C2C12 Muscle Myotubes, but not Kidney Proximal Tubule HK-2 Cells, Elevate Erythritol Synthesis in Response to Oxidative Stress.

Background: As a biomarker, elevated serum erythritol concentrations predict type 2 diabetes and cardiovascular disease onset. Erythritol was recently shown to be a product of human glucose metabolism through the pentose phosphate pathway. The regulation of erythritol synthesis from glucose has been explored in cancer cells but not in nontransformed cells. Objective: The kidneys and skeletal muscle have increased erythritol content in response to dietary sucrose, which suggests that they may significantly contribute to circulating erythritol concentrations. In the present study, we evaluated if conditions that promote erythritol synthesis in cancer cells are consistent in skeletal muscle and kidney cells. Methods: C2C12 myotubules were used as a model for skeletal muscle, and human kidney (HK)-2 human proximal tubule cells were used to model kidney. C2C12 cells were exposed to high- or low-glucose conditions. Both C2C12 and HK-2 cells were exposed to the free radical generator menadione, then intracellular reactive oxygen species (ROS) and erythritol concentrations were measured. Intracellular sorbitol concentrations were also measured because increased polyol flux was also observed after exposure to excess glucose and oxidative stress. Results: Intracellular erythritol concentrations were significantly elevated in C2C12 cells following both high-glucose and menadione treatment. In contrast, HK-2 cells did not increase erythritol synthesis in response to oxidative stress. Generation of ROS through hydrogen peroxide exposure elevated sorbitol concentrations in both C2C12 and HK-2 cells, whereas generation of radicals with menadione treatment did not affect sorbitol production in either cell type. Conclusions: These findings highlight that the factors contributing to elevated erythritol synthesis vary between cell types. More specifically, these studies demonstrate that muscle cells increase erythritol synthesis in response to both high glucose in culture medium and oxidative stress, whereas kidney cells increase erythritol synthesis only in response to high glucose.

Sucrose Intake Elevates Erythritol in Plasma and Urine in Male Mice.

BACKGROUND: Elevated serum erythritol concentration is a predictive biomarker of diabetes and cardiovascular incidence and complications. Erythritol is synthesized endogenously from glucose, but little is known regarding the origin of elevated circulating erythritol in vivo. OBJECTIVES: In vitro evidence indicates that intracellular erythritol is elevated by high-glucose cell culture conditions and that final step of erythritol synthesis is catalyzed by the enzymes sorbitol dehydrogenase (SORD) and alcohol dehydrogenase (ADH) 1. The purpose of this study was to determine whether dietary intake and/or diet-induced obesity affect erythritol synthesis in mice and whether this relationship is modified by the loss of the enzymes SORD or ADH1. METHODS: First, 8-wk-old male Sord+/+, Sord-/-, Adh1+/+, and Adh1-/- mice were fed either low-fat diet (LFD) with 10% fat-derived calories or diet-induced obesity high-fat diet (HFD) with 60% fat-derived calories for 8 wk. Plasma and tissue erythritol concentrations were measured using gas chromatography-mass spectrometry. Second, male wild-type 8-wk-old C57BL/6J mice were fed LFD or HFD with plain drinking water or 30% sucrose water for 8 wk. Blood glucose and plasma and urinary erythritol concentrations were measured in nonfasted and fasted samples. Tissue erythritol was measured after killing. Finally, male Sord+/+ and Sord-/- mice were fed LFD with 30% sucrose water for 2 wk; then, nonfasted plasma, urine, and tissue erythritol concentrations were quantified. RESULTS: Plasma and tissue erythritol concentrations were not affected by loss of Sord or Adh1 in mice fed LFD or HFD. In wild-type mice, consumption of 30% sucrose water significantly elevated plasma and urinary erythritol concentrations on both LFD-fed and HFD-fed mice compared with that of plain water. Sord genotype did not affect plasma or urinary erythritol concentration in response to sucrose feeding, but Sord-/- mice had reduced kidney erythritol content compared with wild-type littermates in response to sucrose. CONCLUSIONS: Sucrose intake, not HFD, elevates erythritol synthesis and excretion in mice. Loss of ADH1 or SORD does not significantly affect erythritol concentration in mice.

Erythritol synthesis is elevated in response to oxidative stress and regulated by the non-oxidative pentose phosphate pathway in A549 cells.

Background: Erythritol is a predictive biomarker of cardiometabolic diseases and is produced from glucose metabolism through the pentose phosphate pathway (PPP). Little is known regarding the regulation of endogenous erythritol synthesis in humans. Objective: In the present study, we investigated the stimuli that promote erythritol synthesis in human lung carcinoma cells and characterized potential points of regulation along the PPP. Methods: Human A549 lung carcinoma cells were chosen for their known ability to synthesize erythritol. A549 cells were treated with potential substrates for erythritol production, including glucose, fructose, and glycerol. Using siRNA knockdown, we assessed the necessity of enzymes G6PD, TKT, TALDO, and SORD for erythritol synthesis. We also used position-specific 13C-glucose tracers to determine whether the carbons for erythritol synthesis are derived directly from glycolysis or through the oxidative PPP. Finally, we assessed if erythritol synthesis responds to oxidative stress using chemical and genetic models. Results: Intracellular erythritol was directly associated with media glucose concentration. In addition, siRNA knockdown of TKT or SORD inhibited erythritol synthesis, whereas siG6PD did not. Both chemically induced oxidative stress and constitutive activation of the antioxidant response transcription factor NRF2 elevated intracellular erythritol. Conclusion: Our findings indicate that in A549 cells, erythritol synthesis is proportional to flux through the PPP and is regulated by non-oxidative PPP enzymes.

Chronic Dietary Erythritol Exposure Elevates Plasma Erythritol Concentration in Mice but Does Not Cause Weight Gain or Modify Glucose Homeostasis.

BACKGROUND: Erythritol is both a common nonnutritive sweetener and an endogenous product of glucose metabolism. Recent reports suggest that elevated plasma erythritol is a predictive biomarker of cardiometabolic disease onset and complications. OBJECTIVES: Although short-term erythritol consumption has been evaluated, the effect of chronically elevated circulating erythritol on adiposity and glucose metabolism has not. This study investigated the effect of longer-term erythritol consumption on weight gain and glucose tolerance in young/adolescent mice. METHODS: Four erythritol supplementation experiments were completed and analyzed separately in male C57BL/6J mice. In experiments 1 and 2, mice aged 8 wk or 20 wk, respectively, were randomly allocated to consume 16% fat diet (LFD) or LFD with 40 g/kg erythritol. In experiments 3 and 4, mice aged 8 wk or 20 wk were fed 45% fat diet (HFD) or HFD with 40 g/kg erythritol (HFD + ERY). In each experiment, we compared the effect of erythritol consumption on plasma erythritol, body weight and composition, glucose tolerance, and brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression. We also investigated relative endogenous tissue erythritol concentrations in a subset of control (LFD or HFD) mice in experiments 1 and 3. RESULTS: There was no effect of erythritol supplementation on body weight or glucose tolerance in experiments 1-3. In experiment 4, in the 20-wk-old mice fed HFD or HFD + ERY, there was a significant interaction of time and erythritol on body weight (P < 0.0001), but the main effect of diet was not significant. Plasma erythritol was elevated 40-fold in mice consuming erythritol-supplemented diets relative to mice consuming LFD or HFD controls. We found no effect of chronic erythritol consumption on BAT UCP1 protein concentrations. Liver and kidney tissue contained significantly higher endogenous erythritol than quadriceps and visceral adipose (P < 0.001) in young mice fed LFD and HFD. CONCLUSIONS: In young/adolescent mice, prolonged erythritol consumption did not significantly affect body weight, composition, or glucose tolerance.

Mammalian metabolism of erythritol: a predictive biomarker of metabolic dysfunction.

PURPOSE OF REVIEW: To summarize recent advances in our understanding of mammalian erythritol metabolism and its use as a predictive biomarker of cardiometabolic disease risk. RECENT FINDINGS: Elevated serum erythritol predicts future central adiposity gain and type 2 diabetes mellitus in healthy adults. Erythritol is a newly recognized human metabolic product of glucose, synthesized through the pentose phosphate pathway. The final conversion of this metabolic pathway is catalyzed by the enzymes sorbitol dehydrogenase and alcohol dehydrogenase 1. Erythritol is also a well characterized nonnutritive sweetener. Recent studies show that dietary erythritol can be metabolized to erythrose or erythronate in humans before excretion. SUMMARY: Elevated serum erythritol predicts risk for cardiometabolic disease, but more research is required to maximize its utility as a biomarker, including characterizing the determinants of endogenous erythritol synthesis from glucose. New insights into dietary erythritol metabolism also highlight the need to evaluate the effects of long-term erythritol consumption.

Unexpected roles for ADH1 and SORD in catalyzing the final step of erythritol biosynthesis.

The low-calorie sweetener erythritol is endogenously produced from glucose through the pentose phosphate pathway in humans. Erythritol is of medical interest because elevated plasma levels of this polyol are predictive for visceral adiposity gain and development of type 2 diabetes. However, the mechanisms behind these associations remain unknown because the erythritol biosynthesis pathway, particularly the enzyme catalyzing the final step of erythritol synthesis (reduction of erythrose to erythritol), is not characterized. In this study, we purified two enzymes from rabbit liver capable of catalyzing the conversion of erythrose to erythritol: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Both recombinant human ADH1 and SORD reduce erythrose to erythritol, using NADPH as a co-factor, and cell culture studies indicate that this activity is primarily NADPH-dependent. We found that ADH1 variants vary markedly in both their affinity for erythrose and their catalytic capacity (turnover number). Interestingly, the recombinant protein produced from the ADH1B2 variant, common in Asian populations, is not active when NADPH is used as a co-factor in vitro We also confirmed SORD contributes to intracellular erythritol production in human A549 lung cancer cells, where ADH1 is minimally expressed. In summary, human ADH1 and SORD catalyze the conversion of erythrose to erythritol, pointing to novel roles for two dehydrogenase proteins in human glucose metabolism that may contribute to individual responses to diet. Proteomics data are available via ProteomeXchange with identifier PXD015178.