DEFINING THE ROLE OF ENDOGENOUS ERYTHRITOL SYNTHESIS IN OBESITY AND DIABETES DEVELOPMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1026400
• Grant No.: 2021-67034-35110
• Cumulative Award Amt.: $180,000.00
• Proposal No.: 2020-09981
• Project Start Date: Jun 1, 2021
• Project End Date: May 31, 2024
• Grant Year: 2021
• Program Code: [A7101]- AFRI Predoctoral Fellowships

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
(N/A)

Non Technical Summary
In two separate prospective cohort studies, increased plasma erythritol predicted risk for weight gain and type 2 diabetes mellitus (T2DM) up to 20 years before disease onset, but the causal mechanisms underlying the association have not been defined. Understanding this relationship is important because erythritol, a four-carbon sugar alcohol, is available from both endogenous and exogenous sources. Erythritol was recently shown to be synthesized from glucose by human metabolism, but this newly uncovered metabolic pathway is largely uncharacterized. Similarly, erythritol is used increasingly as a non-caloric sweetener and food additive because acute doses are rapidly cleared in urine. The effects of chronically elevated erythritol levels have not been assessed, though studies in model systems indicate that erythritol exposure causes changes in energy metabolism gene expression. Our overall objectives are to: 1) define metabolic regulatory mechanisms that govern rates of endogenous erythritol production, and 2) identify "downstream" metabolic targets and signaling pathways influenced by erythritol exposure. Our central hypothesis is that increased erythritol production is an alternative means of metabolizing and disposing of glucose in response to positive energy balance. The central hypothesis will be tested using two specific aims: 1) Define the metabolic determinants that lead to increased endogenous erythritol synthesis, and 2) Determine the effect of elevated plasma erythritol on gene expression. The first aim will employ mouse models of human genetic variants combined with state-of-the-art stable isotope tracer and metabolic function technologies to characterize the genetic and dietary determinants leading to increased plasma erythritol. The second aim will interrogate the relationship between chronic dietary erythritol exposure and changes in metabolic function and metabolic gene expression in obese C57BL6/J mice. The proposed research is innovative in concept, as endogenous erythritol production from glucose is a newly recognized human metabolic pathway. The proposed research is significant in that it will uncover the role of elevated plasma erythritol in the development of T2DM and weight gain and fundamentally advance our understanding of central energy metabolism and its regulation. Ultimately, these studies have the potential to inform use of this novel metabolic pathway for chronic disease prevention and treatment.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	7010	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
7010 - Biological Cell Systems;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

erythritol

nutritional biomarker

obesity

type 2 diabetes mellitus

Goals / Objectives
The enclosed Predoctoral application is a Research project that addresses the AFRI Farm Bill priority area "food safety, nutrition, and health." Erythritol synthesis from glucose is recently discovered pathway that will improve early detection of metabolic dysfunction in humans. In a prospective cohort study, serum erythritol at baseline predicted Type 2 Diabetes Mellitus development at follow-up, up to 20 years later. Elevated serum erythritol is also predictive of central adiposity gain in young adults. Although erythritol is associated with weight gain and diabetes, the determinants of erythritol synthesis and its role in disease progression have not been defined. Our preliminary data show that serum erythritol is responsive to dietary intake. We have also identified two enzymes, sorbitol dehydrogenase (SORD) and alcohol dehydrogenase 1 (ADH1), that convert erythrose to erythritol through the pentose-phosphate pathway. This proposal will use cell culture and animal models to determine how expression of these enzymes and dietary composition effect erythritol synthesis. We will also elucidate how erythritol is associated with diet-induced changes in central metabolism and its "downstream" effects on metabolic regulation. Together, these approaches will fill in gaps in our understanding of human erythritol metabolism and improve its utility as a nutritional biomarker. We will address these goals with the following aims:Specific Aim 1: Define the metabolic determinants that lead to increased endogenous erythritol synthesis.Based on our preliminary data, we expect that diet composition will affect erythritol synthesis. We have also identified two candidate enzymes that catalyze erythritol synthesis in vitro: sorbitol dehydrogenase (SORD) and alcohol dehydrogenase 1 (ADH1).8 We have generated knockout mouse models of both enzymes to assess their role in endogenous erythritol production. We will compare endogenous erythritol production across diets and genotypes using a stable isotope glucose tracer. We will also use stable isotope tracers to assess central carbon metabolism, including PPP flux.Specific Aim 2: Determine the effect of elevated plasma erythritol on gene expression.We will administer dietary erythritol to wildtype mice to elevate blood and tissue erythritol levels. Using RNA sequencing in liver and adipose tissue, we will determine the effect of erythritol on the expression of key genes involved in glucose metabolism, lipogenesis, adipogenesis, and energy expenditure.Objectives are as follows: Measure the metabolic phenotype of Sord-/- and Adh1-/- mice exposed to experimental diets with a range of macronutrient composition, including body weight, food intake, body composition, and glucose tolerance.Determine the plasma and tissue erythritol content of Sord-/- and Adh1-/- mice exposed to experimental diets.In cell culture models, determine the regulatory steps of erythritol synthesis using siRNA knockdowns of SORD, glucose-6-phosphate dehydrogenase, transketolase, and transaldolase under normal glucose and hyperglycemic conditions.In cell culture models, evaluate the response of erythritol synthesis to alternative pentose phosphate pathway stimuli, such as inflammation and oxidative stress.Using metabolic flux analysis, determine how endogenous erythritol production is associated with altered pentose phosphate pathway, glycolytic and TCA-cycle flux in vivo in Sord-/- and Adh1-/- mice.In wildtype C57BL/6J mice fed high-fat diet, determine the effect of erythritol supplementation on weight gain and glucose intolerance.In wildtype C57BL/6J mice fed high-fat diet or high-fat diet with erythritol, measure erythritol-induced changes in gene expression using RNA sequencing and pathway analysis.

Project Methods
Aim 1: Define the metabolic determinants that lead to increased endogenous erythritol synthesis.We will use two mouse knockout models on a C5BL/6J background: a whole-body knockout of Adh1 and a whole-body knockout of Sord. Both mouse models are in-hand and have been shown to be viable and fertile. We will compare wild-type littermates to homozygous knockout Sord and Adh1 mice. SORD and ADH1 catalyze erythritol synthesis in vitro.These models will allow us to evaluate the effects of reduced SORD or ADH1 levels on erythritol production in vivo. Mice will be singly housed and placed on one of four experimental diets for a total of 8 weeks. Experimental diets will include: 1) low-fat, 2) low-fat supplemented with glucose in drinking water, 3) high-fat, and 4) high-fat supplemented with glucose in drinking water. All diets are based on AIN-93G purified diet. Exposing mice to varied levels of both fat and glucose will allow us to determine how the interaction between dietary composition and genotype affects erythritol production and subsequent weight gain. Based on the observed difference in plasma erythritol between high and low-fat diet-fed mice, each group will contain 8 male and 8 female mice to detect significant differences between groups at 90% power with α=0.05. We do not expect to detect sex-specific differences based on genotype or glucose exposure but have powered the study to detect sex-specific differences if present. The following outcomes will be measured:We will measure food intake and body weight twice weekly.Plasma erythritol, isolated from tail vein blood, will be measured by GC-MS biweekly.Body composition will be assessed using nuclear magnetic resonance imaging (NMR) after 2 and 8 weeks of intervention.Glucose metabolism will also be assessed at 2 and 8 weeks using intraperitoneal glucose tolerance testing (IPGTT).After 8 weeks of intervention, mice will be sacrificed, and tissues will be collected. Brown, epidydimal, and inguinal adipose depots will be collected and weighed to assess fat distribution. We will quantify tissue-specific erythritol production using GC-MS.To a subset of mice in each group (n=4 per exposure group to detect at least a 30% difference in erythritol synthesis with α=0.05 and 90% power), we will administer [U13C]-glucose at 2 and 8 weeks of treatment. We will quantify the amount of labeled erythritol in plasma produced from glucose. From these same mice, we will perform metabolic flux analysis (MFA) on plasma and liver, as previously described.12 MFA will be used to determine how endogenous erythritol production is associated with altered pentose phosphate pathway, glycolytic and TCA-cycle flux in vivo. MFA will be completed in collaboration with Dr. Karsten Hiller at the University of Braunschweig.We expect that high fat and/or high glucose diets will increase erythritol synthesis. Sord and Adh1 knockout mice will have lower levels of plasma erythritol and convert less [U13C]-glucose to erythritol than wildtype mice on any diet. We also expect that, compared to animals with lower erythritol levels, animals with high plasma erythritol will have increased flux through the PPP.Aim 2: Determine the effect of elevated plasma erythritol on gene expression.Wildtype C57BL/6J mice will be placed on modified AIN-93G with 45% fat derived calories, or 45% fat diet containing 40g/kg erythritol for 8 weeks. To detect significant differences between groups at 90% power with α=0.05, each group will contain 12 male and 12 female mice. High-fat diet was chosen to model the Western diet, inducing weight gain and glucose intolerance. We will measure food intake and body weight twice weekly for the duration of the study. Total erythritol in plasma will be measured by GC-MS after 2 and 8 weeks of treatment. To assess if elevating plasma erythritol impacts weight or glucose metabolism, we will also measure body composition by NMR and perform glucose tolerance testing at 2 and 8 weeks of treatment. After 8 weeks of intervention, all mice will be sacrificed, and tissues will be collected. RNA will be extracted from adipose and liver tissue then prepped for sequencing, sequenced using the Illumina NextSeq 500 platform, and analyzed for differential expression of genes.We will also use PANTHER Pathway ontology analysis to assess cellular pathways that are regulated by erythritol exposure. We expect that elevating plasma erythritol will upregulate genes related to glucose utilization and energy expenditure, and that elevated plasma erythritol will protect against weight gain and glucose intolerance.Data analysisPhenotype data from Aims 1 and 2 will be analyzed using a random coefficients model to account for repeated measurements of several responses. The responses will be weight, food intake, plasma erythritol, body composition, and glucose tolerance. Independent fixed effects will be time, sex, fat content of diet, glucose exposure level, genotype, and sex; random effect will be the mouse. This analysis will be followed by either Tukey or Bonferroni correction for multiple comparisons. Effects with p<0.05 will be considered significant. Importantly, the Adh1 and Sord studies in Aim 1 will be considered separately. We will work closely with the Cornell Statistical Consulting Unit (see attached letter of support) to use the most rigorous approach possible in statistical analysis.Pitfalls and Alternative ApproachesIn Aim 1, we may find no difference in erythritol production between wildtype and knockout Adh1 and Sord mice. This would indicate that neither ADH1 nor SORD, is rate-limiting in erythritol production. We have also proposed to determine erythritol levels at multiple doses of dietary fat and glucose to understand the relationship between nutrient availability and erythritol production. It is possible that, in Aim 2, erythritol will not cause changes in expression of genes related to glucose or energy expenditure. Because we are using a whole-transcriptome sequencing approach, we will have data on the effect of erythritol on other genes involved in metabolism.

Progress 06/01/21 to 08/18/23

Outputs
Target Audience:The target audience of the presentations and publications produced through this project is researchers in nutrition and metabolism. Changes/Problems:An unexpected outcome of this project was the modest impact of ADH1 and SORD loss on circulating erythritol. Because overall erythritol synthesis is only modestly impacted by loss of ADH1 or SORD, we did not complete the proposed metabolic flux analysis in objective 5. Alternatively, we investigated the relationship between simple sugar intake, genotype, and erythritol. We have determined that excess sugars lead to elevated plasma erythritol. We exposed SORD knockout mice to 30% sugar in drinking water, then measure plasma and urinary erythritol. We observed that loss of SORD did not impact circulating erythritol levels but did cause a 25% reduction in tissue erythritol in mice fed sucrose water. We also did not complete the RNA sequencing in objective 7. Based on our finding that dietary erythritol exposure did not impact body weight or glucose tolerance, we redirected our effort to characterize the dietary factors that promote endogenous erythritol synthesis. What opportunities for training and professional development has the project provided?This project has provided the opportunity for both technical and professional training. Technical training included the use of stable isotope tracers to study glucose metabolism in cell culture, gene modification in cell culture, and targeted detection of metabolites using GC-MS. In mice, technical training included metabolic phenotyping andmeasurement of metabolites in blood, urine, and tissues. Professional development activities have included attending both national and international conferences, attending a course in proteomic and transcriptomic analysis, and participating in Cornell's "NextGen Professors" cohort. Additionally, serving as a Student Interest Group executive member for the American Society of Nutrition provided networking and professional training. One-on-one and group meetings with committee members also have supported my training. How have the results been disseminated to communities of interest?A total of four manuscripts were written and either published or are under review to disseminate research findings to the scientific community. Five abstracts, including four oralpresentations and one poster presentation, were shared with the scientific community at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact: Elevated serum erythritol is a biomarker for chronic disease states including Type 2 Diabetes Mellitus and cardiovascular disease. Human erythritol synthesis from glucose was also recently discovered, but the metabolic pathways leading to elevated serum erythritol are not defined. The overall objective of this project is to define the causes of erythritol production, and the physiological impacts of erythritol synthesis on human metabolism. Defining these mechanisms will improve our use of this predictive biomarker to prevent cardiometabolic diseases. With the support of this fellowship, we have identified that erythritol synthesis increases in response to excess dietary sugars and oxidative stress. These are two known contributors to the development of cardiometabolic diseases. Importantly, we have also determined that chronic exposure to erythritol from dietary sources does not impair metabolism in mice or contribute to weight gain. Throughout the project funding period, these findings have been disseminated to researchers through threefirst-author journal publications, one additional publication under review,and five conference presentations. Objectives: 1. Measure the metabolic phenotype ofSord-/-andAdh1-/-mice exposed to experimental diets with a range of macronutrient composition, including body weight, food intake, body composition, and glucose tolerance. We completed exposure of Sordand Adh1wildtype(+/+) and knockout (-/-) mice to both low-fat diet (LFD) and high-fat diet (HFD) and assessment of their metabolic phenotype. We collected data on body weight, body composition, food intake, glucose tolerance, and adiposity. Adh1 -/- mice show no difference in overall body weight, composition, food intake or glucose tolerance when compared to Sord +/+littermates consuming either LFD or HFD. Sord -/-mice exhibit impaired glucose tolerance compared to Sord +/+littermates on both a LFD and HFD but show no significant difference in body composition. This data was published in the Journal of Nutrition in 2023. 2. Determine the plasma and tissue erythritol content ofSord-/-andAdh1-/-mice exposed to experimental diets. We collected plasma and tissues of mice exposed to HFD or LFD for 8 weeks. We measured plasma erythritol at 2, 5, and 8 weeks of exposure to experimental diets. We measured tissue erythritol following the full 8 weeks of dietary treatment. We found that mice lacking ADH1 do not exhibit any perturbation in erythritol metabolism. Plasma and tissue erythritol content was the same in Adh1 +/+ and -/-mice. Mice lacking SORD exhibit a modest reduction in plasma and tissue erythritol when fed diets containing low or high fat content. This data indicates that in mice, SORD may contribute more to erythritol synthesis than ADH1. Based on our finding that high sucrose water can significantly elevate erythritol synthesis, we also exposed Sord +/+ and Sord -/- mice to low fat diet supplemented with high sucrose water. We measured body weight and food intake and plasma, urinary, and tissue erythritol following two weeks of exposure to sucrose water. We observed that Sord -/- had no impact on circulating erythritol in the presence of high sucrose consumption. Interestingly, tissue erythritol was reduced by 25% in Sord -/- mice compared to Sord +/+ mice. These findings indicate that SORD is not essential for erythritol synthesis but does contribute to tissue erythritol levels. These findings have been published in the Journal of Nutrition in 2023. 3. In cell culture models, determine the regulatory steps of erythritol synthesis using siRNA knockdowns of SORD, glucose-6-phosphate dehydrogenase, transketolase, and transaldolase under normal glucose and hyperglycemic conditions. We used siRNA to knock down the listed enzymes in A549 cells under normal-glucose and high-glucose conditions, then collected intracellular metabolites and measured intracellular erythritol content. We found that under normal glucose conditions, there is no effect on erythritol synthesis when expression of the enzymes SORD, G6PD, TKT, or TALDO is reduced. Contrastingly, in hyperglycemic conditions, reducing the expression of SORD or TKT significantly decreases the synthesis of erythritol. Importantly, this suggests that glucose availability is a stronger regulator of increase erythritol synthesis than pentose phosphate pathway enzyme expression. These findings were published in Frontiers in Nutrition in 2022. 4. In cell culture models, evaluate the response of erythritol synthesis to alternative pentose phosphate pathway stimuli, such as inflammation and oxidative stress. We exposed A549 cells to hydrogen peroxide, a strong oxidative stress inducing agent. We also exposed cells to varying concentrations of sugars, including glucose and fructose. Following treatment, we harvested intracellular metabolites and measured intracellular erythritol. We found that erythritol synthesis in cell culture is directly related to the sugar availability in culture medium. We also observed that erythritol synthesis is elevated in response to oxidative stress. The responses to sugar and oxidative stress are modulated by antioxidant response transcription factor NRF2. These findings connect intracellular erythritol synthesis to two key factors in the progression of cardiometabolic diseases. These findings in A549 cells were published in Frontiers in Nutrition in 2022. To expand on the work in A549 cells, we exposed kidney (HK-2) and skeletal muscle (C2C12) cell models to high glucose media and to reactive oxygen species to determine how intracellular erythritol responds in these physiologically relevant cell types. We measured reactive oxygen species and intracellular erythritol following these exposures. We observed that both skeletal muscle myotubes and kidney proximal tubule cells elevate intracellular erythritol when exposed to high glucose media compared to low glucose media. Skeletal muscle cells, but not kidney cells, elevated erythritol synthesis in response to oxidative stress. These findings confirm that high glucose availability and oxidative stress are cellular factors which promote erythritol synthesis. They also highlight that the mechanisms which promote erythritol synthesis vary by cell/tissue type. These results are presented in a manuscript that is currently under review at Current Developments in Nutrition. 5. Using metabolic flux analysis, determine how endogenous erythritol production is associated with altered pentose phosphate pathway, glycolytic and TCA-cycle fluxin vivoinSord-/-andAdh1-/-mice. Due to the limited changes in endogenous erythritol production in the Sord-/-andAdh1-/-mice, this objective was changed. We instead measured the endogenous erythritol production in wildtype C57BL/6J mice that were fed high or low-fat diet with and without sucrose water. The results of these alternative experiments were published in the Journal of Nutrition in 2023. 6. In wildtype C57BL/6J mice fed high-fat diet, determine the effect of erythritol supplementation on weight gain and glucose intolerance. We exposed 8-week-old or 20-week-old C57BL/6J mice to a low or high-fat diet with or without erythritol supplementation. Body weight and body composition, food intake, andglucose tolerance were measured. We found that erythritol supplementation did not impact weight gain or glucose metabolism in young and middle-aged mice fed a low or high-fat diet. The findings of this objective were published in the Journal of Nutrition in 2021. 7. In wildtype C57BL/6J mice fed high-fat diet or high-fat diet with erythritol, measure erythritol-induced changes in gene expression using RNA sequencing and pathway analysis. Based on our findings that erythritol supplementation did not impact weight gain or glucose intolerance, we did not complete RNA sequencing in these mice.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Ortiz SR, Field MS. C2C12 muscle myotubes, but not kidney proximal tubule HK-2 cells, elevate erythritol synthesis in response to oxidative stress. Current Developments in Nutrition. 2023, Under Review.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The target audience of the presentations and publications produced through this project is researchers in nutrition and metabolism. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided the opportunity for both technical and professional training. Technical training included the use of stable isotope tracers to study glucose metabolism in cell culture, as well as targeted detection of metabolites using GC-MS. Professional development activities have included attending both a national and international conference, attending a course in proteomic and transcriptomic analysis, and participating in Cornell's "NextGen Professors" cohort. In addition to these activities, one-on-one and group meetings with committee members have supported my training. How have the results been disseminated to communities of interest?One newmanuscripthasbeen generatedto disseminate the results of these objectives and is under review with the Journal of Nutrition. Two previouspublications havebeen released addressing the grant objectives. Three additional abstracts have been submitted to conferences. These abstracts were shared with researchers in the field of nutrition and biochemistrythrough oral and poster presentations. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, one additional manuscript containing the results of objective 4 will be drafted and submitted for publication. The results of all objectives will be compiled andsummarized in a dissertation, and the project director will have their PhD conferred.

Impacts
What was accomplished under these goals? Impact: Elevated serum erythritol is a biomarker for chronic disease states including Type 2 Diabetes Mellitus and cardiovascular disease. Human erythritol synthesis from glucose was also recently discovered, but the metabolic pathways leading to elevated serum erythritol are not defined. The overall objective of this project is to define the causes of erythritol production, and the physiological impacts of erythritol synthesis on human metabolism. Defining these mechanisms will improve our use of this predictive biomarker to prevent cardiometabolic diseases. With the support of this fellowship, we have identified that erythritol synthesis increases in response to excess dietary sugars and oxidative stress. These are two known contributors to the development of cardiometabolic diseases. Importantly, we have also determined that chronic exposure to erythritol from dietary sources does not impair metabolism in mice or contribute to weight gain. These findings have been disseminated to researchers through threefirst-author journal publications and five conference presentations. Objectives: Measure the metabolic phenotype ofSord-/-andAdh1-/-mice exposed to experimental diets with a range of macronutrient composition, including body weight, food intake, body composition, and glucose tolerance. This objective was addressed in the previous funding period. Determine the plasma and tissue erythritol content ofSord-/-andAdh1-/-mice exposed to experimental diets. We previously observed that Sord-/- mice had a very modest reduction in plasma erythritol. Based on the finding that high sucrose water can significantly elevate erythritol synthesis, we exposed Sord +/+ and Sord -/- mice to low fat diet supplemented with high sucrose water. We measured body weight and food intake and plasma, urinary, and tissue erythritol following two weeks of exposure to sucrose water. We observed that Sord -/- had no impact on circulating erythritol in the presence of high sucrose consumption. Interestingly, tissue erythritol was reduced by 25% in Sord -/- mice compared to Sord +/+ mice. These findings indicate that SORD is not essential for erythritol synthesis, but does contribute to tissue erythritol levels. These findings have been described in a manuscript currently under review with the Journal of Nutrition. In cell culture models, determine the regulatory steps of erythritol synthesis using siRNA knockdowns of SORD, glucose-6-phosphate dehydrogenase, transketolase, and transaldolase under normal glucose and hyperglycemic conditions. This objective was addressed in the previous funding period. In cell culture models, evaluate the response of erythritol synthesis to alternative pentose phosphate pathway stimuli, such as inflammation and oxidative stress. We previously observed in A549 lung cancer cells that intracellular erythritol is elevated by high glucose media and oxidative stress.To expand on these findings, we exposed kidney (HK-2) and skeletal muscle (C2C12) cell models to high glucose media and to reactive oxygen species to determine how intracellular erythritol responds in these physiologically relevant cell types. We measured reactive oxygen species and intracellular erythritol following these exposures. We observed that both skeletal muscle myotubes and kidney proximal tubule cells elevate intracellular erythritol when exposed to high glucose media compared to low glucose media. Skeletal muscle cells, but not kidney cells, elevated erythritol synthesis in response to oxidative stress. These findings confirm that high glucose availability and oxidative stress are cellular factors which promote erythritol synthesis. They also highlight that the mechanisms which promote erythritol synthesis vary by cell/tissue type. These results will be disseminated in an oral presentation at the annual meeting of the American Society for Nutrition, Nutrition 2023. Using metabolic flux analysis, determine how endogenous erythritol production is associated with altered pentose phosphate pathway, glycolytic and TCA-cycle fluxin vivoinSord-/-andAdh1-/-mice. This objective was not addressed in this funding period. In wildtype C57BL/6J mice fed high-fat diet, determine the effect of erythritol supplementation on weight gain and glucose intolerance. This objective was addressed in the previousfunding period. In wildtype C57BL/6J mice fed high-fat diet or high-fat diet with erythritol, measure erythritol-induced changes in gene expression using RNA sequencing and pathway analysis. This objective was not addressed in this funding period.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Ortiz SR, Field MS. Sucrose intake elevates erythritol in mouse plasma and urine. The Journal of Nutrition. 2023, Under Review.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ortiz SR, Heinz A, Hiller K, Field MS. Erythritol synthesis is elevated in response to oxidative stress and regulated by the non-oxidative pentose phosphate pathway in A549 cells. Frontiers in Nutrition. 2022;9:953056. doi:10.3389/fnut.2022.953056
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Semira R. Ortiz, Martha S. Field, (2022, October). Plasma and urinary erythritol are increased in response to excess dietary sucrose. 3rd International Conference on Precision Nutrition and Metabolism in Public Health and Medicine, Crete, Greece.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Ortiz SR, Field MS, (2023, March). Sord deletion reduces erythritol synthesis in liver and kidney of sucrose-fed mice. ASBMB Discover BMB.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Ortiz SR, Field MS, (2023, July). Erythritol synthesis is elevated by oxidative stress in muscle myotubes. ASN's Nutrition 2023.

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:The target audience of the presentations and publications produced through this project is researchers in nutrition and metabolism. Changes/Problems:An unexpected outcome of this project was the modest impact of ADH1 and SORD loss on circulating erythritol. Because overall erythritol synthesis is only modestly impacted by loss of ADH1 or SORD, we will no longer complete the proposed metabolic flux analysis in objective 5. Alternatively, we plan to investigate the relationship between simple sugar intake, genotype, and erythritol. We have determined that excess sugars lead to elevated plasma erythritol. We expect that exposure to excess sugar will magnify the impact of SORD loss on circulating erythritol. We will expose SORD knockout mice to 30% sugar in drinking water, then measure plasma and urinary erythritol. What opportunities for training and professional development has the project provided?This project has provided the opportunity for both technical and professional training. Technical training included the use of stable isotope tracers to study glucose metabolism in cell culture, as well as targeted detection of metabolites using GC-MS. Professional development activities have included attending both a national and international conference, attending a course in proteomic and transcriptomic analysis, and participating in Cornell's "NextGen Professors" cohort. In addition to these activities, one-on-one and group meetings with committee members have supported my training. How have the results been disseminated to communities of interest?Two manuscripts have been generated to disseminate the results of these objectives. One manuscript is published in the Journal of Nutrition, the second is under review. Two additional abstracts have been submitted to conferences. These abstracts were shared with researchers in the field of nutrition through oral presentations at two international conferences. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, additional data regarding endogenous erythritol synthesis in mice will be analyzed (objectives 1 and 2). A manuscript containing the results of objectives 1 and 2 will be drafted and submitted for publication.

Impacts
What was accomplished under these goals? Impact Statement: Elevated serum erythritol is a biomarker for chronic disease states including Type 2 Diabetes Mellitus and cardiovascular disease. Human erythritol synthesis from glucose was also recently discovered, but the metabolic pathways leading to elevated serum erythritol are not defined. The overall objective of this project is to define the causes of erythritol production, and the physiological impacts of erythritol synthesis on human metabolism. Defining these mechanisms will improve our use of this predictive biomarker to prevent cardiometabolic diseases. With the support of this fellowship, we have identified that erythritol synthesis increases in response to excess dietary sugars and oxidative stress. These are two known contributors to the development of cardiometabolic diseases. Importantly, we have also determined that chronic exposure to erythritol from dietary sources does not impair metabolism in mice or contribute to weight gain. These findings have been disseminated to researchers through two first-author journal publications and two conference presentations. Objectives: Measure the metabolic phenotype of Sord-/- and Adh1-/- mice exposed to experimental diets with a range of macronutrient composition, including body weight, food intake, body composition, and glucose tolerance. We have completed exposure of SORD and ADH1 WT and knockout (KO, or -/-) mice to both low-fat diet (LFD) and high-fat diet (HFD) and assessment of their metabolic phenotype. We collected data on body weight, body composition, food intake, glucose tolerance, and adiposity. ADH1 KO mice show no difference in overall body weight, composition, food intake or glucose tolerance when compared to WT littermates consuming either LFD or HFD. SORD KO mice exhibit impaired glucose tolerance compared to WT littermates on both a LFD and HFD but show no significant difference in body composition. Determine the plasma and tissue erythritol content of Sord-/- and Adh1-/- mice exposed to experimental diets. We collected plasma and tissues of mice exposed to HFD or LFD for 8 weeks. We measured plasma erythritol at 2, 5, and 8 weeks of exposure to experimental diets. We measured tissue erythritol following the full 8 weeks of dietary treatment. We found that mice lacking ADH1 do not exhibit any perturbation in erythritol metabolism. Plasma and tissue erythritol content was the same in ADH1 WT and KO mice. Mice lacking SORD exhibit a modest reduction in plasma and tissue erythritol when fed diets containing low or high fat content. We expect that when exposed to excess dietary sugars, loss of Sord will further blunt erythritol synthesis compared to WT mice, as observed in cell culture models. These data indicate that in mice, SORD may contribute more to erythritol synthesis than ADH1. In cell culture models, determine the regulatory steps of erythritol synthesis using siRNA knockdowns of SORD, glucose-6-phosphate dehydrogenase, transketolase, and transaldolase under normal glucose and hyperglycemic conditions. We used siRNA to knock down the listed enzymes in A549 cells under normal-glucose and high-glucose conditions, then collected intracellular metabolites and measured intracellular erythritol content. We found that under normal glucose conditions, there is no effect on erythritol synthesis when expression of the enzymes SORD, G6PD, TKT, or TALDO is reduced. Contrastingly, in hyperglycemic conditions, reducing the expression of SORD or TKT significantly decreases the synthesis of erythritol. These findings were shared at the 2nd International Conference on Precision Nutrition and Metabolism in Public Health and Medicine, Rhodes, Greece These findings have been submitted in a manuscript to the Journal of Nutrition, (currently under review) and are available as a pre-print on BioRxiv. In cell culture models, evaluate the response of erythritol synthesis to alternative pentose phosphate pathway stimuli, such as inflammation and oxidative stress. We exposed A549 cells to hydrogen peroxide, a strong oxidative stress inducing agent. We also exposed cells to varying concentrations of sugars, including glucose and fructose. Following treatment, we harvested intracellular metabolites and measured intracellular erythritol. We found that erythritol synthesis in cell culture is directly related to the sugar availability in culture medium. We also observed that erythritol synthesis is elevated in response to oxidative stress. The responses to sugar and oxidative stress are modulated by antioxidant response transcription factor NRF2. These findings connect intracellular erythritol synthesis to two key factors in the progression of cardiometabolic diseases. These findings have been submitted to Journal of Nutrition and are currently under review Using metabolic flux analysis, determine how endogenous erythritol production is associated with altered pentose phosphate pathway, glycolytic and TCA-cycle flux in vivo in Sord-/- and Adh1-/- mice. We have not addressed this objective in the current academic year. In wildtype C57BL/6J mice fed high-fat diet, determine the effect of erythritol supplementation on weight gain and glucose intolerance. We found that erythritol supplementation did not impact weight gain or glucose metabolism in young and middle-aged mice fed a low or high-fat diet. The findings of this objective were published in the Journal of Nutrition. In wildtype C57BL/6J mice fed high-fat diet or high-fat diet with erythritol, measure erythritol-induced changes in gene expression using RNA sequencing and pathway analysis. We have not addressed this objective in the current academic year.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Semira R. Ortiz, Martha S. Field, (2021, October). Metabolic regulation of erythritol synthesis, a biomarker of cardiometabolic disease. 2nd International Conference on Precision Nutrition and Metabolism in Public Health and Medicine, Rhodes, Greece.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Ortiz SR, Heinz A, Hiller K, Field MS. Erythritol synthesis in human cells is elevated in response to oxidative stress and regulated by the non-oxidative pentose phosphate pathway. The Journal of Nutrition. 2022, Under Review.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Semira R. Ortiz, Martha S. Field, (2022, March). Urinary erythritol is increased in response to excess dietary sucrose. Nutrition 2022 Live Online.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Ortiz SR, Field MS. Chronic Dietary Erythritol Exposure Elevates Plasma Erythritol Concentration in Mice but Does Not Cause Weight Gain or Modify Glucose Homeostasis. The Journal of Nutrition. 2021;151(8):2114-2124. doi:10.1093/jn/nxab130