cyclists habituated to a long-term low carbohydrate high-fat diet Christopher C. Webster1 , Timothy D. Noakes1 , Shaji K. Chacko2 , Jeroen Swart1 , Tertius A. Kohn1 and James A. H. Smith1 1
Division of Exercise Science and Sports Medicine (ESSM), Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Newlands, South Africa 2 Department of Pediatrics, Children’s Nutrition Research Center, US Department of Agriculture/Agricultural Research Service, Baylor College of Medicine, Houston, TX, USA
Key points
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r Blood glucose is an important fuel for endurance exercise. It can be derived from ingested carbohydrate, stored liver glycogen and newly synthesized glucose (gluconeogenesis).
r We hypothesized that athletes habitually following a low carbohydrate high fat (LCHF) diet r r r
would have higher rates of gluconeogenesis during exercise compared to those who follow a mixed macronutrient diet. We used stable isotope tracers to study glucose production kinetics during a 2 h ride in cyclists habituated to either a LCHF or mixed macronutrient diet. The LCHF cyclists had lower rates of total glucose production and hepatic glycogenolysis but similar rates of gluconeogenesis compared to those on the mixed diet. The LCHF cyclists did not compensate for reduced dietary carbohydrate availability by increasing glucose synthesis during exercise but rather adapted by altering whole body substrate utilization.
Abstract Endogenous glucose production (EGP) occurs via hepatic glycogenolysis (GLY) and gluconeogenesis (GNG) and plays an important role in maintaining euglycaemia. Rates of GLY and GNG increase during exercise in athletes following a mixed macronutrient diet; however, these processes have not been investigated in athletes following a low carbohydrate high fat (LCHF) diet. Therefore, we studied seven well-trained male cyclists that were habituated to either a LCHF (7% carbohydrate, 72% fat, 21% protein) or a mixed diet (51% carbohydrate, 33% fat, 16% protein) for longer than 8 months. After an overnight fast, participants performed a 2 h laboratory ride at 72% of maximal oxygen consumption. Glucose kinetics were measured at rest and during the final 30 min of exercise by infusion of [6,6-2 H2 ]-glucose and the ingestion of 2 H2 O tracers. Rates of EGP and GLY both at rest and during exercise were significantly lower in the LCHF group than the mixed diet group (Exercise EGP: LCHF, 6.0 ± 0.9 mg kg−1 min−1 , Mixed, 7.8 ± 1.1 mg kg−1 min−1 , P < 0.01; Exercise GLY: LCHF, 3.2 ± 0.7 mg kg−1 min−1 , Mixed, 5.3 ± 0.9 mg kg−1 min−1 , P < 0.01). Conversely, no difference was detected in rates of GNG between groups at rest or during exercise (Exercise: LCHF, 2.8 ± 0.4 mg kg−1 min−1 , Mixed, 2.5 ± 0.3 mg kg−1 min−1 , P = 0.15). We conclude that athletes on a LCHF diet do not compensate for reduced glucose availability via higher rates of glucose synthesis compared to athletes on a mixed diet. Instead, GNG remains relatively stable, whereas glucose oxidation and GLY are influenced by dietary factors.
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DOI: 10.1113/JP271934
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(Received 23 November 2015; accepted after revision 23 February 2016; first published online 26 February 2016) Corresponding author J. Smith: Private Bag X3, Rondebosch, Cape Town 7701, South Africa. Email: [email protected] Abbreviations ASA24, automated self-administered 24 h recall; βHB, β-hydroxybutyrate; BMI, body mass index; CV, coefficient of variation; EGP, endogenous glucose production; FFA, free fatty acids; GLY, glycogenolysis; GNG, gluconeogenesis; HR, heart rate; HRmax , maximum heart rate; LCHF, low carbohydrate high fat; MIDA, mass isotopomer distribution analysis; MUFA, monounsaturated fatty acids; PPO, peak power output; PUFA, polyunsaturated fatty acids; Ra , rate of appearance; Rd , rate of disappearance; RER, respiratory exchange ratio; RPE, rating of perceived exertion; V˙ CO2 , volume of carbon dioxide production; V˙ O2 , volume of oxygen uptake; V˙ O2 max , maximal oxygen uptake; V˙ E , minute ventilation.
Introduction Plasma glucose is an important source of energy for exercise and can be derived from the ingestion of exogenous carbohydrate and from glucose produced endogenously by the liver and, to a lesser extent, by the kidneys (Nuttall et al. 2008). As exercise duration and intensity increase, the demand for plasma glucose becomes greater (Romijn et al. 1993; van Loon et al. 2001; Trimmer et al. 2002) such that the termination of exhaustive endurance exercise is often associated with hypoglycaemia (Coyle et al. 1986; Cermak & van Loon, 2013). Thus, the liver plays a crucial role in maintaining glucose homeostasis during endurance exercise, particularly when exogenous carbohydrate is not freely available. Endogenous glucose production (EGP) occurs via two processes: glycogenolysis (GLY), which is the hydrolysis of stored liver glycogen, and gluconeogenesis (GNG), which is the synthesis of new glucose in the liver and kidneys from precursors such as lactate, glycerol, certain amino acids and potentially ketone bodies (Glew, 2010). EGP and particulary hepatic GLY are potently suppressed by carbohydrate ingestion and the consequent rise in plasma glucose and insulin concentrations (Jeukendrup et al. 1999). On the other hand, low plasma glucose and insulin concentrations and a rise in plasma glucagon, catecholamine and/or glucocorticoid concentrations can stimulate EGP. This tight regulation maintains euglycaemia by matching the rate of EGP to the rate at which glucose is taken out of the circulation by tissues. In endurance athletes who eat a conventional high carbohydrate or mixed macronutrient diet, the rate of EGP after an overnight fast doubles from rest to exercise at 50% of maximal oxygen uptake (V˙ O2 max ) (Trimmer et al. 2002) and can increase up to 4-fold from rest to exercise at 75% V˙ O2 max (Emhoff et al. 2013). These changes are the result of higher rates of both hepatic GLY and GNG, although hepatic GLY appears to play the dominant role, particularly as the intensity of exercise increases. Indeed, hepatic GLY has been reported to account for as much as 80% of EGP during exercise at 65% V˙ O2 max (Trimmer et al. 2002; Emhoff et al. 2013).
Rates of hepatic GLY are dependent on hepatic glycogen content (Arkinstall et al. 2004) and are therefore greatest after a period of high carbohydrate feeding (Bisschop et al. 2000), although they gradually decline with fasting (Landau et al. 1996) or as endurance exercise progresses (Ahlborg et al. 1974). Conversely, the main factor that determines the rate of GNG is gluconeogenic substrate delivery to the liver (Miller et al. 2002; Gustavson et al. 2003). For example, lactate is considered the primary precursor for hepatic GNG (Ahlborg et al. 1974; Consoli et al. 1990; Meyer et al. 2002) and becomes especially important as exercise intensity increases and plasma lactate concentrations rise. During very high intensity exercise, however, there is a marked reduction in hepatic blood flow (Wahren et al. 1971), which potentially limits GNG as a result of reduced precursor delivery (Sumida et al. 2006). Glycerol becomes an important precursor during prolonged fasting (Landau et al. 1996; Jensen et al. 2001) or during prolonged exercise when the rates of lipolysis are increased (Ahlborg et al. 1974). Additionally, increased plasma free fatty acid (FFA) concentrations may directly stimulate the production of glucose via GNG (Chen et al. 1999; Roden et al. 2000; Stingl et al. 2001). Clearly, substrate availability can have a profound influence on the nature of EGP; however, it is unclear how altering the macronutrient content of an athlete’s diet affects rates of GNG and hepatic GLY during exercise. This is important because low carbohydrate high fat (LCHF) diets are becoming increasingly popular among athletes as a potential means of improving endurance performance, managing weight or reducing the risk of developing chronic disease (Paoli et al. 2013; Paoli et al. 2015; Volek et al. 2016). Athletes eating a LCHF diet experience a dramatic shift away from carbohydrate towards fat oxidation during exercise (Phinney et al. 1983; Goedecke et al. 1999; Volek et al. 2016). Even so, they are still able to sustain relatively high carbohydrate oxidation rates of between 1.0 and 1.5 g min−1 during prolonged endurance exercise in the fasted state (Lambert et al. 1994; Zajac et al. 2014). These rates are lower than in control athletes eating a mixed macronutrient diet (2.0 to 2.5 g min−1 ) but, in the context of very limited dietary carbohydrate intake, C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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Glucose production during low carbohydrate exercise
it is unclear from where this carbohydrate is derived. Because athletes eating a LCHF diet have reduced muscle and liver glycogen stores (Bergstr¨om et al. 1967; Nilsson & Hultman, 1973; Phinney et al. 1983; Lambert et al. 1994) and often eat less than 50 g of carbohydrate per day, we hypothesized that they will produce substantially more glucose from GNG than athletes eating a diet higher in carbohydrate. Therefore, the present study aimed to determine whether there are differences in rates of EGP, hepatic GLY and total GNG during exercise between groups of endurance-trained cyclists who habitually eat either a mixed macronutrient or LCHF diet. Methods Ethical approval
The present study was approved by the Human Research Ethics Committee of the Faculty of Health Sciences, University of Cape Town, and was performed in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants before they were enrolled in the study. Overview of study
The participants in this cross-sectional study included a group of seven healthy, well-trained male cyclists who had habitually eaten a LCHF diet for at least the previous 8 months, and a group of seven healthy ‘control’ cyclists matched for age, body mass index (BMI), cycling ability and body fat percentage who had habitually eaten a mixed macronutrient diet for at least as long as the LCHF group. To confirm eligibility and to match the two groups, participants underwent a screening phase where in-depth diet history and anthropometry were assessed. They also completed a peak power output (PPO) / V˙ O2 max test on a cycle ergometer and a familiarization trial ride. Eligible participants then performed a 2 h laboratory ride at 55% of their PPO, during which stable isotope tracers were used to study glucose kinetics, whereas indirect calorimetry was used to estimate whole body substrate oxidation. Participants and inclusion/exclusion criteria
Participants were eligible if they were well-trained male cyclists who had at least 2 years of cycling experience and had been actively competing and/or training for at least the past 3 months; were free from known metabolic medical conditions; were not currently taking any medications; were between the ages of 18 and 45 years; had not gained or lost more than 2 kg of body weight for at least the previous 6 weeks; and had not substantially changed their diet composition for the previous 6 months. Participants C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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in the LCHF group were included if they ate less than 50 g of carbohydrate per day or no more than 10% of total calories as carbohydrate. Participants in the mixed diet group were included if they ate more than 350 g of carbohydrate per day or more than 50% of total calories as carbohydrate. Diet assessment
Habitual diet was assessed using in-depth interviews, written questionnaires about their diet history and a non-quantitative food frequency questionnaire. These tools were used to identify participants that we were confident had not significantly changed their diets within the past 6 months and probably fit into either the LCHF or mixed diet groups. Participants who reported conflicting information were not enrolled in the study. Participants then kept a first 3 day diet record, which included at least one exercising day and one non-work day. Participants were counselled how to accurately record the description and quantities of their food intake. With the assistance of the participant, the investigator then entered foods and dietary information from the 3 day record into online Automated Self-Administered 24 hour Recall (ASA24) software, which is based on the USDA Automated Multiple-Pass Method that has been validated previously (Kipnis et al. 2003; Moshfegh et al. 2008). Nutrients of foods not available on ASA24 were entered manually and all nutrient data in the ASA24 reports were checked by the investigator. This dietary record was used to determine eligibility and served as a familiarization for the diet assessment immediately prior to the tracer infusion trial. Participants who qualified for the trial were then asked to maintain their habitual diet for the duration of the study and the investigators were in regular communication with the participants during the trial to check that they only ate foods suitable for their respective diets. In the 3 days prior to the tracer infusion trial, a second detailed diet record was kept by the participant, which was captured into ASA24 on the day of the tracer infusion trial as described above. All reported data are from this second 3 day diet record. Every participant ate an evening meal between 18.30 and 19.00 h the day before the experimental trial. This meal was chosen to be typical of their usual pre-race meal and was consistent with their habitual diet assessment in terms of macronutrient content. The study design allowed glucose kinetics to be investigated under LCHF or mixed diet conditions closely reflecting how the respective participants had adapted to train and race. Maximal exercise test
All exercise trials were performed using the participants’ own bicycles mounted on a cycle ergometer
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(Computrainer Pro 3D; RacerMate, Seattle, Washington, USA), which was calibrated before and after warm-up as described previously (Lamberts et al. 2009). After a 10 min warm-up, an incremental test was started at a work rate of 100 W and the load increased continuously by 20 W min–1 until the participant could no longer sustain a cadence greater than 70 rpm. Minute ventilation volume (V˙ E ) and the volume of oxygen uptake (V˙ O2 ) and carbon dioxide production (V˙ CO2 ) per minute were recorded using a breath-by-breath gas analyser (Jaeger Oxycon Pro, Hoechberg, Germany) (Macfarlane, 2001), which was calibrated immediately before each trial. PPO was calculated as the average workload over the last minute of the test. V˙ O2 max was calculated as the highest V˙ O2 averaged over 15 s. Heart rate (HR) was measured during all exercise trials using a Suunto T6 HR monitor (Suunto Oy, Vantaa, Finland). Maximal heart rate (HRmax ) was calculated as the highest HR averaged over 4 s during the PPO test. Submaximal exercise trials
Participants performed two similar 2 h laboratory rides separated by 72 h. They were required to use motorized transport to travel to the laboratory and were instructed to keep their physical activity to a minimum on the morning of the rides. The first ride served as a familiarization trial. Participants performed a light recovery exercise session in their own time on the day after this familiarization ride but refrained from exercise the day prior to the experimental trial. During the experimental ride, stable isotope tracers were used to study glucose kinetics. Both rides started at a work rate of 100 W less than 55% of the participant’s PPO. The work rate was continuously increased up to 55% of their PPO over the first 12 min and then remained constant at 55% of PPO for the duration of the ride. During the last 5 min of each 15 min interval during the ride, participants were fitted with a respiration gas analysis mask to record V˙ E , V˙ O2 and V˙ CO2 . The first 1 min and last 15 s of each measurement period were excluded from the analysis to ensure that steady-state data were used. HR was recorded continuously during the ride and averaged during the measurement period. Rating of perceived exertion (RPE) was recorded before and after each 5 min respiratory gas collection period using the Borg 6–20 RPE scale (Borg, 1970). Experimental protocol
An overview of the experimental trial is presented in Fig. 1. The day before the experimental trial, a baseline blood sample was collected. That evening, participants ate their usual pre-race meal between 18.30 and 19.00 h, after which they fasted until the end of the experimental ride. Between
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21.00 and 23.00 h, participants ingested 4 g kg−1 body weight 2 H2 O (99.8 atom percent 2 H; Cambridge Isotope Laboratories, Cambridge, MA, USA), which enriched their body water to 0.5 % 2 H2 O (data not shown). The next morning, participants arrived at the laboratory at 06.00 h and remained in a supine position when a cannula was inserted into the antecubital vein of each arm. The right cannula was used to sample blood and the left was used for the infusion of [6,6-2 H2 ]-glucose (99 atom percent 2 H; Cambridge Isotope Laboratories). A heating blanket was placed over the lower right arm to ensure that the sampled blood was arterialized. At 06.30 h, a 6.5 mg kg−1 priming dose of [6,6-2 H2 ]-glucose was injected, followed by a 2 h continuous infusion of [6,6-2 H2 ]-glucose at a rate of 0.055 mg kg−1 min−1 using an automated syringe pump (Travenol Auto Syringe Model 5C; Travenol Laboratories Inc, Hooksett, NH, USA). After 2 h, the [6,6-2 H2 ]-glucose infusion rate was doubled to 0.11 mg kg−1 min−1 and the participant commenced the ride. This [6,6-2 H2 ]glucose infusion, blood sampling and exercise protocol is similar to that used previously (Febbraio et al. 2004) and achieved adequate isotopic equilibrium (data not shown). Sample collection
Blood samples for isotope analysis were collected in EDTA containing tubes prior to the start of [6,6-2 H2 ]-glucose infusion (–120 min); during the final 20 min of the resting infusion period (−20, −10 and 0 min); and during the final 30 min of exercise (90, 105 and 120 min). Blood samples for hormone and substrate analysis were collected immediately prior to exercise (0 min), as well as at 30, 60, 90 and 120 min during exercise. Blood for plasma glucose and lactate analysis was collected in tubes containing fluoride and oxalate; blood for serum FFA and insulin analysis was collected in tubes with a clot activator and gel barrier; blood for plasma glucagon and glycerol analysis was collected in tubes containing EDTA. All blood samples for plasma and serum analysis were centrifuged at 3000 g at 4°C for 10 min immediately after collection. They were then kept frozen on dry ice for the remainder of the trial before being stored at −20 or −80 °C prior to subsequent analysis. Whole blood was used immediately for β-hydroxybutyrate (βHB) analysis. To measure muscle glycogen usage during the ride, a muscle biopsy was collected from the vastus lateralis muscle of the right leg 15 min prior to the start of exercise and from the left leg within 5–10 min of ending exercise. The suction-assisted needle biopsy technique described by Bergstr¨om et al. (1967) and as modified by Evans et al. (1982) was used. Muscle samples were rapidly dissected free of connective tissue and frozen and stored in liquid nitrogen for later analysis.
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Rate of appearance (Ra ) and rate of disappearance (Rd ) of glucose was calculated during the last 20 min of rest (−20, −10 and 0 min) and during the last 30 min of exercise (90, 105 and 120 min) using the non-steady-state equations of Steele modified for use with stable isotopes (Wolfe, 1992): R a = [F − V((C 1 + C 2 )/2)((IE 2 − IE 1 )/(t2 − t1 ))]/ [(IE 1 + IE 2 )/2] R d = R a − V[(C 2 − C 1 )/(t2 − t1 )]
0 h3 10
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Where Ra and Rd are in mg kg–1 min–1 , F is the infusion rate (mg kg–1 min–1 ), V is the blood pool (0.18 l kg−1 ) (Emhoff et al. 2013), C is the plasma concentration of glucose (mg dl–1 ), IE is the M+2 enrichment (molar percent excess) and t is the sampling time (min). The infusion rate was subtracted from Ra to calculate the rate of EGP (mg kg–1 min–1 ).
[6,6-2H2]-glucose
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Rest + [6,6-2H2]-glucose (0.3 μmol/kg/min) –120
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The fraction of EGP derived from GNG (fractional GNG) was estimated from plasma samples using the average deuterium enrichment method (Chacko et al. 2008). The principle behind this technique is that the glucose produced via GNG incorporates hydrogen from body water at all carbon (C) positions (C-1, 2, 3, 4, 5, 6, or 6), whereas glucose derived from GLY incorporates hydrogen ions only at the C-2 position (Wolfe, 1992). Moreover, 2 H incorporation at C-2 is the result of complete 2 H exchange with body water during extensive glucose 6-phosphate to fructose 6-phosphate isomerization and rapidly achieves 2 H enrichment equivalent to that in body water (Landau et al. 1996). Therefore, after ingestion and equilibration of 2 H2 O in the total body water pool, the average deuterium enrichment of glucose at any position besides C-2 reflects glucose produced via gluconeogenic pathways (Chacko et al. 2008). Assuming complete equilibration of plasma 2 H2 O enrichment with deuterium enrichment of glucose at C-2, fractional GNG can be calculated as the ratio of average deuterium enrichment of glucose at C-1, 3, 4, 5, 6 and 6 (i.e. not at C-2) to enrichment of plasma 2 H2 O (Landau et al. 1996; Chacko et al. 2008). To quantify these specific glucose isotope enrichments, selective ion monitoring was performed as described previously (Chacko et al. 2008). Briefly, the pentaacetate derivatives of plasma glucose were prepared and analysed using gas chromatography–mass spectrometry (6890N/5975B inert EI/CI; Agilent Technologies, Wilmington, DE, USA) in the positive chemical ionization mode using methane as the reagent gas. Selective ion monitoring of m/z 170/169 was performed to determine the M+1 enrichment of deuterium in the circulating glucose carbons (C-1, 3, 4, 5, 6 or 6) where M is the base mass 169, representing unlabelled
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glucose (Chacko et al. 2008). To accurately measure the deuterium labelling of glucose from ingested 2 H2 O, the enrichment of M+1 resulting from natural abundance was subtracted. Deuterium enrichment in plasma water was determined by isotope ratio mass spectrometry (Delta+ XL IRMS; Thermo Finnigan, Bremen, Germany) as described previously (Chacko et al. 2008). The M+2 enrichment of glucose derived from infusion of [6,62 H2 ]-glucose was determined from plasma samples using gas chromatography–mass spectrometry in the electron impact ionization mode by selective ion monitoring of m/z 244/242 and was used to measure appearance rate of glucose as described previously (Bier et al. 1977; Chacko & Sunehag, 2010).
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Figure 1. An overview of the experimental trial starting from the day prior to the 2 h ride Clock times (above line) and days or minutes relative to the start of exercise (below line) are shown. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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Fractional GNG was calculated from the average M+1 enrichment of 2 H2 O as: Average M+1 enrichment = (M+1)(2 H)(m/z170/169) /6 Fractional GNG = Average M + 1 enrichment/E 2 H2 O) Where (M+1)(2 H)(m/z 170/169) is the M+1 enrichment of deuterium in glucose measured using m/z 170/169 minus the natural abundance M+1 enrichment, ‘6’ is the number of 2 H labelling sites on the m/z 170/169 fragment of glucose and E 2 H2 O is the deuterium enrichment in plasma water (Chacko et al. 2008). Absolute rates of GNG and hepatic GLY were calculated as: GNG(mg kg−1 min−1 ) = fractional GNG × EGP GLY(mg kg−1 min−1 ) = EGP − GNG Metabolic calculations
Carbohydrate and fat oxidation rates and energy expenditure were calculated from V˙ O2 and V˙ CO2 using the stoichiometric equations and appropriate energy equivalents below, with the assumption that urinary nitrogen excretion rate was negligible (Frayn, 1983): Carbohydrate oxidation rate (g min−1 ) = (4.55 V˙ CO2 ) − (3.21V˙ O2 ) Fat oxidation rate (g min−1 ) = (1.67 V˙ O2 )−(1.67 V˙ CO2 ) 1 g of CHO = 4 kcal; 1 g of fat = 9 kcal Substrate analysis
All sample analyses except for βHB were performed in duplicate and the intra-assay coefficient of variation (CV) is provided for each assay. Plasma glucose (CV, 1.82%) and lactate concentrations (CV, 0.9%) were determined using the glucose oxidase method (YSI 2300; STAT PLUS, Yellow Springs, OH, USA). Serum insulin concentrations (CV, 0.58%) were determined using automated chemiluminescence (Centaur CP system; Siemens Healthcare Diagnostics Inc., New York, NY, USA). Blood βHB concentrations were determined immediately after sampling using a FreeStyle Optium Xceed β-ketone meter and FreeStyle Optium H test strips (Abbott Laboratories, Abbott Park, IL, USA). Serum FFA concentrations (CV, 4.6%) were determined using a commercial kit (FFA half-micro test; Roche Applied Science, Mannheim, Germany) and a Multi-Mode Microplate Reader (BioTek Synergy HT; BioTek Inc., Winooski, VT, USA). Plasma
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glucagon concentrations (CV, 19%) were determined using a commercial kit (Glucagon EIA Kit; Sigma-Aldrich, St Louis, MO, USA). Serum glycerol concentrations (CV, 6.7%) were determined using a commercial kit (Abcam, Cambridge, UK). Muscle glycogen
Muscle glycogen content was measured as glucose equivalents using the glucose oxidase method following the acid hydrolysis of glycogen to glucose (Passonneau & Lauderdale, 1974). Approximately 20 mg of muscle (wet weight) was weighed and freeze dried prior to the analysis. The rate of muscle glycogen utilization (g min–1 ) was calculated using 15 kg as an estimate of leg muscle mass (Janssen et al. 2000). Statistical analysis
Normality was tested using Shapiro-Wilk’s W test and Levine’s test for equality of variance. Where normally distributed, differences between groups were detected using the independent t test. Where not normally distributed, the Mann–Whitney U test was used. Two-way ANOVA for repeated measures was used to determine differences in substrate oxidation rates, substrate and hormone concentrations and muscle glycogen content. Where a significant interaction effect was detected, further statistical analysis was performed using Tukey’s honestly significant post hoc test. P < 0.05 was considered statistically significant. Statistical analysis was performed using Statistica, version 12 (StatSoft, Inc., Tulsa, OK, USA). Results Participant and dietary characteristics
As intended, the two groups were well matched for age, weight, BMI, body fat percentage, PPO and V˙ O2 max (Table 1). The macronutrient composition of the diets during the 3 days prior to the experimental trial was 21% protein, 72% fat and 7% carbohydrate in the LCHF group and 16% protein, 33% fat and 51% carbohydrate in the mixed diet group. The LCHF group averaged between 15 and 82 g day−1 of total carbohydrate (including dietary fibre) compared to between 272 and 561 g day−1 in the mixed diet group (Table 2). Whether expressed in absolute terms (g day–1 ) or relative to body weight (g kg–1 day–1 ), there was no difference in energy intake or protein intake between groups. Despite the LCHF group eating almost twice the amount of total fat, intakes of polyunsaturated fatty acids (PUFA) were not significantly different between groups (P = 0.91) (Table 2). The LCHF group consumed
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Table 1. Participant characteristics for the LCHF and mixed diet groups LCHF Age (years) Weight (kg) BMI (kg m–2 ) Body fat (%) HRmax (beats min–1 ) V˙ O2 max (ml min–1 ) V˙ O2 max (ml kg–1 min–1 ) PPO (W) PPO (W kg–1 )
36 78 23.6 10 184 4683 61 369 4.8
± ± ± ± ± ± ± ± ±
6 (29–44) 9 (65–90) 1.8 (20–27) 3 (7–14) 5 (176–193) 445 (4019–5388) 5 (52–68) 27 (322–422) 0.4 (4.0–5.2)
Mixed 32 74 23.4 10 182 4573 63 367 5.0
± ± ± ± ± ± ± ± ±
5 (24–40) 8 (62–89) 2.0 (20–27) 3 (7–17) 8 (172–193) 483 (3770–5054) 8 (46–73) 38 (291–412) 0.4 (4.3–5.4)
P 0.24 0.41 0.76 0.90 0.54 0.69 0.61 0.93 0.34
Values are presented as the mean ± SD (range); n = 7 per group. P values were determined by an independent t test.
Table 2. Dietary characteristics for the LCHF and mixed diet groups LCHF Time on diet (months) Energy intake (kcal day–1 ) Protein (g day–1 ) Fat (g day–1 ) Carbohydrate (g day–1 ) Sugars (g day–1 ) Fibre (g day–1 ) Saturated fat (g day–1 ) MUFA (g day–1 ) PUFA (g day–1 )
13 2866 147 231 50 25 13 97 90 26
± ± ± ± ± ± ± ± ± ±
6 (8–24) 296 (2310–3190) 35 (78–192) 21 (201–256) 20 (15–82) 15 (5–53) 6 (4–18) 11 (85–118) 20 (70–124) 7 (16–36)
Mixed 107 3187 131 120 394 173 42 39 44 26
± ± ± ± ± ± ± ± ± ±
115 (9–360) 941 (2387–4916) 51 (77–194) 52 (65–216) 102 (272–561) 60 (89–264) 17 (16–72) 15 (18–124) 20 (24–84) 15 (12–53)
P 0.18 0.41 0.50
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Gluconeogenesis during endurance exercise in cyclists habituated to a long-term low carbohydrate high-fat diet Christopher C. Webster1 , Timothy D. Noakes1 , Shaji K. Chacko2 , Jeroen Swart1 , Tertius A. Kohn1 and James A. H. Smith1 1
Division of Exercise Science and Sports Medicine (ESSM), Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Newlands, South Africa 2 Department of Pediatrics, Children’s Nutrition Research Center, US Department of Agriculture/Agricultural Research Service, Baylor College of Medicine, Houston, TX, USA
Key points
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r Blood glucose is an important fuel for endurance exercise. It can be derived from ingested carbohydrate, stored liver glycogen and newly synthesized glucose (gluconeogenesis).
r We hypothesized that athletes habitually following a low carbohydrate high fat (LCHF) diet r r r
would have higher rates of gluconeogenesis during exercise compared to those who follow a mixed macronutrient diet. We used stable isotope tracers to study glucose production kinetics during a 2 h ride in cyclists habituated to either a LCHF or mixed macronutrient diet. The LCHF cyclists had lower rates of total glucose production and hepatic glycogenolysis but similar rates of gluconeogenesis compared to those on the mixed diet. The LCHF cyclists did not compensate for reduced dietary carbohydrate availability by increasing glucose synthesis during exercise but rather adapted by altering whole body substrate utilization.
Abstract Endogenous glucose production (EGP) occurs via hepatic glycogenolysis (GLY) and gluconeogenesis (GNG) and plays an important role in maintaining euglycaemia. Rates of GLY and GNG increase during exercise in athletes following a mixed macronutrient diet; however, these processes have not been investigated in athletes following a low carbohydrate high fat (LCHF) diet. Therefore, we studied seven well-trained male cyclists that were habituated to either a LCHF (7% carbohydrate, 72% fat, 21% protein) or a mixed diet (51% carbohydrate, 33% fat, 16% protein) for longer than 8 months. After an overnight fast, participants performed a 2 h laboratory ride at 72% of maximal oxygen consumption. Glucose kinetics were measured at rest and during the final 30 min of exercise by infusion of [6,6-2 H2 ]-glucose and the ingestion of 2 H2 O tracers. Rates of EGP and GLY both at rest and during exercise were significantly lower in the LCHF group than the mixed diet group (Exercise EGP: LCHF, 6.0 ± 0.9 mg kg−1 min−1 , Mixed, 7.8 ± 1.1 mg kg−1 min−1 , P < 0.01; Exercise GLY: LCHF, 3.2 ± 0.7 mg kg−1 min−1 , Mixed, 5.3 ± 0.9 mg kg−1 min−1 , P < 0.01). Conversely, no difference was detected in rates of GNG between groups at rest or during exercise (Exercise: LCHF, 2.8 ± 0.4 mg kg−1 min−1 , Mixed, 2.5 ± 0.3 mg kg−1 min−1 , P = 0.15). We conclude that athletes on a LCHF diet do not compensate for reduced glucose availability via higher rates of glucose synthesis compared to athletes on a mixed diet. Instead, GNG remains relatively stable, whereas glucose oxidation and GLY are influenced by dietary factors.
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DOI: 10.1113/JP271934
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(Received 23 November 2015; accepted after revision 23 February 2016; first published online 26 February 2016) Corresponding author J. Smith: Private Bag X3, Rondebosch, Cape Town 7701, South Africa. Email: [email protected] Abbreviations ASA24, automated self-administered 24 h recall; βHB, β-hydroxybutyrate; BMI, body mass index; CV, coefficient of variation; EGP, endogenous glucose production; FFA, free fatty acids; GLY, glycogenolysis; GNG, gluconeogenesis; HR, heart rate; HRmax , maximum heart rate; LCHF, low carbohydrate high fat; MIDA, mass isotopomer distribution analysis; MUFA, monounsaturated fatty acids; PPO, peak power output; PUFA, polyunsaturated fatty acids; Ra , rate of appearance; Rd , rate of disappearance; RER, respiratory exchange ratio; RPE, rating of perceived exertion; V˙ CO2 , volume of carbon dioxide production; V˙ O2 , volume of oxygen uptake; V˙ O2 max , maximal oxygen uptake; V˙ E , minute ventilation.
Introduction Plasma glucose is an important source of energy for exercise and can be derived from the ingestion of exogenous carbohydrate and from glucose produced endogenously by the liver and, to a lesser extent, by the kidneys (Nuttall et al. 2008). As exercise duration and intensity increase, the demand for plasma glucose becomes greater (Romijn et al. 1993; van Loon et al. 2001; Trimmer et al. 2002) such that the termination of exhaustive endurance exercise is often associated with hypoglycaemia (Coyle et al. 1986; Cermak & van Loon, 2013). Thus, the liver plays a crucial role in maintaining glucose homeostasis during endurance exercise, particularly when exogenous carbohydrate is not freely available. Endogenous glucose production (EGP) occurs via two processes: glycogenolysis (GLY), which is the hydrolysis of stored liver glycogen, and gluconeogenesis (GNG), which is the synthesis of new glucose in the liver and kidneys from precursors such as lactate, glycerol, certain amino acids and potentially ketone bodies (Glew, 2010). EGP and particulary hepatic GLY are potently suppressed by carbohydrate ingestion and the consequent rise in plasma glucose and insulin concentrations (Jeukendrup et al. 1999). On the other hand, low plasma glucose and insulin concentrations and a rise in plasma glucagon, catecholamine and/or glucocorticoid concentrations can stimulate EGP. This tight regulation maintains euglycaemia by matching the rate of EGP to the rate at which glucose is taken out of the circulation by tissues. In endurance athletes who eat a conventional high carbohydrate or mixed macronutrient diet, the rate of EGP after an overnight fast doubles from rest to exercise at 50% of maximal oxygen uptake (V˙ O2 max ) (Trimmer et al. 2002) and can increase up to 4-fold from rest to exercise at 75% V˙ O2 max (Emhoff et al. 2013). These changes are the result of higher rates of both hepatic GLY and GNG, although hepatic GLY appears to play the dominant role, particularly as the intensity of exercise increases. Indeed, hepatic GLY has been reported to account for as much as 80% of EGP during exercise at 65% V˙ O2 max (Trimmer et al. 2002; Emhoff et al. 2013).
Rates of hepatic GLY are dependent on hepatic glycogen content (Arkinstall et al. 2004) and are therefore greatest after a period of high carbohydrate feeding (Bisschop et al. 2000), although they gradually decline with fasting (Landau et al. 1996) or as endurance exercise progresses (Ahlborg et al. 1974). Conversely, the main factor that determines the rate of GNG is gluconeogenic substrate delivery to the liver (Miller et al. 2002; Gustavson et al. 2003). For example, lactate is considered the primary precursor for hepatic GNG (Ahlborg et al. 1974; Consoli et al. 1990; Meyer et al. 2002) and becomes especially important as exercise intensity increases and plasma lactate concentrations rise. During very high intensity exercise, however, there is a marked reduction in hepatic blood flow (Wahren et al. 1971), which potentially limits GNG as a result of reduced precursor delivery (Sumida et al. 2006). Glycerol becomes an important precursor during prolonged fasting (Landau et al. 1996; Jensen et al. 2001) or during prolonged exercise when the rates of lipolysis are increased (Ahlborg et al. 1974). Additionally, increased plasma free fatty acid (FFA) concentrations may directly stimulate the production of glucose via GNG (Chen et al. 1999; Roden et al. 2000; Stingl et al. 2001). Clearly, substrate availability can have a profound influence on the nature of EGP; however, it is unclear how altering the macronutrient content of an athlete’s diet affects rates of GNG and hepatic GLY during exercise. This is important because low carbohydrate high fat (LCHF) diets are becoming increasingly popular among athletes as a potential means of improving endurance performance, managing weight or reducing the risk of developing chronic disease (Paoli et al. 2013; Paoli et al. 2015; Volek et al. 2016). Athletes eating a LCHF diet experience a dramatic shift away from carbohydrate towards fat oxidation during exercise (Phinney et al. 1983; Goedecke et al. 1999; Volek et al. 2016). Even so, they are still able to sustain relatively high carbohydrate oxidation rates of between 1.0 and 1.5 g min−1 during prolonged endurance exercise in the fasted state (Lambert et al. 1994; Zajac et al. 2014). These rates are lower than in control athletes eating a mixed macronutrient diet (2.0 to 2.5 g min−1 ) but, in the context of very limited dietary carbohydrate intake, C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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it is unclear from where this carbohydrate is derived. Because athletes eating a LCHF diet have reduced muscle and liver glycogen stores (Bergstr¨om et al. 1967; Nilsson & Hultman, 1973; Phinney et al. 1983; Lambert et al. 1994) and often eat less than 50 g of carbohydrate per day, we hypothesized that they will produce substantially more glucose from GNG than athletes eating a diet higher in carbohydrate. Therefore, the present study aimed to determine whether there are differences in rates of EGP, hepatic GLY and total GNG during exercise between groups of endurance-trained cyclists who habitually eat either a mixed macronutrient or LCHF diet. Methods Ethical approval
The present study was approved by the Human Research Ethics Committee of the Faculty of Health Sciences, University of Cape Town, and was performed in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants before they were enrolled in the study. Overview of study
The participants in this cross-sectional study included a group of seven healthy, well-trained male cyclists who had habitually eaten a LCHF diet for at least the previous 8 months, and a group of seven healthy ‘control’ cyclists matched for age, body mass index (BMI), cycling ability and body fat percentage who had habitually eaten a mixed macronutrient diet for at least as long as the LCHF group. To confirm eligibility and to match the two groups, participants underwent a screening phase where in-depth diet history and anthropometry were assessed. They also completed a peak power output (PPO) / V˙ O2 max test on a cycle ergometer and a familiarization trial ride. Eligible participants then performed a 2 h laboratory ride at 55% of their PPO, during which stable isotope tracers were used to study glucose kinetics, whereas indirect calorimetry was used to estimate whole body substrate oxidation. Participants and inclusion/exclusion criteria
Participants were eligible if they were well-trained male cyclists who had at least 2 years of cycling experience and had been actively competing and/or training for at least the past 3 months; were free from known metabolic medical conditions; were not currently taking any medications; were between the ages of 18 and 45 years; had not gained or lost more than 2 kg of body weight for at least the previous 6 weeks; and had not substantially changed their diet composition for the previous 6 months. Participants C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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in the LCHF group were included if they ate less than 50 g of carbohydrate per day or no more than 10% of total calories as carbohydrate. Participants in the mixed diet group were included if they ate more than 350 g of carbohydrate per day or more than 50% of total calories as carbohydrate. Diet assessment
Habitual diet was assessed using in-depth interviews, written questionnaires about their diet history and a non-quantitative food frequency questionnaire. These tools were used to identify participants that we were confident had not significantly changed their diets within the past 6 months and probably fit into either the LCHF or mixed diet groups. Participants who reported conflicting information were not enrolled in the study. Participants then kept a first 3 day diet record, which included at least one exercising day and one non-work day. Participants were counselled how to accurately record the description and quantities of their food intake. With the assistance of the participant, the investigator then entered foods and dietary information from the 3 day record into online Automated Self-Administered 24 hour Recall (ASA24) software, which is based on the USDA Automated Multiple-Pass Method that has been validated previously (Kipnis et al. 2003; Moshfegh et al. 2008). Nutrients of foods not available on ASA24 were entered manually and all nutrient data in the ASA24 reports were checked by the investigator. This dietary record was used to determine eligibility and served as a familiarization for the diet assessment immediately prior to the tracer infusion trial. Participants who qualified for the trial were then asked to maintain their habitual diet for the duration of the study and the investigators were in regular communication with the participants during the trial to check that they only ate foods suitable for their respective diets. In the 3 days prior to the tracer infusion trial, a second detailed diet record was kept by the participant, which was captured into ASA24 on the day of the tracer infusion trial as described above. All reported data are from this second 3 day diet record. Every participant ate an evening meal between 18.30 and 19.00 h the day before the experimental trial. This meal was chosen to be typical of their usual pre-race meal and was consistent with their habitual diet assessment in terms of macronutrient content. The study design allowed glucose kinetics to be investigated under LCHF or mixed diet conditions closely reflecting how the respective participants had adapted to train and race. Maximal exercise test
All exercise trials were performed using the participants’ own bicycles mounted on a cycle ergometer
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(Computrainer Pro 3D; RacerMate, Seattle, Washington, USA), which was calibrated before and after warm-up as described previously (Lamberts et al. 2009). After a 10 min warm-up, an incremental test was started at a work rate of 100 W and the load increased continuously by 20 W min–1 until the participant could no longer sustain a cadence greater than 70 rpm. Minute ventilation volume (V˙ E ) and the volume of oxygen uptake (V˙ O2 ) and carbon dioxide production (V˙ CO2 ) per minute were recorded using a breath-by-breath gas analyser (Jaeger Oxycon Pro, Hoechberg, Germany) (Macfarlane, 2001), which was calibrated immediately before each trial. PPO was calculated as the average workload over the last minute of the test. V˙ O2 max was calculated as the highest V˙ O2 averaged over 15 s. Heart rate (HR) was measured during all exercise trials using a Suunto T6 HR monitor (Suunto Oy, Vantaa, Finland). Maximal heart rate (HRmax ) was calculated as the highest HR averaged over 4 s during the PPO test. Submaximal exercise trials
Participants performed two similar 2 h laboratory rides separated by 72 h. They were required to use motorized transport to travel to the laboratory and were instructed to keep their physical activity to a minimum on the morning of the rides. The first ride served as a familiarization trial. Participants performed a light recovery exercise session in their own time on the day after this familiarization ride but refrained from exercise the day prior to the experimental trial. During the experimental ride, stable isotope tracers were used to study glucose kinetics. Both rides started at a work rate of 100 W less than 55% of the participant’s PPO. The work rate was continuously increased up to 55% of their PPO over the first 12 min and then remained constant at 55% of PPO for the duration of the ride. During the last 5 min of each 15 min interval during the ride, participants were fitted with a respiration gas analysis mask to record V˙ E , V˙ O2 and V˙ CO2 . The first 1 min and last 15 s of each measurement period were excluded from the analysis to ensure that steady-state data were used. HR was recorded continuously during the ride and averaged during the measurement period. Rating of perceived exertion (RPE) was recorded before and after each 5 min respiratory gas collection period using the Borg 6–20 RPE scale (Borg, 1970). Experimental protocol
An overview of the experimental trial is presented in Fig. 1. The day before the experimental trial, a baseline blood sample was collected. That evening, participants ate their usual pre-race meal between 18.30 and 19.00 h, after which they fasted until the end of the experimental ride. Between
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21.00 and 23.00 h, participants ingested 4 g kg−1 body weight 2 H2 O (99.8 atom percent 2 H; Cambridge Isotope Laboratories, Cambridge, MA, USA), which enriched their body water to 0.5 % 2 H2 O (data not shown). The next morning, participants arrived at the laboratory at 06.00 h and remained in a supine position when a cannula was inserted into the antecubital vein of each arm. The right cannula was used to sample blood and the left was used for the infusion of [6,6-2 H2 ]-glucose (99 atom percent 2 H; Cambridge Isotope Laboratories). A heating blanket was placed over the lower right arm to ensure that the sampled blood was arterialized. At 06.30 h, a 6.5 mg kg−1 priming dose of [6,6-2 H2 ]-glucose was injected, followed by a 2 h continuous infusion of [6,6-2 H2 ]-glucose at a rate of 0.055 mg kg−1 min−1 using an automated syringe pump (Travenol Auto Syringe Model 5C; Travenol Laboratories Inc, Hooksett, NH, USA). After 2 h, the [6,6-2 H2 ]-glucose infusion rate was doubled to 0.11 mg kg−1 min−1 and the participant commenced the ride. This [6,6-2 H2 ]glucose infusion, blood sampling and exercise protocol is similar to that used previously (Febbraio et al. 2004) and achieved adequate isotopic equilibrium (data not shown). Sample collection
Blood samples for isotope analysis were collected in EDTA containing tubes prior to the start of [6,6-2 H2 ]-glucose infusion (–120 min); during the final 20 min of the resting infusion period (−20, −10 and 0 min); and during the final 30 min of exercise (90, 105 and 120 min). Blood samples for hormone and substrate analysis were collected immediately prior to exercise (0 min), as well as at 30, 60, 90 and 120 min during exercise. Blood for plasma glucose and lactate analysis was collected in tubes containing fluoride and oxalate; blood for serum FFA and insulin analysis was collected in tubes with a clot activator and gel barrier; blood for plasma glucagon and glycerol analysis was collected in tubes containing EDTA. All blood samples for plasma and serum analysis were centrifuged at 3000 g at 4°C for 10 min immediately after collection. They were then kept frozen on dry ice for the remainder of the trial before being stored at −20 or −80 °C prior to subsequent analysis. Whole blood was used immediately for β-hydroxybutyrate (βHB) analysis. To measure muscle glycogen usage during the ride, a muscle biopsy was collected from the vastus lateralis muscle of the right leg 15 min prior to the start of exercise and from the left leg within 5–10 min of ending exercise. The suction-assisted needle biopsy technique described by Bergstr¨om et al. (1967) and as modified by Evans et al. (1982) was used. Muscle samples were rapidly dissected free of connective tissue and frozen and stored in liquid nitrogen for later analysis.
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Rate of appearance (Ra ) and rate of disappearance (Rd ) of glucose was calculated during the last 20 min of rest (−20, −10 and 0 min) and during the last 30 min of exercise (90, 105 and 120 min) using the non-steady-state equations of Steele modified for use with stable isotopes (Wolfe, 1992): R a = [F − V((C 1 + C 2 )/2)((IE 2 − IE 1 )/(t2 − t1 ))]/ [(IE 1 + IE 2 )/2] R d = R a − V[(C 2 − C 1 )/(t2 − t1 )]
0 h3 10
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Where Ra and Rd are in mg kg–1 min–1 , F is the infusion rate (mg kg–1 min–1 ), V is the blood pool (0.18 l kg−1 ) (Emhoff et al. 2013), C is the plasma concentration of glucose (mg dl–1 ), IE is the M+2 enrichment (molar percent excess) and t is the sampling time (min). The infusion rate was subtracted from Ra to calculate the rate of EGP (mg kg–1 min–1 ).
[6,6-2H2]-glucose
h3
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The fraction of EGP derived from GNG (fractional GNG) was estimated from plasma samples using the average deuterium enrichment method (Chacko et al. 2008). The principle behind this technique is that the glucose produced via GNG incorporates hydrogen from body water at all carbon (C) positions (C-1, 2, 3, 4, 5, 6, or 6), whereas glucose derived from GLY incorporates hydrogen ions only at the C-2 position (Wolfe, 1992). Moreover, 2 H incorporation at C-2 is the result of complete 2 H exchange with body water during extensive glucose 6-phosphate to fructose 6-phosphate isomerization and rapidly achieves 2 H enrichment equivalent to that in body water (Landau et al. 1996). Therefore, after ingestion and equilibration of 2 H2 O in the total body water pool, the average deuterium enrichment of glucose at any position besides C-2 reflects glucose produced via gluconeogenic pathways (Chacko et al. 2008). Assuming complete equilibration of plasma 2 H2 O enrichment with deuterium enrichment of glucose at C-2, fractional GNG can be calculated as the ratio of average deuterium enrichment of glucose at C-1, 3, 4, 5, 6 and 6 (i.e. not at C-2) to enrichment of plasma 2 H2 O (Landau et al. 1996; Chacko et al. 2008). To quantify these specific glucose isotope enrichments, selective ion monitoring was performed as described previously (Chacko et al. 2008). Briefly, the pentaacetate derivatives of plasma glucose were prepared and analysed using gas chromatography–mass spectrometry (6890N/5975B inert EI/CI; Agilent Technologies, Wilmington, DE, USA) in the positive chemical ionization mode using methane as the reagent gas. Selective ion monitoring of m/z 170/169 was performed to determine the M+1 enrichment of deuterium in the circulating glucose carbons (C-1, 3, 4, 5, 6 or 6) where M is the base mass 169, representing unlabelled
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glucose (Chacko et al. 2008). To accurately measure the deuterium labelling of glucose from ingested 2 H2 O, the enrichment of M+1 resulting from natural abundance was subtracted. Deuterium enrichment in plasma water was determined by isotope ratio mass spectrometry (Delta+ XL IRMS; Thermo Finnigan, Bremen, Germany) as described previously (Chacko et al. 2008). The M+2 enrichment of glucose derived from infusion of [6,62 H2 ]-glucose was determined from plasma samples using gas chromatography–mass spectrometry in the electron impact ionization mode by selective ion monitoring of m/z 244/242 and was used to measure appearance rate of glucose as described previously (Bier et al. 1977; Chacko & Sunehag, 2010).
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Figure 1. An overview of the experimental trial starting from the day prior to the 2 h ride Clock times (above line) and days or minutes relative to the start of exercise (below line) are shown. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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Fractional GNG was calculated from the average M+1 enrichment of 2 H2 O as: Average M+1 enrichment = (M+1)(2 H)(m/z170/169) /6 Fractional GNG = Average M + 1 enrichment/E 2 H2 O) Where (M+1)(2 H)(m/z 170/169) is the M+1 enrichment of deuterium in glucose measured using m/z 170/169 minus the natural abundance M+1 enrichment, ‘6’ is the number of 2 H labelling sites on the m/z 170/169 fragment of glucose and E 2 H2 O is the deuterium enrichment in plasma water (Chacko et al. 2008). Absolute rates of GNG and hepatic GLY were calculated as: GNG(mg kg−1 min−1 ) = fractional GNG × EGP GLY(mg kg−1 min−1 ) = EGP − GNG Metabolic calculations
Carbohydrate and fat oxidation rates and energy expenditure were calculated from V˙ O2 and V˙ CO2 using the stoichiometric equations and appropriate energy equivalents below, with the assumption that urinary nitrogen excretion rate was negligible (Frayn, 1983): Carbohydrate oxidation rate (g min−1 ) = (4.55 V˙ CO2 ) − (3.21V˙ O2 ) Fat oxidation rate (g min−1 ) = (1.67 V˙ O2 )−(1.67 V˙ CO2 ) 1 g of CHO = 4 kcal; 1 g of fat = 9 kcal Substrate analysis
All sample analyses except for βHB were performed in duplicate and the intra-assay coefficient of variation (CV) is provided for each assay. Plasma glucose (CV, 1.82%) and lactate concentrations (CV, 0.9%) were determined using the glucose oxidase method (YSI 2300; STAT PLUS, Yellow Springs, OH, USA). Serum insulin concentrations (CV, 0.58%) were determined using automated chemiluminescence (Centaur CP system; Siemens Healthcare Diagnostics Inc., New York, NY, USA). Blood βHB concentrations were determined immediately after sampling using a FreeStyle Optium Xceed β-ketone meter and FreeStyle Optium H test strips (Abbott Laboratories, Abbott Park, IL, USA). Serum FFA concentrations (CV, 4.6%) were determined using a commercial kit (FFA half-micro test; Roche Applied Science, Mannheim, Germany) and a Multi-Mode Microplate Reader (BioTek Synergy HT; BioTek Inc., Winooski, VT, USA). Plasma
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glucagon concentrations (CV, 19%) were determined using a commercial kit (Glucagon EIA Kit; Sigma-Aldrich, St Louis, MO, USA). Serum glycerol concentrations (CV, 6.7%) were determined using a commercial kit (Abcam, Cambridge, UK). Muscle glycogen
Muscle glycogen content was measured as glucose equivalents using the glucose oxidase method following the acid hydrolysis of glycogen to glucose (Passonneau & Lauderdale, 1974). Approximately 20 mg of muscle (wet weight) was weighed and freeze dried prior to the analysis. The rate of muscle glycogen utilization (g min–1 ) was calculated using 15 kg as an estimate of leg muscle mass (Janssen et al. 2000). Statistical analysis
Normality was tested using Shapiro-Wilk’s W test and Levine’s test for equality of variance. Where normally distributed, differences between groups were detected using the independent t test. Where not normally distributed, the Mann–Whitney U test was used. Two-way ANOVA for repeated measures was used to determine differences in substrate oxidation rates, substrate and hormone concentrations and muscle glycogen content. Where a significant interaction effect was detected, further statistical analysis was performed using Tukey’s honestly significant post hoc test. P < 0.05 was considered statistically significant. Statistical analysis was performed using Statistica, version 12 (StatSoft, Inc., Tulsa, OK, USA). Results Participant and dietary characteristics
As intended, the two groups were well matched for age, weight, BMI, body fat percentage, PPO and V˙ O2 max (Table 1). The macronutrient composition of the diets during the 3 days prior to the experimental trial was 21% protein, 72% fat and 7% carbohydrate in the LCHF group and 16% protein, 33% fat and 51% carbohydrate in the mixed diet group. The LCHF group averaged between 15 and 82 g day−1 of total carbohydrate (including dietary fibre) compared to between 272 and 561 g day−1 in the mixed diet group (Table 2). Whether expressed in absolute terms (g day–1 ) or relative to body weight (g kg–1 day–1 ), there was no difference in energy intake or protein intake between groups. Despite the LCHF group eating almost twice the amount of total fat, intakes of polyunsaturated fatty acids (PUFA) were not significantly different between groups (P = 0.91) (Table 2). The LCHF group consumed
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Table 1. Participant characteristics for the LCHF and mixed diet groups LCHF Age (years) Weight (kg) BMI (kg m–2 ) Body fat (%) HRmax (beats min–1 ) V˙ O2 max (ml min–1 ) V˙ O2 max (ml kg–1 min–1 ) PPO (W) PPO (W kg–1 )
36 78 23.6 10 184 4683 61 369 4.8
± ± ± ± ± ± ± ± ±
6 (29–44) 9 (65–90) 1.8 (20–27) 3 (7–14) 5 (176–193) 445 (4019–5388) 5 (52–68) 27 (322–422) 0.4 (4.0–5.2)
Mixed 32 74 23.4 10 182 4573 63 367 5.0
± ± ± ± ± ± ± ± ±
5 (24–40) 8 (62–89) 2.0 (20–27) 3 (7–17) 8 (172–193) 483 (3770–5054) 8 (46–73) 38 (291–412) 0.4 (4.3–5.4)
P 0.24 0.41 0.76 0.90 0.54 0.69 0.61 0.93 0.34
Values are presented as the mean ± SD (range); n = 7 per group. P values were determined by an independent t test.
Table 2. Dietary characteristics for the LCHF and mixed diet groups LCHF Time on diet (months) Energy intake (kcal day–1 ) Protein (g day–1 ) Fat (g day–1 ) Carbohydrate (g day–1 ) Sugars (g day–1 ) Fibre (g day–1 ) Saturated fat (g day–1 ) MUFA (g day–1 ) PUFA (g day–1 )
13 2866 147 231 50 25 13 97 90 26
± ± ± ± ± ± ± ± ± ±
6 (8–24) 296 (2310–3190) 35 (78–192) 21 (201–256) 20 (15–82) 15 (5–53) 6 (4–18) 11 (85–118) 20 (70–124) 7 (16–36)
Mixed 107 3187 131 120 394 173 42 39 44 26
± ± ± ± ± ± ± ± ± ±
115 (9–360) 941 (2387–4916) 51 (77–194) 52 (65–216) 102 (272–561) 60 (89–264) 17 (16–72) 15 (18–124) 20 (24–84) 15 (12–53)
P 0.18 0.41 0.50
