Human peripancreatic adipose tissue paracrine signaling impacts insulin secretion, blood flow, and gene transcription.

CONTEXT: Adipose tissue accumulation around non-adipose tissues is associated with obesity and metabolic disease. One relatively unstudied depot is peripancreatic adipose tissue (PAT) that accumulates in obesity and insulin resistance and may impact beta cell function. Pancreatic lipid accumulation and PAT content are negatively related to metabolic outcomes in humans, but these studies are limited by the inability to pursue mechanisms. OBJECTIVE: We obtained PAT from human donors through the Human Pancreas Analysis Program to evaluate differences in paracrine signaling compared to subcutaneous adipose tissue (SAT), as well as effects of the PAT secretome on aortic vasodilation, human islet insulin secretion, and gene transcription using RNAseq. RESULTS: PAT had greater secretion of IFN-γ and most inflammatory eicosanoids compared to SAT. Secretion of adipokines negatively related to metabolic health were also increased in PAT compared to SAT. We found no overall effects of PAT compared to SAT on human islet insulin secretion, however, insulin secretion was suppressed after PAT exposure from men compared to women. Vasodilation was significantly dampened by PAT conditioned media, an effect explained almost completely by PAT from men and not women. Islets treated with PAT showed selective changes in lipid metabolism pathways while SAT altered cellular signaling and growth. RNAseq analysis showed changes in islet gene transcription impacted by PAT compared to SAT, with the biggest changes found between PAT based on sex. CONCLUSION: The PAT secretome is metabolically negative compared to SAT, and impacts islet insulin secretion, blood flow, and gene transcription in a sex dependent manner.

Maternal Docosahexaenoic Acid Supplementation Alters Maternal and Fetal Docosahexaenoic Acid Status and Placenta Phospholipids in Pregnancies Complicated by High Body Mass Index.

INTRODUCTION: An optimal fetal supply of docosahexaenoic acid (DHA) is critical for normal brain development. The relationship between maternal DHA intake and DHA delivery to the fetus is complex and is dependent on placental handling of DHA. Little data exist on placental DHA levels in pregnancies supplemented with the recommended dose of 200 mg/d. Our objective was to determine how prenatal DHA at the recommended 200 mg/d impacts maternal, placental, and fetal DHA status in both normal-weight and high-BMI women compared to women taking no supplements. METHODS: Maternal blood, placenta, and cord blood were collected from 30 healthy pregnant women (BMI 18.9-43.26 kg/m2) giving birth at term. Red blood cells (RBCs) and villous tissue were isolated, and lipids were extracted to determine DHA content by LC-MS/MS. Data were analyzed by supplement group (0 vs. 200 mg/d) and maternal BMI (normal weight or high BMI) using two-way ANOVA. We measured maternal choline levels in maternal and cord plasma samples. RESULTS: Supplementation with 200 mg/d DHA significantly increased (p < 0.05) maternal and cord RBC DHA content only in pregnancies complicated by high BMI. We did not find any impact of choline levels on maternal or cord RBC phospholipids. There were no significant differences in total placental DHA content by supplementation or maternal BMI (p > 0.05). Placental levels of phosphatidylinositol (PI) and phosphatidic acid containing DHA species were higher (p < 0.05) in high-BMI women without DHA supplementation compared to both normal-BMI and high-BMI women taking DHA supplements. CONCLUSION: Maternal DHA supplementation at recommended doses cord increased RBC DHA content only in pregnancies complicated by higher BMI. Surprisingly, we found that obesity was related to an increase in placental PI and phosphatidic acid species, which was ameliorated by DHA supplementation. Phosphatidic acid activates placental mTOR, which regulates amino acid transport and may explain previous findings of the impact of DHA on placental function. Current recommendations for DHA supplementation may not be achieving the goal of improving fetal DHA levels in normal-weight women.

Oxidised phosphatidylcholine induces sarcolemmal ceramide accumulation and insulin resistance in skeletal muscle.

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.

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

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.

Synthesis of phospholipids in human placenta.

INTRODUCTION: Placental phospholipid synthesis is critical for the expansion of the placental exchange surface area and for production of signaling molecules. Despite their importance, it is not yet established which enzymes involved in the de novo synthesis and remodeling of placental phospholipids are expressed and active in the human placenta. METHODS: We identified phospholipid synthesis enzymes by immunoblotting in placental homogenates and immunofluorescence in placenta tissue sections. Primary human trophoblast (PHT) cells from term healthy placentas (n = 10) were cultured and exposed to 13C labeled fatty acids (16:0, 18:1 and 18:2 n-6, 22:6 n-3) for 2 and 24 h. Three phospholipid classes; phosphatidic acid, phosphatidylcholine, and lysophosphatidylcholine containing 13C fatty acids were quantified by Liquid Chromatography with tandem mass spectrometry (LC/MS-MS). RESULTS: Acyl transferase and phospholipase enzymes were detected in human placenta homogenate and primarily expressed in the syncytiotrophoblast. Three representative 13C fatty acids (16:0, 18:1 and 18:2 n-6) were incorporated rapidly into phosphatidic acid in trophoblasts, but 13C labeled docosahexaenoic acid (DHA; 22:6 n-3) incorporation was not detected. 13C DHA was incorporated into phosphatidylcholine. Lysophosphatidylcholine containing all four 13C labeled fatty acids were found in high abundance. CONCLUSIONS: Phospholipid synthesis and remodeling enzymes are present in the syncytiotrophoblast. 13C labeled fatty acids were rapidly incorporated into cellular phospholipids. 13C DHA was incorporated into phospholipids through the remodeling pathway rather than by de novo synthesis. These understudied pathways are highly active and critical for structure and function of the placenta.

Knockdown of Placental Major Facilitator Superfamily Domain Containing 2a in Pregnant Mice Reduces Fetal Brain Growth and Phospholipid Docosahexaenoic Acid Content.

INTRODUCTION: Docosahexaenoic acid (DHA) is an n-3 long chain polyunsaturated fatty acid critical for fetal brain development that is transported to the fetus from the mother by the placenta. The lysophosphatidylcholine (LPC) transporter, Major Facilitator Superfamily Domain Containing 2a (MFSD2a), is localized in the basal plasma membrane of the syncytiotrophoblast of the human placenta, and MFSD2a expression correlates with umbilical cord blood LPC-DHA levels in human pregnancy. We hypothesized that placenta-specific knockdown of MFSD2a in pregnant mice reduces phospholipid DHA accumulation in the fetal brain. METHODS: Mouse blastocysts (E3.5) were transduced with an EGFP-expressing lentivirus containing either an shRNA targeting MFSD2a or a non-coding sequence (SCR), then transferred to pseudopregnant females. At E18.5, fetuses were weighed and their placenta, brain, liver and plasma were collected. MFSD2a mRNA expression was determined by qPCR in the brain, liver and placenta and phospholipid DHA was quantified by LC-MS/MS. RESULTS: MFSD2a-targeting shRNA reduced placental mRNA MFSD2a expression by 38% at E18.5 (n = 45, p < 0.008) compared with SCR controls. MFSD2a expression in the fetal brain and liver were unchanged. Fetal brain weight was reduced by 13% (p = 0.006). Body weight, placenta and liver weights were unaffected. Fetal brain phosphatidyl choline and phosphatidyl ethanolamine DHA content was lower in fetuses with placenta-specific MFSD2a knockdown. CONCLUSIONS: Placenta-specific reduction in expression of the LPC-DHA transporter MFSD2a resulted in reduced fetal brain weight and lower phospholipid DHA content in the fetal brain. These data provide mechanistic evidence that placental MFSD2a mediates maternal-fetal transfer of LPC-DHA, which is critical for brain growth.

Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

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.

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.

Mitochondrial electron transport chain, ceramide and Coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

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. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results 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 (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.

Altered Cord Blood Lipid Concentrations Correlate with Birth Weight and Doppler Velocimetry of Fetal Vessels in Human Fetal Growth Restriction Pregnancies.

Fetal growth restriction (FGR) is associated with short- and long-term morbidity, often with fetal compromise in utero, evidenced by abnormal Doppler velocimetry of fetal vessels. Lipids are vital for growth and development, but metabolism in FGR pregnancy, where fetuses do not grow to full genetic potential, is poorly understood. We hypothesize that triglyceride concentrations are increased in placentas and that important complex lipids are reduced in cord plasma from pregnancies producing the smallest babies (birth weight < 5%) and correlate with ultrasound Dopplers. Dopplers (umbilical artery, UA; middle cerebral artery, MCA) were assessed longitudinally in pregnancies diagnosed with estimated fetal weight (EFW) < 10% at ≥29 weeks gestation. For a subset of enrolled women, placentas and cord blood were collected at delivery, fatty acids were extracted and targeted lipid class analysis (triglyceride, TG; phosphatidylcholine, PC; lysophosphatidylcholine, LPC; eicosanoid) performed by LCMS. For this sub-analysis, participants were categorized as FGR (Fenton birth weight, BW ≤ 5%) or SGA "controls" (Fenton BW > 5%). FGRs (n = 8) delivered 1 week earlier (p = 0.04), were 29% smaller (p = 0.002), and had 133% higher UA pulsatility index (PI, p = 0.02) than SGAs (n = 12). FGR plasma TG, free arachidonic acid (AA), and several eicosanoids were increased (p < 0.05); docosahexaenoic acid (DHA)-LPC was decreased (p < 0.01). Plasma TG correlated inversely with BW (p < 0.05). Plasma EET, non-esterified AA, and DHA correlated inversely with BW and directly with UA PI (p < 0.05). Placental DHA-PC and AA-PC correlated directly with MCA PI (p < 0.05). In fetuses initially referred for inadequate fetal growth (EFW < 10%), those with BW ≤ 5% demonstrated distinctly different cord plasma lipid profiles than those with BW > 5%, which correlated with Doppler PIs. This provides new insights into fetal lipidomic response to the FGR in utero environment. The impact of these changes on specific processes of growth and development (particularly fetal brain) have not been elucidated, but the relationship with Doppler PI may provide additional context for FGR surveillance, and a more targeted approach to nutritional management of these infants.

Human placental lipid content and lipid metabolic enzyme abundance in obesity and across gestation.

Changes in placental lipid metabolism influence the delivery of lipids critical for fetal development and fetal requirements for lipids change across gestation. We hypothesized that placental lipid content and metabolic enzyme protein levels increase across gestation and are elevated in obesity. Placentas (4-40 weeks' gestation) were collected from control (body mass index, BMI = 18.5-24.9, n=37) and obese (BMI > 30, n=19) pregnant women. Trophoblast villous tissue was homogenized and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) for phospholipid and triacylglycerol (TAG) analysis and western blot for protein quantification. The placental content of TAG species and nine of 35 identified phosphatidylcholines (PC) were significantly higher (P<0.05) in first trimester (28-79%, 10-47%, respectively). Furthermore, two TAG and three PC differed by maternal BMI and were significantly increased (P<0.05) in the obese group in first trimester (72-87%, 88-119%, respectively). Placental protein abundance of glycerol-2-phosphate (GPAT3) and 1-acyl-sn-glycerol-3-phosphate acyltransferase 2 (AGPAT2), involved in de novo synthesis of PC and TAG, were higher (P<0.05) in the first trimester (66 and 74%, respectively). The protein abundance of the PC-remodeling enzyme PLA2G4c was also higher (63%) in first trimester (P<0.05). In conclusion, the placental content of many phospholipid and TAG species and the protein level of associated synthesis enzymes are higher in first-trimester human placenta. The high PC content may be related to the rapid membrane expansion in early pregnancy and the low placental oxygen tension may promote the accumulation of tissue TAGs in first trimester. Maternal obesity had only limited impact on placental lipid content and metabolic enzyme protein abundance.

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.

Serum dihydroceramides correlate with insulin sensitivity in humans and decrease insulin sensitivity in vitro.

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.

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.

Maternal obesity causes fetal cardiac hypertrophy and alters adult offspring myocardial metabolism in mice.

Obesity in pregnant women causes fetal cardiac dysfunction and increases offspring cardiovascular disease risk, but its effect on myocardial metabolism is unknown. We hypothesized that maternal obesity alters fetal cardiac expression of metabolism-related genes and shifts offspring myocardial substrate preference from glucose towards lipids. Female mice were fed control or obesogenic diets before and during pregnancy. Fetal hearts were studied in late gestation (embryonic day (E) 18.5; term ≈ E21), and offspring were studied at 3, 6, 9 or 24 months postnatally. Maternal obesity increased heart weight and peroxisome proliferator activated receptor gamma (Pparg) expression in female and male fetuses and caused left ventricular diastolic dysfunction in the adult offspring. Cardiac dysfunction worsened progressively with age in female, but not male, offspring of obese dams, in comparison to age-matched control animals. In 6-month-old offspring, exposure to maternal obesity increased cardiac palmitoyl carnitine-supported mitochondrial respiration in males and reduced myocardial 18 F-fluorodeoxyglucose uptake in females. Cardiac Pparg expression remained higher in adult offspring of obese dams than control dams and was correlated with contractile and metabolic function. Maternal obesity did not affect cardiac palmitoyl carnitine respiration in females or 18 F-fluorodeoxyglucose uptake in males and did not alter cardiac 3 H-oleic acid uptake, pyruvate respiration, lipid content or fatty acid/glucose transporter abundance in offspring of either sex. The results support our hypothesis and show that maternal obesity affects offspring cardiac metabolism in a sex-dependent manner. Persistent upregulation of Pparg expression in response to overnutrition in utero might underpin programmed cardiac impairments mechanistically and contribute to cardiovascular disease risk in children of women with obesity. KEY POINTS: Obesity in pregnant women causes cardiac dysfunction in the fetus and increases lifelong cardiovascular disease risk in the offspring. In this study, we showed that maternal obesity in mice induces hypertrophy of the fetal heart in association with altered expression of genes related to nutrient metabolism. Maternal obesity also alters cardiac metabolism of carbohydrates and lipids in the adult offspring. The results suggest that overnutrition in utero might contribute to increased cardiovascular disease risk in children of women with obesity.

Targeting Fat Oxidation in Mouse Prostate Cancer Decreases Tumor Growth and Stimulates Anti-Cancer Immunity.

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.

Lipidomic characterization and localization of phospholipids in the human lung.

Lipids play a central role in lung physiology and pathology; however, a comprehensive lipidomic characterization of human pulmonary cells relevant to disease has not been performed. The cells involved in lung host defense, including alveolar macrophages (AMs), bronchial epithelial cells (BECs), and alveolar type II cells (ATIIs), were isolated from human subjects and lipidomic analysis by LC-MS and LC-MS/MS was performed. Additionally, pieces of lung tissue from the same donors were analyzed by MALDI imaging MS in order to determine lipid localization in the tissue. The unique distribution of phospholipids in ATIIs, BECs, and AMs from human subjects was accomplished by subjecting the large number of identified phospholipid molecular species to univariant statistical analysis. Specific MALDI images were generated based on the univariant statistical analysis data to reveal the location of specific cell types within the human lung slice. While the complex composition and function of the lipidome in various disease states is currently poorly understood, this method could be useful for the characterization of lipid alterations in pulmonary disease and may aid in a better understanding of disease pathogenesis.

G2A Signaling Dampens Colitic Inflammation via Production of IFN-γ.

Proinflammatory consequences have been described for lysophosphatidylcholine, a lipid product of cellular injury, signaling via the G protein-coupled receptor G2A on myeloid and lymphoid inflammatory cells. This prompted the hypothesis that genetic deletion of G2A would limit intestinal inflammation in a mouse model of colitis induced by dextran sodium sulfate. Surprisingly, G2A(-/-) mice exhibited significantly worsened colitis compared with wild-type mice, as demonstrated by disease activity, colon shortening, histology, and elevated IL-6 and IL-5 in colon tissues. Investigation of inflammatory cells recruited to inflamed G2A(-/-) colons showed significantly more TNF-α(+) and Ly6C(hi)MHCII(-) proinflammatory monocytes and eosinophils than in wild-type colons. Both monocytes and eosinophils were pathogenic as their depletion abolished the excess inflammation in G2A(-/-) mice. G2A(-/-) mice also had less IFN-γ in inflamed colon tissues than wild-type mice. Fewer CD4(+) lymphocytes were recruited to inflamed G2A(-/-) colons, and fewer colonic lymphocytes produced IFN-γ upon ex vivo stimulation. Administration of IFN-γ to G2A(-/-) mice during dextran sodium sulfate exposure abolished the excess colitic inflammation and reduced colonic IL-5 and eosinophil numbers to levels seen in wild-type mice. Furthermore, IFN-γ reduced the numbers of TNF-α(+) monocyte and enhanced their maturation from Ly6C(hi)MHCII(-) to Ly6C(int)MHCII(+) Taken together, the data suggest that G2A signaling serves to dampen intestinal inflammation via the production of IFN-γ, which, in turn, enhances monocyte maturation to a less inflammatory program and ultimately reduces eosinophil-induced injury of colonic tissues.

Phospholipid Ozonation Products Activate the 5-Lipoxygenase Pathway in Macrophages.

Ozone is a highly reactive environmental toxicant that can react with the double bonds of lipids in pulmonary surfactant. This study was undertaken to investigate the proinflammatory properties of the major lipid-ozone product in pulmonary surfactant, 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0/9al-PC), with respect to eicosanoid production. A dose-dependent increase in the formation of 5-lipoxygenase (5-LO) products was observed in murine resident peritoneal macrophages (RPM) and alveolar macrophages (AM) upon treatment with 16:0/9al-PC. In contrast, the production of cyclooxygenase (COX) derived eicosanoids did not change from basal levels in the presence of 16:0/9al-PC. When 16:0/9al-PC and the TLR2 ligand, zymosan, were added to RPM or AM, an enhancement of 5-LO product formation along with a concomitant decrease in COX product formation was observed. Neither intracellular calcium levels nor arachidonic acid release was influenced by the addition of 16:0/9al-PC to RPM. Results from mitogen-activated protein kinase (MAPK) inhibitor studies and direct measurement of phosphorylation of MAPKs revealed that 16:0/9al-PC activates the p38 MAPK pathway in RPM, which results in the activation of 5-LO. Our results indicate that 16:0/9al-PC has a profound effect on the eicosanoid pathway, which may have implications in inflammatory pulmonary disease states where eicosanoids have been shown to play a role.