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As the consumer interest and market for plant-based meat alternatives grows, understanding the nutritional differences between alternative and traditional meats is essential. This article describes an untargeted gas chromatography–mass spectrometry (GC–MS)-based metabolomics approach that compares the chemical profiles of a popular plant-based meat alternative and grass-fed ground beef using a GC system coupled to a GC–MS device. The samples were derivatized to simplify the chromatographic process and render the polar metabolites more volatile for GC–MS analysis. Statistical and multivariate analysis of the acquired and processed GC–MS data revealed that 90% of the annotated compounds differed between the plant-based alternative meat and the grass-fed ground beef samples. The ground beef and plant-based products each contained several compounds that were found in much smaller quantities, or not at all, in the other product. These results indicate differences in organic composition even though the nutritional labels on the back of the products were nearly identical. Heat maps, principal component analysis (PCA) score plots, variable importance plots (VIPs), and the clustering of compounds into metabolite classes provided further insights into the differences between the two types of meat products. The biological significance of the comparative data was studied using online databases and pathway analysis tools.

Hypersecretion of amylin, a β-cell hormone that regulates satiation, is common in individuals with prediabetes and is associated with pancreatic amyloid deposition and type-2 diabetes. Evidence has emerged that increased circulating levels of amyloid-forming human amylin may potentially impair brain function. Because mouse amylin is non-amyloidogenic, we generated transgenic mice with conditional pancreatic expression of amyloid-forming human amylin to study how in vivo knockdown of human amylin expression influences brain function during the development of type-2 diabetes. Males and females were fed a high-fat diet starting at 3 months of age to induce amylin hypersecretion and glucose dysregulation. Males developed hyperglycemia at 5 months of age, whereas females showed glucose dysregulation 3-4 months later. At 5 months of age, human amylin-expressing male mice were randomly assigned to either amylin downregulation group (by peritoneal tamoxifen injection) or control group (maintained amylin expression) (n = 10/group). Two months later, we assessed brain function with the novel object recognition test and performed comparative non-targeted metabolomics and global RNA-seq analyses of hippocampal tissue. Mice with downregulated human amylin show enhanced recognition memory index (p < 0.001) and lower blood glucose levels (p < 0.001) compared to those that continued to express human amylin. This was associated with increased hippocampal levels of glycolysis metabolites, including lactic acid (p < 0.01), glucose-6-phosphate (p = 0.06), and fructose (p = 0.07). Hippocampal gene-expression patterns between the two mouse groups revealed extensive compensatory changes in gene expression related to glucose metabolism. In conclusion, amylin downregulation in diabetic mice improves systemic glucose homeostasis and memory. Molecular processes associated with improved memory involve increased hippocampal glycolytic fluxes and compensatory gene expression.

Insulin resistance (IR) in adolescents with obesity is associated with a sex-dependent metabolic 'signature' comprising the branched-chain amino acids (BCAAs), glutamate/glutamine, C3/C5 acylcarnitines and uric acid. Here, we compared the levels of branched-chain α-keto acids (BCKAs) and glutamate/glutamine, which are the byproducts of BCAA catabolism and uric acid among adolescents with obesity prior to and following a 6-month lifestyle-intervention program.Fasting plasma samples from 33 adolescents with obesity (16 males, 17 females, aged 12-18 year) were analysed by flow-injection tandem MS and LC-MS/MS. Multiple linear regression models were used to correlate changes in BCKAs, glutamate/glutamine and uric acid with changes in weight and insulin sensitivity as assessed by HOMA-IR, adiponectin and the ratio of triglyceride (TG) to HDL. In predictive models, BCKAs, glutamate/glutamine and uric acid at baseline were used as explanatory variables.Baseline BCKAs, glutamate/glutamine and uric acid were higher in males than females despite comparable BMI-metrics. Following lifestyle-intervention, α-keto-β-methylvalerate (α-KMV, a metabolic by product of isoleucine) decreased in males but not in females. The ratio of BCKA/BCAA trended lower in males. In the cohort as a whole, BCKAs correlated positively with the ratio of TG to HDL at baseline and HOMA-IR at 6-month-follow-up. Glutamate/glutamine was positively associated with HOMA-IR at baseline and 6-month-follow-up. A reduction in BCKAs was associated with an increase in adiponectin, and those with higher BCKAs at baseline had higher adiponectin levels at 6-month-follow-up. Interestingly those adolescents with higher uric acid levels at baseline had greater reduction in weight.BCKAs and glutamate/glutamine may serve as biomarkers of IR in adolescents with obesity, and uric acid might serve as a predictor of weight loss in response to lifestyle-intervention. Differential regulation of BCAA catabolism in adolescent males and females implicates critical roles for sex steroids in metabolic homeostasis.

Objectives We have previously demonstrated that insulin resistance (IR) in youth is associated with elevated levels of branched-chain amino acids (BCAAs). BCAAs compete with aromatic amino acids (AAA) including tryptophan, the precursor of serotonin, for uptake into β-cells and other tissues via the large neutral amino acid transporter. Serotonin has been reported to increase β-cell mass and glucose-dependent insulin secretion. In this study we have explored how BCAA, tryptophan (one of the AAA), and a subset of their metabolites are modulated in youth-onset T2D. Based on our prior studies in neuronal BCAA metabolism, we hypothesized that elevated BCAA could induce diversion of tryptophan metabolism towards production of kynurenine rather than serotonin in youth with T2D. To test this, we analyzed 24-hour urine samples and compared levels of byproducts of BCAAs and tryptophan metabolism in obese youth with T2D with those in non-diabetic obese and lean youth of comparable age, pubertal status and ethnicity. Methods 56 non-diabetic adolescents with overweight/obesity ("obese"), 42 adolescents with T2D ("T2D"), and 43 normal weight controls ("lean"), ages 12-21 years-old were studied. Weight, height, BMI, BMI% were extracted from medical charts. Body fat percent (BF%) was measured by TANITA. We also measured metabolites derived from BCAA catabolism, including the branched-chain ketoacids (BCKAs), and tryptophan metabolism, including intermediates of the serotonergic and kynurenine pathways, in spot and 24-hour urine samples by liquid chromatography/tandem-mass spectrometry (LC/MS-MS). Levels were normalized to urine creatinine. Group differences were assessed by Kruskal Wallis or ANOVA. Results Groups were comparable for age (obese 14.8 +/- 1.9; T2D 15.7 +/- 2.1 and lean 14.9 +/- 1.9-yr), pubertal status, and ethnicity. Youth with T2D were predominantly female (T2D, 28 F, 14 M; obese 33 F, 23 M and lean, 17 F, 26 M), and had highest BF% (obese 37.3 +/- 9.5; T2D 42.9 +/- 9.9; lean 20.1 +/- 6.3%; p=2.58e-22). In 24-hour urine samples, BCKAs, tryptophan, and kynurenine levels were higher in T2D (p=0.0002, p= 0.0045 and p= 0.00009 respectively) than in either lean controls or nondiabetic youth with obesity; in contrast, there were no differences between lean controls and non-diabetic youth with obesity. The levels of 5-HIAA, the principal metabolite of serotonin, were comparable across groups; however, the ratio of kynurenine/tryptophan was higher (p= 0.0112) in youth with T2D and the ratios of 5-HIAA/ tryptophan (p=0.027) and 5-HIAA/Kynurenine (p=0.0067) were lower compared to the other two groups. Those ratios were comparable between lean controls and non-diabetic youth with obesity. Conclusions Increased BCKAs are accompanied by diversion of tryptophan metabolism from the serotonin pathway to the kynurenine pathway, suggesting perturbations in both BCAA and AAA metabolism in youth-onset T2D. These alterations could contribute to development of beta-cell dysfunction and progression to T2D in youth. Presentation: Sunday, June 12, 2022 12:00 p.m. - 12:15 p.m.

Background Type 2 diabetes mellitus (DM) is associated with an increased risk of fracture despite a paradoxically higher average bone mineral density. Additional markers of fracture risk among older adults with DM are needed to identify at-risk individuals. Method The MURDOCK study is an ongoing, longitudinal cohort study, initiated in 2007, of residents in Cabarrus and surrounding counties, based in Kannapolis, North Carolina. At study enrollment, participants completed medical and health questionnaires, and provided biospecimen samples for the biorepository. In this nested case-control analysis, incident fractures since study enrollment among MURDOCK participants were identified by self-reported survey and by query of the electronic medical records (EMR). Fracture cases were matched 2: 1 by age, gender, race/ethnicity, and BMI to participants without incident fracture. Stored biorepository serum samples were analyzed for both conventional metabolites (non-esterified fatty acids (NEFA), triglycerides, lactate, and ketones) and targeted metabolomics (amino acids and acylcarnitines), using the Beckman DxC 600 clinical analyzer and flow injection electrospray ionization tandem mass spectrometry, respectively. Principal components analysis (PCA) was used to identify relevant profiles for metabolomics data. The association between incident fracture and metabolic profile was assessed using conditional logistic regression, stratified by matched group and controlled for multiple confounders including tobacco and alcohol use, medical comorbidities (e.g., coronary artery disease, congestive heart failure, chronic kidney disease, rheumatoid arthritis) and medications (e.g., insulin, metformin, thiazolidinediones). Results 107 incident fractures were identified by self-reported questionnaire and EMR query; 210 controls were matched. The study population was predominantly female (71.8%) and white (90.6%) with mean (SD) age 65.0 (7.7) years and BMI 31.8 (6.2) kg/m2. PCA identified 2 amino acid (AA) profiles: profile 1 (AA1) consisted of the branched chain amino acids, phenylalanine and tyrosine; profile 2 (AA2) consisted of serine, arginine, asparagine/aspartate (Asx) and glutamine/glutamate (Glx). After controlling for multiple risk factors, AA2 was significantly associated with incident fracture (OR 1.72, 95% CI: 1.17–2.54). NEFA were associated with lower odds of fracture (OR 0.06, 95% CI: 0.01–0.31). There was no association of fracture with other conventional metabolites, acylcarnitine factors, nor AA1. Conclusion Given association of NEFA and AA2 with incident fracture, our results indicate novel biomarkers, as well as potential mechanisms of fracture risk among older adults with DM. Higher level of NEFA was associated with lower fracture odds; though not further classified in this study, prior studies have shown direct effects of polyunsaturated and monounsaturated fatty acids on osteoclasts. Higher level of AA2, including the amino acids Glx and Asx, was associated with increased fracture odds. Prior studies have shown direct effects of Glx on both osteoblasts and osteoclasts, and Glx and Asx have also been associated with muscle mass and strength. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.

Objectives In healthy, normal - weight adults there is a circadian rhythm in blood pressure (BP) and plasma catecholamines (epinephrine and norepinephrine): BP and the levels of norepinephrine and epinephrine decline during sleep, followed by a rapid increase during the early morning hours. Studies in adults with Type 2 diabetes (T2D) show blunted reductions in BP and plasma catecholamine levels during sleep that are associated with increased risks for heart failure, stroke, myocardial infarction, and sudden death. In this study, we explored if the circadian rhythm in catecholamines is disrupted in youth-onset T2D. We hypothesized that increased sympatho-adrenal activity during sleep differentiates youth with T2D from non-diabetic overweight/obese and lean youth of comparable age, pubertal status, and ethnicity. To that end, we measured urine catecholamines in fasting – morning-spot urines and 24-hour urine samples. Methods 56 non-diabetic adolescents with overweight/obesity ("obese"), 42 adolescents with T2D ("T2D"), and 43 normal-weight controls ("lean"); aged 12-21 years, were studied. None was diagnosed with hypertension, and none was on antihypertensive treatment. Weight, height, BMI, BMI%, and BP were extracted from medical charts. Body fat percent (BF%) was measured by TANITA. Stress levels were assessed using PHQ-2 and PHQ-9 questionnaires. Fractionated free urine catecholamines (epinephrine, norepinephrine, and dopamine) were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS-MS) in both spot and 24-hour urines, normalized to urinary creatinine. The ratio of fasting morning urine catecholamines to 24-hour urine catecholamines was calculated to assess circadian variation in catecholamines. Group differences were assessed by Kruskal-Wallis or ANOVA. Results Groups were comparable for age (obese 14.8 +/- 1.9; T2D 15.7 +/- 2.1 and lean 14.9 +/- 1.9-yr), pubertal status, and ethnicity. Obese youth with and without T2D were predominantly female (T2D, 28 F, 14 M; obese 33 F, 23 M; lean, 17 F, 26 M); those with T2D had highest BF% (obese 37.3 +/- 9.5; T2D 42.9 +/- 9.9; lean 20.1 +/- 6.3%; p=2.58e-22) and systolic blood pressure (obese 115.36 +/- 11.54; T2D 127.83 +/- 12.08 and lean 111.65 +/- 9.13 mmHg; p=5.49e-10). Fractionated free urine catecholamines (epinephrine, norepinephrine and dopamine) were comparable across groups in 24-hour urines. However, fasting morning epinephrine levels and the ratio of fasting morning/24-hour epinephrine were higher in T2D (p=0.0035, and p=0.035, respectively) than in either lean or nondiabetic youth with obesity. There were no differences in morning urine catecholamines between lean controls and non-diabetic youth with obesity. Conclusions Our results suggest a disrupted circadian rhythm in catecholamines in youth-onset T2D, with a blunted overnight fall in epinephrine levels. Higher levels of epinephrine levels at night in youth with T2D might be associated with, or predispose to, hypertension and long-term cardiovascular complications. Presentation: Sunday, June 12, 2022 11:30 a.m. - 11:45 a.m.

Hypersecretion of amylin, a β-cell hormone that regulates satiation, is common in individuals with prediabetes and is associated with pancreatic amyloid deposition and type-2 diabetes. Evidence has emerged that increased circulating levels of amyloid-forming human amylin may potentially impair brain function. Because mouse amylin is non-amyloidogenic, we generated transgenic mice with conditional pancreatic expression of amyloid-forming human amylin to study how in vivo knockdown of human amylin expression influences brain function during the development of type-2 diabetes. Males and females were fed a high-fat diet starting at 3 months of age to induce amylin hypersecretion and glucose dysregulation. Males developed hyperglycemia at 5 months of age, whereas females showed glucose dysregulation 3-4 months later. At 5 months of age, human amylin-expressing male mice were randomly assigned to either amylin downregulation (by peritoneal tamoxifen injection) or control (maintained amylin expression) groups (n = 10/group) . Two months later, we assessed brain function with a novel object recognition test and performed comparative non-targeted metabolomics and global RNA-seq analyses of hippocampal tissue. Mice with downregulated human amylin showed enhanced recognition memory index (p < 0.001) and lower blood glucose levels (p < 0.001) compared to those that continued to express human amylin. This was associated with increased hippocampal levels of glycolysis metabolites, including lactic acid (p < 0.01) , glucose-6-phosphate (p = 0.06) , and fructose (p = 0.07) . Hippocampal gene-expression patterns between the two mouse groups revealed extensive compensatory changes in gene expression related to glucose metabolism. In conclusion, amylin downregulation in diabetic mice improves systemic glucose homeostasis and memory. Molecular processes associated with improved memory involve increased hippocampal glycolytic fluxes and compensatory gene expression. Disclosure V.Gopal viswanathan: None. M.Muehlbauer: None. J.R.Bain: Other Relationship; CardioEphEx, LLC. F.Despa: None. N.Verma: None. E.D.Winford: None. D.Kotiya: None. L.E.Radulescu: None. N.Leibold: None. K.C.Chen: None. S.Despa: None. D.A.Hill: None. Funding National Institutes of Health NS116058, AG057290, AG053999

Introduction/Background Type 1 diabetes (T1D) is associated with decreased skeletal muscle mass (SMM) and impaired muscle function in adults. Despite the importance of skeletal muscle in insulin action and metabolic homeostasis, our understanding of skeletal muscle health in children with T1D is very limited. Here we used a novel, non-invasive methodology to compare muscle mass (MM) in children with T1D and sex and age-matched healthy children without diabetes. Objectives We hypothesized that: (a) children with T1D have lower MM than age, sex, and BMI-matched children without T1D; (b) MM in children with T1D correlates with insulin sensitivity and glycemic control. Methods Weight (BW), height, BMI%, body fat% (BF%), daily insulin requirement, CGM data, HbA1c, and total and HMW adiponectin were measured in 24 young children (15 females, 9 males; age 6-11 years) with T1D. BF% and fat-free-mass (FFM) was measured by bioelectrical impedance. Insulin sensitivity was assessed by total and HMW-adiponectin and daily insulin requirements. Glycemic control was assessed by glucose time in range, mean blood glucose, and HbA1c. MM was measured using the D3creatine dilution method in 22 of the children with T1D (14 females, 8 males) and, in a separate study, in 34 children without T1D (17 females and 17 males). This method involves an oral 10 mg dose of deuterated creatine (methyl-d3, D3Cr). Creatine is converted in vivo to creatinine, including D3Cr to D3creatinine, in muscle by an irreversible, nonenzymatic reaction and then excreted. A single fasting urine sample is collected to determine D3creatinine enrichment. MM/BW (%) was calculated to normalize for body weight. Results There were strong positive associations between MM and body weight (r=0.85, p Conclusions This is the first study to quantitate MM directly in young children with T1D. Our results demonstrate a lower %MM in 6–11-year-old pre-pubertal children with T1D with stable and reasonable glycemic control. The cause for the lower % MM in T1D is not clear; however future studies could examine the rates of muscle protein and fat synthesis and breakdown in healthy and diabetic children along with their relations to insulin administration, metabolic control, growth, and pubertal development. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

This study investigated the mechanistic basis behind the reported superior efficacy of high-avidity CD8 T cells. CMV and HIV are both chronic viral infections, but while CMV-specific CD8 T cells can mediate lifelong viral control, in untreated HIV infection HIV-specific CD8 T cells progressively lose function. Using in vitro studies of human cells, we show that avidity-dependent downregulation of the CD8 co-receptor directly programs metabolism, due to a novel association between CD8 and the glucose transporter GLUT1. We used flow cytometry to profile ex vivo and virus-specific CD8 T cells from HIV-infected individuals on antiretroviral therapy. Ex vivo, cells expressing low levels of CD8 (CD8dim) expressed more CD69 but less cell surface GLUT1, and took up less glucose (2-NBDG) than CD8bright T cells. Following antigen stimulation, CD3, CD8, and GLUT1 were downregulated from the cell surface in an avidity-dependent manner. CMV-specific CD8 T cells, which were of higher avidity, downregulated these proteins to a greater extent than lower-avidity HIV-specific CD8 T cells. GLUT1 downregulation strongly correlated with CD8 but not CD3 downregulation. Antibody-mediated downregulation of CD8 from the cell surface resulted in reduced glucose uptake and increased fatty acid (Bodipy) uptake, independent of CD3. Finally, CD3, CD8, and GLUT1 downregulation by HIV-specific CD8 T cells was impaired following viral escape mutations that reduced CD8 T cell avidity. We confirmed this finding in a transduction setting with a single clonal TCR. Our data reveal a novel function of the CD8 co-receptor, linking the avidity and metabolism of CD8 T cells.

A case of unexplained anemia of 10 yrs. duration is presented. Although all evidence led to the gastrointestinal tract as the site of blood loss, the jejunum was not implicated until the 5th admission to the hospital. A summary of the diagnostic aides from the standpoint of history, laboratory and roentgenology is given in attempting to establish the diagnosis preoperatively.