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Objective: The objective of this study was to explore how dietary macronutrient composition influences postprandial appetite hormone responses and subsequent energy intake., Methods: A total of 20 adults (mean [SEM], age 30 [1] years, BMI 27.8 [1.3] kg/m 2 , n = 8 with normal weight, n = 6 with overweight, n = 6 with obesity) consumed a low-fat (LF) diet (10% fat, 75% carbohydrate) and a low-carbohydrate (LC) diet (10% carbohydrate, 75% fat) for 2 weeks each in an inpatient randomized crossover design. At the end of each diet, participants consumed isocaloric macronutrient-representative breakfast test meals, and 6-h postprandial responses were measured. Ad libitum energy intake was measured for the rest of the day., Results: The LC meal resulted in greater mean postprandial plasma active glucagon-like peptide-1 (GLP-1; LC: 6.44 [0.78] pg/mL, LF: 2.46 [0.26] pg/mL; p < 0.0001), total glucose-dependent insulinotropic polypeptide (GIP; LC: 578 [60] pg/mL, LF: 319 [37] pg/mL; p = 0.0004), and peptide YY (PYY; LC: 65.6 [5.6] pg/mL, LF: 50.7 [3.8] pg/mL; p = 0.02), whereas total ghrelin (LC: 184 [25] pg/mL, LF: 261 [47] pg/mL; p = 0.0009), active ghrelin (LC: 91 [9] pg/mL, LF: 232 [28] pg/mL; p < 0.0001), and leptin (LC: 26.9 [6.5] ng/mL, LF: 35.2 [7.5] ng/mL; p = 0.01) were lower compared with LF. Participants ate more during LC at lunch (244 [85] kcal; p = 0.01) and dinner (193 [86] kcal; p = 0.04), increasing total subsequent energy intake for the day compared with LF (551 [103] kcal; p < 0.0001)., Conclusions: In the short term, endogenous gut-derived appetite hormones do not necessarily determine ad libitum energy intake., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society.)

Objective: We investigated how changes in 24-h respiratory exchange ratio (RER) and substrate oxidation during fasting versus an energy balance condition influence subsequent ad libitum food intake., Methods: Forty-four healthy, weight-stable volunteers (30 male and 14 female; mean [SD], age 39.3 [11.0] years; BMI 31.7 [8.3] kg/m 2 ) underwent 24-h energy expenditure measurements in a respiratory chamber during energy balance (50% carbohydrate, 30% fat, and 20% protein) and 24-h fasting. Immediately after each chamber stay, participants were allowed 24-h ad libitum food intake from computerized vending machines., Results: Twenty-four-hour RER decreased by 9.4% (95% CI: -10.4% to -8.5%; p < 0.0001) during fasting compared to energy balance, reflecting a decrease in carbohydrate oxidation (mean [SD], -2.6 [0.8] MJ/day; p < 0.0001) and an increase in lipid oxidation (2.3 [0.9] MJ/day; p < 0.0001). Changes in 24-h RER and carbohydrate oxidation in response to fasting were correlated with the subsequent energy intake such that smaller decreases in fasting 24-h RER and carbohydrate oxidation, but not lipid oxidation, were associated with greater energy intake after fasting (r = 0.31, p = 0.04; r = 0.40, p = 0.007; and r = -0.27, p = 0.07, respectively)., Conclusions: Impaired metabolic flexibility to fasting, reflected by an inability to transition away from carbohydrate oxidation, is linked with increased energy intake., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Objective: Elevated rates of gluconeogenesis are an early pathogenic feature of youth-onset type 2 diabetes (Y-T2D), but targeted first-line therapies are suboptimal, especially in African American (AA) youth. We evaluated glucose-lowering mechanisms of metformin and liraglutide by measuring rates of gluconeogenesis and β-cell function after therapy in AA Y-T2D., Methods: In this parallel randomized clinical trial, 22 youth with Y-T2D-age 15.3 ± 2.1 years (mean ± SD), 68% female, body mass index (BMI) 40.1 ± 7.9 kg/m2, duration of diagnosis 1.8 ± 1.3 years-were randomized to metformin alone (Met) or metformin + liraglutide (Lira) (Met + Lira) and evaluated before and after 12 weeks. Stable isotope tracers were used to measure gluconeogenesis [2H2O] and glucose production [6,6-2H2]glucose after an overnight fast and during a continuous meal. β-cell function (sigma) and whole-body insulin sensitivity (mSI) were assessed during a frequently sampled 2-hour oral glucose tolerance test., Results: At baseline, gluconeogenesis, glucose production, and fasting and 2-hour glucose were comparable in both groups, though Met + Lira had higher hemoglobin A1C. Met + Lira had a greater decrease from baseline in fasting glucose (-2.0 ± 1.3 vs -0.6 ± 0.9 mmol/L, P = .008) and a greater increase in sigma (0.72 ± 0.68 vs -0.05 ± 0.71, P = .03). The change in fractional gluconeogenesis was similar between groups (Met + Lira: -0.36 ± 9.4 vs Met: 0.04 ± 12.3%, P = .9), and there were no changes in prandial gluconeogenesis or mSI. Increased glucose clearance in both groups was related to sigma (r = 0.63, P = .003) but not gluconeogenesis or mSI., Conclusion: Among Y-T2D, metformin with or without liraglutide improved glycemia but did not suppress high rates of gluconeogenesis. Novel therapies that will enhance β-cell function and target the elevated rates of gluconeogenesis in Y-T2D are needed., (Published by Oxford University Press on behalf of the Endocrine Society 2023.)

Background: It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury., Methods: C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1 -/- knockout and ( Mkp1 +/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test., Results: ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro , ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo , female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes., Conclusion: Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Walter, Montoya-Durango, Rodriguez, Wang, Zhang, Chariker, Rouchka, Maldonado, Bennett, McClain, Barve and Gobejishvili.)

Exercise improves chronic inflammation and is recommended as a first-line medical or behavioral treatment for OSA with obesity. We examined whether the effects of an exercise program on inflammatory blood markers differed according to severity of OSA among obese adults. Overweight (BMI > 27 kg/m 2 ) adults were evaluated for OSA using overnight polysomnography and subsequently classified as exhibiting no-to-mild OSA (AHI < 15 events/hour) or moderate-to-severe OSA (AHI ≥ 15 events/hour). Cardiorespiratory fitness, body composition assessed by DXA, fasting metabolic parameters and adipokines (i.e., glucose, insulin, leptin and adioponectin), and multiple markers of inflammation (i.e., CRP, IL-4, IL-8 and TNF-α) were measured at baseline (Pre) and following a 6-week (3 days per week) comprehensive exercise program (Post). Ten adults (Age: 48 ± 8 years; W:6; M:4) with no/mild OSA and 12 adults (Age: 54 ± 8 years; W:5; M:7) with moderate/severe OSA completed all aspects of the trial. No significant differences in age, cardiorespiratory fitness, body composition, fasting metabolic parameters and most inflammatory markers were observed between groups at baseline. Exercise training decreased total fat mass (Pre: 41,167 ± 13,315 g; Post: 40,311 ± 12,657 g; p  = 0.008), leptin (Pre: 26.7 ± 29.6 pg/ml; Post: 22.7 ± 19.4 pg/ml; p  = 0.028) and adiponectin (Pre: 16.6 ± 10.9 µg/ml; Post: 11.0 ± 10.6 µg/ml; p  = 0.004) in those with moderate/severe OSA. Among those with no/mild OSA, exercise training resulted in a decrease in total fat mass (Pre = 37,332 ± 20,258 g; Post: 37,068 ± 18,268 g, p  = 0.037). These data suggest that while 6 weeks of exercise reduced adipokines in those with moderate-to-severe OSA, it was not sufficient to improve common markers of inflammation among overweight adults with OSA., Competing Interests: Conflict of interestKK declares that she has no conflict of interest. HP declares that she has no conflict of interest. SP declares that he has no conflict of interest. CP declares that he has no conflict of interest. DD declares that he has no conflict of interest., (© The Author(s), under exclusive licence to Japanese Society of Sleep Research 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Context: Childhood overnutrition is associated with increased growth and bone mineral density (BMD) vs the opposite for undernutrition. The role of insulin receptor (InsR) signaling in these phenotypes is unclear. Rare disease patients with hyperinsulinemia and impaired InsR function (homozygous [-/-] or heterozygous [+/-] INSR pathogenic variants, type B insulin resistance [TBIR]) model increased InsR signaling, while patients with intact InsR function (congenital generalized lipodystrophy, CGL) model decreased InsR signaling., Objective: This work aimed to understand mechanisms whereby InsR signaling influences growth., Methods: A cross-sectional comparison was conducted of CGL (N = 23), INSR-/- (N = 13), INSR+/- (N = 17), and TBIR (N = 8) at the National Institutes of Health. Main outcome measures included SD scores (SDS) for height, body mass index, insulin-like growth factor (IGF)-1, and BMD, and IGF binding proteins (IGFBP)-1 and -3., Results: INSR-/- vs CGL had higher insulin (median 266 [222-457] vs 33 [15-55] mcU/mL), higher IGFBP-1 (72 350 [55 571-103 107] vs 6453 [1634-26 674] pg/mL), lower BMI SDS (-0.7 ± 1.1 vs 0.5 ± 0.9), lower height SDS (-1.9[-4.3 to -1.3] vs 1.1 [0.5-2.5]), lower BMD SDS (-1.9 ± 1.4 vs 1.9 ± 0.7), and lower IGFBP-3 (0.37 [0.19-1.05] vs 2.00 [1.45-2.67] μg/mL) (P < .05 for all). INSR +/- were variable. Remission of TBIR lowered insulin and IGFBP-1, and increased IGF-1 and IGFBP-3 (P < .05)., Conclusion: Patients with hyperinsulinemia and impaired InsR function exhibit impaired growth and lower BMD, whereas elevated InsR signaling (CGL) causes accelerated growth and higher BMD. These patients demonstrate that insulin action through the InsR stimulates direct anabolic effects in bone and indirect actions through the growth hormone (GH)-IGF-1 axis. TBIR patients exhibit abnormalities in the GH axis that resolve when InsR signaling is restored, supporting a causal relationship between InsR and GH axis signaling., (Published by Oxford University Press on behalf of the Endocrine Society 2023.)

Purpose: Patients with multiple endocrine neoplasia type 1 (MEN1) are predisposed to develop duodenopancreatic neuroendocrine tumors (dpNETs), and metastatic dpNET is the primary cause of disease-related mortality. Presently, there is a paucity of prognostic factors that can reliably identify patients with MEN1-related dpNETS who are at high risk of distant metastasis. In the current study, we aimed to establish novel circulating molecular protein signatures associated with disease progression., Experimental Design: Mass spectrometry-based proteomic profiling was conducted on plasmas procured through an international collaboration between MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht from a cohort of 56 patients with MEN1 [14 with distant metastasis dpNETs (cases) and 42 with either indolent dpNETs or no dpNETs (controls)]. Findings were compared to proteomic profiles generated from serially collected plasmas from a mouse model of Men1-pancreatic neuroendocrine tumors (Men1fl/flPdx1-CreTg) and control mice (Men1fl/fl)., Results: A total of 187 proteins were found to be elevated in MEN1 patients with distant metastasis compared to controls, including 9 proteins previously associated with pancreatic cancer and other neuronal proteins. Analyses of mouse plasmas revealed 196 proteins enriched for transcriptional targets of oncogenic MYCN, YAP1, POU5F1, and SMAD that were associated with disease progression in Men1fl/flPdx1-CreTg mice. Cross-species intersection revealed 19 proteins positively associated with disease progression in both human patients and in Men1fl/flPdx1-CreTg mice., Conclusions: Our integrated analyses identified novel circulating protein markers associated with disease progression in MEN1-related dpNET., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Cyclic nucleotides are second messengers, which play significant roles in numerous biological processes. Previous work has shown that cAMP and cGMP signaling regulates various pathways in liver cells, including Kupffer cells, hepatocytes, hepatic stellate cells, and cellular components of hepatic sinusoids. Importantly, it has been shown that cAMP levels and enzymes involved in cAMP homeostasis are affected by alcohol. Although the role of cyclic nucleotide signaling is strongly implicated in several pathological pathways in liver diseases, studies describing the changes in genes regulating cyclic nucleotide metabolism in ALD are lacking., Methods: Male C57B/6 mice were used in an intragastric model of alcohol-associated steatohepatitis (ASH). Liver injury, inflammation, and fibrogenesis were evaluated by measuring plasma levels of injury markers, liver tissue cytokines, and gene expression analyses. Liver transcriptome analysis was performed to examine the effects of alcohol on regulators of cyclic AMP and GMP levels and signaling. cAMP and cGMP levels were measured in mouse livers as well as in livers from healthy human donors and patients with alcohol-associated hepatitis (AH)., Results: Our results show significant changes in several phosphodiesterases (PDEs) with specificity to degrade cAMP (Pde4a, Pde4d, and Pde8a) and cGMP (Pde5a, Pde6d, and Pde9a), as well as dual-specificity PDEs (Pde1a and Pde10a) in ASH mouse livers. Adenylyl cyclases (ACs) 7 and 9, which are responsible for cAMP generation, were also affected by alcohol. Importantly, adenosine receptor 1, which has been implicated in the pathogenesis of liver diseases, was significantly increased by alcohol. Adrenoceptors 1 and 3 (Adrb), which couple with stimulatory G protein to regulate cAMP and cGMP signaling, were significantly decreased. Additionally, beta arrestin 2, which interacts with cAMP-specific PDE4D to desensitize G-protein-coupled receptor to generate cAMP, was significantly increased by alcohol. Notably, we observed that cAMP levels are much higher than cGMP levels in the livers of humans and mice; however, alcohol affected them differently. Specifically, cGMP levels were higher in patients with AH and ASH mice livers compared with controls. As expected, these changes in liver cyclic nucleotide signaling were associated with increased inflammation, steatosis, apoptosis, and fibrogenesis., Conclusions: These data strongly implicate dysregulated cAMP and cGMP signaling in the pathogenesis of ASH. Future studies to identify changes in these regulators in a cell-specific manner could lead to the development of novel targeted therapies for ASH.

Objective: This study aimed to investigate human fetal exposure to non-nutritive sweeteners (NNS) by analyzing amniotic fluid and umbilical cord blood., Study Design: Concentrations of four NNS (acesulfame-potassium [ace-K], saccharin, steviol glucuronide, and sucralose) were measured in amniotic fluid ( n  = 13) and cord blood samples ( n  = 15) using liquid chromatography-mass spectrometry. Amniotic fluid samples were obtained for research purposes at the time of term elective cesarean birth or clinically indicated third trimester amnioreduction at Mercy Hospital for Women (Melbourne, Australia). All except four women were in the fasting state. Cord blood samples were obtained from an independent cohort of newborns whose mothers were enrolled in a separate clinical trial at the National Institutes of Health., Results: Ten of 13 amniotic fluid samples contained at least one NNS (ace-K, saccharin, steviol glucuronide, and/or sucralose). Maximum amniotic fluid NNS concentrations of ace-K, saccharin, steviol glucuronide, and sucralose were 78.9, 55.9, 93.5, and 30.6 ng/mL, respectively. Ace-K and saccharin were present in 100% and 80% of the cord blood samples, with maximal concentrations of 6.5 and 2.7 ng/mL, respectively. Sucralose was not detected and steviol glucuronide was not measurable in any of the cord blood samples., Conclusion: Our results provide evidence of human transplacental transmission of NNS. Based on results predominantly obtained from rodent models, we speculate that NNS exposure may adversely influence the offsprings' metabolic health. Well-designed, prospective clinical trials are necessary to understand the impact of NNS intake during pregnancy on human development and long-term health., Key Points: · NNS consumption during pregnancy has increased in recent years.. · Maternal NNS intake during pregnancy is associated with preterm birth and higher infant weight gain in epidemiologic studies.. · In rodents, in utero NNS exposure induces metabolic abnormalities in mothers and their offspring, alters offspring gut microbiota composition, and promotes sweet taste preference in adulthood.. · It is presently unknown whether and to what degree maternal NNS ingestion in humans leads to direct in utero exposure.. · This study provides the first evidence of in utero NNS exposure in humans and highlights the urgent need to investigate clinical consequences of early life NNS exposure on metabolism, weight, taste preference, and general health.., Competing Interests: None declared., (Thieme. All rights reserved.)

Objective: This study investigated whether interindividual variance in diet-induced metabolic flexibility is explained by differences in gut hormone concentrations., Methods: A total of 69 healthy volunteers with normal glucose regulation underwent 24-hour assessments of respiratory quotient (RQ) in a whole-room indirect calorimeter during eucaloric feeding (EBL; 50% carbohydrate, 30% fat) and then, in a crossover design, during 24-hour fasting and three normal-protein (20%) overfeeding diets (200% energy requirements). Metabolic flexibility was defined as the change in 24-hour RQ from EBL during standard (50% carbohydrate), high-fat (60%), and high-carbohydrate (75%) overfeeding diets. Plasma concentrations of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) after an overnight fast were measured prior to and after each diet., Results: Compared with EBL, on average, 24-hour RQ decreased by ~4% during high-fat overfeeding, whereas it increased by ~4% during standard overfeeding and by ~9% during high-carbohydrate overfeeding. During high-carbohydrate overfeeding, but not during any other overfeeding diet or fasting, increased GLP-1 concentration was associated with increased RQ (r = 0.44, p < 0.001), higher/lower carbohydrate/lipid oxidation rates (r = 0.34 and r = -0.51, both p < 0.01), respectively, and increased plasma insulin concentration (r = 0.38, p = 0.02)., Conclusions: Increased GLP-1 concentration following high-carbohydrate overfeeding associated with a greater shift to carbohydrate oxidation, suggesting that GLP-1 may be implicated in diet-induced metabolic flexibility to carbohydrate overload., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA.)

Omicron is currently the dominant SARS-CoV-2 variant and several sublineages have emerged. Questions remain about the impact of previous SARS-CoV-2 exposure on cross-variant immune responses elicited by the SARS-CoV-2 Omicron sublineage BA.2 compared to BA.1. Here we show that without previous history of COVID-19, BA.2 infection induces a reduced immune response against all variants of concern (VOC) compared to BA.1 infection. The absence of ACE2 binding in sera of previously naïve BA.1 and BA.2 patients indicates a lack of meaningful neutralization. In contrast, anti-spike antibody levels and neutralizing activity greatly increased in the BA.1 and BA.2 patients with a previous history of COVID-19. Transcriptome analyses of peripheral immune cells showed significant differences in immune response and specific antibody generation between BA.1 and BA.2 patients as well as significant differences in the expression of specific immune genes. In summary, prior infection status significantly impacts the innate and adaptive immune response against VOC following BA.2 infection., Competing Interests: The authors declare no competing interests.

Background: Partial lipodystrophy (PL) syndromes involve deficiency of adipose tissue, causing severe insulin resistance and hypertriglyceridemia. Apolipoprotein C-III (apoC-III) is elevated in PL and is thought to contribute to hypertriglyceridemia by inhibiting lipoprotein lipase (LPL)., Objective: We hypothesized that volanesorsen, an antisense oligonucleotide to apoC-III, would decrease apoC-III, increase LPL activity, and lower triglycerides in PL., Methods: Five adults with PL enrolled in a 16-week placebo-controlled, randomized, double blind study of volanesorsen, 300 mg weekly, followed by 1-year open label extension., Results: Within-subject effects of volanesorsen before and after 16 weeks of active drug are reported due to small sample size. From week 0 to 16, apoC-III decreased from median (25 th , 75 th %ile) 380 (246, 600) to 75 (26, 232) ng/mL, and triglycerides decreased from 503 (330, 1040) to 116 (86, 355) mg/dL while activation of LPL by subjects' serum increased from 21 (20, 25) to 36 (29, 42) nEq/mL*min. Although, A1c did not change, peripheral and hepatic insulin sensitivity (glucose disposal and suppression of glucose production during hyperinsulinemic clamp) increased and palmitate turnover decreased. After 32-52 weeks of volanesorsen, liver fat decreased. Common adverse events included injection site reactions and decreased platelets., Conclusions: In PL, volanesorsen decreased apoC-III and triglycerides, in part through an LPL dependent mechanism, and may improve insulin resistance and hepatic steatosis., Competing Interests: Declaration of Competing Interest In-kind research support for this study (including study drug, analysis of pharmacokinetics and lipids, database management, patient remuneration and visiting nurse support) were provided by Ionis Pharmaceuticals and Akcea Therapeutics. All authors have declared that no conflict of interest exists., (Copyright © 2022. Published by Elsevier Inc.)

Objective: Physiological systems responsible for water homeostasis and energy metabolism are interconnected. This study hypothesized altered responses to dehydration including thirst, ad libitum water intake, and copeptin in men with obesity., Methods: Forty-two men (22 lean and 20 with obesity) were stimulated by a 2-hour hypertonic saline infusion and a 24-hour water deprivation. In each dehydrating condition, thirst, ad libitum water intake after dehydration, and urinary and hormonal responses including copeptin were assessed., Results: After each dehydration condition, ad libitum water intake was similar between both groups (p > 0.05); however, those with obesity reported feeling less thirsty (p < 0.05) and had decreased copeptin response and higher urinary sodium concentrations when stressed (p < 0.05). Angiotensin II, aldosterone, atrial and brain natriuretic peptides, and apelin concentrations did not differ by adiposity group and did not explain the different thirst or copeptin responses in men with obesity. However, leptin was associated with copeptin response in lean individuals during the hypertonic saline infusion (p < 0.05), but the relationship was diminished in those with obesity., Conclusions: Diminished thirst and copeptin responses are part of the obesity phenotype and may be influenced by leptin. Adiposity may impact pathways regulating thirst and vasopressin release, warranting further investigation., (© 2022 The Obesity Society (TOS). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Context: A greater decrease in 24-hour energy expenditure (24hEE) during short-term fasting is indicative of a thrifty phenotype., Objective: As ghrelin and the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis are implicated in the regulation of energy intake and metabolism, we investigated whether ghrelin, GH, and IGF-1 concentrations mediate the fasting-induced decrease in 24hEE that characterizes thriftiness., Methods: In 47 healthy individuals, 24hEE was measured in a whole-room indirect calorimeter both during 24-hour eucaloric and fasting conditions. Plasma total ghrelin, GH, and IGF-1 concentrations were measured by enzyme-linked immunosorbent assay after an overnight fast the morning before and after each 24-hour session., Results: During 24-hour fasting, on average 24hEE decreased by 8.0% (P < .001), GH increased by ~5-fold (P < .001), whereas ghrelin (mean +23 pg/mL) and IGF-1 were unchanged (both P ≥ .19) despite a large interindividual variability in ghrelin change (SD 150 pg/mL). Greater fasting-induced increase in ghrelin was associated with a greater decrease in 24hEE during 24-hour fasting (r = -0.42, P = .003), such that individuals who increased ghrelin by 200 pg/mL showed an average decrease in 24hEE by 55 kcal/day., Conclusion: Short-term fasting induced selective changes in the ghrelin/GH/IGF-1 axis, specifically a ghrelin-independent GH hypersecretion that did not translate into increased IGF-1 concentrations. Greater increase in ghrelin after 24-hour fasting was associated with greater decrease in 24hEE, indicating ghrelin as a novel biomarker of increased energy efficiency of the thrifty phenotype., (Published by Oxford University Press on behalf of the Endocrine Society 2022.)

Trophic interactions between microbes are postulated to determine whether a host microbiome is healthy or causes predisposition to disease. Two abundant taxa, the Gram-negative heterotrophic bacterium Bacteroides thetaiotaomicron and the methanogenic archaeon Methanobrevibacter smithii, are proposed to have a synergistic metabolic relationship. Both organisms play vital roles in human gut health; B. thetaiotaomicron assists the host by fermenting dietary polysaccharides, whereas M. smithii consumes end-stage fermentation products and is hypothesized to relieve feedback inhibition of upstream microbes such as B. thetaiotaomicron. To study their metabolic interactions, we defined and optimized a coculture system and used software testing techniques to analyze growth under a range of conditions representing the nutrient environment of the host. We verify that B. thetaiotaomicron fermentation products are sufficient for M. smithii growth and that accumulation of fermentation products alters secretion of metabolites by B. thetaiotaomicron to benefit M. smithii. Studies suggest that B. thetaiotaomicron metabolic efficiency is greater in the absence of fermentation products or in the presence of M. smithii. Under certain conditions, B. thetaiotaomicron and M. smithii form interspecies granules consistent with behavior observed for syntrophic partnerships between microbes in soil or sediment enrichments and anaerobic digesters. Furthermore, when vitamin B 12 , hematin, and hydrogen gas are abundant, coculture growth is greater than the sum of growth observed for monocultures, suggesting that both organisms benefit from a synergistic mutual metabolic relationship. IMPORTANCE The human gut functions through a complex system of interactions between the host human tissue and the microbes which inhabit it. These diverse interactions are difficult to model or examine under controlled laboratory conditions. We studied the interactions between two dominant human gut microbes, B. thetaiotaomicron and M. smithii, using a seven-component culturing approach that allows the systematic examination of the metabolic complexity of this binary microbial system. By combining high-throughput methods with machine learning techniques, we were able to investigate the interactions between two dominant genera of the gut microbiome in a wide variety of environmental conditions. Our approach can be broadly applied to studying microbial interactions and may be extended to evaluate and curate computational metabolic models. The software tools developed for this study are available as user-friendly tutorials in the Department of Energy KBase.

Heterologous ChAdOx1-BNT162b2 vaccination induces a stronger immune response than BNT162b2-BNT162b2. Here, we investigated the molecular transcriptome, germline allelic variants of immunoglobulin loci, and anti-Omicron antibody levels in 46 office and lab workers from the Republic of Korea following ChAdOx1-BNT162b2 vaccination. Anti-spike-specific IgG antibody levels against the ancestral SARS-CoV-2 strain increased from 70 AU/ml to 14,000 AU/ml to 142,000 AU/ml one, three and seven days following the second vaccination. Titers against VOC, including Omicron, were two-fold to three-fold lower, yet higher than those measured following BNT162b2-BNT162b2 vaccination. RNA-seq of peripheral immune cells demonstrated activation of interferon pathways with increased IGHV clonal transcripts encoding neutralizing antibodies. scRNA-seq revealed enriched B cell and CD4 + T cell responses in both ChAdOx1-BNT162b2 and BNT162b2-BNT162b2 recipients, but a stronger clonal expansion of memory B cells with ChAdOx1-BNT162b2. In summary, heterologous ChAdOx1-BNT162b2 provides an innate and adaptive immune response that exceeds homologous BNT162b2 vaccination., Competing Interests: The authors declare no competing interests.

Antibody response following Omicron infection is reported to be less robust than that to other variants. Here we investigated how prior vaccination and/or prior infection modulates that response. Disease severity, antibody responses and immune transcriptomes were characterized in four groups of Omicron-infected outpatients (n=83): unvaccinated/no prior infection, vaccinated/no prior infection, unvaccinated/prior infection and vaccinated/prior infection. The percentage of patients with asymptomatic or mild disease was highest in the vaccinated/no prior infection group (87%) and lowest in the unvaccinated/no prior infection group (47%). Significant anti-Omicron spike antibody levels and neutralizing activity were detected in the vaccinated group immediately after infection but were not present in the unvaccinated/no prior infection group. Within two weeks, antibody levels against Omicron, increased. Omicron neutralizing activity in the vaccinated group exceeded that of the prior infection group. No increase in neutralizing activity in the unvaccinated/no prior infection group was seen. The unvaccinated/prior infection group showed an intermediate response. We then investigated the early transcriptomic response following Omicron infection in these outpatient populations and compared it to that found in unvaccinated hospitalized patients with Alpha infection. Omicron infected patients showed a gradient of transcriptional response dependent upon whether or not they were previously vaccinated or infected. Vaccinated patients showed a significantly blunted interferon response as compared to both unvaccinated Omicron infected outpatients and unvaccinated Alpha infected hospitalized patients typified by the response of specific gene classes such as OAS and IFIT that control anti-viral responses and IFI27, a predictor of disease outcome., Competing Interests: Authors LK, MF, YC, CJ, and PK are employed by TyrolPath Obrist Brunhuber GmbH, Zams, Austria. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lee, Knabl, Walter, Knabl, Dai, Füßl, Caf, Jeller, Knabl, Obermoser, Baurecht, Kaiser, Zabernigg, Wurdinger, Furth and Hennighausen.)

Background & Aims: Hepatic energy metabolism is a dynamic process modulated by multiple stimuli. In nonalcoholic fatty liver disease (NAFLD), human studies typically focus on the static fasting state. We hypothesized that unique postprandial alterations in hepatic lipid metabolism are present in NAFLD., Methods: In a prospective clinical study, 37 patients with NAFLD and 10 healthy control subjects ingested a standardized liquid meal with pre- and postprandial blood sampling. Postprandial plasma lipid kinetics were characterized at the molecular lipid species level by untargeted lipidomics, cluster analysis, and lipid particle isolation, then confirmed in a mouse model., Results: There was a specific increase of multiple plasma diacylglycerol (DAG) species at 4 hours postprandially in patients with NAFLD but not in controls. This was replicated in a nonalcoholic steatohepatitis mouse model, where postprandial DAGs increased in plasma and concomitantly decreased in the liver. The increase in plasma DAGs appears early in the disease course, is dissociated from NAFLD severity and obesity, and correlates with postprandial insulin levels. Immunocapture isolation of very low density lipoprotein in human samples and stable isotope tracer studies in mice revealed that elevated postprandial plasma DAGs reflect hepatic secretion of endogenous, rather than meal-derived lipids., Conclusions: We identified a selective insulin-related increase in hepatic secretion of endogenously derived DAGs after a mixed meal as a unique feature of NAFLD. DAGs are known to be lipotoxic and associated with atherosclerosis. Although it is still unknown whether the increased exposure to hepatic DAGs contributes to extrahepatic manifestations and cardiovascular risk in NAFLD, our study highlights the importance of extending NAFLD research beyond the fasting state., (Published by Elsevier Inc.)

Knowledge about the impact of prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the elderly on mRNA vaccination response is needed to appropriately address the demand for additional vaccinations in this vulnerable population. Here, we show that octogenarians, a high-risk population, mount a sustained SARS-CoV-2 spike-specific immunoglobulin G (IgG) antibody response for 15 months following infection. This response boosts antibody levels 35-fold upon receiving a single dose of BNT162b2 mRNA vaccine 15 months after recovery from coronavirus disease 2019 (COVID-19). In contrast, antibody responses in naive individuals boost only 6-fold after a second vaccine. Spike-specific angiotensin-converting enzyme 2 (ACE2) antibody binding responses in the previously infected octogenarians following two vaccine doses exceed those found in a naive cohort after two doses. RNA sequencing (RNA-seq) demonstrates activation of interferon-induced genetic programs, which persist only in the previously infected. A preferential increase of specific immunoglobulin G heavy chain variable (IGHV) clonal transcripts that are the basis of neutralizing antibodies is observed only in the previously infected nuns., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Context: Leptin replacement with metreleptin improves glycemia and hypertriglyceridemia in severely hypoleptinemic patients with generalized lipodystrophy (GLD), but its effects are variable in partially leptin-deficient patients with partial lipodystrophy (PLD)., Objective: Compare 3 leptin assays (Study I); identify diagnostic performance of leptin assays to detect responders to metreleptin for each assay (Study II)., Design: Study I: cross-sectional analysis of average bias between leptin assays. Study II: retrospective analysis of diagnostic accuracy of potential leptin cut points to detect clinical responders to metreleptin., Setting: National Institutes of Health; University of Michigan., Participants and Interventions: Study I: Metreleptin-naïve patients with lipodystrophy (GLD, n = 33, PLD, n = 67) and healthy volunteers (n = 239). Study II: GLD (n = 66) and PLD (n = 84) patients treated with metreleptin for 12 months., Outcome Measures: Leptin concentrations by Millipore radioimmunoassay (RIA), Millipore enzyme-linked immunosorbent assay (MELISA), and R&D Systems enzyme-linked immunosorbent assay (RDELISA). Response to metreleptin therapy was defined as either reduction ≥1.0% in A1c or ≥30% in serum triglycerides., Results: RDELISA measured 3.0 ± 9.5 ng/mL higher than RIA; MELISA measured 11.0 ± 17.8 and 14.0 ±19.2 less than RIA and RDELISA, respectively. Leptin by RIA, MELISA, and RDELISA modestly predicted metreleptin response in GLD + PLD [receiver operating characteristic (ROC) area under the curve (AUC) 0.74, 0.69, and 0.71, respectively; P < 0.01 for all] with lower predictive power in PLD (ROC AUC 0.63, 0.61 and 0.65, respectively; P > 0.05 for all). The only reproducible cut point identified on sensitivity analyses was RIA leptin 7.2 ng/mL (sensitivity 56%; specificity 78%)., Conclusions: Three common leptin assays are not interchangeable, and a reliable cut point to select responders to metreleptin was not identified., (Published by Oxford University Press on behalf of the Endocrine Society 2021.)

Background: Along with the rising incidence of obesity, there has been an increase in patients diagnosed with early-onset colorectal cancer (<50 years old). In colorectal cancer, worse patient survival is associated with certain cytokine expression and downregulation of peroxisome proliferator activated receptor gamma expression. The effects of the obesity hormone leptin and macrophage-specific metabolite itaconate on these mechanisms are poorly understood. We investigated their impact on peroxisome proliferator activated receptor gamma and macrophage cytokine expression in vitro., Methods: M2-like macrophages were treated with either leptin, 4-octyl itaconate, or dimethyl itaconate in a dose- and time-dependent manner. Gene expression after treatment with 4 doses (D1-4) of each compound was analyzed at 4 time points (3, 6, 18, and 24 hours)., Results: Peroxisome proliferator activated receptor gamma was downregulated after 4-octyl itaconate treatment at 18 hours (FC -32.67, P ≤ .001). Interleukin-8 was upregulated after leptin and dimethyl itaconate treatment at 6 hours (FC 26.35 at D4, P ≤ .001, and FC 23.26 at D3, P = .006). Dimethyl itaconate upregulated IL-1β at 24 hours (FC 18.00 at D4, P ≤ .001). Tumor necrosis factor-α showed maximum downregulation after 4-octyl itaconate at 18 hours (FC -103.25 at D4, P ≤ .001)., Conclusions: Itaconate downregulates peroxisome proliferator activated receptor gamma as a tumor-suppressing factor and upregulates anti-inflammatory cytokines in M2-like macrophages. Itaconate provides a link between obesity and colorectal cancer and may be a key regulator in early-onset colorectal cancer., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Context: Duodenopancreatic neuroendocrine tumors (dpNETs) frequently occur in patients with multiple endocrine neoplasia type 1 (MEN1), and metastatic dpNET is the primary cause of disease-related mortality. There is a need for biomarkers that can identify patients with MEN1-related dpNETs that are at high risk of developing distant metastasis. Polyamines have tumor-promoting roles in several cancer types., Objective: We hypothesized that MEN1-dpNET-related disease progression is associated with elevated levels of circulating polyamines., Methods: Through an international collaboration between The University of Texas MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht, plasma polyamine levels were assessed using mass spectrometry in 84 patients with MEN1 (20 with distant metastatic dpNETs [patients] and 64 with either indolent dpNETs or no dpNETs [controls]). A mouse model of MEN1-pNET, Men1fl/flPdx1-CreTg, was used to test time-dependent changes in plasma polyamines associated with disease progression., Results: A 3-marker plasma polyamine signature (3MP: N-acetylputrescine, acetylspermidine, and diacetylspermidine) distinguished patients with metastatic dpNETs from controls in an initial set of plasmas from the 3 participating centers. The fixed 3MP yielded an area under the curve of 0.84 (95% CI, 0.62-1.00) with 66.7% sensitivity at 95% specificity for distinguishing patients from controls in an independent test set from MDACC. In Men1fl/flPdx1-CreTg mice, the 3MP was elevated early and remained high during disease progression., Conclusion: Our findings provide a basis for prospective testing of blood-based polyamines as a potential means for monitoring patients with MEN1 for harboring or developing aggressive disease., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nucleoside analogue (NA) therapy for chronic hepatitis B (CHB) is associated with improved clinical outcomes, but usually requires long-term use. Whether treatment can be safely withdrawn and the factors associated with post-withdrawal outcome are not well defined. To assess long-term outcomes after stopping antiviral therapy, patients with hepatitis B e antigen (HBeAg)-negative CHB who had received antiviral therapy for 4 or more years with hepatitis B virus (HBV) DNA (≤100 IU/mL) were prospectively withdrawn from antiviral therapy and monitored monthly for the initial 6 months and every 3 months thereafter. Those with clinical relapse were retreated according to severity of relapse. Fifteen patients were withdrawn from lamivudine (4), adefovir (5), or a combination of the two (6) after a mean treatment duration of 8.4 years. The mean age was 45 years, 13 were male, and 8 were initially HBeAg-positive before treatment. After a mean follow-up of 6.6 years, outcomes differed by pretreatment HBeAg status. All patients who were HBeAg+ before treatment experienced virological relapse (8 of 8); 6 of 8 experienced clinical relapse; 4 of 8 had ALT flares; 5 of 8 required re-initiation of treatment, one of whom cleared hepatitis B surface antigen (HBsAg); and 3 of 8 remained off treatment, one of whom cleared HBsAg. In contrast, 4 of 7 patients who were HBeAg-negative before treatment experienced virological relapse, 3 of 7 experienced clinical relapse, and 1 of 7 had an alanine aminotransferase (ALT) flare. None restarted treatment, and 4 of 7 cleared HBsAg. Low pre-withdrawal HBsAg level was predictive of HBsAg loss. Conclusion: NA therapy can be safely withdrawn with long-term remission and high rates of HBsAg loss in most HBeAg-negative patients without cirrhosis. Patients who were initially HBeAg+ should not be withdrawn from treatment, because clinical relapse was frequent and often severe., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Background: The hepatokine fibroblast growth factor 21 (FGF21) influences eating behavior and sugar consumption in rodent models. However, whether circulating FGF21 concentration is associated with food and soda intake in humans is still unclear., Objective: We investigated whether fasting plasma FGF21 concentration is associated with objective measures of ad libitum food intake and soda consumption., Methods: Healthy individuals [n = 109; 69 men, aged 34 ± 10 y; BMI (kg/m2): 30.4 ± 7.7; body fat by DXA: 30.5% ± 8.9%] with available plasma for hormonal measurements participated in an inpatient cohort study to objectively quantify ad libitum food and soda intake for 3 d using an automated and reproducible vending machine paradigm. Fasting plasma FGF21 concentration was measured by ELISA prior to ad libitum feeding., Results: Fasting FGF21 concentration was inversely associated with daily soda intake (R = -0.22, P = 0.02 adjusted for demographics and anthropometrics), such that an interindividual difference of 200 pg/mL was associated with an average lower soda consumption by 68 kcal/d. Conversely, no associations were observed with total daily energy intake or macronutrient intake (all P > 0.17)., Conclusions: Higher plasma fasting FGF21 concentration is associated with lower ad libitum soda intake. Although this inverse correlation does not imply causation, the present results support the putative role of FGF21 in the reward pathways regulating sugar consumption in humans. This trial was registered at www.clinicaltrials.gov as NCT00342732., (Published by Oxford University Press on behalf of the American Society for Nutrition 2021.)

Background: Dopamine, a key neurotransmitter in the autonomic nervous system participating in the homeostatic balance between sympathetic and parasympathetic divisions, is involved in food intake regulation. Objective: We investigated whether dopamine is altered by acute fasting or overfeeding diets with varying macronutrient content. Design: Ninety-nine healthy subjects underwent 24-h dietary interventions including eucaloric feeding, fasting, and five different overfeeding diets in a crossover design. Overfeeding diets (200% of eucaloric requirements) included one diet with 3%-protein (low-protein high-fat overfeeding-LPF: 46%-fat), three diets with 20%-protein, and a diet with 30%-protein (44%-fat). Urine was collected for 24 h and urinary dopamine concentration was quantified by high-performance liquid chromatography. Plasma pancreatic polypeptide (PP) concentration, an indirect marker of parasympathetic activity, was measured prior to and after each diet after an overnight fast. Results: During 24-h of fasting, dopamine decreased on average by ~14% compared to eucaloric conditions, whereas PP increased by two-fold (both p < 0.001). Lower dopamine during 24-h fasting correlated with increased PP (r = -0.40, p < 0.001). Similarly, on average urinary dopamine decreased during LPF by 14% ( p < 0.001) and lower dopamine correlated with increased PP (r = -0.31, p = 0.01). No changes in dopamine and PP concentrations were observed during other overfeeding diets (all p > 0.05). Conclusions: Dopamine concentrations decrease during short-term fasting and overfeeding with a low-protein diet. As both dietary conditions have in common protein deficit, the correlation between dopamine and PP suggests a compensatory mechanism underlying the shift from sympathetic to parasympathetic drive during dietary protein deprivation.

Objective: Erythropoietin (EPO), the cytokine required for erythropoiesis, contributes to metabolic regulation of fat mass and glycemic control. EPO treatment in mice on high-fat diets (HFD) improved glucose tolerance and decreased body weight gain via reduced fat mass in males and ovariectomized females. The decreased fat accumulation with EPO treatment during HFD in ovariectomized females was abrogated with estradiol supplementation, providing evidence for estrogen-related gender-specific EPO action in metabolic regulation. In this study, we examined the cross-talk between estrogen mediated through estrogen receptor α (ERα) and EPO for the regulation of glucose metabolism and fat mass accumulation., Methods: Wild-type (WT) mice and mouse models with ERα knockout (ERα-/-) and targeted deletion of ERα in adipose tissue (ERα adipoKO ) were used to examine EPO treatment during high-fat diet feeding and after diet-induced obesity., Results: ERα-/- mice on HFD exhibited increased fat mass and glucose intolerance. EPO treatment on HFD reduced fat accumulation in male WT and ERα-/- mice and female ERα-/- mice but not female WT mice. EPO reduced HFD increase in adipocyte size in WT mice but not in mice with deletion of ERα independent of EPO-stimulated reduction in fat mass. EPO treatment also improved glucose and insulin tolerance significantly greater in female ERα-/- mice and female ERα adipoKO compared with WT controls. Increased metabolic activity by EPO was associated with browning of white adipocytes as shown by reductions in white fat-associated genes and induction of brown fat-specific uncoupling protein 1 (UCP1)., Conclusions: This study clearly identified the role of estrogen signaling in modifying EPO regulation of glucose metabolism and the sex-differential EPO effect on fat mass regulation. Cross-talk between EPO and estrogen was implicated for metabolic homeostasis and regulation of body mass in female mice., (Published by Elsevier GmbH.)

The carbohydrate-insulin model of obesity posits that high-carbohydrate diets lead to excess insulin secretion, thereby promoting fat accumulation and increasing energy intake. Thus, low-carbohydrate diets are predicted to reduce ad libitum energy intake as compared to low-fat, high-carbohydrate diets. To test this hypothesis, 20 adults aged 29.9 ± 1.4 (mean ± s.e.m.) years with body mass index of 27.8 ± 1.3 kg m -2 were admitted as inpatients to the National Institutes of Health Clinical Center and randomized to consume ad libitum either a minimally processed, plant-based, low-fat diet (10.3% fat, 75.2% carbohydrate) with high glycemic load (85 g 1,000 kcal -1 ) or a minimally processed, animal-based, ketogenic, low-carbohydrate diet (75.8% fat, 10.0% carbohydrate) with low glycemic load (6 g 1,000 kcal -1 ) for 2 weeks followed immediately by the alternate diet for 2 weeks. One participant withdrew due to hypoglycemia during the low-carbohydrate diet. The primary outcomes compared mean daily ad libitum energy intake between each 2-week diet period as well as between the final week of each diet. We found that the low-fat diet led to 689 ± 73 kcal d -1 less energy intake than the low-carbohydrate diet over 2 weeks (P < 0.0001) and 544 ± 68 kcal d -1 less over the final week (P < 0.0001). Therefore, the predictions of the carbohydrate-insulin model were inconsistent with our observations. This study was registered on ClinicalTrials.gov as NCT03878108 .

Context: Lipodystrophy syndromes cause hypertriglyceridemia that improves with leptin treatment using metreleptin. Mechanisms causing hypertriglyceridemia and improvements after metreleptin are incompletely understood., Objective: Determine relationship of circulating lipoprotein lipase (LPL) modulators with hypertriglyceridemia in healthy controls and in patients with lipodystrophy before and after metreleptin., Methods: Cross-sectional comparison of patients with lipodystrophy (generalized lipodystrophy n = 3; partial lipodystrophy n = 11) vs age/sex-matched healthy controls (n = 28), and longitudinal analyses in patients before and after 2 weeks and 6 months of metreleptin. The study was carried out at the National Institutes of Health, Bethesda, Maryland. Outcomes were LPL stimulators apolipoprotein (apo) C-II and apoA-V and inhibitors apoC-III and angiopoietin-like proteins (ANGPTLs) 3, 4, and 8; ex vivo activation of LPL by plasma., Results: Patients with lipodystrophy were hypertriglyceridemic and had higher levels of all LPL stimulators and inhibitors vs controls except for ANGPTL4, with >300-fold higher ANGPTL8, 4-fold higher apoC-III, 3.5-fold higher apoC-II, 1.9-fold higher apoA-V, 1.6-fold higher ANGPTL3 ( P < .05 for all). At baseline, all LPL modulators except ANGPLT4 positively correlated with triglycerides. Metreleptin decreased apoC-II and apoC-III after 2 weeks and 6 months, and decreased ANGPTL8 after 6 months (P < 0.05 for all). Plasma from patients with lipodystrophy caused higher ex vivo LPL activation vs hypertriglyceridemic control plasma ( P < .0001), which did not change after metreleptin., Conclusion: Elevations in LPL inhibitors apoC-III and ANGPTL8 may contribute to hypertriglyceridemia in lipodystrophy, and may mediate reductions in circulating and hepatic triglycerides after metreleptin. These therefore are strong candidates for therapies to lower triglycerides in these patients., (Published by Oxford University Press on behalf of the Endocrine Society 2020.)

Aims/hypothesis: Metformin is the only approved oral agent for youth with type 2 diabetes but its mechanism of action remains controversial. Recent data in adults suggest a primary role for the enteroinsular pathway, but there are no data in youth, in whom metformin efficacy is only ~50%. Our objectives were to compare incretin concentrations and rates of glucose production and gluconeogenesis in youth with type 2 diabetes before and after short-term metformin therapy compared with peers with normal glucose tolerance (NGT)., Methods: This is a case-control observational study in youth with type 2 diabetes who were not on metformin (n = 18) compared with youth with NGT (n = 10) who were evaluated with a 2 day protocol. A 75 g OGTT was administered to measure intact glucagon-like 1 peptide (iGLP-1), gastric inhibitory polypeptide (GIP) and peptide YY (PYY). Insulinogenic index (IGI) and whole-body insulin sensitivity were calculated using glucose and insulin levels from the OGTT. Basal rates of gluconeogenesis ( 2 H 2 O), glucose production ([6,6- 2 H 2 ]glucose) and whole-body lipolysis ([ 2 H 5 ]glycerol) were measured after an overnight fast on study day 2. Youth with type 2 diabetes (n = 9) were subsequently evaluated with an identical 2 day protocol after 3 months on the metformin study., Results: Compared with individuals with NGT, those with type 2 diabetes had higher fasting (7.8 ± 2.5 vs 5.1 ± 0.3 mmol/l, mean ± SD p = 0.002) and 2 h glucose concentrations (13.8 ± 4.5 vs 5.9 ± 0.9 mmol/l, p = 0.001), higher rates of absolute gluconeogenesis (10.0 ± 1.7 vs 7.2 ± 1.1 μmol [kg fat-free mass (FFM)] -1  min -1 , p < 0.001) and whole-body lipolysis (5.2 ± 0.9 vs 4.0 ± 1.4 μmol kg FFM -1  min -1 , p < 0.01), but lower fasting iGLP-1 concentrations (0.5 ± 0.5 vs 1.3 ± 0.7 pmol/l, p < 0.01). Metformin decreased 2 h glucose (pre metformin 11.4 ± 2.8 vs post metformin 9.9 ± 1.9 mmol/l, p = 0.04) and was associated with ~20-50% increase in IGI (median [25th-75th percentile] pre 1.39 [0.89-1.47] vs post 1.43 [0.88-2.70], p = 0.04), fasting iGLP-1 (pre 0.3 ± 0.2 vs post 1.0 ± 0.7 pmol/l, p = 0.02), 2 h iGLP (pre 0.4 ± 0.2 vs post 1.2 ± 0.9 pmol/l, p = 0.06), fasting PYY (pre 6.3 ± 2.2 vs post 10.5 ± 4.3 pmol/l, p < 0.01) and 2 h PYY (pre 6.6 ± 2.9 vs post 9.0 ± 4.0 pmol/l, p < 0.01). There was no change in BMI, insulin sensitivity or GIP concentrations pre vs post metformin. There were no differences pre vs post metformin in rates of glucose production (15.0 ± 3.9 vs 14.9 ± 2.2 μmol kg FFM -1  min -1 , p = 0.84), absolute gluconeogenesis (9.9 ± 1.8 vs 9.7 ± 1.7 μmol kg FFM -1  min -1 , p = 0.76) or whole-body lipolysis (5.0 ± 0.7 vs 5.3 ± 1.3 μmol kg FFM -1  min -1 , p = 0.20). Post metformin iGLP-1 and PYY concentrations in youth with type 2 diabetes were comparable to levels in youth with NGT., Conclusions/interpretation: Overall, the improved postprandial blood glucose levels and increase in incretins observed in the absence of changes in insulin sensitivity and gluconeogenesis, support an enteroinsular mechanistic pathway in youth with type 2 diabetes treated with short-term metformin.

Background: Adaptive thermogenesis during prolonged energy deficit refers to the greater than expected reduction in energy expenditure (EE) independent of concomitant loss of metabolically active body mass., Objective: As inter-individual variability in the magnitude of adaptive thermogenesis may influence the extent of energy deficit thereby predicting the amount of weight reduction, we investigated whether early adaptive thermogenesis is a determinant of weight loss after 6 weeks of daily 50% caloric restriction in an inpatient setting., Design and Methods: The current study reports the results of an exploratory, secondary analysis in overweight but otherwise healthy subjects (n = 11, 7 men, 35 ± 9y, BMI = 40 ± 7 kg/m 2 , body fat = 63.3 ± 5.3%). Body composition and 24-h EE (24hEE) measurement in a whole-room indirect calorimeter were used to calculate the magnitude of adaptive thermogenesis while on caloric restriction after 1, 3 and 6 weeks. Energy deficit during caloric restriction was quantified via food, stool, and urine bomb calorimetry. Fasting hormonal concentrations (FT4, FT3, FGF21, leptin) were obtained at baseline and at weeks 3 and 6 during caloric restriction., Results: The magnitude of adaptive thermogenesis in 24hEE after 1 week of caloric restriction was -178 ± 137 kcal/day (mean ± SD), was overall stable during and following caloric restriction, and demonstrated remarkable intra-individual consistency. A relatively greater decrease in 24hEE of 100 kcal/d after 1 week of caloric restriction was associated on average with reduced energy deficit by 8195 kcal over 6 weeks and predicted 2.0 kg less weight loss, of which 0.5 kg was fat mass, after 6 weeks. No correlations were found between hormonal concentrations and weight loss., Conclusions: The extent of weight loss is influenced by the magnitude of adaptive thermogenesis in the early stage of caloric restriction. Although these results need replication in larger study groups with adequate statistical power, targeting adaptive thermogenesis may help to optimize long-term interventions in obesity therapy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Published by Elsevier Inc.)

The detection of rare mutational targets in plasma (liquid biopsy) has emerged as a promising tool for the assessment of patients with cancer. We determined the presence of cell-free DNA containing the BRAFV600E mutations (cf BRAFV600E ) in plasma samples from 57 patients with papillary thyroid cancer (PTC) with somatic BRAFV600E mutation-positive primary tumors using microfluidic digital PCR, and co-amplification at lower denaturation temperature (COLD) PCR. Mutant cf BRAFV600E alleles were detected in 24/57 (42.1%) of the examined patients. The presence of cf BRAFV600E was significantly associated with tumor size ( p = 0.03), multifocal patterns of growth ( p = 0.03), the presence of extrathyroidal gross extension ( p = 0.02) and the presence of pulmonary micrometastases ( p = 0.04). In patients with low-, intermediate- and high-risk PTCs, cf BRAFV600E was detected in 4/19 (21.0%), 8/22 (36.3%) and 12/16 (75.0%) of cases, respectively. Patients with detectable cfBRAFV600E were characterized by a 4.68 times higher likelihood of non-excellent response to therapy, as compared to patients without detectable cf BRAF V600E (OR (odds ratios), 4.68; 95% CI (confidence intervals)) 1.26-17.32; p = 0.02). In summary, the combination of digital polymerase chain reaction (dPCR) with COLD-PCR enables the detection of BRAFV600E in the liquid biopsy from patients with PTCs and could prove useful for the identification of patients with PTC at an increased risk for a structurally or biochemically incomplete or indeterminate response to treatment., Competing Interests: The authors declare no conflict of interest.

De novo lipogenesis (DNL) plays a role in the development of hepatic steatosis. In humans with lipodystrophy, reduced adipose tissue causes lower plasma leptin, insulin resistance, dyslipidemia, and ectopic triglyceride (TG) accumulation. We hypothesized that recombinant leptin (metreleptin) for 6 months in 11 patients with lipodystrophy would reduce DNL by decreasing insulin resistance and glycemia, thus reducing circulating TG and hepatic TG. The percentage of TG in TG-rich lipoprotein particle (TRLP-TG) derived from DNL (%DNL) was measured by deuterium incorporation from body water into palmitate. At baseline, DNL was elevated, similar to levels previously shown in obesity-associated nonalcoholic fatty liver disease (NAFLD). After metreleptin, DNL decreased into the normal range. Similarly, absolute DNL (TRLP-TG × %DNL) decreased by 88% to near-normal levels. Metreleptin improved peripheral insulin sensitivity (hyperinsulinemic-euglycemic clamp) and lowered hemoglobin A1c and hepatic TG. Both before and after metreleptin, DNL positively correlated with insulin resistance, insulin doses, and hepatic TG, supporting the hypothesis that hyperinsulinemia stimulates DNL and that elevated DNL is integral to the pathogenesis of lipodystrophy-associated NAFLD. These data suggest that leptin-mediated improvement in insulin sensitivity increases clearance of blood glucose by peripheral tissues, reduces hepatic carbohydrate flux, and lowers insulinemia, resulting in DNL reductions and improvements in hepatic steatosis and dyslipidemia.

BACKGROUNDPostreceptor insulin resistance (IR) is associated with hyperglycemia and hepatic steatosis. However, receptor-level IR (e.g., insulin receptor pathogenic variants, INSR) causes hyperglycemia without steatosis. We examined 4 pathologic conditions of IR in humans to examine pathways controlling lipid metabolism and gluconeogenesis.METHODSCross-sectional study of severe receptor IR (INSR, n = 7) versus postreceptor IR that was severe (lipodystrophy, n = 14), moderate (type 2 diabetes, n = 9), or mild (obesity, n = 8). Lipolysis (glycerol turnover), hepatic glucose production (HGP), gluconeogenesis (deuterium incorporation from body water into glucose), hepatic triglyceride (magnetic resonance spectroscopy), and hepatic fat oxidation (plasma β-hydroxybutyrate) were measured.RESULTSLipolysis was 2- to 3-fold higher in INSR versus all other groups, and HGP was 2-fold higher in INSR and lipodystrophy versus type 2 diabetes and obesity (P < 0.001), suggesting severe adipose and hepatic IR. INSR subjects had a higher contribution of gluconeogenesis to HGP, approximately 77%, versus 52% to 59% in other groups (P = 0.0001). Despite high lipolysis, INSR subjects had low hepatic triglycerides (0.5% [interquartile range 0.1%-0.5%]), in contrast to lipodystrophy (10.6% [interquartile range 2.8%-17.1%], P < 0.0001). β-hydroxybutyrate was 2- to 7-fold higher in INSR versus all other groups (P < 0.0001), consistent with higher hepatic fat oxidation.CONCLUSIONThese data support a key pathogenic role of adipose tissue IR to increase glycerol and FFA availability to the liver in both receptor and postreceptor IR. However, the fate of FFA diverges in these populations. In receptor-level IR, FFA oxidation drives gluconeogenesis rather than being reesterified to triglyceride. In contrast, in postreceptor IR, FFA contributes to both gluconeogenesis and hepatic steatosis.TRIAL REGISTRATIONClinicalTrials.gov NCT01778556, NCT00001987, and NCT02457897.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases, US Department of Agriculture/Agricultural Research Service 58-3092-5-001.

A greater decrease in 24-h energy expenditure (24 EE) during 24-h fasting defines a "thriftier" metabolic phenotype prone to weight gain during overfeeding and resistant to weight loss during caloric restriction. As the thermogenic response to mild cold exposure (COLD) may similarly characterize this human phenotype identified by acute fasting conditions, we analyzed changes in 24 EE and sleeping metabolic rate (SLEEP) in a whole-room indirect calorimeter during 24-h fasting at thermoneutrality (24°C) and during energy balance both at thermoneutrality (24°C) and mild cold (19°C) in 20 healthy volunteers (80% male; aged 36.6 ± 11.4 years; percentage body fat 34.8 ± 10.5%). Greater decrease in 24 EE during fasting (thriftier phenotype) was associated with less increase in 24 EE during COLD (i.e., less cold-induced thermogenesis). Greater decreases in plasma fibroblast growth factor 21 (FGF21) after 24-h fasting and after COLD were highly correlated and associated with greater decreases in SLEEP in both conditions. We conclude that the metabolic responses to short-term fasting and COLD are associated with and mediated by the liver-derived hormone FGF21. Thus, the 24 EE response to COLD further identifies the "thrifty" versus "spendthrift" phenotype, providing an additional setting to investigate the physiological mechanisms underlying the human metabolic phenotype and characterizing the individual susceptibility to weight change., (© 2020 by the American Diabetes Association.)

Objective: The relationship between adipocyte size and ad libitum energy intake has not been previously examined. This study hypothesized an inverse relationship between adipocyte size and daily energy intake (DEI)., Methods: Seventy healthy adults (39 men and 31 women; BMI 30.0  [SD 6.3]) underwent dual-energy x-ray absorptiometry and subcutaneous fat biopsies from the abdomen and thigh. Osmium-fixed adipocytes were sized with a Coulter counter. Volunteers self-selected food from a vending machine paradigm as the only source of energy intake over 3 days as inpatients. Volunteers also had 24-hour respiratory quotient (RQ) measured in a whole-room indirect calorimeter., Results: In women, the large cell peak diameter of the thigh depot was greater than that of the abdominal depot (Δ = +15.8 μm; P < 0.0001). In women, thigh peak diameter was inversely associated with DEI (β = -264.7 kcal/d per 10-μm difference; P = 0.03) after adjusting for demographics and body composition. The thigh peak diameter in women was associated with 24-hour RQ (r = -0.47, P = 0.04) after adjusting for demographics, body composition, and 24-hour energy balance. These associations did not extend to men or the abdominal depot., Conclusions: In women, thigh adipocyte size was associated with reduced DEI and 24-hour RQ, indicating a special role for thigh fat in women. This depot-specific sexual dimorphism indicates common regulation of energy intake and adipocyte size in the thigh region of women., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Purpose: The human thrifty phenotype hypothesis presupposes that lower 24-hour (24h) energy expenditure (24EE) during famine preserves body mass and promotes survival. The prevailing view defines thrifty individuals as having a lower 24EE during fasting. However, it is also plausible that the greater decline in 24EE during fasting in thrifty individuals is due to higher 24EE during energy balance conditions (ENBAL). Herein, we provide evidence that this is indeed the case., Methods: In 108 healthy subjects, 24EE was measured in a whole-room indirect calorimeter both during ENBAL and 24h fasting conditions. Subjects were categorized as thrifty or spendthrift based on the median value (-162 kcal/day) of the difference in 24EE (adjusted for body composition) between fasting and ENBAL conditions. Concomitant 24h urinary catecholamines were assessed by liquid chromatography-mass spectrometry., Results: Compared to ENBAL, 24EE decreased during 24h fasting by 172 kcal/day (standard deviation = 93; range, -470 to 122). A greater-than-median decrease in 24EE ("thriftier" phenotype) was due to higher 24EE during ENBAL (+124 kcal/day; P < 0.0001) but not to lower 24EE during fasting (P = 0.35). Greater fasting-induced increase in epinephrine was associated with concomitant lower decrease in 24EE (r = 0.27; P = 0.006)., Main Conclusion: The greater decrease in 24EE during acute fasting (which characterizes the thrifty phenotype) is not due to reduced metabolic rate during fasting but to a relatively higher 24EE during feeding conditions, and this decrease in 24EE during fasting is accompanied by a smaller increase in epinephrine. These results recharacterize the prevailing view of the short-term 24EE responses that define the human metabolic phenotypes. Clinical Trials: NCT00523627, NCT00687115, NCT02939404., (Published by Oxford University Press on behalf of the Endocrine Society 2020.)

BACKGROUNDMirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatment of overactive bladder. Encouraging preclinical results suggest that β3-AR agonists could also improve obesity-related metabolic disease by increasing brown adipose tissue (BAT) thermogenesis, white adipose tissue (WAT) lipolysis, and insulin sensitivity.METHODSWe treated 14 healthy women of diverse ethnicities (27.5 ± 1.1 years of age, BMI of 25.4 ± 1.2 kg/m2) with 100 mg mirabegron (Myrbetriq extended-release tablet, Astellas Pharma) for 4 weeks in an open-label study. The primary endpoint was the change in BAT metabolic activity as measured by [18F]-2-fluoro-d-2-deoxy-d-glucose (18F-FDG) PET/CT. Secondary endpoints included resting energy expenditure (REE), plasma metabolites, and glucose and insulin metabolism as assessed by a frequently sampled intravenous glucose tolerance test.RESULTSChronic mirabegron therapy increased BAT metabolic activity. Whole-body REE was higher, without changes in body weight or composition. Additionally, there were elevations in plasma levels of the beneficial lipoprotein biomarkers HDL and ApoA1, as well as total bile acids. Adiponectin, a WAT-derived hormone that has antidiabetic and antiinflammatory capabilities, increased with acute treatment and was 35% higher upon completion of the study. Finally, an intravenous glucose tolerance test revealed higher insulin sensitivity, glucose effectiveness, and insulin secretion.CONCLUSIONThese findings indicate that human BAT metabolic activity can be increased after chronic pharmacological stimulation with mirabegron and support the investigation of β3-AR agonists as a treatment for metabolic disease.TRIAL REGISTRATIONClinicaltrials.gov NCT03049462.FUNDINGThis work was supported by grants from the Intramural Research Program of the NIDDK, NIH (DK075112, DK075116, DK071013, and DK071014).

Direct evidence in humans for the impact of the microbiome on nutrient absorption is lacking. We conducted an extended inpatient study using two interventions that we hypothesized would alter the gut microbiome and nutrient absorption. In each, stool calorie loss, a direct proxy of nutrient absorption, was measured. The first phase was a randomized cross-over dietary intervention in which all participants underwent in random order 3 d of over- and underfeeding. The second was a randomized, double-blind, placebo-controlled pharmacologic intervention using oral vancomycin or matching placebo (NCT02037295). Twenty-seven volunteers (17 men and 10 women, age 35.1 ± 7.3, BMI 32.3 ± 8.0), who were healthy other than having impaired glucose tolerance and obesity, were enrolled and 25 completed the entire trial. The primary endpoints were the effects of dietary and pharmacological intervention on stool calorie loss. We hypothesized that stool calories expressed as percentage of caloric intake would increase with underfeeding compared with overfeeding and increase during oral vancomycin treatment. Both primary endpoints were met. Greater stool calorie loss was observed during underfeeding relative to overfeeding and during vancomycin treatment compared with placebo. Key secondary endpoints were to evaluate the changes in gut microbial community structure as evidenced by amplicon sequencing and metagenomics. We observed only a modest perturbation of gut microbial community structure with under- versus overfeeding but a more widespread change in community structure with reduced diversity with oral vancomycin. Increase in Akkermansia muciniphila was common to both interventions that resulted in greater stool calorie loss. These results indicate that nutrient absorption is sensitive to environmental perturbations and support the translational relevance of preclinical models demonstrating a possible causal role for the gut microbiome in dietary energy harvest.

Background: African Americans (AAs) are at a higher risk for developing type 2 diabetes compared with non-Hispanic whites (NHWs). The causal role of β-cell glucose sensitivity (β-GS) and insulin clearance in hyperinsulinemia in AA adults is unclear., Objective: Using a cross-sectional study design, we compared β-cell function and insulin clearance in nondiabetic AAs (n = 36) and NHWs (n = 47) after a mixed meal test (MMT)., Methods: Insulin secretion rate, glucose sensitivity, rate sensitivity, and insulin sensitivity during MMT were derived from a mathematical model. Levels of insulin-degrading enzyme (IDE) and carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1), key players in insulin clearance, were measured (by enzyme-linked immunosorbent assay) in hepatic cytosolic fractions from age-, sex-, and body mass index-matched AA and NHW cadaveric donors (n = 10)., Results: Fasting and mean postprandial plasma glucose levels were similar in both ethnic groups. AAs had significantly higher fasting and mean postprandial plasma insulin levels. However, fasting ISR, total insulin output, and insulin sensitivity during MMT were not different between the groups. β-GS and rate sensitivity were higher in AAs. Fasting and meal plasma insulin clearance were lower in AAs. Hepatic levels of IDE and CEACAM-1 were similar in AAs and NHWs. Hepatic IDE activity was significantly lower in AAs., Conclusions: In this study, lower insulin clearance contributes to higher plasma insulin levels in AAs. Reduced insulin clearance may be explained by lower IDE activity levels in AAs. Further confirmatory studies are needed to investigate diminished insulin clearance in AAs as a result of lower IDE activity levels., (Published by Oxford University Press on behalf of the Endocrine Society 2020.)

The ability to switch fuels for oxidation in response to changes in macronutrient composition of diet (metabolic flexibility) may be informative of individuals' susceptibility to weight gain. Seventy-nine healthy, weight-stable participants underwent 24-h assessments of energy expenditure and respiratory quotient (RQ) in a whole-room calorimeter during energy balance (EBL) (50% carbohydrate, 30% fat) and then during 24-h fasting and three 200% overfeeding diets in a crossover design. Metabolic flexibility was defined as the change in 24-h RQ from EBL during fasting and standard overfeeding (STOF) (50% carbohydrate, 30% fat), high-fat overfeeding (HFOF) (60% fat, 20% carbohydrate), and high-carbohydrate overfeeding (HCOF) (75% carbohydrate, 5% fat) diets. Free-living weight change was assessed after 6 and 12 months. Compared with EBL, RQ decreased on average by 9% during fasting and by 4% during HFOF but increased by 4% during STOF and by 8% during HCOF. A smaller decrease in RQ, reflecting a smaller increase in lipid oxidation rate, during HFOF but not during the other diets predicted greater weight gain at both 6 and 12 months. An impaired metabolic flexibility to acute HFOF can identify individuals prone to weight gain, indicating that an individual's capacity to oxidize dietary fat is a metabolic determinant of weight change., (© 2019 by the American Diabetes Association.)

Background: Analysis of plasma circulating cell-free DNA integrity (cfDI) has emerged as a promising tool in the diagnosis of malignant vs. benign tumors. There is limited data on the role of cfDI in thyroid cancer. The goal of this study was to analyze cfDI as a biomarker of malignancy in patients with cytologically indeterminate thyroid nodules. Methods: The cfDI was measured in the plasma of patients with cytologically indeterminate thyroid nodules. All patients underwent plasma collection within 24-72 h before surgical treatment for thyroid nodules. Additionally, samples were collected from seven patients via the vein draining the thyroid and peripheral vein during surgery. Quantitative real-time PCR was performed on the isolated cell-free DNA using two different primer sets (115 and 247 bp) to amplify consensus ALU sequences. The cfDI was calculated as the ratio of ALU247 to ALU115. Results: All data are given as median [25th-75th percentile]. The study group consisted of 67 patients with 100 nodules, 80.6% (54/67) women, aged 43 [33-60] years. There was no difference in cfDI between 29 patients with benign nodules (0.49 [0.41-0.59]) and 38 patients with malignant lesions (0.45 [0.36-0.57], p = 0.19). There was no difference in cfDI in the vein draining the thyroid (0.47 [0.24-1.05]) and peripheral vein (0.48 [0.36-0.56], p = 0.44). In comparison to thyroid cancer patients, patients with benign nodules were characterized by significantly higher concentrations of ALU115 (1,064 [529-2,960] vs. 411 [27-1,049] ng/ml; p = 0.002) and ALU247 (548 [276-1,894] vs. 170 [17-540] ng/ml; p = 0.0005), most likely because benign tumors were larger (3, [1.8-4.1 cm]) than malignant lesions (0.7 [0.23-1.45], p < 0.0001). Women had significantly lower cfDI (0.45 [0.27-0.54]) than men (0.56 [0.44-0.8], p = 0.011). Conclusion: The cfDI measured in the vein draining the thyroid is similar to the cfDI measured in the antecubital vein, validating cfDI measurements by peripheral liquid biopsy. Analysis of cfDI needs to be stratified by patients gender. In contrast to its diagnostic utility in aggressive cancers, cfDI has limited utility as a biomarker of malignancy in cytologically indeterminate thyroid nodules., (Copyright © 2019 Thakur, Tobey, Daley, Auh, Walter, Patel, Nilubol, Kebebew, Patel, Jensen, Vasko and Klubo-Gwiezdzinska.)

Background: A greater decrease in 24-hour energy expenditure (EE) during fasting and a smaller increase in 24-hour EE during low-protein overfeeding (metabolic "thrifty" phenotype) predict weight gain. As thyroid hormones (TH) are implicated in energy intake and metabolism, we assessed whether: (i) TH concentrations are altered by 24-hour fasting or overfeeding diets with varying protein content and (ii) diet-related changes in TH correlate with concomitant changes in EE. Methods: Fifty-eight euthyroid healthy subjects with normal glucose regulation underwent 24-hour dietary interventions including fasting, eucaloric feeding, and five overfeeding diets in a crossover design within a whole-room indirect calorimeter to measure the 24-hour EE. Overfeeding diets (200% of energy requirements) included three diets with 20% protein, one diet with 3% protein (low-protein overfeeding diet [LPF]: 46% fat), and one diet with 30% protein (high-protein overfeeding diet [HPF]: 44% fat, n  = 51). Plasma free thyroxine (fT4), free triiodothyronine (fT3), and fibroblast growth factor 21 (FGF21) concentrations were measured after overnight fast the morning of and after each diet. Results: On average, fT4 increased by 8% (+0.10 ng/dL, 95% confidence interval [CI 0.07-0.13], p  < 0.0001) and fT3 decreased by 6% (-0.17 pg/mL [CI -0.27 to -0.07], p  = 0.001) after 24-hour fasting, whereas both fT4 and fT3 decreased by 5% (-0.07 ng/dL [CI -0.11 to -0.04], p  < 0.0001) and 4% (-0.14 pg/mL [CI -0.24 to -0.04], p  = 0.008) following HPF, respectively. Greater decreases in fT3 after HPF are associated with larger decreases in FGF21 ( r  = 0.40, p  = 0.005). Following LPF, the mean fT3 increased by 6% (+0.14 pg/mL [CI 0.05-0.2], p  = 0.003) with no change in fT4 ( p  = 0.7). No changes in TH were observed after normal-protein overfeeding diets (all p  > 0.1). No associations were observed between TH concentrations and diet-related changes in 24-hour EE during any diet (all p  > 0.07). Conclusions: Acute (200%) short-term (24 hours) changes in food intake induce small changes in TH concentrations only after diets with low (0% fasting and 3% protein overfeeding) or high (30% protein overfeeding) protein content. The fT3-FGF21 association after high-protein overfeeding suggests a role for TH in inhibiting FGF21 secretion by the liver during protein excess. These results indicate that TH are involved in protein metabolism; however, they do not mediate the short-term EE response to diets that characterize the metabolic phenotypes and determine the individual susceptibility to weight gain.