In this study, the effect of carbohydrate restriction on flux through the metabolic pathways of hepatic glucose production and the TCA cycle were simultaneously assessed by isotopomer analysis of glucose using 2H and 13C NMR spectroscopy. We found that carbohydrate restriction increased the rate of gluconeogenesis and decreased the rate of glycogenolysis. However, the observed increase in gluconeogenesis in the low-carbohydrate group was solely the result of increased GNGPEP rather than GNGglycerol. Despite the energetic investment required to increase GNGPEP, TCA cycle flux in the low-carbohydrate group was similar to the low-calorie group, indicating similar rates of energy generation. Interestingly, in the groups consuming carbohydrate as a significant proportion of their diet (weight-stable, low-calorie), the TCA cycle alone provided sufficient energy to drive gluconeogenesis regardless of whether the gluconeogenic substrate was assumed to be lactate or alanine. This was not the case in individuals undergoing carbohydrate restriction, indicating that a reorganization of hepatic energy metabolism occurred in tandem with the changes in hepatic carbohydrate metabolism.
Among previous studies of carbohydrate restriction, it remained unclear which gluconeogenic precursors were primarily responsible for increased gluconeogenesis. Evidence of a negative correlation between alanine conversion to glucose and dietary carbohydrate content suggests that anaplerosis and GNGPEP
are increased with decreased dietary intake of carbohydrate (28
). However, increased fat oxidation during carbohydrate restriction might be expected to increase availability of the gluconeogenic precursor glycerol (10
). In the present study, we showed that the increase in gluconeogenesis associated with carbohydrate restriction is due to the induction of GNGPEP
. This suggests that in fasted human subjects undergoing weight loss, the elevated gluconeogenesis associated with carbohydrate restriction is driven by substrates such as lactate or amino acids. While it seems likely this increase is due to amplified protein turnover, we could not rule out enhanced cycling of lactate from the periphery back to liver (Cori Cycle) as a source of increased gluconeogenesis. Plasma lactate levels were similar between the two weight loss groups (); however, these were static measurements and gave no insight into the rate of production of lactate by muscle and uptake by liver. Likewise, protein turnover measurements were not performed so the contribution of amino acids also remains unknown. However, it is interesting to note that individuals on a low-carbohydrate diet increased their protein intake in favor of fat (), possibly as method to stave off non-dietary protein breakdown for the formation of glucose.
The contribution of glycerol as a substrate for gluconeogenesis was surprisingly unresponsive to dietary macronutrient composition. Though GNGglycerol
appeared to be numerically higher in the low-carbohydrate group, this failed to reach statistical significance. It is possible that the small sample size of the study and/or the sensitivity of our technique limited our ability to detect modest changes in this measure. However, prior data in fasting man suggests that gluconeogenesis from glycerol occurs at a relatively fixed rate (30
). Our findings would further support this observation. Indeed, insulin levels were similar between the groups undergoing dietary restriction, suggesting that rates of peripheral lipolysis were also similar (). This was somewhat surprising as prior data in lean individuals clearly demonstrates a reduction in insulin levels and increase in free fatty acid levels as a result of carbohydrate restriction (31
). However, the present data is akin to that of Allick et al. in which overweight/obese individuals with diabetes maintained similar insulin and free fatty acid levels regardless of dietary macronutrient composition (32
). Further studies are needed to verify this finding.
It should be noted that prior studies assessing the impact of carbohydrate restriction on hepatic glucose metabolism show that the main effect is a reduction in hepatic glucose output, predominantly via a reduction in glycogenolysis (31
). This is in contrast to the present study in which hepatic glucose production was similar between the dietary groups. The low-carbohydrate group was able to maintain hepatic glucose production at the levels observed for the weight-stable and low-calorie groups by increasing GNGPEP
to match the reduction in glycogenolysis. This observation is reminiscent of “hepatic autoregulation” by which endogenous glucose production remains unchanged in the setting of altered gluconeogenesis or glycogenolysis because the two pathways tend to compensate for each other (28
). This finding may also be the result of the much larger intake of dietary protein in the low-carbohydrate group (~34%) as compared to prior studies (11-15%) (31
), possibly yielding an enhanced supply of gluconeogenic substrate.
The multi-tracer approach used in the present study allowed for the simultaneous assessment of both hepatic glucose production as well as TCA cycle flux. Knowledge of metabolic flux through these pathways provided insight into the relationship between hepatic glucose and energy metabolism (). Energetic coupling of GNGPEP
and the TCA cycle was observed as a correlation between TCA cycle flux and PEPCK flux (), the metabolic pathway responsible for the delivery of substrate for gluconeogenesis. It is, however, intriguing that the increased GNGPEP
in the low-carbohydrate group was not associated with increased TCA cycle flux (i.e., energy production). Indeed, assuming net glucose synthesis predominated (i.e., alanine or other amino acids acted as the gluconeogenic substrate as opposed to lactate), energy production in the TCA cycle would be unable to meet the energetic demands of gluconeogenesis in the low-carbohydate group. This would suggest a greater reliance on sources of energy upstream from the terminal oxidation of fat in the TCA cycle in this group, possibly β-oxidation/ketogenesis. Prior data has demonstrated a relationship between GNGPEP
and ketogenesis: in general, ketogenesis parallels the rate of gluconeogenesis under most circumstances (30
). Indeed, static measurements of ketone bodies were markedly higher in subjects undergoing carbohydrate restriction, indicating that the availability of acetyl-CoA to HMG-CoA synthase may have been greater. However, absolute rates of fatty acid delivery to liver as well as ketone body production were not measured, limiting our ability to further interpret the above findings.
Every attempt was made to equalize caloric intake between the two dietary restriction groups. However, a trend was noted towards decreased caloric intake in the group undergoing carbohydrate restriction (). It is possible that the differences observed between these two groups are solely the result of differences in caloric intake and weight loss. However, prior data obtained under weight-stable, isocaloric conditions showed similar changes in hepatic glucose metabolism in lean individuals (11
). Likewise, fractional and absolute glucose production was similar between the weight-stable and low-calorie group despite the difference in energy balance between the two. It should also be noted that the present study was not designed to determine the effectiveness of these two weight-loss diets in weight reduction, but was simply designed to assess hepatic metabolism under the differing macronutrient compositions during negative energy balance. Additionally, it was our desire to examine changes in hepatic metabolism under conditions likely to be encountered in a clinical setting; hence, dietary choices of the subjects were more varied than what would be encountered in a strict physiologic study.
In conclusion, we have shown that the sources from which endogenous glucose is produced are dependent upon dietary macronutrient composition. Carbohydrate restriction yields a decreased rate of glycogenolysis and an increased rate of gluconeogenesis compared to calorie restriction. We have shown for the first time that this increased rate of hepatic gluconeogenesis is the result of an increased rate of utilization of substrates like lactate and amino acids, but not glycerol. Additionally, the TCA cycle appears to be the energetic patron of GNGPEP
, as TCA cycle flux and PEPCK flux were highly correlated. Furthermore, it appears that the shift in glucose metabolism associated with a low carbohydrate diet leads to an increased contribution of energy generated outside of the TCA cycle to gluconeogenesis. This shift is consistent with enhanced β-oxidation/ketogenesis, which could be beneficial in individuals with NAFLD due to enhanced disposal of hepatic triglyceride. These findings may explain, in part, the correlation between carbohydrate intake and severity of liver disease in individuals with NAFLD (7
). Understanding the alterations to cellular energetics that occur with simple macronutrient manipulation may be important for understanding and treating NAFLD and other metabolic disorders associated with obesity (34