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Objective: To assess insulin action on peripheral glucose utilization and nonesterified fatty acid (NEFA) suppression as a predictor of coronary artery calcification (CAC) in patients with type 1 diabetes and nondiabetic controls.Research Design and Methods: Insulin action was measured by a three-stage hyperinsulinemic-euglycemic clamp (4, 8, and 40 mU/m²/min) in 87 subjects from the Coronary Artery Calcification in Type 1 Diabetes cohort (40 diabetic, 47 nondiabetic; mean age 45 ± 8 years; 55% female).Results: Peripheral glucose utilization was lower in subjects with type 1 diabetes compared with nondiabetic controls: glucose infusion rate (mg/kg FFM/min) = 6.19 ± 0.72 vs. 12.71 ± 0.66, mean ± SE, P < 0.0001, after adjustment for age, sex, BMI, fasting glucose, and final clamp glucose and insulin. Insulin-induced NEFA suppression was also lower in type 1 diabetic compared with nondiabetic subjects: NEFA levels (μM) during 8 mU/m²/min insulin infusion = 370 ± 27 vs. 185 ± 25, P < 0.0001, after adjustment for age, sex, BMI, fasting glucose, and time point insulin. Lower glucose utilization and higher NEFA levels, correlated with CAC volume (r = -0.42, P < 0.0001 and r = 0.41, P < 0.0001, respectively) and predicted the presence of CAC (odds ratio [OR] = 0.45, 95% CI = 0.22-0.93, P = 0.03; OR = 2.4, 95% CI = 1.08-5.32, P = 0.032, respectively). Insulin resistance did not correlate with GHb or continuous glucose monitoring parameters.Conclusions: Type 1 diabetic patients are insulin resistant compared with nondiabetic subjects, and the degree of resistance is not related to current glycemic control. Insulin resistance predicts the extent of coronary artery calcification and may contribute to the increased risk of cardiovascular disease in patients with type 1 diabetes as well as subjects without diabetes. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Intracellular ceramide accumulation in specific cellular compartments is a potential mechanism explaining muscle insulin resistance in the pathogenesis of type 2 diabetes. Muscle sarcolemmal ceramide accumulation negatively impacts insulin sensitivity in humans, but the mechanism explaining this localised accumulation is unknown. Previous reports revealed that circulating oxidised LDL is elevated in serum of individuals with obesity and type 2 diabetes. Oxidised phosphatidylcholine, which is present in oxidised LDL, has previously been linked to ceramide pathway activation, and could contribute to localised ceramide accumulation in skeletal muscle. We hypothesised that oxidised phosphatidylcholine inversely correlates with insulin sensitivity in serum, and induces sarcolemmal ceramide accumulation and decreases insulin sensitivity in muscle., Methods: We used LC-MS/MS to quantify specific oxidised phosphatidylcholine species in serum from a cross-sectional study of 58 well-characterised individuals spanning the physiological range of insulin sensitivity. We also performed in vitro experiments in rat L6 myotubes interrogating the role of specific oxidised phosphatidylcholine species in promoting sarcolemmal ceramide accumulation, inflammation and insulin resistance in skeletal muscle cells., Results: Human serum oxidised phosphatidylcholine levels are elevated in individuals with obesity and type 2 diabetes, inversely correlated with insulin sensitivity, and positively correlated with sarcolemmal C18:0 ceramide levels in skeletal muscle. Specific oxidised phosphatidylcholine species, particularly 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), increase total ceramide and dihydroceramide and decrease total sphingomyelin in the sarcolemma of L6 myotubes by de novo ceramide synthesis and sphingomyelinase activation. POVPC also increases inflammatory signalling and causes insulin resistance in L6 myotubes., Conclusions/interpretation: These data suggest that circulating oxidised phosphatidylcholine species promote ceramide accumulation and decrease insulin sensitivity in muscle, help explain localised sphingolipid accumulation and muscle inflammatory response, and highlight oxidised phosphatidylcholine species as potential targets to combat insulin resistance., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Our objective was to interrogate mesenchymal stem cell (MSC) lipid metabolism and gestational exposures beyond maternal body mass that may contribute to child obesity risk. MSCs were cultured from term infants of mothers with obesity (n = 16) or normal weight (n = 15). In MSCs undergoing myogenesis in vitro, we used lipidomics to distinguish phenotypes by unbiased cluster analysis and lipid challenge (24-hour excess fatty acid [24hFA]). We measured MSC AMP-activated protein kinase (AMPK) activity and fatty acid oxidation (FAO), and a composite index of maternal glucose, insulin, triglycerides, free fatty acids, TNF-α, and high-density lipoprotein and total cholesterol in fasting blood from mid and late gestation (~17 and ~27 weeks, respectively). We measured child adiposity at birth (n = 29), 4-6 months (n = 29), and 4-6 years (n = 13). Three MSC clusters were distinguished by triacylglycerol (TAG) stores, with greatest TAGs in Cluster 2. All clusters increased acylcarnitines and TAGs with 24hFA, although Cluster 2 was more pronounced and corresponded to AMPK activation and FAO. Maternal metabolic markers predicted MSC clusters and child adiposity at 4-6 years (both highest in Cluster 3). Our data support the notion that MSC phenotypes are predicted by comprehensive maternal metabolic milieu exposures, independent of maternal BMI, and suggest utility as an at-birth predictor for child adiposity, although validation with larger longitudinal samples is warranted.

Physical activity, including structured exercise, is associated with favorable health-related chronic disease outcomes. Although there is evidence of various molecular pathways that affect these responses, a comprehensive molecular map of these molecular responses to exercise has not been developed. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) is a multicenter study designed to isolate the effects of structured exercise training on the molecular mechanisms underlying the health benefits of exercise and physical activity. MoTrPAC contains both a preclinical and human component. The details of the human studies component of MoTrPAC that include the design and methods are presented here. The human studies contain both an adult and pediatric component. In the adult component, sedentary participants are randomized to 12 wk of Control, Endurance Exercise Training, or Resistance Exercise Training with outcomes measures completed before and following the 12 wk. The adult component also includes recruitment of highly active endurance-trained or resistance-trained participants who only complete measures once. A similar design is used for the pediatric component; however, only endurance exercise is examined. Phenotyping measures include weight, body composition, vital signs, cardiorespiratory fitness, muscular strength, physical activity and diet, and other questionnaires. Participants also complete an acute rest period (adults only) or exercise session (adults, pediatrics) with collection of biospecimens (blood only for pediatrics) to allow for examination of the molecular responses. The design and methods of MoTrPAC may inform other studies. Moreover, MoTrPAC will provide a repository of data that can be used broadly across the scientific community. NEW & NOTEWORTHY The Molecular Transducers of Physical Activity Consortium (MoTrPAC) will be the first large trial to isolate the effects of structured exercise training on the molecular mechanisms underlying the health benefits of exercise and physical activity. By generating a compendium of the molecular responses to exercise, MoTrPAC will lay the foundation for a new era of biomedical research on Precision Exercise Medicine. Presented here is the design, protocols, and procedures for the MoTrPAC human studies.

Elevated skeletal muscle diacylglycerols (DAGs) and ceramides can impair insulin signaling, and acylcarnitines (acylCNs) reflect impaired mitochondrial fatty acid oxidation, thus, the intramuscular lipid profile is indicative of insulin resistance. Acute (i.e., postprandial) hyperinsulinemia has been shown to elevate lipid concentrations in healthy muscle and is an independent risk factor for type 2 diabetes (T2D). However, it is unclear how the relationship between acute hyperinsulinemia and the muscle lipidome interacts across metabolic phenotypes, thus contributing to or exacerbating insulin resistance. We therefore investigated the impact of acute hyperinsulinemia on the skeletal muscle lipid profile to help characterize the physiological basis in which hyperinsulinemia elevates T2D risk. In a cross-sectional comparison, endurance athletes ( n = 12), sedentary lean adults ( n = 12), and individuals with obesity ( n = 13) and T2D ( n = 7) underwent a hyperinsulinemic-euglycemic clamp with muscle biopsies. Although there were no significant differences in total 1,2-DAG fluctuations, there was a 2% decrease in athletes versus a 53% increase in T2D during acute hyperinsulinemia ( P = 0.087). Moreover, C18 1,2-DAG species increased during the clamp with T2D only, which negatively correlated with insulin sensitivity ( P < 0.050). Basal muscle C18:0 total ceramides were elevated with T2D ( P = 0.029), but not altered by clamp. Acylcarnitines were universally lowered during hyperinsulinemia, with more robust reductions of 80% in athletes compared with only 46% with T2D (albeit not statistically significant, main effect of group, P = 0.624). Similar fluctuations with acute hyperinsulinemia increasing 1,2 DAGs in insulin-resistant phenotypes and universally lowering acylcarnitines were observed in male mice. In conclusion, acute hyperinsulinemia elevates muscle 1,2-DAG levels with insulin-resistant phenotypes. This suggests a possible dysregulation of intramuscular lipid metabolism in the fed state in individuals with low insulin sensitivity, which may exacerbate insulin resistance. NEW & NOTEWORTHY Postprandial hyperinsulinemia is a risk factor for type 2 diabetes and may increase muscle lipids. However, it is unclear how the relationship between acute hyperinsulinemia and the muscle lipidome interacts across metabolic phenotypes, thus contributing to insulin resistance. We observed that acute hyperinsulinemia elevates muscle 1,2-DAGs in insulin-resistant phenotypes, whereas ceramides were unaltered. Insulin-mediated acylcarnitine reductions are also hindered with high-fat feeding. The postprandial period may exacerbate insulin resistance in metabolically unhealthy phenotypes.

Subcutaneous white adipose tissue (scWAT) is a dynamic storage and secretory organ that regulates systemic homeostasis, yet the impact of endurance exercise training (ExT) and sex on its molecular landscape is not fully established. Utilizing an integrative multi-omics approach, and leveraging data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we show profound sexual dimorphism in the scWAT of sedentary rats and in the dynamic response of this tissue to ExT. Specifically, the scWAT of sedentary females displays -omic signatures related to insulin signaling and adipogenesis, whereas the scWAT of sedentary males is enriched in terms related to aerobic metabolism. These sex-specific -omic signatures are preserved or amplified with ExT. Integration of multi-omic analyses with phenotypic measures identifies molecular hubs predicted to drive sexually distinct responses to training. Overall, this study underscores the powerful impact of sex on adipose tissue biology and provides a rich resource to investigate the scWAT response to ExT., (© 2024. Battelle Memorial Institute.)

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders., Competing Interests: AD, SM, KC, LC, JK, NT, XL, MA, JM, AD, AG, SZ, KZ, BB, JB No competing interests declared, AR, JB is an employee of Eli Lilly. The author has no other competing interests to declare, DJ Senior editor, eLife, (© 2023, Diaz-Vegas et al.)

Background: Exposure to intrauterine obesity can disrupt clock gene rhythmicity in animal models. The aim of this pilot study was to determine if maternal obesity alters rhythmic expression of core clock in mesenchymal stem cells (MSCs) from umbilical cords of human infants born to mothers with obesity (Ob-MSC) vs. normal weight (NW-MSC)., Methods: We compared in vitro rhythmic expression patterns of core clock ( BMAL1 , CLOCK , PER2 ) and clock-output ( NR1D1 ), components in undifferentiated Ob-MSCs ( n = 3) vs. NW-MSCs ( n = 3). MSCs were harvested every 2 h, following a dexamethasone shock, for 30 h. Adipogenesis or myogenesis was induced in vitro and markers of adipogenesis and fat storage were assessed, respectively., Results: We detected significant rhythmicity in expression patterns of BMAL1 , PER2 , and NR1D1 at the group level in Ob- and NW-MSCs ( p < 0.05). PER2 oscillatory amplitude was 3-fold higher in Ob-MSCs vs. NW-MSCs ( p < 0.006). During adipogenesis, Ob-MSCs had higher PPAR γ protein content ( p = 0.04) vs. NW-MSC. During myogenesis, Ob-MSCs had higher saturated triacylglycerols ( p = 0.04) vs. NW-MSC., Conclusion: Rhythmic expressions of BMAL1 , PER2 , and NR1D1 are detectable in undifferentiated MSCs. Higher PER2 oscillatory amplitude was paralleled by higher markers of fat storage during differentiation in Ob-MSCs vs. NW-MSCs, and supports that the core clock and cellular metabolism may be linked in infant MSCs.

Maternal consumption of a Western-style diet (mWD) during pregnancy alters fatty acid metabolism and reduces insulin sensitivity in fetal skeletal muscle. The long-term impact of these fetal adaptations and the pathways underlying disordered lipid metabolism are incompletely understood. Therefore, we tested whether a mWD chronically fed to lean, insulin-sensitive adult Japanese macaques throughout pregnancy and lactation would impact skeletal muscle oxidative capacity and lipid metabolism in adolescent offspring fed a postweaning (pw) Western-style diet (WD) or control diet (CD). Although body weight was not different, retroperitoneal fat mass and subscapular skinfold thickness were significantly higher in pwWD offspring consistent with elevated fasting insulin and glucose. Maximal complex I (CI)-dependent respiration in muscle was lower in mWD offspring in the presence of fatty acids, suggesting that mWD impacts muscle integration of lipid with nonlipid oxidation. Abundance of all five oxidative phosphorylation complexes and VDAC, but not ETF/ETFDH, were reduced with mWD, partially explaining the lower respiratory capacity with lipids. Muscle triglycerides increased with pwWD; however, the fold increase in lipid saturation, 1,2-diacylglycerides, and C18 ceramide compared between pwCD and pwWD was greatest in mWD offspring. Reductions in CI abundance and VDAC correlated with reduced markers of oxidative stress, suggesting that these reductions may be an early-life adaptation to mWD to mitigate excess reactive oxygen species. Altogether, mWD, independent of maternal obesity or insulin resistance, results in sustained metabolic reprogramming in offspring muscle despite a healthy diet intervention., Article Highlights: In lean, active adolescent offspring, a postweaning Western-style diet (pwWD) leads to shifts in body fat distribution that are associated with poorer insulin sensitivity. Fatty acid-linked oxidative metabolism was reduced in skeletal muscles from offspring exposed to maternal Western-style diet (mWD) even when weaned to a healthy control diet for years. Reduced oxidative phosphorylation complex I-V and VDAC1 abundance partially explain decreased skeletal muscle respiration in mWD offspring. Prior exposure to mWD results in greater fold increase with pwWD in saturated lipids and bioactive lipid molecules (i.e. ceramide and sphingomyelin) associated with insulin resistance., (© 2023 by the American Diabetes Association.)

Stimulation of adipocyte β-adrenergic receptors (β-ARs) induces expression of uncoupling protein 1 (UCP1), promoting nonshivering thermogenesis. Association of β-ARs with a lysine-myristoylated form of A kinase-anchoring protein 12 (AKAP12, also known as gravin-α) is required for downstream signaling that culminates in UCP1 induction. Conversely, demyristoylation of gravin-α by histone deacetylase 11 (HDAC11) suppresses this pathway. Whether inhibition of HDAC11 in adipocytes is sufficient to drive UCP1 expression independently of β-ARs is not known. Here, we demonstrate that adipocyte-specific deletion of HDAC11 in mice leads to robust induction of UCP1 in adipose tissue (AT), resulting in increased body temperature. These effects are mimicked by treating mice in vivo or human AT ex vivo with an HDAC11-selective inhibitor, FT895. FT895 triggers biphasic, gravin-α myristoylation-dependent induction of UCP1 protein expression, with a noncanonical acute response that is posttranscriptional and independent of protein kinase A (PKA), and a delayed response requiring PKA activity and new Ucp1 mRNA synthesis. Remarkably, HDAC11 inhibition promotes UCP1 expression even in models of adipocyte catecholamine resistance where β-AR signaling is blocked. These findings define cell-autonomous, multimodal roles for HDAC11 as a suppressor of thermogenesis, and highlight the potential of inhibiting HDAC11 to therapeutically alter AT phenotype independently of β-AR stimulation.

Sphingolipids are thought to promote skeletal muscle insulin resistance. Deoxysphingolipids (dSLs) are atypical sphingolipids that are increased in the plasma of individuals with type 2 diabetes and cause β-cell dysfunction in vitro. However, their role in human skeletal muscle is unknown. We found that dSL species are significantly elevated in muscle of individuals with obesity and type 2 diabetes compared with athletes and lean individuals and are inversely related to insulin sensitivity. Furthermore, we observed a significant reduction in muscle dSL content in individuals with obesity who completed a combined weight loss and exercise intervention. Increased dSL content in primary human myotubes caused a decrease in insulin sensitivity associated with increased inflammation, decreased AMPK phosphorylation, and altered insulin signaling. Our findings reveal a central role for dSL in human muscle insulin resistance and suggest dSLs as therapeutic targets for the treatment and prevention of type 2 diabetes., Article Highlights: Deoxysphingolipids (dSLs) are atypical sphingolipids elevated in the plasma of individuals with type 2 diabetes, and their role in muscle insulin resistance has not been investigated. We evaluated dSL in vivo in skeletal muscle from cross-sectional and longitudinal insulin-sensitizing intervention studies and in vitro in myotubes manipulated to synthesize higher dSLs. dSLs were increased in the muscle of people with insulin resistance, inversely correlated to insulin sensitivity, and significantly decreased after an insulin-sensitizing intervention; increased intracellular dSL concentrations cause myotubes to become more insulin resistant. Reduction of muscle dSL levels is a potential novel therapeutic target to prevent/treat skeletal muscle insulin resistance., (© 2023 by the American Diabetes Association.)

Intermuscular adipose tissue (IMAT) is a distinct adipose depot described in early reports as a 'fatty replacement' or 'muscle fat infiltration' that was linked to ageing and neuromuscular disease. Later studies quantifying IMAT with modern in vivo imaging methods (computed tomography and magnetic resonance imaging) revealed that IMAT is proportionately higher in men and women with type 2 diabetes mellitus and the metabolic syndrome than in people without these conditions and is associated with insulin resistance and poor physical function with ageing. In parallel, agricultural research has provided extensive insight into the role of IMAT and other muscle lipids in muscle (that is, meat) quality. In addition, studies using rodent models have shown that IMAT is a bona fide white adipose tissue depot capable of robust triglyceride storage and turnover. Insight into the importance of IMAT in human biology has been limited by the dearth of studies on its biological properties, that is, the quality of IMAT. However, in the past few years, investigations have begun to determine that IMAT has molecular and metabolic features that distinguish it from other adipose tissue depots. These studies will be critical to further decipher the role of IMAT in health and disease and to better understand its potential as a therapeutic target., (© 2022. Springer Nature Limited.)

Aims/hypothesis: Although insulin resistance often leads to type 2 diabetes mellitus, its early stages are often unrecognised, thus reducing the probability of successful prevention and intervention. Moreover, treatment efficacy is affected by the genetics of the individual. We used gene expression profiles from a cross-sectional study to identify potential candidate genes for the prediction of diabetes risk and intervention response., Methods: Using a multivariate regression model, we linked gene expression profiles of human skeletal muscle and intermuscular adipose tissue (IMAT) to fasting glucose levels and glucose infusion rate. Based on the expression patterns of the top predictive genes, we characterised and compared individual gene expression with clinical classifications using k-nearest neighbour clustering. The predictive potential of the candidate genes identified was validated using muscle gene expression data from a longitudinal intervention study., Results: We found that genes with a strong association with clinical measures clustered into three distinct expression patterns. Their predictive values for insulin resistance varied substantially between skeletal muscle and IMAT. Moreover, we discovered that individual gene expression-based classifications may differ from classifications based predominantly on clinical variables, indicating that participant stratification may be imprecise if only clinical variables are used for classification. Of the 15 top candidate genes, ST3GAL2, AASS, ARF1 and the transcription factor SIN3A are novel candidates for predicting a refined diabetes risk and intervention response., Conclusion/interpretation: Our results confirm that disease progression and successful intervention depend on individual gene expression states. We anticipate that our findings may lead to a better understanding and prediction of individual diabetes risk and may help to develop individualised intervention strategies., (© 2023. The Author(s).)

Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Adipose tissue secretions are depot-specific and vary based on anatomical location. Considerable attention has been focused on visceral (VAT) and subcutaneous (SAT) adipose tissue with regard to metabolic disease, yet our knowledge of the secretome from these depots is incomplete. We conducted a comprehensive analysis of VAT and SAT secretomes in the context of metabolic function. Conditioned media generated using SAT and VAT explants from individuals with obesity were analyzed using proteomics, mass spectrometry, and multiplex assays. Conditioned media were administered in vitro to rat hepatocytes and myotubes to assess the functional impact of adipose tissue signaling on insulin responsiveness. VAT secreted more cytokines (IL-12p70, IL-13, TNF-α, IL-6, and IL-8), adipokines (matrix metalloproteinase-1, PAI-1), and prostanoids (TBX2, PGE2) compared with SAT. Secretome proteomics revealed differences in immune/inflammatory response and extracellular matrix components. In vitro, VAT-conditioned media decreased hepatocyte and myotube insulin sensitivity, hepatocyte glucose handling, and increased basal activation of inflammatory signaling in myotubes compared with SAT. Depot-specific differences in adipose tissue secretome composition alter paracrine and endocrine signaling. The unique secretome of VAT has distinct and negative impact on hepatocyte and muscle insulin action., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Serum ceramides, especially C16:0 and C18:0 species, are linked to CVD risk and insulin resistance, but details of this association are not well understood. We performed this study to quantify a broad range of serum sphingolipids in individuals spanning the physiologic range of insulin sensitivity and to determine if dihydroceramides cause insulin resistance in vitro. As expected, we found that serum triglycerides were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals. Serum ceramides were not significantly different within groups but, using all ceramide data relative to insulin sensitivity as a continuous variable, we observed significant inverse relationships between C18:0, C20:0, and C22:0 species and insulin sensitivity. Interestingly, we found that total serum dihydroceramides and individual species were significantly greater in individuals with obesity and T2D compared with athletes and lean individuals, with C18:0 species showing the strongest inverse relationship to insulin sensitivity. Finally, we administered a physiological mix of dihydroceramides to primary myotubes and found decreased insulin sensitivity in vitro without changing the overall intracellular sphingolipid content, suggesting a direct effect on insulin resistance. These data extend what is known regarding serum sphingolipids and insulin resistance and show the importance of serum dihydroceramides to predict and promote insulin resistance in humans., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Adipose tissue secretes an abundance of lipid and protein mediators, and this secretome is depot-specific, with local and systemic effects on metabolic regulation. Intermuscular adipose tissue (IMAT) accumulates within the skeletal muscle compartment in obesity, and is associated with insulin resistance and metabolic disease. While the human IMAT secretome decreases insulin sensitivity in vitro, its composition is entirely unknown. The current study was conducted to investigate the composition of the human IMAT secretome, compared to that of the subcutaneous (SAT) and visceral adipose tissue (VAT) depots. IMAT, SAT, and VAT explants from individuals with obesity were used to generate conditioned media. Proteomics analysis of conditioned media was performed using multiplex proximity extension assays, and eicosanoid analysis using liquid chromatography-tandem mass spectrometry. Compared to SAT and/or VAT, IMAT secreted significantly more cytokines (IL2, IL5, IL10, IL13, IL27, FGF23, IFNγ and CSF1) and chemokines (MCP1, IL8, CCL11, CCL20, CCL25 and CCL27). Adipokines hepatocyte growth factor and resistin were secreted significantly more by IMAT than SAT or VAT. IMAT secreted significantly more eicosanoids (PGE 2, TXB 2 , 5-HETE, and 12-HETE) compared to SAT and/or VAT. In the context of obesity, IMAT is a distinct adipose tissue with a highly immunogenic and inflammatory secretome, and given its proximity to skeletal muscle, may be critical to glucose regulation and insulin resistance., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Preclinical rodent and nonhuman primate models investigating maternal obesity have highlighted the importance of the intrauterine environment in the development of insulin resistance in offspring; however, it remains unclear if these findings can be translated to humans. To investigate possible intrauterine effects in humans, we isolated mesenchymal stem cells (MSCs) from the umbilical cord tissue of infants born to mothers of normal weight or mothers with obesity. Insulin-stimulated glycogen storage was determined in MSCs undergoing myogenesis in vitro. There was no difference in insulin action based on maternal obesity. However, maternal free fatty acid (FFA) concentration, cord leptin, and intracellular triglyceride content were positively correlated with insulin action. Furthermore, MSCs from offspring born to mothers with elevated FFAs displayed elevated activation of the mTOR signaling pathway. Taken together, these data suggest that infants born to mothers with elevated lipid availability have greater insulin action in MSCs, which may indicate upregulation of growth and lipid storage pathways during periods of maternal overnutrition., (© 2022 by the American Diabetes Association.)

Alterations in adipose tissue composition and function are associated with obesity and contribute to the development of type 2 diabetes. While the significance of this relationship has been cemented, our understanding of the multifaceted role of adipose tissue in metabolic heath and disease continues to evolve and expand. Heterogenous populations of cells that make up adipose tissue throughout the body generate diverse secretomes containing a mosaic of bioactive compounds with vast structural and signaling capabilities. While there are many reports highlighting the important role of adipose tissue endocrine signaling in insulin resistance and type 2 diabetes, the direct, local, paracrine effect of adipose tissue has received less attention. Recent studies have begun to underscore the importance of considering anatomically discrete adipose depots for their specific impact on local microenvironments and metabolic function in neighboring tissues as well as regulation of whole-body physiology. This article highlights the important role of adipose tissue paracrine signaling on metabolic function and insulin sensitivity in nearby tissues and organs, specifically focusing on visceral, pancreatic, subcutaneous, intermuscular, and perivascular adipose tissue depots., (© 2022 by the American Diabetes Association.)

Purpose: To train and test a machine learning model to automatically measure mid-thigh muscle cross-sectional area (CSA) to provide rapid estimation of appendicular lean mass (ALM) and predict knee extensor torque of obese adults., Methods: Obese adults [body mass index (BMI) = 30-40 kg/m 2 , age = 30-50 years] were enrolled for this study. Participants received full-body dual-energy X-ray absorptiometry (DXA), mid-thigh MRI, and completed knee extensor and flexor torque assessments via isokinetic dynamometer. Manual segmentation of mid-thigh CSA was completed for all MRI scans. A convolutional neural network (CNN) was created based on the manual segmentation to develop automated quantification of mid-thigh CSA. Relationships were established between the automated CNN values to the manual CSA segmentation, ALM via DXA, knee extensor, and flexor torque., Results: A total of 47 obese patients were enrolled in this study. Agreement between the CNN-automated measures and manual segmentation of mid-thigh CSA was high (>0.90). Automated measures of mid-thigh CSA were strongly related to the leg lean mass ( r = 0.86, p < 0.001) and ALM ( r = 0.87, p < 0.001). Additionally, mid-thigh CSA was strongly related to knee extensor strength ( r = 0.76, p < 0.001) and moderately related to knee flexor strength ( r = 0.48, p = 0.002)., Conclusion: CNN-measured mid-thigh CSA was accurate compared to the manual segmented values from the mid-thigh. These values were strongly predictive of clinical measures of ALM and knee extensor torque. Mid-thigh MRI may be utilized to accurately estimate clinical measures of lean mass and function in obese adults., Competing Interests: Conflict of Interest: 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.

Study Objectives: Insufficient sleep is believed to promote positive energy balance (EB) and weight gain. Increasing weekend sleep duration to "recover" from weekday sleep loss is common, yet little is known regarding how weekend recovery sleep influences EB. We conducted a randomized controlled trial to assess how: (1) 2 days and 8 days of insufficient sleep and (2) ad libitum weekend recovery sleep impact EB (energy intake [EI] - energy expenditure [EE])., Methods: Following ten baseline days with 9 h per night sleep opportunities, participants completed one of three 10-day experimental protocols with ad libitum EI: control (9 h sleep opportunities; n = 8; 23 ± 5 years [mean ± SD]); sleep restriction (SR; 5 h sleep opportunities; n = 14; 25 ± 5 years); sleep restriction with weekend recovery sleep (SR + WR; 5 days insufficient sleep, 2 days ad libitum weekend recovery sleep, 3 days recurrent insufficient sleep; n = 14; 27 ± 4 years)., Results: Twenty-four hour EB increased (p < 0.001; main effect) by an average of 797.7 ± 96.7 (±SEM) kcal during the 10-day experimental protocol versus baseline with no significant differences between groups. Percent change from baseline in 24 h-EE was higher (p < 0.05) on day 2 of insufficient sleep (SR and SR + WR groups; 10 ± 1%) versus adequate sleep (control group; 4 ± 3%)., Conclusions: In this between-group study, the effects of adequate sleep and insufficient sleep, with or without or weekend recovery sleep, on 24 h-EB were similar. Examining EB and body weight changes using within-subject cross-over designs and "free-living" conditions outside the laboratory (e.g. sleep extension) are needed to advance our understanding of the links between insufficient sleep, weekend recovery sleep and weight-gain., (© Sleep Research Society 2021. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Prior data demonstrated three weeks of sleep restriction and concurrent circadian disruption uncoupled bone turnover markers (BTMs), indicating decreased bone formation and no change or increased bone resorption. The effect of insufficient sleep with or without ad libitum weekend recovery sleep on BTMs is unknown., Methods: BTMs were measured in stored serum from 20 healthy adults randomized to one of three study groups consisting of a control group (N = 3 men; 9 h/night) or one of two nocturnal sleep restriction groups in an inpatient laboratory environment. One Sleep Restriction group ("SR"; N = 9; 4 women) had 5 h sleep opportunity per night for nine nights. The other sleep restriction group had an opportunity for ad libitum Weekend Recovery sleep ("WR"; N = 8; 4 women) after four nights of 5 h sleep opportunity per night. Food intake was energy balanced at baseline and ad libitum thereafter. Fasted morning BTM levels and hourly 24 h melatonin levels were obtained on study days 3 (baseline), 5 (after 1 night of sleep restriction for WR and SR), and 11 (after a sleep restricted workweek with weekend recovery sleep in WR or 7 nights of sleep restriction in SR). Linear mixed-effects modeling was used to examine the effect of study duration (e.g., change over time), study condition, age, and sex on BTMs. Pearson correlations were used to determine associations between changes in BTMs and changes in weight and morning circadian misalignment (i.e., duration of high melatonin levels after wake time)., Results: There was no significant difference between the three study groups in change over time (p ≥ 0.4 for interaction between assigned group and time for all BTMs), adjusted for age and sex. There was no significant change in N-terminal propeptide of procollagen type I (P1NP), osteocalcin, or C-telopeptide of type I collagen (CTX) from baseline to day 11 (all p ≥ 0.3). In women <25 years old, there was a non-significant decline in P1NP from day 3 to day 5 (= -15.74 ± 7.80 ng/mL; p = 0.06). Change in weight and morning circadian misalignment from baseline to day 11 were correlated with statistically non-significant changes in BTMs (all p ≤ 0.05)., Conclusion: In this small secondary analysis, we showed that nine nights of prescribed sleep restriction with or without weekend recovery sleep and ad libitum food intake did not alter BTMs. It is possible that age, sex, weight change and morning circadian misalignment modify the effects of sleep restriction on bone metabolism., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Adipose tissues are not homogeneous and show site-specific properties. An elusive and understudied adipose tissue depot, most likely due to its limited accessibility, is the intermuscular adipose tissue (IMAT) depot. Adipose tissue is a pliable organ with the ability to adapt to its physiological context, yet whether that adaptation is harmful or beneficial in the IMAT depot remains to be explored in humans. Potential reasons for IMAT accumulation in humans being deleterious or beneficial include 1 ) sex and related circulating hormone levels, 2 ) race and ethnicity, and 3 ) lifestyle factors (e.g., diet and physical activity level). IMAT quantity per se may not be the driving factor in the etiology of insulin resistance and type 2 diabetes, but rather the quality of the IMAT itself is the true puppeteer. Adipose tissue quality likely influences its secreted factors, which are also likely to influence metabolism of surrounding tissues. The advent of molecular assessments such as transcriptome sequencing (RNAseq), assay for transposase-accessible chromatin using sequencing (ATACseq), and DNA methylation at the single-cell and single-nucleus levels, as well as the potential for ultrasound-guided biopsies specifically for IMAT, will permit more sophisticated investigations of human IMAT and dramatically advance our understanding of this enigmatic adipose tissue., (© 2021 by the American Diabetes Association.)

Recent studies suggest that the accumulation of atypical, 1-deoxysphingolipids that lack the C1 hydroxyl group may be associated with diabetic neuropathy (DN). We hypothesized that specific plasma 1-deoxysphingolipids associate with DN severity, and that alterations in plasma serine and alanine associate with 1-deoxysphingolipid elevation in patients with type 2 diabetes (T2D). We examined individual 1-deoxysphingolipid species using LC/MS/MS in plasma samples from 75 individuals including lean controls (LC, n = 19), those with obesity (n = 19), obesity with T2D without DN (ob/T2D, n = 18), and obesity with T2D with DN (Ob/T2D/DN, n = 19). We observed a step wise increase in 1-deoxydihydroceramides across these four groups (spearman correlation coefficient r = 0.41, p = 0.0002). Mean total concentrations of 1-deoxydihydroceramides, and most individual 1-deoxydihydroceramide species, were higher in ob/T2D/DN versus LC group (8.939 vs. 5.195 pmol/100 μL for total 1-deoxydihydroceramides p = 0.005). No significant differences in 1-deoxydihydroceramides were observed between the ob/T2D and ob/T2D/DN groups. l-alanine was higher and l-serine lower in ob/T2D/DN versus LC groups (326.2 vs. 248.0 μM, p = 0.0086 and 70.2 vs. 89.8 μM, p = 0.0110), consistent with a potential contribution of these changes to the observed 1-deoxysphingolipids profiles. 1-deoxydihydroceramides correlated inversely with leg intraepidermal nerve fiber density (CC -0.40, p = 0.003). These findings indicate that 1-deoxydihydroceramides may be important biomarkers and/or mediators of DN., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: V·F, Z.S, S·S, H·K, B·C·B, E.L.F, and R.J.E.B have no conflicts of interest. C·B Dr. Callaghan consults for a PCORI grant, DynaMed, and performs medical legal consultations including consultations for the Vaccine Injury Compensation Program., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Sex differences in insulin sensitivity are present throughout the life-span, with men having a higher prevalence of insulin resistance and diabetes compared with women. Differences in lean mass, fat mass, and fat distribution-particularly ectopic fat-have all been postulated to contribute to the sexual dimorphism in diabetes risk. Emerging data suggest ectopic lipid composition and subcellular localization are most relevant; however, it is not known whether they explain sex differences in obesity-induced insulin resistance., Methods: To address this gap, this study evaluated insulin sensitivity and subcellular localization of intramuscular triacylglycerol, diacylglycerol, and sphingolipids as well as muscle acylcarnitines and serum lipidomics in people with obesity., Results: Insulin sensitivity was significantly lower in men (P < 0.05); however, no sex differences were found in localization of intramuscular triacylglycerol, diacylglycerol, or sphingolipids in skeletal muscle. In contrast, men had higher total muscle acylcarnitine (P < 0.05) and long-chain muscle acylcarnitine (P < 0.05), which were related to lower insulin sensitivity (r = -0.42, P < 0.05). Men also displayed higher serum ceramide (P = 0.05) and lysophosphatidylcholine (P < 0.01)., Conclusions: These data reveal novel sex-specific associations between lipid species involved in the coupling of mitochondrial fatty acid transport, β-oxidation, and tricarboxylic acid cycle flux that may provide therapeutic targets to improve insulin sensitivity., (© 2021 The Obesity Society.)

Objective: Few studies have examined the effects of participants' diet and activity prior to sample collection on metabolomics profiles, and results have been conflicting. We compared the effects of overnight fasting with or without 3 days of standardized diet and restricted physical activity on the human blood metabolome, and examined the effects of these protocols on our ability to detect differences in metabolomics profiles in adolescent girls with obesity and polycystic ovary syndrome (PCOS) vs. sex and BMI-matched controls., Methods: This was a cross-sectional study of 16 adolescent girls with obesity and PCOS and 5 sex and BMI-matched controls. Fasting plasma metabolomic profiles were measured twice in each participant: once without preceding restriction of physical activity or control of macronutrient content ("typical fasting visit"), and again after 12 h of monitored inpatient fasting with 3 days of standardized diet and avoidance of vigorous exercise ("controlled fasting visit"). Moderated paired t-tests with FDR correction for multiple testing and multilevel sparse partial least-squares discriminant analysis (sPLS-DA) were used to examine differences between the 2 visits and to compare the PCOS and control groups with the 2 visits combined and again after stratifying by visit., Results: Twenty-three known metabolites were significantly different between the controlled fasting and typical fasting visits. Hypoxanthine and glycochenodeoxycholic acid had the largest increases in relative abundance at the controlled fasting visit compared to the typical fasting visit, while oleoyl-glycerol and oleamide had the largest increases in relative abundance at the typical fasting visit compared to the controlled fasting visit. sPLS-DA showed excellent discrimination between the 2 visits; however, when the samples from the 2 visits were combined, differences between the PCOS and control groups could not be detected. After stratifying by visit, discrimination of PCOS status was improved., Conclusions: There were differences in fasting metabolomic profiles following typical fasting vs monitored fasting with preceding restriction of physical activity and control of macronutrient content, and combining samples from the two visits obscured differences by PCOS status. In studies performing metabolomics analysis, careful attention should be paid to acute diet and activity history. Depending on the sample size of the study and the expected effect size of the outcomes of interest, control of diet and physical activity beyond typical outpatient fasting may not be required., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

Aims/hypothesis: Subcellular localisation is an important factor in the known impact of bioactive lipids, such as diacylglycerol and sphingolipids, on insulin sensitivity in skeletal muscle; yet, the role of localised intramuscular triacylglycerol (IMTG) is yet to be described. Excess accumulation of IMTG in skeletal muscle is associated with insulin resistance, and we hypothesised that differences in subcellular localisation and composition of IMTG would relate to metabolic health status in humans., Methods: We evaluated subcellular localisation of IMTG in lean participants, endurance-trained athletes, individuals with obesity and individuals with type 2 diabetes using LC-MS/MS of fractionated muscle biopsies and insulin clamps., Results: Insulin sensitivity was significantly different between each group (athletes>lean>obese>type 2 diabetes; p < 0.001). Sarcolemmal IMTG was significantly greater in individuals with obesity and type 2 diabetes compared with lean control participants and athletes, but individuals with type 2 diabetes were the only group with significantly increased saturated IMTG. Sarcolemmal IMTG was inversely related to insulin sensitivity. Nuclear IMTG was significantly greater in individuals with type 2 diabetes compared with lean control participants and athletes, and total and saturated IMTG localised in the nucleus had a significant inverse relationship with insulin sensitivity. Total cytosolic IMTG was not different between groups, but saturated cytosolic IMTG species were significantly increased in individuals with type 2 diabetes compared with all other groups. There were no significant differences between groups for IMTG concentration in the mitochondria/endoplasmic reticulum., Conclusions/interpretation: These data reveal previously unknown differences in subcellular IMTG localisation based on metabolic health status and indicate the influence of sarcolemmal and nuclear IMTG on insulin sensitivity. Additionally, these studies suggest saturated IMTG may be uniquely deleterious for muscle insulin sensitivity. Graphical abstract.

Lipid catabolism represents an Achilles heel in prostate cancer (PCa) that can be exploited for therapy. CPT1A regulates the entry of fatty acids into the mitochondria for beta-oxidation and its inhibition has been shown to decrease PCa growth. In this study, we examined the pharmacological blockade of lipid oxidation with ranolazine in TRAMPC1 PCa models. Oral administration of ranolazine (100 mg/Kg for 21 days) resulted in decreased tumor CD8 + T-cells Tim3 content, increased macrophages, and decreased blood myeloid immunosuppressive monocytes. Using multispectral staining, drug treatments increased infiltration of CD8 + T-cells and dendritic cells compared to vehicle. Functional studies with spleen cells of drug-treated tumors co-cultured with TRAMPC1 cells showed increased ex vivo T-cell cytotoxic activity, suggesting an anti-tumoral response. Lastly, a decrease in CD4 + and CD8 + T-cells expressing PD1 was observed when exhausted spleen cells were incubated with TRAMPC1 Cpt1a-KD compared to the control cells. These data indicated that genetically blocking the ability of the tumor cells to oxidize lipid can change the activation status of the neighboring T-cells. This study provides new knowledge of the role of lipid catabolism in the intercommunication of tumor and immune cells, which can be extrapolated to other cancers with high CPT1A expression.

Skeletal muscle fat infiltration (known as myosteatosis) is an ectopic fat depot that increases with aging and is recognized to negatively correlate with muscle mass, strength, and mobility and disrupt metabolism (insulin resistance, diabetes). An interdisciplinary workshop convened by the National Institute on Aging Division of Geriatrics and Clinical Gerontology on September 2018, discussed myosteatosis in the context of skeletal muscle function deficit (SMFD). Its purpose was to gain a better understanding of the roles of myosteatosis in aging muscles and metabolic disease, particularly its potential determinants and clinical consequences, and ways of properly assessing it. Special attention was given to functional status and standardization of measures of body composition (including the value of D 3 -creatine dilution method) and imaging approaches [including ways to better use dual-energy X-ray absorptiometry (DXA) through the shape and appearance modeling] to assess lean mass, sarcopenia, and myosteatosis. The workshop convened innovative new areas of scientific relevance to light such as the effect of circadian rhythms and clock disruption in skeletal muscle structure, function, metabolism, and potential contribution to increased myosteatosis. A muscle-bone interaction perspective compared mechanisms associated with myosteatosis and bone marrow adiposity. Potential preventive and therapeutic approaches highlighted ongoing work on physical activity, myostatin treatment, and calorie restriction. Myosteatosis' impact on cancer survivors raised new possibilities to identify its role and to engage in cross-disciplinary collaboration. A wide range of research opportunities and challenges in planning for the most appropriate study design, interpretation, and translation of findings into clinical practice were discussed and are presented here., (Copyright © 2020 Correa-de-Araujo, Addison, Miljkovic, Goodpaster, Bergman, Clark, Elena, Esser, Ferrucci, Harris-Love, Kritchevsky, Lorbergs, Shepherd, Shulman and Rosen.)

Accumulation of lipid in skeletal muscle is thought to be related to the development of insulin resistance and type 2 diabetes. Initial work in this area focused on accumulation of intramuscular triglyceride; however, bioactive lipids such as diacylglycerols and sphingolipids are now thought to play an important role. Specific species of these lipids appear to be more negative toward insulin sensitivity than others. Adding another layer of complexity, localization of lipids within the cell appears to influence the relationship between these lipids and insulin sensitivity. This article summarizes how accumulation of total lipids, specific lipid species, and localization of lipids influence insulin sensitivity in humans. We then focus on how these aspects of muscle lipids are impacted by acute and chronic aerobic and resistance exercise training. By understanding how exercise alters specific species and localization of lipids, it may be possible to uncover specific lipids that most heavily impact insulin sensitivity., (© 2020 by the American Diabetes Association.)

Objective: This study aimed to phenotype and compare adipose, hepatic, and muscle insulin sensitivity (IS) in a diet- and physical activity-controlled cohort of normoglycemic youth with obesity with that of participants without obesity (controls) to distinguish early metabolic abnormalities in pediatric obesity., Methods: Thirty-eight participants (17 in the control group [BMI < 85th percentile] and 21 youth with obesity [BMI ≥ 95th percentile]; age: 12-21 years; 76% female; Tanner stage 4-5; sedentary) were enrolled. Tissue-specific IS was measured using a four-phase hyperinsulinemic-euglycemic clamp with glucose and glycerol isotope tracers to assess suppression of endogenous glucose release and lipolysis by insulin. Intramyocellular lipid content was assessed by 1 H-magnetic resonance spectroscopy, and hepatic fat fraction (HFF) and visceral fat were assessed by magnetic resonance imaging. Calf-muscle mitochondrial activity was measured with exercise-stimulated 31 P-magnetic resonance spectroscopy., Results: Youth with obesity had higher HFF (P < 0.001), visceral fat (P = 0.024), and intramyocellular lipid content (P = 0.017) and lower muscle (glucose clearance rate [P < 0.001]), adipose (P < 0.0001), and hepatic IS (P < 0.003). Mitochondria postexercise response was not different. In participants with obesity, muscle IS inversely correlated with HFF (r = 0.700, P = 0.002) and suppressed free fatty acid concentrations (r = -0.65, P = 0.003)., Conclusions: Inactive normoglycemic youth with obesity had decreased muscle, adipose, and hepatic IS. Free fatty acids and liver fat were inversely associated with muscle IS, which argues for lipid-targeted interventions., (© 2019 The Obesity Society.)

Context: Type 1 diabetes in adolescence is characterized by insulin deficiency and insulin resistance (IR), both thought to increase cardiovascular disease risk. We previously demonstrated that adolescents with type 1 diabetes have adipose, hepatic, and muscle IR, and that metformin lowers daily insulin dose, suggesting improved IR. However, whether metformin improves IR in muscle, hepatic, or adipose tissues in type 1 diabetes was unknown., Objective: Measure peripheral, hepatic, and adipose insulin sensitivity before and after metformin or placebo therapy in youth with obesity with type 1 diabetes., Design: Double-blind, placebo-controlled clinical trial., Setting: Multi-center at eight sites of the T1D Exchange Clinic Network., Participants: A subset of 12- to 19-year-olds with type 1 diabetes (inclusion criteria: body mass index ≥85th percentile, HbA1c 7.5% to 9.9%, insulin dosing ≥0.8 U/kg/d) from a larger trial (NCT02045290) were enrolled., Intervention: Participants were randomized to 3 months of metformin (N = 19) or placebo (N = 18) and underwent a three-phase hyperinsulinemic euglycemic clamp with glucose and glycerol isotope tracers to assess tissue-specific IR before and after treatment., Main Outcome Measures: Peripheral insulin sensitivity, endogenous glucose release, rate of lipolysis., Results: Between-group differences in change in insulin sensitivity favored metformin regarding whole-body IR [change in glucose infusion rate 1.3 (0.1, 2.4) mg/kg/min, P = 0.03] and peripheral IR [change in metabolic clearance rate 0.923 (-0.002, 1.867) dL/kg/min, P = 0.05]. Metformin did not impact insulin suppression of endogenous glucose release (P = 0.12). Adipose IR was not assessable with traditional methods in this highly IR population., Conclusions: Metformin appears to improve whole-body and peripheral IR in youth who are overweight/obese with type 1 diabetes., (Copyright © 2019 Endocrine Society.)

Objective: Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance (IR) and visceral adiposity in adults and boys, but girls with NAFLD are understudied. We sought to evaluate adipose, liver, and skeletal muscle insulin sensitivity in obese adolescent females with or without hepatic steatosis (HS) (intrahepatic triglyceride (IHTG) content >5.5%) along with cardiometabolic components typically associated with IR., Study Design: 73 obese adolescent girls at high risk for NAFLD were enrolled. Participants underwent fasting labs, an MRI to measure IHTG and visceral fat, 31 phosphorous MR spectroscopy for muscle mitochondrial function, 1 H MR spectroscopy for intramyocellular lipid (IMCL), bicycle ergometry to assess VO 2 peak and a 4-phase hyperinsulinemic euglycemic clamp with isotope tracers to measure hepatic and peripheral IR. 29 participants had HS [age 15 yrs(13,16), BMI%ile 98.7(97.4,99.0), IHTG 10.4%(8.0,13.5)] and 44 did not [age 15 yrs(13,17), BMI%ile 98.5(96.2,99.0), IHTG 2.0%(1.1,3.0)]., Results: During hyperinsulinemia, participants with HS vs. non-HS had failure to suppress free fatty acids (p = 0.008), endogenous glucose release (p = 0.002), and a lower glucose metabolic rate of disappearance (Rd) (p = 0.012). Girls with NALFD also had higher visceral fat (p < 0.001), systolic blood pressure (p = 0.026), triglycerides (p = 0.02), ALT (p < 0.01) and white blood cell count (p < 0.01), and lower adiponectin (p = 0.02). There was no difference between girls with and without HS in systemic glycerol turnover measured with glycerol release, or in IMCL, mitochondrial function or VO 2 peak., Conclusions: Obese adolescent girls with HS have evidence of multi-tissue IR, visceral adiposity, inflammation and multiple components of the metabolic syndrome, arguing for close cardiometabolic surveillance over time of girls with HS., (© 2019 The Author(s).)

Polycystic ovarian syndrome (PCOS) is associated with insulin resistance (IR) and altered muscle mitochondrial oxidative phosphorylation. IR in adults with obesity and diabetes is associated with changes in amino acid, free fatty acid (FFA), and mitochondrial acylcarnitine (AC) metabolism. We sought to determine whether these metabolites are associated with IR and/or androgens in youth-onset PCOS. We enrolled obese girls with PCOS [ n = 15, 14.5 yr (SD 1.6), %BMI 98.5 (SD 1.0)] and without PCOS [ n = 6, 13.2 yr (SD 1.2), %BMI 98.0 (SD 1.1)]. Insulin sensitivity was assessed by hyperinsulinemic euglycemic clamp. Untargeted metabolomics of plasma was performed while fasting and during hyperinsulinemia. Fasting arginine, glutamine, histidine, lysine, phenylalanine, and tyrosine were higher ( P < 0.04 for all but P < 0.001 for valine), as were glutamine and histidine during hyperinsulinemia ( P < 0.03). Higher valine during hyperinsulinemia was associated with IR ( r = 0.59, P = 0.006). Surprisingly, end-clamp AC C4 was lower in PCOS, and lower C4 was associated with IR ( r = -0.44, P = 0.04). End-clamp FFAs of C14:0, C16:1, and C18:1 were higher in PCOS girls, and C16:1 and C18:1 strongly associated with IR ( r = 0.73 and 0.53, P < 0.01). Free androgen index related negatively to short-, medium-, and long-chain AC ( r = -0.41 to -0.71, P < 0.01) but not FFA or amino acids. Obese girls with PCOS have a distinct metabolic signature during fasting and hyperinsulinemia. As in diabetes, IR related to valine and FFAs, with an unexpected relationship with AC C4, suggesting unique metabolism in obese girls with PCOS.

Intermuscular adipose tissue (IMAT) is negatively related to insulin sensitivity, but a causal role of IMAT in the development of insulin resistance is unknown. IMAT was sampled in humans to test for the ability to induce insulin resistance in vitro and characterize gene expression to uncover how IMAT may promote skeletal muscle insulin resistance. Human primary muscle cells were incubated with conditioned media from IMAT, visceral (VAT), or subcutaneous adipose tissue (SAT) to evaluate changes in insulin sensitivity. RNAseq analysis was performed on IMAT with gene expression compared with skeletal muscle and SAT, and relationships to insulin sensitivity were determined in men and women spanning a wide range of insulin sensitivity measured by hyperinsulinemic-euglycemic clamp. Conditioned media from IMAT and VAT decreased insulin sensitivity similarly compared with SAT. Multidimensional scaling analysis revealed distinct gene expression patterns in IMAT compared with SAT and muscle. Pathway analysis revealed that IMAT expression of genes in insulin signaling, oxidative phosphorylation, and peroxisomal metabolism related positively to donor insulin sensitivity, whereas expression of macrophage markers, inflammatory cytokines, and secreted extracellular matrix proteins were negatively related to insulin sensitivity. Perilipin 5 gene expression suggested greater IMAT lipolysis in insulin-resistant individuals. Combined, these data show that factors secreted from IMAT modulate muscle insulin sensitivity, possibly via secretion of inflammatory cytokines and extracellular matrix proteins, and by increasing local FFA concentration in humans. These data suggest IMAT may be an important regulator of skeletal muscle insulin sensitivity and could be a novel therapeutic target for skeletal muscle insulin resistance.

People commonly increase sleep duration on the weekend to recover from sleep loss incurred during the workweek. Whether ad libitum weekend recovery sleep prevents metabolic dysregulation caused by recurrent insufficient sleep is unknown. Here, we assessed sleep, circadian timing, energy intake, weight gain, and insulin sensitivity during sustained insufficient sleep (9 nights) and during recurrent insufficient sleep following ad libitum weekend recovery sleep. Healthy, young adults were randomly assigned to one of three groups: (1) control (CON; 9-h sleep opportunities, n = 8), (2) sleep restriction without weekend recovery sleep (SR; 5-h sleep opportunities, n = 14), and (3) sleep restriction with weekend recovery sleep (WR; insufficient sleep for 5-day workweek, then 2 days of weekend recovery, then 2 nights of insufficient sleep, n = 14). For SR and WR groups, insufficient sleep increased after-dinner energy intake and body weight versus baseline. During ad libitum weekend recovery sleep, participants cumulatively slept ∼1.1 h more than baseline, and after-dinner energy intake decreased versus insufficient sleep. However, during recurrent insufficient sleep following the weekend, the circadian phase was delayed, and after-dinner energy intake and body weight increased versus baseline. In SR, whole-body insulin sensitivity decreased ∼13% during insufficient sleep versus baseline, and in WR, whole-body, hepatic, and muscle insulin sensitivity decreased ∼9%-27% during recurrent insufficient sleep versus baseline. Furthermore, during the weekend, total sleep duration was lower in women versus men, and energy intake decreased to baseline levels in women but not in men. Our findings suggest that weekend recovery sleep is not an effective strategy to prevent metabolic dysregulation associated with recurrent insufficient sleep., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Adolescents with type 2 diabetes (T2D) have severe insulin resistance (IR) secondary to obesity, genetics, and puberty, and IR predicts metabolic comorbidities. Adults with T2D have multitissue IR, which has guided therapeutic developments, but this is not established in youth. We sought to assess adipose, hepatic, and peripheral insulin sensitivity in adolescents with and without T2D. Twenty-seven youth with T2D [age: 15.6 ± 0.4 yr; female: 78%; body mass index (BMI) percentile: 96.1 (52.6, 95.9), late puberty; hemoglobin A1c (HbA1c) 7.3% (6.2, 10.1)] and 21 controls of similar BMI, pubertal stage, and habitual activity were enrolled. Insulin action was measured with a four-phase hyperinsulinemic-euglycemic clamp (basal, 10, 16, and 80 mU·m -2 ·min -1 for studying adipose, hepatic, and peripheral IR, respectively) with glucose and glycerol isotope tracers. Total fat mass, fat-free mass, liver fat fraction, and visceral fat were measured with dual-energy x-ray absorptiometry (DXA) and MRI, respectively. Free fatty acids (FFAs), lipid profile, and inflammatory markers were also measured. Adolescents with T2D had higher lipolysis ( P = 0.012), endogenous glucose production ( P < 0.0001), and lower glucose clearance ( P = 0.002) during hyperinsulinemia than controls. In T2D, peripheral IR positively correlated to FFA ( P < 0.001), inflammatory markers, visceral ( P = 0.004) and hepatic fat ( P = 0.007); hepatic IR correlated with central obesity ( P = 0.004) and adipose IR ( P = 0.003). Youth with T2D have profound multitissue IR compared with BMI-equivalent youth without T2D. The development of multitissue interactions appears crucial to the pathogenesis of T2D. Therapeutic targets on multitissue IR may be of benefit, deserving of further research.

HPLC-MS/MS has enabled the quantitative analysis of complex mixtures of lipid molecular species. Several separate analyses, using methods that have been optimized for individual lipid classes, provide good lipidomic profiles, but may not be desirable for laboratories constrained by available instrumentation and wanting a higher throughput. Here we describe two methods using binary gradient HiLiC HPLC and triple quadrupole MS that together provide a lipidomic profile for lipids of interest in type 2 diabetes research. Methods for analysis of molecular species of diacylglycerol, ceramide, dihydroceramide, sphingosine, glucosyl- and lactosylceramide, sphingomyelin, and acylcarnitine from skeletal muscle and primary culture cells are described.

Ras-related C3 botulinum toxin substrate 1 (Rac1) is required for normal insulin-stimulated glucose transport in skeletal muscle and evidence indicates Rac1 may be negatively regulated by lipids. We investigated if insulin-stimulated activation of Rac1 (i.e., Rac1-GTP binding) is impaired by accumulation of diacylglycerols (DAG) and ceramides in cultured muscle cells. Treating L6 myotubes with 100 nmol/L insulin resulted in increased Rac1-GTP binding that was rapid (occurring within 2 min), relatively modest (+38 ± 19% vs. basal, P < 0.001), and short-lived, returning to near-basal levels within 15 min of continuous treatment. Incubating L6 myotubes overnight in 500 μmol/L palmitate increased the accumulation of DAG and ceramides (P < 0.05 vs. no fatty acid control). Despite significant accumulation of lipids, insulin-stimulated Rac1-GTP binding was not impaired during palmitate treatment (P = 0.39 vs. no fatty acid control). Nevertheless, phosphorylation of Rac1 effector protein p21-activated kinase (PAK) was attenuated in response to palmitate treatment (P = 0.02 vs. no fatty acid control). Palmitate treatment also increased inhibitory phosphorylation of insulin receptor substrate-1 and attenuated insulin-stimulated phosphorylation of Akt at both Thr308 and Ser473 (all P < 0.05 vs. no fatty acid control). Such signaling impairments resulted in near complete inhibition of insulin-stimulated translocation of glucose transporter protein 4 (GLUT4; P = 0.10 vs. basal during palmitate treatment). In summary, our finding suggests that Rac1 may not undergo negative regulation by DAG or ceramides. We instead provide evidence that attenuated PAK phosphorylation and impaired GLUT4 translocation during palmitate-induced insulin resistance can occur independent of defects in insulin-stimulated Rac1-GTP binding., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Objective: Non-alcoholic fatty liver disease (NAFLD) risk begins in utero in offspring of obese mothers. A critical unmet need in this field is to understand the pathways and biomarkers underlying fetal hepatic lipotoxicity and whether maternal dietary intervention during pregnancy is an effective countermeasure., Methods: We utilized a well-established non-human primate model of chronic, maternal, Western-style diet induced obesity (OB-WSD) compared with mothers on a healthy control diet (CON) or a subset of OB-WSD mothers switched to the CON diet (diet reversal; OB-DR) prior to and for the duration of the next pregnancy. Fetuses were studied in the early 3rd trimester., Results: Fetuses from OB-WSD mothers had higher circulating triglycerides (TGs) and lower arterial oxygenation suggesting hypoxemia, compared with fetuses from CON and OB-DR mothers. Hepatic TG content, oxidative stress (TBARs), and de novo lipogenic genes were increased in fetuses from OB-WSD compared with CON mothers. Fetuses from OB-DR mothers had lower lipogenic gene expression and TBARs yet persistently higher TGs. Metabolomic profiling of fetal liver and serum (umbilical artery) revealed distinct separation of CON and OB-WSD groups, and an intermediate phenotype in fetuses from OB-DR mothers. Pathway analysis identified decreased tricarboxylic acid cycle intermediates, increased amino acid (AA) metabolism and byproducts, and increased gluconeogenesis, suggesting an increased reliance on AA metabolism to meet energy needs in the liver of fetuses from OB-WSD mothers. Components in collagen synthesis, including serum protein 5-hydroxylysine and hepatic lysine and proline, were positively correlated with hepatic TGs and TBARs, suggesting early signs of fibrosis in livers from the OB-WSD group. Importantly, hepatic gluconeogenic and arginine related intermediates and serum levels of lactate, pyruvate, several AAs, and nucleotide intermediates were normalized in the OB-DR group. However, hepatic levels of CDP-choline and total ceramide levels remained high in fetuses from OB-DR mothers., Conclusions: Our data provide new metabolic evidence that, in addition to fetal hepatic steatosis, maternal WSD creates fetal hypoxemia and increases utilization of AAs for energy production and early activation of gluconeogenic pathways in the fetal liver. When combined with hyperlipidemia and limited antioxidant activity, the fetus suffers from hepatic oxidative stress and altered intracellular metabolism which can be improved with maternal diet intervention. Our data reinforce the concept that multiple "first hits" occur in the fetus prior to development of obesity and demonstrate new biomarkers with potential clinical implications for monitoring NAFLD risk in offspring., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Context: Adolescents with type 1 diabetes (T1D) have difficulty obtaining optimal glucose control, which may relate to insulin resistance (IR), especially during puberty. Moreover, IR increases the risk for cardiovascular disease, the leading cause of death in T1D. However, the tissue specificity of IR in adolescents with T1D has not been fully phenotyped., Objective: To assess adipose, hepatic, and peripheral insulin sensitivity in adolescents with and without T1D., Design and Setting: Thirty-five youth with T1D [median age, 16 (first and third quartiles, 14, 17) years; 53% female; median body mass index (BMI) percentile, 82nd (55th, 96th); late puberty; median hemoglobin A1c, 8.3% (7.3%, 9.4%)] and 22 nondiabetic youth of similar age, BMI, pubertal stage, and level of habitual physical activity were enrolled. Insulin action was measured with a four-phase hyperinsulinemic euglycemic clamp (basal and 10, 16, and 80 mU/m2/min) with glucose and glycerol isotope tracers., Results: Adolescents with T1D had a significantly higher rate of lipolysis (P < 0.0001) and endogenous glucose production (P < 0.001) and lower peripheral glucose uptake (glucose rate of disappearance, 6.9 ± 2.9 mg/kg/min for patients with T1D vs 11.3 ± 3.3 for controls; P < 0.0001) during hyperinsulinemia compared with controls. In youth with T1D, glucose rate of disappearance correlated with free fatty acid at the 80-mU/m2/min phase (P = 0.005), markers of inflammation (IL-6; P = 0.012), high-sensitivity C-reactive protein (P = 0.001), and leptin (P = 0.008)], but not hemoglobin A1c., Conclusions: Adolescents with T1D have adipose, hepatic and peripheral IR. This IR occurs regardless of obesity and metabolic syndrome features. Youth with T1D may benefit from interventions directed at improving IR in these tissues, and this area requires further research.

Obese adolescent girls are at increased risk for type 2 diabetes, characterized by defects in insulin secretion and action. We sought to determine if later glucose peak timing (>30 minutes), 1-hour glucose >155 mg/dl, or monophasic pattern of glucose excursion during an oral glucose tolerance test (OGTT) reflect a worse cardiometabolic risk profile. Post-pubertal overweight/obese adolescent girls without diabetes were studied (N = 88; age, 15.2 ± 0.2 years; body mass index percentile, 97.7 ± 0.5). All participants completed an OGTT and body composition measures. Thirty-two girls had a four-phase hyperinsulinemic euglycemic clamp with isotope tracers, vascular imaging, and muscle mitochondrial assessments. Participants were categorized by glucose peak timing (≤30 min = early; >30 min = late), 1-hour glucose concentration (±155 mg/dL) and glucose pattern (monophasic, biphasic). Girls with a late (N = 54) vs earlier peak (n = 34) timing had higher peak glucose ( P < 0.001) and insulin ( P = 0.023), HbA1c ( P = 0.021); prevalence of hepatic steatosis (62% vs 26%; P = 0.003) and lower oral disposition index ( P < 0.001) and glucagon-like peptide-1 response ( P = 0.037). When classified by 1-hour glucose, group differences were similar to peak timing, but minimal when classified by glucose pattern. In the >155 mg/dL group only, peripheral insulin sensitivity and fasting free fatty acids were worse. A later glucose peak or >155 mg/dL 1-hour glucose predicts metabolic disease risk in obese adolescent girls. This may defect incretin effects and first phase insulin response, and muscle and adipose insulin resistance.

Objective: To test the hypothesis that multitissue deficits in insulin sensitivity are greater among women than men with type 1 diabetes compared to respective controls., Research Design and Methods: Three-stage hyperinsulinemic-euglycemic clamps (4, 8, 40 mU/m 2 /min) were performed on 41 people with type 1 diabetes and 47 adults without diabetes (mean ± SD age 46 ± 8). Infusions of [1- 13 C]palmitate, [1,1,2,3,3- 2 H 2 ]glycerol, and [6,6- 2 H 2 ]glucose isotope tracers were used to determine free fatty acid (FFA), glycerol, and glucose kinetics in 52 of these participants (25 M and 27 W)., Results: There was no difference in age or BMI by type 1 diabetes status in either sex. Free fatty acid rate of appearance (FFA Ra) was higher in both sexes with type 1 diabetes compared to those without diabetes during stages 1 and 2. The same was seen with glycerol for stages 1 and 2. During stage 3 glucose rate of disappearance (Rd) was lower in those with type 1 diabetes among both sexes. All had sex by type 1 diabetes interactions with greater deficits in insulin sensitivity in women. While there was no sex by diabetes interaction in regards to glucose rate of appearance (Ra), those with type 1 diabetes had a higher glucose Ra than those without diabetes., Conclusions: We found that type 1 diabetes affected adipose and skeletal muscle insulin sensitivity to a greater extent in women than in men, perhaps contributing to the greater relative increase in cardiovascular risk in women with type 1 diabetes., (Copyright © 2018. Published by Elsevier Inc.)

Background: Accumulation of diacylglycerol (DAG) and sphingolipids is thought to promote skeletal muscle insulin resistance by altering cellular signaling specific to their location. However,the subcellular localization of bioactive lipids in human skeletal muscle is largely unknown., Methods: We evaluated subcellular localization of skeletal muscle DAGs and sphingolipids in lean individuals (n = 15), endurance-trained athletes (n = 16), and obese men and women with (n = 12) and without type 2 diabetes (n = 15). Muscle biopsies were fractionated into sarcolemmal, cytosolic, mitochondrial/ER, and nuclear compartments. Lipids were measured using liquid chromatography tandem mass spectrometry, and insulin sensitivity was measured using hyperinsulinemic-euglycemic clamp., Results: Sarcolemmal 1,2-DAGs were not significantly related to insulin sensitivity. Sarcolemmal ceramides were inversely related to insulin sensitivity, with a significant relationship found for the C18:0 species. Sarcolemmal sphingomyelins were also inversely related to insulin sensitivity, with the strongest relationships found for the C18:1, C18:0, and C18:2 species. In the mitochondrial/ER and nuclear fractions, 1,2-DAGs were positively related to, while ceramides were inversely related to, insulin sensitivity. Cytosolic lipids as well as 1,3-DAG, dihydroceramides, and glucosylceramides in any compartment were not related to insulin sensitivity. All sphingolipids but only specific DAGs administered to isolated mitochondria decreased mitochondrial state 3 respiration., Conclusion: These data reveal previously unknown differences in subcellular localization of skeletal muscle DAGs and sphingolipids that relate to whole-body insulin sensitivity and mitochondrial function in humans. These data suggest that whole-cell concentrations of lipids obscure meaningful differences in compartmentalization and suggest that subcellular localization of lipids should be considered when developing therapeutic interventions to treat insulin resistance., Funding: National Institutes of Health General Clinical Research Center (RR-00036), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01DK089170), NIDDK (T32 DK07658), and Colorado Nutrition Obesity Research Center (P30DK048520).

Intramuscular triglyceride (IMTG) concentration is elevated in insulin-resistant individuals and was once thought to promote insulin resistance. However, endurance-trained athletes have equivalent concentration of IMTG compared with individuals with type 2 diabetes, and have very low risk of diabetes, termed the "athlete's paradox." We now know that IMTG synthesis is positively related to insulin sensitivity, but the exact mechanisms for this are unclear. To understand the relationship between IMTG synthesis and insulin sensitivity, we measured IMTG synthesis in obese control subjects, endurance-trained athletes, and individuals with type 2 diabetes during rest, exercise, and recovery. IMTG synthesis rates were positively related to insulin sensitivity, cytosolic accumulation of DAG, and decreased accumulation of C18:0 ceramide and glucosylceramide. Greater rates of IMTG synthesis in athletes were not explained by alterations in FFA concentration, DGAT1 mRNA expression, or protein content. IMTG synthesis during exercise in Ob and T2D indicate utilization as a fuel despite unchanged content, whereas IMTG concentration decreased during exercise in athletes. mRNA expression for genes involved in lipid desaturation and IMTG synthesis were increased after exercise and recovery. Further, in a subset of individuals, exercise decreased cytosolic and membrane di-saturated DAG content, which may help explain insulin sensitization after acute exercise. These data suggest IMTG synthesis rates may influence insulin sensitivity by altering intracellular lipid localization, and decreasing specific ceramide species that promote insulin resistance.

Objective: Obese girls with polycystic ovarian syndrome (PCOS) have decreased insulin sensitivity (IS), muscle mitochondrial dysfunction and increased liver fat, which may contribute to their increased risk for type 2 diabetes. Less is known regarding normal-weight girls with PCOS., Methods: Normal-weight girls with PCOS [n =18, age 15.9 ± 1.8 years, body mass index (BMI) percentile 68 ± 18] and normal-weight controls (NWC; n = 20; age 15.0 ± 2.1 years, BMI percentile 60 ± 21) were studied. Tissue-specific IS was assessed with a four-phase hyperinsulinemic-euglycemic clamp with isotope tracers and a 2-hour oral glucose tolerance test (OGTT). Hepatic fat was determined using magnetic resonance imaging. Postexercise muscle mitochondrial function was assessed with 31 P MR spectroscopy., Results: Both groups had similar demographics, anthropomorphics, physical attributes, habitual physical activity levels and fasting laboratory values, except for increased total testosterone and DHEAS in PCOS. Clamp-assessed peripheral IS was lower in PCOS (10.4 ± 2.4 mg/kg/min vs 12.7 ± 2.1; P = 0.024). The 120-minute OGTT insulin and glucose concentrations were higher in PCOS (114 IU/mL ± 26 vs 41 ± 25, P = <0.001 and 119 ± 22 mg/dL vs 85 ± 23, P = 0.01, respectively). Muscle mitochondrial ADP and phosphocreatine time constants were slower in PCOS. Despite a higher percentage liver fat in PCOS, hepatic IS was similar between groups, as was adipose IS., Conclusions: Normal-weight girls with PCOS have decreased peripheral IS and muscle mitochondrial dysfunction, abnormal glucose disposal, relative postprandial hyperinsulinemia, and increased hepatic fat compared to NWC. Despite a normal BMI, multiple aspects of metabolism appear altered in normal-weight girls with PCOS.

Understanding the dynamics of autophagy and apoptosis crosstalk in cancer progression remains a challenging task. Here, we reported how the autophagy protein ATG14 induces lipophagy-mediated mitochondrial apoptosis. The overexpression of ATG14 in HeLa cells inhibited cell viability and increased mitochondrial apoptosis and endoplasmic reticulum (ER) stress. Furthermore, inhibition of this ATG14-induced autophagy promoted apoptosis. ATG14 overexpression resulted in the accumulation of free fatty acids (FFA), with a concomitant decrease in the number of lipid droplets. Our data showed that ER stress induced by ATG14 was due to the lipophagy-mediated FFA accumulation, which resulted in ROS-dependent mitochondrial stress leading to apoptosis. Inhibition of lipophagy in HeLa-ATG14 cells enhanced the cellular viability and rescued them from lipotoxicity. Mechanistically, we found that ATG14 interacted with Ulk1 and LC3, and knock down of Ulk1 prevented the lipidation of LC3 and autophagy in HeLa-ATG14 cells. We also identified a phosphatidylethanolamine (PE) binding region in ATG14, and the addition of Ulk1 to Hela-ATG14 cells decreased the ATG14-PE interaction. Lastly, confocal microscopy studies showed that the decrease in ATG14-PE binding was concomitant with the increase in LC3 lipidation over time, confirming the importance of Ulk1 to sort PE to LC3 during ATG14 mediated lipophagy induction. In conclusion, ATG14 and Ulk1 interact to induce lipophagy resulting in FFA accumulation leading to ER stress-mediated apoptosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: Increased liver fat and type 2 diabetes are prevalent in women with polycystic ovarian syndrome (PCOS) and cause excess mortality, yet little is known about their development during adolescence. The objective of this study was to measure hepatic steatosis and related metabolic contributors in girls with obesity, with and without PCOS., Methods: Nondiabetic adolescents with obesity, 41 with PCOS (PCOS; age 15.0 [13.0-16.0] years, BMI 35.2 ± 0.61 kg/m 2 ) and 30 without PCOS (OB; age 14.5 [13.0-17.0], BMI 33.2 ± 1.8), were studied. Visceral and liver fat were assessed with MRI. Serum measures included androgens and 16:1 and 18:1 N7 fatty acids specific to de novo lipogenesis. Adipose, hepatic, and peripheral insulin sensitivity (IS) were assessed with a four-phase hyperinsulinemic-euglycemic clamp with isotope tracers., Results: Forty-nine percent of the PCOS group had hepatic steatosis versus fourteen percent of the OB group (P = 0.02), and the PCOS group had higher N7 (43 ± 4 vs. 29 ± 5 nmol/g; P = 0.02). Peripheral IS was lower in PCOS (9.4 [7.2-12.3] vs. 14.5 [13.1-18.05 mg/lean kg/min]; P < 0.001) as was hepatic (P = 0.006) and adipose IS (P = 0.005). Percent liver fat correlated with N7 (R = 0.46, P = 0.02) and visceral fat (R = 0.42, P < 0.001), not androgens or peripheral IS., Conclusions: Nearly 50% of nondiabetic girls with PCOS and obesity have hepatic steatosis, which relates to visceral fat and lipogenesis, but not to IS or androgens., Competing Interests: The authors declare no conflict of interest, (© 2016 The Obesity Society.)