Intramuscular diacylglycerol accumulates with acute hyperinsulinemia in insulin-resistant phenotypes.

Elevated skeletal muscle diacylglycerols (DAG) and ceramides can impair insulin signaling, and acylcarnitines (acylCN) reflect impaired fatty acid oxidation, thus the intramuscular lipid profile is indicative of insulin resistance. Acute (i.e., postprandial) hyperinsulinemia has been shown to elevate lipids in healthy muscle and is an independent risk factor for type 2 diabetes (T2D). It is unclear how the relationship between acute hyperinsulinemia and the muscle lipidome interacts, thus contributing to or exacerbating insulin resistance. We investigated the impact of acute hyperinsulinemia on the muscle lipidome in order to help characterize the physiological basis in which hyperinsulinemia elevates T2D risk. 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. While there were no significant differences in total 1,2-DAG fluctuations, there was a 2% decrease in athletes versus a 53% increase in T2D. C18 1,2-DAGs increased during the clamp with T2D only, which negatively correlated with insulin sensitivity. Basal muscle C18:0 ceramides were elevated with T2D, but not altered by clamp. Acylcarnitines were universally lowered during hyperinsulinemia, with more robust reductions of 80% in athletes compared to only 46% with T2D. 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.

Deoxysphingolipids: Atypical Skeletal Muscle Lipids Related to Insulin Resistance in Humans That Decrease Insulin Sensitivity In Vitro.

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 ß-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.

Deoxysphingolipids - atypical skeletal muscle lipids related to insulin resistance in humans that decrease insulin sensitivity in vitro.

Sphingolipids are thought to promote skeletal muscle insulin resistance. 1-Deoxysphingolipids (dSL) are atypical sphingolipids that are increased in plasma of individuals with type 2 diabetes and cause ß-cell dysfunction in vitro. However, their role in human skeletal muscle in unknown. We found that dSL species are significantly elevated in muscle of individuals with obesity and type 2 diabetes compared to 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 AMP-activated kinase (AMPK) phosphorylation, and altered insulin signaling. Our findings reveal a central role for dSL in human muscle insulin resistance and suggest dSL as therapeutic targets for the treatment and prevention of type 2 diabetes.

Skeletal muscle and intermuscular adipose tissue gene expression profiling identifies new biomarkers with prognostic significance for insulin resistance progression and intervention response.

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.

Quantifying the inflammatory secretome of human intermuscular adipose tissue.

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 (PGE2, TXB2 , 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.

Exploring Visceral and Subcutaneous Adipose Tissue Secretomes in Human Obesity: Implications for Metabolic Disease.

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.

Utilization of Mid-Thigh Magnetic Resonance Imaging to Predict Lean Body Mass and Knee Extensor Strength in Obese Adults.

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/m2, 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.

Keeping It Local in Metabolic Disease: Adipose Tissue Paracrine Signaling and Insulin Resistance.

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.

Subcellular localisation and composition of intramuscular triacylglycerol influence insulin sensitivity in humans.

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.

Compensatory eating behaviors in male and female rats in response to exercise training.

Exercise is often used as a strategy for weight loss maintenance. In preclinical models, we have shown that exercise may be beneficial because it counters the biological drive to regain weight. However, our studies have demonstrated sex differences in the response to exercise in this context. In the present study, we sought to better understand why females and males exhibit different compensatory food eating behaviors in response to regular exercise. Using a forced treadmill exercise paradigm, we measured weight gain, energy expenditure, food intake in real time, and the anorectic effects of leptin. The 4-wk exercise training resulted in reduced weight gain in males and sustained weight gain in females. In male rats, exercise decreased intake, whereas it increased food intake in females. Our results suggest that the anorectic effects of leptin were not responsible for these sex differences in appetite in response to exercise. If these results translate to the human condition, they may reveal important information for the use and application of regular exercise programs.

Palmitic acid triggers inflammatory responses in N42 cultured hypothalamic cells partially via ceramide synthesis but not via TLR4.

A high-fat diet induces hypothalamic inflammation in rodents which, in turn, contributes to the development of obesity by eliciting both insulin and leptin resistance. However, the mechanism by which long-chain saturated fatty acids trigger inflammation is still contentious. To elucidate this mechanism, the effect of fatty acids on the expression of the pro-inflammatory cytokines IL-6 and TNFα was investigated in the mHypoE-N42 hypothalamic cell line (N42). N42 cells were treated with lauric acid (LA) and palmitic acid (PA). PA challenge was carried out in the presence of either a TLR4 inhibitor, a ceramide synthesis inhibitor (L-cycloserine), oleic acid (OA) or eicosapentaenoic acid (EPA). Intracellular ceramide accumulation was quantified using LC-ESI-MS/MS. PA but not LA upregulated IL-6 and TNFα. L-cycloserine, OA and EPA all counteracted PA-induced intracellular ceramide accumulation leading to a downregulation of IL-6 and TNFα. However, a TLR4 inhibitor failed to inhibit PA-induced upregulation of pro-inflammatory cytokines.In conclusion, PA induced the expression of IL-6 and TNFα in N42 neuronal cells independently of TLR4 but, partially, via ceramide synthesis with OA and EPA being anti-inflammatory by decreasing PA-induced intracellular ceramide build-up. Thus, ceramide accumulation represents one on the mechanisms by which PA induces inflammation in neurons.

Trisomy 21 activates the kynurenine pathway via increased dosage of interferon receptors.

Trisomy 21 (T21) causes Down syndrome (DS), affecting immune and neurological function by ill-defined mechanisms. Here we report a large metabolomics study of plasma and cerebrospinal fluid, showing in independent cohorts that people with DS produce elevated levels of kynurenine and quinolinic acid, two tryptophan catabolites with potent immunosuppressive and neurotoxic properties, respectively. Immune cells of people with DS overexpress IDO1, the rate-limiting enzyme in the kynurenine pathway (KP) and a known interferon (IFN)-stimulated gene. Furthermore, the levels of IFN-inducible cytokines positively correlate with KP dysregulation. Using metabolic tracing assays, we show that overexpression of IFN receptors encoded on chromosome 21 contribute to enhanced IFN stimulation, thereby causing IDO1 overexpression and kynurenine overproduction in cells with T21. Finally, a mouse model of DS carrying triplication of IFN receptors exhibits KP dysregulation. Together, our results reveal a mechanism by which T21 could drive immunosuppression and neurotoxicity in DS.

Metformin Improves Peripheral Insulin Sensitivity in Youth With Type 1 Diabetes.

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.

Intermuscular adipose tissue directly modulates skeletal muscle insulin sensitivity in humans.

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.

SerpinA3N is a novel hypothalamic gene upregulated by a high-fat diet and leptin in mice.

BACKGROUND: Energy homeostasis is regulated by the hypothalamus but fails when animals are fed a high-fat diet (HFD), and leptin insensitivity and obesity develops. To elucidate the possible mechanisms underlying these effects, a microarray-based transcriptomics approach was used to identify novel genes regulated by HFD and leptin in the mouse hypothalamus. RESULTS: Mouse global array data identified serpinA3N as a novel gene highly upregulated by both a HFD and leptin challenge. In situ hybridisation showed serpinA3N expression upregulation by HFD and leptin in all major hypothalamic nuclei in agreement with transcriptomic gene expression data. Immunohistochemistry and studies in the hypothalamic clonal neuronal cell line, mHypoE-N42 (N42), confirmed that alpha 1-antichymotrypsin (α1AC), the protein encoded by serpinA3, is localised to neurons and revealed that it is secreted into the media. SerpinA3N expression in N42 neurons is upregulated by palmitic acid and by leptin, together with IL-6 and TNFα, and all three genes are downregulated by the anti-inflammatory monounsaturated fat, oleic acid. Additionally, palmitate upregulation of serpinA3 in N42 neurons is blocked by the NFκB inhibitor, BAY11, and the upregulation of serpinA3N expression in the hypothalamus by HFD is blunted in IL-1 receptor 1 knockout (IL-1R1 -/- ) mice. CONCLUSIONS: These data demonstrate that serpinA3 expression is implicated in nutritionally mediated hypothalamic inflammation.

Intracellular localization of diacylglycerols and sphingolipids influences insulin sensitivity and mitochondrial function in human skeletal muscle.

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).