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.

Maternal diet alters long-term innate immune cell memory in fetal and juvenile hematopoietic stem and progenitor cells in nonhuman primate offspring.

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.

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.

In Vitro Circadian Clock Gene Expression Assessments in Mesenchymal Stem Cells from Human Infants: A Pilot Study.

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 PPARγ 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.

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.

Infant Mesenchymal Stem Cell Insulin Action Is Associated With Maternal Plasma Free Fatty Acids, Independent of Obesity Status: The Healthy Start Study.

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.

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.

Myosteatosis in the Context of Skeletal Muscle Function Deficit: An Interdisciplinary Workshop at the National Institute on Aging.

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 D3-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.

Amino acid and fatty acid metabolomic profile during fasting and hyperinsulinemia in girls with polycystic ovarian syndrome.

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.

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).

ATG14 facilitated lipophagy in cancer cells induce ER stress mediated mitoptosis through a ROS dependent pathway.

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.

Hepatic Steatosis is Common in Adolescents with Obesity and PCOS and Relates to De Novo Lipogenesis but not Insulin Resistance.

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/m2 ) 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.

Biomarkers of Ectopic Fat Deposition: The Next Frontier in Serum Lipidomics.

CONTEXT: Strong evidence suggests that ectopic fat rather than fat mass per se drives risk for type 2 diabetes. Nonetheless, biomarkers of ectopic fat have gone unexplored. OBJECTIVE: To determine the utility of serum lipidomics to predict ectopic lipid deposition. DESIGN: Cross-sectional. SETTING: The Clinical Translational Research Center at the University of Colorado Anschutz Medical Campus. PARTICIPANTS: Endurance-trained athletes (n = 15, 41 ± 0.9 y old; body mass index 24 ± 0.6 kg/m(2)) and obese people with or without type 2 diabetes (n = 29, 42 ± 1.4 y old; body mass index 32 ± 2.5 kg/m(2)). INTERVENTION: Blood sampling and skeletal muscle biopsy. MAIN OUTCOME MEASURES: Multivariable models determined the ability of serum lipids to predict intramuscular (im) lipid accumulation of triacylglycerol (TAG), diacylglycerol (DAG), and ceramide (liquid chromatography tandem mass spectroscopy). RESULTS: Among people with obesity, serum ganglioside C22:0 and lactosylceramide C14:0 predicted muscle TAG (overall model R(2) = 0.48), whereas serum DAG C36:1 and free fatty acid (FFA) C18:4 were strong predictors of muscle DAG (overall model R(2) = 0.77), as were serum TAG C58:5, FFA C14:2 and C14:3, phosphotidylcholine C38:1, and cholesterol ester C24:1 to predict muscle ceramide (overall model R(2) = 0.85). Among endurance-trained athletes, serum FFA C14:1 and sphingosine were significant predictors of muscle TAG (overall model R(2) = 0.81), whereas no models could predict intramuscular DAG or ceramide in this group. CONCLUSIONS: Different serum lipids predict intramuscular TAG accumulation in obese people vs athletes. The ability of serum lipidomics to predict intramuscular DAG and ceramide in insulin-resistant humans may prove a new biomarker to determine risk for diabetes.

Early life exposure to maternal insulin resistance has persistent effects on hepatic NAFLD in juvenile nonhuman primates.

The origins of nonalcoholic fatty liver disease (NAFLD) may lie in early intrauterine exposures. Here we examined the maternal response to chronic maternal high-fat (HF) diet and the impact of postweaning healthy diet on mechanisms for NAFLD development in juvenile nonhuman primate (NHP) offspring at 1 year of age. Pregnant females on HF diet were segregated as insulin resistant (IR; HF+IR) or insulin sensitive (IS; HF+IS) compared with control (CON)-fed mothers. HF+IR mothers have increased body mass, higher triglycerides, and increased placental cytokines. At weaning, offspring were placed on a CON or HF diet. Only offspring from HF+IR mothers had increased liver triglycerides and upregulated pathways for hepatic de novo lipid synthesis and inflammation that was irreversible upon switching to a healthy diet. These juvenile livers also showed a combination of classical and alternatively activated hepatic macrophages and natural killer T cells, in the absence of obesity or insulin resistance. Our findings suggest that maternal insulin resistance, including elevated triglycerides, insulin, and weight gain, initiates dysregulation of the juvenile hepatic immune system and development of de novo lipogenic pathways that persist in vitro and may be an irreversible "first hit" in the pathogenesis of NAFLD in NHP.

Impaired contractile recovery after low-flow myocardial ischemia in a porcine model of metabolic syndrome.

Clinical metabolic syndrome conveys a poor prognosis in patients with acute coronary syndrome, not fully accounted for by the extent of coronary atherosclerosis. To explain this observation, we determined whether postischemic myocardial contractile and metabolic function are impaired in a porcine dietary model of metabolic syndrome without atherosclerosis. Micropigs (n = 28) were assigned to a control diet (low fat, no added sugars) or an intervention diet (high saturated fat and simple sugars, no added cholesterol) for 7 mo. The intervention diet produced obesity, hypertension, dyslipidemia, and impaired glucose tolerance, but not atherosclerosis. Under open-chest, anesthetized conditions, pigs underwent 45 min of low-flow myocardial ischemia and 120 min of reperfusion. In both diet groups, contractile function was similar at baseline and declined similarly during ischemia. However, after 120 min of reperfusion, regional work recovered to 21 ± 12% of baseline in metabolic syndrome pigs compared with 61 ± 13% in control pigs (P = 0.01). Ischemia-reperfusion caused a progressive decline in mechanical/metabolic efficiency (regional work/O2 consumption) in metabolic syndrome hearts, but not in control hearts. Metabolic syndrome hearts demonstrated altered fatty acyl composition of cardiolipin and increased Akt phosphorylation in both ischemic and nonischemic regions, suggesting tonic activation. Metabolic syndrome hearts used more fatty acid than control hearts (P = 0.03). When fatty acid availability was restricted by prior insulin exposure, differences between groups in postischemic contractile recovery and mechanical/metabolic efficiency were eliminated. In conclusion, pigs with characteristics of metabolic syndrome demonstrate impaired contractile and metabolic recovery after low-flow myocardial ischemia. Contributory mechanisms may include remodeling of cardiolipin, abnormal activation of Akt, and excessive utilization of fatty acid substrates.

Novel and reversible mechanisms of smoking-induced insulin resistance in humans.

Smoking is the most common cause of preventable morbidity and mortality in the United States, in part because it is an independent risk factor for the development of insulin resistance and type 2 diabetes. However, mechanisms responsible for smoking-induced insulin resistance are unclear. In this study, we found smokers were less insulin sensitive compared with controls, which increased after either 1 or 2 weeks of smoking cessation. Improvements in insulin sensitivity after smoking cessation occurred with normalization of IRS-1(ser636) phosphorylation. In muscle cell culture, nicotine exposure significantly increased IRS-1(ser636) phosphorylation and decreased insulin sensitivity, recapitulating the phenotype of smoking-induced insulin resistance in humans. The two pathways known to stimulate IRS-1(ser636) phosphorylation (p44/42 mitogen-activated protein kinase [MAPK] and mammalian target of rapamycin [mTOR]) were both stimulated by nicotine in culture. Inhibition of mTOR, but not p44/42 MAPK, during nicotine exposure prevented IRS-1(ser636) phosphorylation and normalized insulin sensitivity. These data indicate nicotine induces insulin resistance in skeletal muscle by activating mTOR. Therapeutic agents designed to oppose skeletal muscle mTOR activation may prevent insulin resistance in humans who are unable to stop smoking or are chronically exposed to secondhand smoke.

Progestin modulates the lipid profile and sensitivity of breast cancer cells to docetaxel.

Progestins induce lipid accumulation in progesterone receptor (PR)-positive breast cancer cells. We speculated that progestin-induced alterations in lipid biology confer resistance to chemotherapy. To examine the biology of lipid loaded breast cancer cells, we used a model of progestin-induced lipid synthesis. T47D (PR-positive) and MDA-MB-231 (PR-negative) cell lines were used to study progestin response. Oil red O staining of T47D cells treated with progestin showed lipid droplet formation was PR dependent, glucose dependent and reduced sensitivity to docetaxel. This protection was not observed in PR-negative MDA-MB-231 cells. Progestin treatment induced stearoyl CoA desaturase-1 (SCD-1) enzyme expression and chemical inhibition of SCD-1 diminished lipid droplets and cell viability, suggesting the importance of lipid stores in cancer cell survival. Gas chromatography/mass spectroscopy analysis of phospholipids from progestin-treated T47D cells revealed an increase in unsaturated fatty acids, with oleic acid as most abundant. Cells surviving docetaxel treatment also contained more oleic acid in phospholipids, suggesting altered membrane fluidity as a potential mechanism of chemoresistance mediated in part by SCD-1. Lastly, intact docetaxel molecules were present within progestin induced lipid droplets, suggesting a protective quenching effect of intracellular lipid droplets. Our studies suggest the metabolic adaptations produced by progestin provide novel metabolic targets for future combinatorial therapies for progestin-responsive breast cancers.

Altered intramuscular lipid metabolism relates to diminished insulin action in men, but not women, in progression to diabetes.

Whether sex differences in intramuscular triglyceride (IMTG) metabolism underlie sex differences in the progression to diabetes are unknown. Therefore, the current study examined IMTG concentration and fractional synthesis rate (FSR) in obese men and women with normal glucose tolerance (NGT) vs. those with prediabetes (PD). PD (n = 13 men and 7 women) and NGT (n = 7 men and 12 women) groups were matched for age and anthropometry. Insulin action was quantified using a hyperinsulinemic-euglycemic clamp with infusion of [6,6-(2)H(2)]-glucose. IMTG concentration was measured by gas chromatography/mass spectrometry (GC/MS) and FSR by GC/combustion isotope ratio MS (C-IRMS), from muscle biopsies taken after infusion of [U-(13)C]palmitate during 4 h of rest. In PD men, the metabolic clearance rate (MCR) of glucose was lower during the clamp (4.71 ± 0.77 vs. 8.62 ± 1.26 ml/kg fat-free mass (FFM)/min, P = 0.04; with a trend for lower glucose rate of disappearance (Rd), P = 0.07), in addition to higher IMTG concentration (41.2 ± 5.0 vs. 21.2 ± 3.4 µg/mg dry weight, P ≤ 0.01), lower FSR (0.21 ± 0.03 vs. 0.42 ± 0.06 %/h, P ≤ 0.01), and lower oxidative capacity (P = 0.03) compared to NGT men. In contrast, no difference in Rd, IMTG concentration, or FSR was seen in PD vs. NGT women. Surprisingly, glucose Rd during the clamp was not different between NGT men and women (P = 0.25) despite IMTG concentration being higher (42.6 ± 6.1 vs. 21.2 ± 3.4 µg/mg dry weight, P = 0.03) and FSR being lower (0.23 ± 0.04 vs. 0.42 ± 0.06 %/h, P = 0.02) in women. Alterations in IMTG metabolism relate to diminished insulin action in men, but not women, in the progression toward diabetes.

Increased intramuscular lipid synthesis and low saturation relate to insulin sensitivity in endurance-trained athletes.

Intramuscular triglyceride (IMTG) has received considerable attention as a potential mechanism promoting insulin resistance. Endurance-trained athletes have high amounts of IMTG but are insulin sensitive, suggesting IMTG content alone does not change insulin action. Recent data suggest increased muscle lipid synthesis protects against fat-induced insulin resistance. We hypothesized that rates of IMTG synthesis at rest would be increased in athletes compared with controls. Eleven sedentary men and 11 endurance-trained male cyclists participated in this study. An intravenous glucose tolerance test was performed to assess insulin action. After 3 days of dietary control and an overnight fast, [13C16]palmitate was infused at 0.0174 micromol.kg(-1).min(-1) for 4 h, followed by a muscle biopsy to measure isotope incorporation into IMTG and diacylglycerol. Compared with controls, athletes were twice as insulin sensitive (P=0.004) and had a significantly greater resting IMTG concentration (athletes: 20.4+/-1.6 microg IMTG/mg dry wt, controls: 14.5+/-1.8 microg IMTG/mg dry wt, P=0.04) and IMTG fractional synthesis rate (athletes: 1.56+/-0.37%/h, controls: 0.61+/-0.15%/h, P=0.03). Stearoyl-CoA desaturase 1 mRNA expression (P=0.02) and protein content (P=0.03) were also significantly greater in athletes. Diacylglycerol, but not IMTG, saturation was significantly less in athletes compared with controls (P=0.002). These data indicate endurance-trained athletes have increased synthesis rates of skeletal muscle IMTG and decreased saturation of skeletal muscle diacylglycerol. Increased synthesis rates are not due to recovery from exercise and are likely adaptations to chronic endurance exercise training.