The effect of short-term fasting on liver and skeletal muscle lipid, glucose, and energy metabolism in healthy women and men.

Fasting promotes triglyceride (TG) accumulation in lean tissues of some animals, but the effect in humans is unknown. Additionally, fasting lipolysis is sexually dimorphic in humans, suggesting that lean tissue TG accumulation and metabolism may differ between women and men. This study investigated lean tissue TG content and metabolism in women and men during extended fasting. Liver and muscle TG content were measured by magnetic resonance spectroscopy during a 48-h fast in healthy men and women. Whole-body and hepatic carbohydrate, lipid, and energy metabolism were also evaluated using biochemical, calorimetric, and stable isotope tracer techniques. As expected, postabsorptive plasma fatty acids (FAs) were higher in women than in men but increased more rapidly in men with the onset of early starvation. Concurrently, sexual dimorphism was apparent in lean tissue TG accumulation during the fast, occurring in livers of men but in muscles of women. Despite differences in lean tissue TG distribution, men and women had identical fasting responses in whole-body and hepatic glucose and oxidative metabolism. In conclusion, TG accumulated in livers of men but in muscles of women during extended fasting. This sexual dimorphism was related to differential fasting plasma FA concentrations but not to whole body or hepatic utilization of this substrate.

admission on day 0 until 12:00 on day 2. Between 08:30 and 09:00 (day 1 and 2), all subjects underwent measurement of respiratory quotient using a Delta Trak II indirect calorimeter (Sensormedics, Yorba Linda, CA). Hepatic and intramyocellular TG content were measured on admission and at 12:00 on day 1 and 2. A subset of participants (women = 6, men = 7) underwent additional studies using stable isotope tracers. Between 22:00 and 09:00 (day 0-1), subjects received two tracers orally: [U- 13 C]propionate ( ‫ف‬ 1200 mg) at 08:30 and divided doses of 70% [ 2 H]water (5 g/kg body water, calculated as 60% of body weight in men and 50% of body weight in women) at 22:00, 02:00, and 06:00. Subjects were allowed to drink 0.5% [ 2 H]water ad libitum for the remainder of the fast. Subjects were then given a 2.25 mg/kg bolus of [3, 4-13 C] glucose intravenously followed by a 2-hour infusion (0.0225 mg/ kg/min). At the end of the infusion period, a 50-ml blood draw was performed. These procedures were repeated on day 2 of the fast, except for overnight loading with 70% [ 2 H]water.

Blood samples
Every 4 h, 3 ml of blood was drawn from each subject. Blood was collected in nonheparinized, EDTA-containing tubes and immediately centrifuged to isolate plasma. Samples were immediately frozen and maintained at Ϫ 80°C, after which they were thawed once and analyzed. Plasma glucose, cholesterol, TG, and high-density lipoprotein cholesterol (HDL-c) concentrations were determined using a Vitros 250 spectrophotmetric analyzer (Ortho-Clinical Diagnostics, Rochester, NY). Enzyme-linked immunosorbent assay kits were used to measure plasma insulin, leptin, and adiponectin concentrations (Millipore, Billerica, MA) as well as plasma-free fatty acid (FA) concentrations (Wako Chemicals USA, Richmond, VA). Plasma ketone bodies were determined using a commercial kit (Wako Chemicals, Richmond, VA). Other chemistries were performed by an outside laboratory (Quest Diagnostics, Madison, NJ).

Measurement of hepatic glucose and energy metabolism
Plasma was extracted with perchloric acid and the glucose was purifi ed as previously described ( 17,18 ). Purifi ed plasma glucose was converted to 1,2-isopropylidene glucofuranose (monoacetone glucose) before 2 H and 13 C NMR analysis as detailed previously (17)(18)(19). Samples were analyzed on a 14.1 Tesla Varian Inova spectrometer (Varian Instruments, Palo Alto, CA) equipped with a 3 mm broadband probe tuned to 92 MHz for 2 H spectra or 150 MHz for 13 C spectra. Resonance areas were determined using ACD/Labs 12.0 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada).
The relative deuterium enrichments in glucose H2, H5 and H6s were assessed by 2 H-NMR and these values were used to of adipose FAs to liver and muscle may also alter glucose homeostasis: 1 ) increased FA oxidation in liver stimulates gluconeogenesis and 2 ) increased intramyocellular TGs may reduced insulin sensitivity and impair glucose disposal (11)(12)(13)(14)(15). The impact of fasting-induced free FA availability on lean tissue TG content and glucose homeostasis in normal women and men is unknown. The present study investigated changes in lean tissue TG content during a 48-h fast in healthy men and women using proton magnetic resonance spectroscopy ( 1 H-MRS) and related these fi ndings to changes in carbohydrate, lipid, and energy metabolism determined via biochemical, calorimetric, and MRS-based stable isotope tracer measurements. Multiple stable isotope tracers were used to evaluate the fasting response of hepatic glucose production, glycogenolysis, gluconeogenesis, and metabolic pathways of the tricarboxylic acid (TCA) cycle. Women and men were examined separately to determine whether known sexually dimorphic fasting lipid metabolism ( 16 ) extends to lean tissue lipid accumulation and metabolism during fasting.

Participants
Healthy individuals were recruited for study at the University of Texas Southwestern Medical Center. The study population was composed of nine women and nine men whose characteristics are presented in Table 1 . All of the women studied were premenopausal, and two were taking oral contraceptives. The protocol and consent form were approved by the UTSW Institutional Review Board, and all participants provided written informed consent before enrollment.

Statistical analysis
Statistical analyses were performed using SigmaPlot 11.0 (Systat Software, Inc., San Jose, CA). Differences between the two groups were evaluated using the unpaired t -tests. Proportions were evaluated with the Chi-square test. The signifi cance of trends over the fast was determined using 1-factor repeatedmeasures ANOVA. Comparisons of multiple measurements between groups were performed using 2-factor repeated-measures ANOVA. Tukey's test was used for post hoc analysis of signifi cant fi ndings on ANOVA. Unless otherwise indicated, values are presented as median and interquartile range. Statistical signifi cance was taken at P < 0.05.

Changes in plasma metabolite concentrations over a 48-h fast
A comparison of metabolic parameters in women and men over the 48-h fast is presented in Table 2 . Consistent with prior reports, differences in lipid metabolites were apparent between the sexes ( 16 ). There was a small but signifi cant rise in plasma cholesterol with fasting that was independent of sex. Plasma TGs were highest in the immediate postprandial period and declined with fasting in women and men; however, a signifi cant disordinal interaction was encountered between sex and duration of fasting. Men had higher plasma TGs after 4 h of fasting but determine the fractional contribution of gluconeogenesis and glycogenolysis to endogenous glucose production as previously detailed ( 17 ). Pathways intersecting the TCA cycle were evaluated by 13 C-NMR analysis of glucose C2 isotopomers formed as a consequence of metabolism of [U- 13 C]propionate ( 20 ). Assumptions regarding this model have been previously reported ( 21 ). Endogenous glucose production was measured by dilution of [3, [4][5][6][7][8][9][10][11][12][13] C]glucose as previously described ( 22 ). Fractional glycogenolysis and gluconeogenesis measured by 2 H-NMR was normalized with pyruvate carboxylase (PC)/phosphoenolpyruvate carboxykinase (PEPCK), pyruvate cycling and gluconeogenesis relative to TCA cycle fl ux measured by 13 C-NMR and the rate of endogenous glucose production (µmol/kg/min) was used to calculate the absolute fl uxes through each of these pathways ( 23 ).

Measurement of hepatic and intramyocellular TG
Planning images were obtained and localized 1 H-NMR spectra were acquired using a 3.0 Tesla ACHIEVA whole-body MR system (Philips Medical Systems, Best, The Netherlands) with subjects in the supine position. For liver, a 2 × 2 × 2 cm volume of interest was positioned in the right hepatic lobe, avoiding major blood vessels, intra-hepatic bile ducts, and the lateral margins of liver. For muscle, a 2 × 2 × 2 cm volume of interest was positioned in the soleus, ensuring consistent orientation of muscle fi bers and avoiding major blood vessels, bone, and subcutaneous fat. Soleus muscle was chosen due to the predominance of oxidative (type II) fi bers in this muscle group ( 8 ) and the larger anticipated TG signal relative to other muscle groups ( 24 ). After the system was tuned and shimmed, spectra were collected using a combination of whole-body and SENSE torso (liver) or knee (muscle) coils for radio frequency signal transmission and reception and a STEAM sequence with interpulse delay Tr = 1.6 s, spin echo time Te = 14 ms, mixing time Tm = 18 ms, 16 acquisitions, and 2048 data points over a 1,500-Hz spectral width. The TG content is expressed as a percent of the methylene groups resonance at 1.40  Values are median with interquartile range. Data for the entire 48-h period were analyzed by 2-factor repeated-measures ANOVA. ALT, alanine aminotransferase; AST, aspartate aminotransferase. Conversions: total cholesterol and VLDL-TG (mg/dl) × 0.02586 = mmol/l; glucose (mg/dl) × 0.05551 = mmol/l . adiponectin, and transaminases were also comparable between the sexes. Plasma leptin was signifi cantly higher in women than in men. Taken together, these data are consistent with a sexually dimorphic induction of lipolysis and FA metabolism over the fasting period.

Changes in calorimetric measurements with fasting
Inasmuch as the differential changes in lipidemia in response to fasting suggested distinct substrate oxidation in women and men, we investigated whole-body fuel sources using indirect calorimetry. Regardless of sex, CO 2 production (VCO 2 ) was unchanged from day 1  Women had lower VCO 2 ( P = 0.027), VO 2 ( P = 0.006), and energy expenditure ( P = 0.008) when compared with men; however, these differences were not present when normalized to lean body weight. The lack of maintained a lower concentration for the majority of the fast. Plasma-free FA concentrations were higher in women than men from 4 to 24 h of fasting ( Fig. 1A ; average 24 h values shown). Although there was a signifi cant rise in plasma-free FAs in both sexes between 20 and 32 h of fasting, the rate of increase was 4-fold greater in men than in women ( Fig. 1B ) . Despite the sex differences in plasma FA concentrations, there was no signifi cant difference in plasma ketones, with each sex exhibiting similar ketosis during the fasting period ( Fig. 1C, D ). We also examined the relationship between plasma-free FA and ketones in each gender ( Fig. 1E, F ; average 24 h values shown). AN-COVA revealed a signifi cant positive within-subjects correlation between these metabolites in women ( r = 0.654; P < 0.001) and men ( r = 0.606; P < 0.001) over the 48-h fast. As a group, women also exhibited a signifi cant positive relationship between plasma-free FA and ketone concentrations over the initial ( r = 0.799; P = 0.008) and fi nal ( r = 0.732; P = 0.025) day of the fast. However, a similar between subjects relationship was not present in men during either 24-h period (initial, r = Ϫ 0.259; P = 0.501; fi nal, r = Ϫ 0.031; P = 0.937). Plasma lactate, glucose, insulin, 4-and 24-h data, there was no signifi cant difference in intramyocellular TGs between women and men and no signifi cant change in levels over this period. However, an ordinal interaction was encountered from 24 to 48 h of fasting ( P = 0.028). Post hoc analysis revealed that women had a signifi cant increase in intramyocellular TGs by 48 h of fasting ( P < 0.001), whereas men did not ( P = 0.127). Together, MRS of muscle and liver indicated a sexually dimorphic response of lean tissue lipid accumulation during fasting, with women but not men favoring a modest accumulation of lipid in skeletal muscle during short-term fasting and men but not women favoring a modest accumulation of lipid in liver.

Endogenous glucose production, glycogenolysis, and gluconeogenesis during fasting
We next examined how short-term fasting altered hepatic glucose metabolism in a subset of subjects using stable difference in respiratory quotients between the sexes throughout the fast suggested that the altered metabolic response to fasting was not mediated by differential wholebody substrate oxidation.

Effect of fasting on lean tissue TG content
Given the increased FA metabolism during fasting, we determined if a redistribution of this substrate as stored TGs was apparent in lean tissues. Hepatic TG content was similar in women and men at admission to the CTRC (0.9% [0.6-1.9%] vs. 0.8% [0.2-1.2%]; P = 0.254). Fig. 2A and 2B show the relative change in liver TGs for women and men from 4 to 24 h and from 24 to 48 h of fasting. Together these data demonstrated a signifi cant disordinal interaction between sex and duration of fasting for hepatic TG content ( P = 0.047). The interaction increased in signifi cance when the hepatic TG content after 4 and 24 h of fasting was compared between women and men separately ( P = 0.017); however, neither the interaction nor main effects were signifi cant on separate comparison of the 24 and 48 h fasting data. Closer inspection of hepatic TG content at 4 and 24 h of fasting demonstrated that men experienced a modest but signifi cant increase in liver fat ( P = 0.043), whereas liver fat levels in women tended to decrease ( P = 0.092).
Intramyocellular TG content was also comparable between women and men at the onset of fasting (2.4% [1.9-3.5] vs. 1  Relative change in lean tissue TG content with fasting in women and men. Measurements were obtained using a Philips 3.0 Tesla ACHIEVA wholebody MR system and a SENSE torso or knee coil with volume selective spectroscopy (STEAM, 2 × 2 × 2 cm voxel). Data were analyzed using 2-factor repeatedmeasures ANOVA. A and B: A signifi cant disordinal interaction between sex and duration of fasting for hepatic TG content was encountered ( P = 0.047). This was due to a signifi cant increase in hepatic TG content from 4 to 24 h in men ( P = 0.043) in conjunction with a tendency for levels in women to decrease ( P = 0.092). Further changes in hepatic TG content from 24 to 48 h of fasting were not apparent in women or men. C and D: No signifi cant change in intramyocellular TG content was apparent in either sex from 4 to 24 h of fasting. Intramyocellular TG content exhibited an ordinal interaction between sex and duration of fasting ( P < 0.001) from 24 to 48 h due to a significant increase in intramyocellular TGs in women but not in men ( P = 0.127). Results were similar when the single apparent outlier among the women was excluded from the analysis. * P < 0.05; *** P < 0.001. glycogenolysis ( P < 0.001) ( Fig. 3A and 3B). Although the fraction of circulating glucose derived from gluconeogenesis increased signifi cantly with fasting ( P < 0.001), the absolute rate of gluconeogenesis remained constant throughout the fast ( P = 0.704). In addition, the contribution of the precursor from which glucose was synthesized (glycerol vs. lactate/amino acids) remained constant throughout the fast ( P > 0.05). Similar results were obtained when the data were normalized to lean body weight (data not shown). These data demonstrated that, despite isotope tracers (women = 6, men = 7). Enrollment characteristics of the subset did not differ from that of the study population (data not shown). Subjects were loaded orally with [ 2 H]water to determine the origins of endogenous glucose production (EGP). In addition, subjects received a 2-h tracer infusion of [3,4-13 C]glucose to determine rates of EGP. No differences in hepatic metabolic fl uxes were observed between women or men, so the data were combined to examine the effect of fasting on these pathways. EGP declined with fasting duration due to reduced Fig. 3. Change in hepatic glucose and energy metabolism with fasting. Measurements were obtained using a combination of 2 H and 13 C (oral and intravenous) stable isotope tracers and NMR spectroscopy. No differences in hepatic metabolic fl uxes were observed between men or women, so the data were combined to examine the effect of fasting on these pathways. Data were analyzed using 1-factor repeated-measures ANOVA and are presented as mean with SEM. A: The metabolic pathways studied included hepatic glucose production, composed of glycogenolysis, as well as gluconeogenesis occurring from glycerol, lactate, and amino acids. The techniques used also allowed pathways centered on the TCA cycle to be investigated, including cycle turnover, anaplerosis (PC-PEPCK fl ux), and pyruvate cycling. B: Endogenous glucose production declined signifi cantly from 24 to 48 h of fasting. This reduction was solely due a decrease in glycogenolysis (white) ( P < 0.001). Gluconeogenesis occurring from glycerol (dark gray) as well as lactate/amino acids (light gray) did not change over the fasting period (fractional values [inset] are presented as mean and SD). C: Flux through PC and PEPCK tended to decline from 24 to 48 h of fasting. Nonetheless, the rate of anaplerosis (PC-PEPCK fl ux) was several-fold higher than the actual rate of gluconeogenesis occurring from OAA (light gray) (day 1: 3.6-fold vs. day 2: 2.6-fold; P = 0.009). The excess production of OAA and PEP was accounted for by pyruvate cycling (dots), a futile cycle usually attributed to pyruvate kinase and malic enzyme activity, with pyruvate returning to the TCA cycle via pyruvate carboxylase. The rate of pyruvate cycling decreased signifi cantly with fasting ( P = 0.029), completely accounting for the decrease in anaplerotic fl ux. D: In contrast with a massive induction of ketones with fasting and in spite of an increase in FA availability as the duration of fasting increased, TCA cycle turnover (black) diminished signifi cantly from 24 to 48 h of fasting. * P < 0.05; *** P < 0.001. and tissue TG distribution during extended fasting, glucose and oxidative metabolism were remarkably similar between women and men ( Fig. 1C, 1D, and 3D ). As expected, increased plasma-free FAs and a near 20-fold induction of ketonemia during 48 h of fasting ( Table 2 ) was accompanied by a 20% reduction in plasma glucose and a 25% decrease in glucose production and utilization. This reduction in glucose production was secondary to suppressed glycogenolysis ( Fig. 3B ), with no compensatory increase in gluconeogenesis. Despite increased hepatic fat oxidation, as indicated by the onset of ketosis, energy production by the TCA cycle declined signifi cantly with fasting as hepatic redox state became progressively reduced ( Fig. 3D ).
The fi ndings of the present study are reminiscent of differences in fasting metabolism between male C57BL/6J and SJL/J mice previously reported by Guan et al. ( 6 ). Like women, SJL/J mice were resistant to fasting-induced increases in liver TGs. This resistance was due to enhanced uptake and oxidation of free FA by skeletal muscle during fasting, sparing liver from FA overload. Consistent with enhanced uptake and storage of FA by skeletal muscle, women demonstrated a signifi cant increase in intramyocellular TG content with fasting that was not observed in men ( Fig. 3 ). It has been shown that fatty acid transporter (FAT/CD36) and ␤ -oxidation enzyme protein content is higher in skeletal muscle of women ( 29,30 ). Likewise, Mittendorfer et al. ( 31 ) have shown signifi cantly greater lean tissue free FA uptake and oxidation in untrained women during exercise-induced lipolysis. As with SJL/J mice, the lack of increase in hepatic TG content with fasting in women may be explained, in part, by a greater capacity for uptake and processing of circulating FA by skeletal muscle.
The failure of serum concentrations of free FA to increase with fasting in SJL/J mice in conjunction with a modest increase in serum ketone bodies ( ‫ف‬ 30% of C57BL/6J) was the initial clue that most free FAs released from adipose tissue were removed by skeletal muscle before entering the portal circulation ( 6 ). In contrast, women have basally higher lipolysis and postabsorptive plasma-free FA concentrations than men ( 32-34 ) ( Fig. 2B ) even though hepatic TG content tended to decline. However, we also observed a more rapid and greater induction of plasma-free FA in men after 20-32 h of fasting ( Fig. 1B ). This comparably blunted rise in free FA in women during this period may have contributed to the relatively static levels of hepatic TG during the 48-h fast. In support of this, women with the lowest serum-free FA concentration by 48 h of fasting ( r = 0.900; P < 0.001) or the smallest increase in serum-free FAs ( r = 0.683; P = 0.036) experienced a decrease in hepatic TGs. Together, these metabolic attributes may explain the relative resistance of women to accumulation of hepatic TGs with fasting. These fi ndings may provide insight into the observation that the risk of pathologic accumulation of hepatic TGs (fatty liver disease) is modestly, but signifi cantly, higher in men ( 35 ). To our knowledge, these are the fi rst data demonstrating sexual dimorphism in the hepatic response to lipid excess in humans. differences in hepatic lipid accumulation, the rate of gluconeogenesis from all precursors was remarkably stable over the entirety of the fast in women and in men.

Effect of fasting on mitochondrial anaplerosis in liver
To examine the impact of fasting on hepatic anaplerosis, we analyzed the 13 C isotopomers of plasma glucose formed from the [U- 13 C]propionate by 13 C NMR. This approach measures fl ux through the combined pathways of pyruvate carboxylase (PC) and phosphoenolypyruvate carboxykinase (PEPCK) ( 20,21 ). Anaplerosis tended to decrease with fasting ( Fig. 3A, C ), suggesting that there was an unexpected decrease in PC-PEPCK fl ux. Nonetheless, the rate of anaplerosis was several-fold higher than the actual rate of gluconeogenesis occurring from oxaloacetate (OAA) (day 1: 3.6-fold vs. day 2: 2.6-fold; P = 0.009). As previously described ( 21,27 ), the excess production of OAA and PEP was accounted for by pyruvate cycling, a futile cycle usually attributed to pyruvate kinase (PEP → pyruvate) and malic enzyme activity (OAA → malate → pyruvate), with pyruvate returning to the TCA cycle via PC (pyruvate → OAA) ( Fig. 3A ). The rate of pyruvate cycling decreased signifi cantly with fasting ( P = 0.029), completely accounting for the decrease in anaplerotic fl ux. These data indicated that constant gluconeogenesis from OAA in the fasting human ( P = 0.492) is maintained by decreased pyruvate kinase or malic enzyme activity rather than an induction of PC and/or PEPCK fl ux.

Hepatic TCA cycle activity with fasting
To determine the effect of short-term fasting on terminal substrate oxidation in liver, we assessed hepatic TCA cycle activity by 2 H and 13 C isotopomer analysis of glucose ( 28 ). In contrast to marked fasting ketosis and in spite of an increase in FA availability, TCA cycle turnover diminished during fasting ( P = 0.021) ( Fig. 3A, D ). Moreover, there was no difference in TCA cycle activity between the sexes at any time during the fast, even when differences in lean body weight were taken into account.

DISCUSSION
In this study, we examined the effect of fasting on lipid and glucose metabolism in healthy women (n = 9) and men (n = 9), examining for the fi rst time physiologic changes that occur in hepatic TG content. The classic metabolic effects of fasting began after 24 h and were marked by a rapid rise in plasma-free FA and ketones with a concomitant decrease in plasma glucose, insulin, and leptin. Consistent with prior reports ( 16 ), signifi cant differences in plasma-free FA concentration were apparent between the sexes ( Fig. 1A, B ), with women having higher free FAs in the postabsorptive period and men having a 4-fold greater rate of increase in concentration over a 48-h fast. In vivo 1 H-MRS also revealed a previously unknown sexually dimorphic response in lean tissue distribution of lipids during a 48-h fast, with more TG accumulation in liver of men ( Fig. 2A ) but in muscle of women ( Fig. 2D ). Despite sex differences in plasma FA concentrations Gluconeogenic fl ux is increased by hepatic lipid oxidation ( 14,15 ) via the production of gluconeogenic cofactors (i.e., ATP, NADH) and allosteric activation of PC by acetyl-CoA ( 45,46 ). However, the initiation of gluconeogenic fl ux by oxidative metabolism appears to apply strictly to TCA cycle oxidation rather than to ␤ -oxidation per se ( 13 ). Despite increased ␤ -oxidation and massive ketosis during fasting, suppression of TCA cycle activity ( Fig. 3D ) was attended by a decline in PC-PEPCK fl ux ( Fig. 3A and  3C ). Only a concomitant decrease in pyruvate cycling prevented a decline in gluconeogenesis. Pyruvate cycling has contributions from malic enzyme and/or pyruvate kinase ( 21 ). Both are important in liver, but malic enzyme activity is unresponsive to nutritional state ( 47 ), whereas pyruvate kinase activity is suppressed 4-fold during fasting ( 48 ). Regardless, substrate cycling provides an important form of metabolic control under rapidly changing environmental conditions ( 49 ). In this case, decreased hepatic pyruvate cycling in fasting humans may support a metabolic buffering system to assure ample availability of PEP and therefore gluconeogenic potential regardless of nutritional state.
In conclusion, lean tissue TG content increased in a sexually dimorphic manner with short-term fasting. The resistance of women to fasting-induced increases in hepatic TG content may be related to the blunted induction of plasma-free FA during extended fasting and an increased propensity for uptake and storage of plasma FAs by skeletal muscle. Our fi ndings may indicate a greater susceptibility of men to pathological accrual of hepatic TGs, providing a potential explanation for the greater prevalence of hepatic steatosis among men ( 35 ). Likewise, these fi ndings may help to explain differences in nonoxidative FA metabolism ( 3 ) among women and men. In contrast, no sex differences were observed in hepatic and whole-body glucose or energy metabolism. This fi nding suggests that the major sex differences in hepatic FA metabolism are at the level of reesterifi cation and/or lipoprotein export. Short-term fasting did not lead to increased rates of gluconeogenesis and total endogenous glucose production decreased due to diminishing glycogenolysis during the fast. Reduced TCA cycle fl ux during fasting ketosis appears to limit PC-PEPCK fl ux, but gluconeogenesis was spared by reduced pyruvate cycling, a potentially important metabolic control point for gluconeogenesis.
FA esterifi cation is greater in women than in men during the postabsorptive period ( 3 ). The rise in intramyocellular TG content in women but not in men supports this fi nding and suggests that skeletal muscle is an important site for reesterifi cation of lipolytic FAs in women. Koutsari et al.
( 3 ) discounted muscle as a possible site for sex differences in nonoxidative FA disposal because women in that study did not demonstrate insulin resistance. In the present study, women had a greater insulin excursion upon refeeding after the 48-h fast, consistent with impaired insulin sensitivity. However, this sex difference was not present in the postprandial period, suggesting that the increased insulin excursion in women was related to higher concentrations of plasma FAs, intramyocellular TGs, or both ( 36 ). The lack of increased intramyocellular TG in men who fasted in our study contrasts with data presented by Stannard et al. ( 10 ); however, those subjects were endurance-trained athletes and the duration of the fast was 24 h greater.
Despite sex differences in FA metabolism and distribution to lean tissues with fasting, whole-body and hepatic energy metabolism were similar between women and men. We found no evidence for differences in respiratory quotient or VO 2 per lean mass between the sexes. Plasma ketone body concentrations were similar throughout the fast ( Fig. 1C ), suggesting that men and women have similar rates of ketogenesis ( 37 ). Similarly, there were no differences in the rates of hepatic oxidative metabolism at the level of the TCA cycle ( Fig. 3A, D ). Among all subjects, there was a reciprocal relationship between ketosis and TCA cycle activity over the course of the fast: Ketosis increased, whereas TCA cycle turnover decreased. Similar fi ndings have been found in animal studies ( 38 ) and have been attributed to the inhibition of citrate synthase by high ATP and suppression of the forward dehydrogenase reactions of the TCA cycle by mitochondrial redox (NADH:NAD + ) ( 39 ). Indeed, liver became progressively more reduced over the 48-h fast, as indicated by a steady increase in the ␤ -hydroxybutyrate/acetoacetate ratio in both sexes ( P < 0.001). Taken together, these data suggest that the uptake of FA by liver for delivery to mitochondria to undergo ␤ -oxidation followed by terminal oxidation in the TCA cycle and/or ketogenesis is remarkably similar in women and men.
The key difference between the sexes appears to be in the FA esterifi cation and lipoprotein export pathways.
The transition to fasting ketosis occurred in tandem with a decline in EGP. This decrease was caused by reduced glycogenolysis ( Fig. 3A, B ), similar to previous studies in fasting humans ( 40,41 ). In contrast, gluconeogenesis and its precursors (glycerol versus lactate/amino acids) was stable across 48 h of fasting, similar to other studies in fasting humans (as reviewed in Ref. 42 ). However, this fi nding differs from the expected transcriptional upregulation of genes, such as PEPCK by fasting ( 43 ). Because PC-PEPCK fl ux tended to decline during fasting, the transcriptional regulation of PEPCK appears less important for controlling gluconeogenic fl ux than previously thought ( 12,44 ) and may serve mainly to defend the pathway against shrinking gluconeogenic substrate supply and/or altered lipid metabolism.