Elevated FFA Research Articles

Circulating beta‐hydroxybutyrate levels in advanced heart failure with reduced ejection fraction: Determinants and prognostic impact

AbstractAimsPatients with heart failure (HF) display metabolic alterations, including heightened ketogenesis, resulting in increased beta‐hydroxybutyrate (β‐OHB) formation. We aimed to investigate the determinants and prognostic impact of circulating β‐OHB levels in patients with advanced HF and reduced ejection fraction (HFrEF).Methods and resultsA total of 867 patients with advanced HFrEF (age 57 ± 11 years, 83% male, 45% diabetic, 60% New York Heart Association class III), underwent clinical and echocardiographic examination, circulating metabolite assessment, and right heart catheterization (n = 383). The median β‐OHB level was 64 (interquartile range [IQR] 33–161) μmol/L (normal 0–74 μmol/L). β‐OHB levels correlated with increased markers of lipolysis (free fatty acids [FFA]), higher natriuretic peptides, worse pulmonary haemodynamics, and lower humoral regulators of ketogenesis (insulin/glucagon ratio). During a median follow‐up of 1126 (IQR 410–1781) days, there were 512 composite events, including 324 deaths, 81 left ventricular assist device implantations and 107 urgent cardiac transplantations. In univariable Cox regression, increased β‐OHB levels (T3 vs. T1: hazard ratio [HR] 1.39, 95% confidence interval [CI] 1.13–1.72, p = 0.002) and elevated FFA levels (T3 vs. T1: HR 1.39, 95% CI 1.09–1.79, p = 0.008) were both predictors of a worse prognosis. In multivariable Cox analysis evaluating the simultaneous associations of FFA and β‐OHB levels with outcomes, only FFA levels remained significantly associated with adverse outcomes.ConclusionsIn patients with advanced HFrEF, increased plasma β‐OHB correlate with FFA levels, worse right ventricular function, greater neurohormonal activation and other markers of HF severity. The association between plasma β‐OHB and adverse outcomes is eliminated after accounting for FFA levels, suggesting that increased β‐OHB is a consequence reflecting heightened lipolytic state, rather than a cause of worsening HF.

75-OR: Cold-Responsive Proteolytic Rewiring of Mitochondria Safeguards White Fat Thermogenic Remodeling

Mitochondrial proteases are emerging as key regulators of mitochondrial plasticity acting both as protein quality surveillance and regulatory enzymes. It remains unclear, however, whether the regulated mitochondrial proteolysis is mechanistically linked to cell identity switch such as white-to-beige conversion of adipocytes. Here we report that cold-responsive mitochondrial proteolysis is a prerequisite for conversion from white-to-beige adipocytes during white adipose tissue (WAT) browning. MitoTimer reporter and chemical thiol probe analysis reveal that thermogenic stimulation selectively promotes mitochondrial proteostasis in WAT via mitochondrial protease LONP1 both in vivo and in vitro. Disruption of LONP1-dependent proteolysis substantially impairs cold or b3-AR agonist induced adipocyte identity programming, evidenced by the remarkably attenuated formation of UCP1+ multilocular adipocytes in IWAT from Lonp1 f/f/Adipoq-Cre (LONP1 AKO) mice. LONP1 AKO mice exposed to cold had lower body temperature, elevated serum TG and FFA levels. Chow-fed LONP1 AKO mice also developed glucose intolerance. “AdipoChaser” system “pulse-chase” lineage tracing experiments confirm that LONP1 ablation markedly blocks the direct cell fate programming of pre-existing mature adipocytes. Importantly, augmented LONP1 expression corrects aging-related impairments in white-to-beige adipocytes conversion. 10-month-old adipocyte-specific Lonp1 overexpression mice had significantly lower body weight and fat mass as a result of elevated energy expenditure. Thus, the identification of LONP1 as a critical safeguard of WAT beiging holds promise for translational applications in enhancing white-to-beige adipocytes conversion capacity relevant to a variety of metabolic disease states and during aging. Disclosure T.Fu: None. W.Sun: None. Z.Zhou: None. Z.Xu: None. M.Yan: None. L.Yang: None. Y.Yin: None. Z.Gan: None.

367-OR: E-cigarette Vaping Exposure Increases Adipose 11ß-HSD1 Expression and Disturbs Glucose Homeostasis in Mice

Electronic cigarette (E-cigs) use may increase the prediabetic risk, but the underlying mechanisms remain underexplored. Pre-receptor activation of glucocorticoids (GCs) via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in adipose tissues has been identified as an important mediator of insulin resistance and type 2 diabetes. However, little is known about the effects of E-cig vaping on 11β-HSD1 and glucose homeostasis. To address this issue, we conducted animal model studies with mice exposed to aerosolized PBS, nicotine-free or nicotine-containing E-cigs, with concurrent exposure to either vehicle or the GC receptor (GR) antagonist RU486. We observed that exposure of E-cig vaping nicotine for 4 weeks increased 11β-HSD1 expression and induced lipase HSL and ATGL abundance within the adipose tissues with induction of hypercortisolemia in response to elevated plasma nicotine levels in mice compared with those of aerosolized E-cig vaping vehicle or PBS controls. Induction of adipose 11β-HSD1 was correlated with elevated the expression of hepatic gluconeogenic enzymes PEPCK and G6Pase and corresponded to the elevated fasting glucose levels. Moreover, E-cig vaping nicotine also increased glucose intolerance, but decreased the glucose-lowing effects of insulin and elevated plasma FFA levels in mice compared with PBS controls. However, E-cig vapor without nicotine did not affect these metabolic parameters. Furthermore, RU486 treatment attenuated E-cig vaping nicotine-induced adipose 11β-HSD1 and lipase expression. In addition, RU486 also attenuated the E-cig-mediated activation of hepatic PEPCK and G6Pase and decreased plasma FFA levels, but did not change the elevated plasma corticosterone levels in mice on E-cig vaping nicotine. These data indicate that E-cig vapor exposure harmfully affects glucose homeostasis and these effects may arise, in part, from aerosol nicotine-induced adipose 11β-HSD1 and GR expression. Disclosure J.Wang: None. T.Friedman: None. M.Jiang: None. Y.Liu: None. J.Liu: None. Y.Wang: None. S.C.J.Kim: None. E.Espinal: None. R.Yang: None. H.Zhong: None. X.M.Shao: None. K.Lutfy: None. Funding Tobacco-Related Disease Research Program (T31IR1603)

Reduced glucose tolerance and insulin sensitivity after prolonged exercise in endurance athletes.

The purpose of this study was to 1. investigate if glucose tolerance is affected after one acute bout of different types of exercise; 2. assess if potential differences between two exercise paradigms are related to changes in mitochondrial function; and 3. determine if endurance athletes differ from nonendurance-trained controls in their metabolic responses to the exercise paradigms. Nine endurance athletes (END) and eight healthy nonendurance-trained controls (CON) were studied. Oral glucose tolerance tests (OGTT) and mitochondrial function were assessed on three occasions: in the morning, 14 h after an overnight fast without prior exercise (RE), as well as after 3 h of prolonged continuous exercise at 65% of VO2 max (PE) or 5 × 4 min at ~95% of VO2 max (HIIT) on a cycle ergometer. Glucose tolerance was markedly reduced in END after PE compared with RE. END also exhibited elevated fasting serum FFA and ketones levels, reduced insulin sensitivity and glucose oxidation, and increased fat oxidation during the OGTT. CON showed insignificant changes in glucose tolerance and the aforementioned measurements compared with RE. HIIT did not alter glucose tolerance in either group. Neither PE nor HIIT affected mitochondrial function in either group. END also exhibited increased activity of 3-hydroxyacyl-CoA dehydrogenase activity in muscle extracts vs. CON. Prolonged exercise reduces glucose tolerance and increases insulin resistance in endurance athletes the following day. These findings are associated with an increased lipid load, a high capacity to oxidize lipids, and increased fat oxidation.

Quercetin Reduces Lipid Accumulation in a Cell Model of NAFLD by Inhibiting De Novo Fatty Acid Synthesis through the Acetyl-CoA Carboxylase 1/AMPK/PP2A Axis.

Dysregulation of de novo lipogenesis (DNL) has recently gained strong attention as being one of the critical factors that contribute to the assessment of non-alcoholic fatty liver disease (NAFLD). NAFLD is often diagnosed in patients with dyslipidemias and type 2 diabetes; thus, an interesting correlation can be deduced between high hematic free fatty acids and glucose excess in the DNL dysregulation. In the present study, we report that, in a cellular model of NAFLD, the coexistence of elevated glucose and FFA conditions caused the highest cellular lipid accumulation. Deepening the molecular mechanisms of the DNL dysregulation—RT-qPCR and immunoblot analysis demonstrated increased expression of mitochondrial citrate carrier (CiC), cytosolic acetyl-CoA carboxylase 1 (ACACA), and diacylglycerol acyltransferase 2 (DGAT2) involved in fatty acids and triglycerides synthesis, respectively. XBP-1, an endoplasmic reticulum stress marker, and SREBP-1 were the transcription factors connected to the DNL activation. Quercetin (Que), a flavonoid with strong antioxidant properties, and noticeably reduced the lipid accumulation and the expression of SREBP-1 and XBP-1, as well as of their lipogenic gene targets in steatotic cells. The anti-lipogenic action of Que mainly occurs through a strong phosphorylation of ACACA, which catalyzes the committing step in the DNL pathway. The high level of ACACA phosphorylation in Que-treated cells was explained by the intervention of AMPK together with the reduction of enzymatic activity of PP2A phosphatase. Overall, our findings highlight a direct anti-lipogenic effect of Que exerted through inhibition of the DNL pathway by acting on ACACA/AMPK/PP2A axis; thus, suggesting this flavonoid as a promising molecule for the NAFLD treatment.

Regulations of Free Fatty Acids and Diabetic Parameters in Drug Naïve Subjects with Type 2 Diabetes Treated with Canagliflozin Monotherapy.

The objective of this study is to investigate the regulations of FFA with canagliflozin in relation to metabolic parameters. Drug naïve subjects with T2DM were administered 50-100 mg/day canagliflozin monotherapy (n=70) for 3 months. Significant correlations between the changes of (Δ) FFA and Δadipo-IR (R=0.496), but no correlations between ΔFFA and ΔHOMA-R were observed. The subjects were divided into three groups with similar numbers according to Δ FFA: group A: highest tertile: (ΔFFA=38.7%, n=23); group B: intermediate tertile: (ΔFFA=2%, n=23); group C: lowest tertile: (ΔFFA=-36%, n=24). Metabolic parameters were compared between group A and group C. At baseline, FFA was higher in group C than group A (p<0.002). Greater degrees of HbA1c reduction and increases of insulin were observed in group C than group A (both p<0.05). In group A, significant reductions of BMI (-2.6%) and HOMA-R (-30%) were seen. In group C, significant reductions of non-HDL-C (-6.2%), UA (-7.6%) or adipo-IR (-28.7%), and increases of HOMA-B (+85.6%) were observed. Taken together, 1) certain population treated with canagliflozin showed decreased FFA. 2) beta-cell function increased while atherogenic cholesterol, UA and adipo-IR decreased in those with reduced FFA. Better glycemic efficacies were seen in these populations. 3) body weight and whole body insulin resistance (HOMA-R) significantly decreased in those with elevated FFA. 4) FFA is linked to adipose insulin resistance (adipo-IR), while it does not appear to impact whole body insulin resistance (HOMA-R).

Follistatin-like 1 as a Novel Adipomyokine Related to Insulin Resistance and Physical Activity.

Follistatin-like protein-1 (FSTL-1) is considered to be an adipokine or myokine that could be a potential regulator of metabolism. Our purpose is to investigate the relationship between circulating FSTL-1 levels and insulin resistance (IR) in type 2 diabetes mellitus (T2DM) and to identify the regulatory factors. FSTL-1 expression in C57BL/6J and db/db mice was examined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blots. Serum FSTL-1 levels were measured by enzyme-linked immunosorbent assay in 298 T2DM patients and 202 healthy controls. Changes in the circulating FSTL-1 level were observed during the oral glucose tolerance test, EHC (euglycemic-hyperinsulinemic clamp), lipid infusion, acute exercise, and cold-exposure test. We found that FSTL-1 protein expression in the adipose tissue of db/db mice was significantly higher than that of wild-type mice. Importantly, circulating FSTL-1 levels in T2DM and overweight/obese participants were higher than those in healthy and lean individuals, and was related to HOMA-IR, adiponectin, and obesity- and metabolism-related parameters. In the intervention study, 45 minutes of physical activity was found to significantly increase the circulating FSTL-1 concentration in young, healthy participants. Further, FSTL-1 protein expression in adipose tissue rose dramatically in response to physical activity in mice. Hyperinsulinemia during EHC and acute elevated FFA induced by lipid infusion resulted in a significant decrease in the circulating FSTL-1 levels. However, no change was found in the circulating FSTL-1 levels in response to the oral glucose challenge or cold-exposure test. FSTL-1 may be an adipomyokine associated with insulin resistance and physical activity, and circulating FSTL-1 levels are increased in patients with T2DM.

Rapid Accumulation of Triacylglycerol in the Liver is a Component of the Acute Exercise Response and May Serve a Protective Role Against Lipotoxicity

We have previously shown that the content of triacylglycerol (TAG) in the liver transiently increases after a single bout of exercise in untrained mice, and this phenomenon has been observed in human subjects as well. This discovery depicted a new phenomenon in the metabolic exercise response. Here we propose the term “lipogenic flexibility” to describe this ability to rapidly alter hepatic TAG content in response to FFA supply. To explore lipogenic flexibility after exercise, we performed lipidomics analysis on liver samples obtained 3 hours after exercise and the following day. Female mice performed a single bout of continuous exercise (CE), high intensity interval exercise (HIIE), or no exercise (CON). The total concentration of hepatic TAG rose 3 hours after exercise (P &lt; 0.05 for both CE and HIIE vs CON), but the total concentration of all other measured lipid classes remained unchanged after exercise (NSD). Hepatic TAG declined back to control levels by the day after exercise. We performed correlation analysis to test the association of TAG (an inert lipid depot) with lipotoxic lipid classes in the liver. Hepatic TAG was positively correlated with hepatic diacylglycerol (DAG) in CON (P &lt; 0.05); however, during the hepatic TAG pool expansion after exercise, liver TAG and DAG were no longer significantly correlated with one another (NSD). Exhibiting lipogenic flexibility after exercise appears to break the equilibration between DAG and TAG, allowing fatty acids to be drawn toward the inert lipid pool (TAG) and away from the lipotoxic lipid pool (DAG). The results suggest that the TAG pool actively expands to buffer elevated FFA supply after exercise and may act to prevent lipotoxicity.Support or Funding InformationThis work was supported by the Faculty Research Grant Program of Rutgers University, the American Diabetes Association grant # 7‐13‐JF‐27‐BR to G Henderson, and the Purdue University College of Health and Human Sciences.

Quality Assessment of Oil Used for Frying of Potato Chips: In the Case of Wolkite University Community

Frying is an old and widely used method of cooking and processing food. Typically, a food is immersed in heated oil for a short duration in a process known as immersion-oil frying. Consumers tend to associate color with flavor, safety, storage time, nutrition, and level of satisfaction, due to the fact that color correlates well with physical, chemical, and sensorial evaluations of food quality. Indicators of poor oil quality include elevated FFA, low smoke point, change of color, low iodine value, peroxide value, total polar material, high foaming properties and increased viscosity. Deep frying and the use of recycled oil for many times is a commonly practiced in commercial and sometimes in domestic cooking processes. The way of this cooking method may create a lipid peroxidation product which brings harm to human health. Quality assessment of oil used for frying of potato chips in the case of Wolkite university community studied. The result of physicochemical properties of oil were investigated from sample selected randomly around Wolkite town and these are free fatty acid, peroxide value, smoking point and iodine value of recycled oils was higher than the recommended values. The strength of current evidence makes it reasonable to recommend complete avoidance of fried foods or at most infrequent to moderate fried food consumption within the context of an overall healthy dietary pattern around Wolkite town. Generally, the present study shows the recycled oil used were not as standards of FAO and also the people of fried food makers were not having awareness about the impacts of recycled oils.

Herd factors influencing free fatty acid concentrations in bulk tank milk.

Free fatty acid (FFA) concentrations can be elevated in raw milk due to improper handling and management at the dairy farm, and high concentrations of FFA can lead to off flavors in milk. This study aimed to describe how the herd production system, milking system, feeding and technological factors impact on FFA concentrations in bulk tank milk. FFA concentrations in bulk milk samples from 259 organic and 3326 conventional herds were analyzed by FT-IR during one year. The FFA content was significantly lower in bulk milk from organic than conventional herds. This was most evident during the summer half-year when the organic cows graze pasture. Bulk milk from automatic milking systems (AMS) and tie-stalls contained greater concentrations of FFA than any other milking parlor systems. In AMS, high milking frequency was found to be the most significant contributor to elevated FFA content in milk. Moreover, a strong interaction was found between milking interval and production system (organic vs. conventional). The technical factors, pre-cooling, onset time for cooling after milk inlet, contact between milk and agitation also impacted on the FFA concentration, whereas other technical factors including centrifugal pump type, length and height of pumping line and type of AMS manufacturer were found to be without significant effect on FFA. Feeding variables, based on feeding plans and evaluation, only explained a small part of the variation in bulk milk FFA. Overall, this study demonstrated that AMS compared to other milking system contributes significantly to increased FFA concentration in bulk tank milk, and within AMS high milking frequency contributes to increased FFA concentration.

The effect of apelin-13 on pancreatic islet beta cell mass and myocardial fatty acid and glucose metabolism of experimental type 2 diabetic rats

Apelin, a new identified adipokine, and its G protein–coupled receptor named APJ are widely expressed in various tissues. Apelin has been found to play important roles in the physiopathology of multiple diseases. Our aim is to assess the effect of long-term apelin treatment on serum insulin level and pancreatic islet beta-cell mass in the late stage of type 2 diabetes without hyperinsulinemia and to investigate the role of apelin in myocardial fatty acid and glucose metabolism. In the present study, the high-fat diet fed-streptozotocin-induced experimental type 2 diabetic rats were given once daily intraperitoneal injection of apelin-13 (0.1 μmol/kg) for 10 weeks. We observed that apelin significantly improved serum insulin reduction and reduced hyperglycemia. Histologic analysis showed that long-term apelin treatment significantly increased pancreatic islet beta cell mass. Exogenous apelin failed to change dyslipidaemia of type 2 diabetic rats. Apelin treatment markedly decreased elevated myocardial FFA and glycogen content. Treatment of type 2 diabetic rats with apelin markedly reduced increased gene expressions of the cardiac fatty acid transporter CD36, CPT-1, and Peroxisome proliferator-activated receptor (PPAR)-α. Whereas the gene levels of citrate synthase and peroxisome proliferator–activated receptor γ coactivator 1-α (PGC1-α), a transcriptional coactivator, mediating mitochondrial biogenesis in heart were unaltered in response to exogenous apelin. Taken together, longer-term apelin treatment prevented pancreatic beta-cell loss or failure in experimental type 2 diabetic rats. Apelin can regulate myocardial metabolism. Apelin reduced myocadial fatty acid uptake and oxidation through inhibiting PPAR-α but did not affect myocardial mitochondrial biogenesis in type 2 diabetic rats.

Metabolomic Responses to Acute Aerobic and Anaerobic Exercise Bouts

PURPOSE: To explore the acute serum metabolomic responses following single bouts of aerobic and anaerobic exercise in differentially-trained subjects. METHODS: Subjects (N = 40) were equally distributed into one of 4 groups (n = 10) based on a combination of sex [male (M) or female (F)] and training history [endurance (E) or resistance (R) trained] (M = 24 + 4 y, 160.5 + 17.2 kg, 12.8 + 5.7 %BF; F= 22 + 2 y 135.5 + 15.8 kg, 23.3 + 4.8 %BF). On separate days, 45 min aerobic (A) or weight-training (W) exercises were performed. Serum was collected pre, 0 & 60 min post exercise (T0, T1 & T2), and analyzed via UHPLC/MS for identification of 754 biochemicals. Principle components analysis (PCA) was used to define metabolite profiles. RMANOVA’s for sex, training status, exercise type, and time were run with significance set at P < .05. RESULTS: Both A and W increased glycolysis (A = 3.5 + .7 fold; W = 3.9 + 1.0 fold; P<.05), with a significantly greater activation for RW, P<.05. TCA activity (AVG = 1.5 + 0.3 & 1.9 + 0.3 fold; P<.05) also increased. Downstream TCA intermediates (succinate, fumarate, & malate) were increased at T1, particularly for succinate in E (P<.05), and returned to baseline by T2. During exercise, A increased fat metabolism as evidenced by elevation of multiple FFAs and acylcarnitines (i.e., palmitate increased 2.0 + 0.4 v 1.0 + 0.1 fold in A v W) and elevated ketone bodies at T1 in RA & EA. Across exercise conditions, E showed relatively lower FFA and BCAA catabolism than R at T2 (E v R changes from baseline: stearate =1.4 + 0.3 v 1.4 + 0.2 fold; 3-hydroxyisobutyrate = 1.8 + 0.2 v 2.2 + 0.1, P<.05) and generally faster returns towards baseline for all metabolites. Sex-dependent differences in global metabolite profiles were more pronounced in E than R groups, including elevated FFA’s and muted TCA intermediate levels and BCAA catabolism in FE during both A and W. CONCLUSION: The biological response to exercise is dictated by the metabolic demand of the exercise and the physiology of the exerciser, allowing exercise type and individual variation to alter the exercise metabolome. Data support greater TCA capacity in E and greater glycolytic power in R leading to differential fuel selection during exercise, particularly with matched mode. Females displayed lower TCA activity, yet higher FFA oxidation with sex differences most apparent in the E groups.

Longer-term impact of hemiparetic stroke on skeletal muscle metabolism—A pilot study

BackgroundHemiparetic stroke leads to structural and metabolic alterations of skeletal muscle tissue, thereby contributing to functional impairment associated with stroke. In situ metabolic processes at tissue level in skeletal muscle have not been investigated. We hypothesize that muscular metabolic capacity is limited after hemiparetic stroke, and that changes affect rather the paretic than non-paretic limb. MethodsNine male hemiparetic stroke survivors (age, 62±8years; BMI, 28±4kg/m2; median stroke latency, 23months ranging from 7 to 34months poststroke) underwent dynamic in situ measurements of carbohydrate and lipid metabolism at fasting condition and during oral glucose tolerance testing, using bilateral microdialysis. Results were compared to 8 healthy male subjects of similar age and BMI. ResultsTissue perfusion, fasting and postprandial profiles of interstitial metabolites glucose, pyruvate, lactate and glycerol did not differ between paretic and non-paretic muscle. Patients displayed higher fasting and postprandial dialysate glycerol levels compared to controls (P<0.001) with elevated plasma FFA (fasting FFA; 0.63±0.23 vs. 0.29±0.17mmol/L; P=0.004). Glycolytic activity was higher in patients vs. controls, with increased lactate production upon glucose load (P<0.001). ConclusionsAn elevated lipolytic and glycolytic activity on tissue level suggests an impaired substrate metabolism with blunted oxidative metabolism in bilateral skeletal muscle in patients after hemiparetic stroke. Muscular metabolic properties did not differ between paretic and non-paretic leg. Further work is needed to investigate the clinical impact of this impaired muscular metabolic capacity in post-stroke patients.

Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the absence vs. presence of E. Mechanisms underlying translation of E-contingent A2 neuron responses to FD into regulatory signaling remain to be determined. © 2016 Wiley Periodicals, Inc.

Unexpected severe consequences of Pikfyve deletion by aP2- or Aq-promoter-driven Cre expression for glucose homeostasis and mammary gland development.

Systemic deficiency of PIKfyve, the evolutionarily conserved phosphoinositide kinase synthesizing cellular PtdIns5P and PtdIns(3,5)P2 and implicated in insulin signaling, causes early embryonic death in mice. In contrast, mice with muscle‐specific Pikfyve disruption have normal lifespan but exhibit early‐age whole‐body glucose intolerance and muscle insulin resistance, thus establishing the key role of muscle PIKfyve in glucose homeostasis. Fat and muscle tissues control postprandial glucose clearance through different mechanisms, raising questions as to whether adipose Pikfyve disruption will also trigger whole‐body metabolic abnormalities, and if so, what the mechanism might be. To clarify these issues, here we have characterized two new mouse models with adipose tissue disruption of Pikfyve through Cre recombinase expression driven by adipose‐specific aP2‐ or adiponectin (Aq) promoters. Whereas both mouse lines were ostensibly normal until adulthood, their glucose homeostasis and systemic insulin sensitivity were severely dysregulated. These abnormalities stemmed in part from accelerated fat‐cell lipolysis and elevated serum FFA. Intriguingly, aP2‐Cre‐PIKfyvefl/fl but not Aq‐Cre‐PIKfyvefl/fl females had severely impaired pregnancy‐induced mammary gland differentiation and lactogenesis, consistent with aP2‐Cre‐mediated Pikfyve excision in nonadipogenic tissues underlying this defect. Intriguingly, whereas mammary glands from postpartum control and Aq‐Cre‐PIKfyvefl/fl mice or ex vivo mammary gland explants showed profound upregulation of PIKfyve protein levels subsequent to prolactin receptor activation, such increases were not apparent in aP2‐Cre‐PIKfyvefl/fl females. Collectively, our data identify for the first time that adipose tissue Pikfyve plays a key role in the mechanisms regulating glucose homeostasis and that the PIKfyve pathway is critical in mammary epithelial differentiation during pregnancy and lactogenesis downstream of prolactin receptor signaling.

Clinical characteristics and heterogeneity in patients with ketosis-prone diabetes

Objective To investigate the clinical characteristics,peripheral insulin sensitivity,and β-cell function in patients with ketosis-prone diabetes(KPD).Methods Thirty-one patients with newly diagnosed ketosisprone diabetes were admitted to West China Hospital from January 2004 to December 2009.They were divided into 2 groups according to their body mass index (BMI):OB-KPD (BMI ≥ 25 kg/m2,n =22) and Lean-KPD (BMI ＜ 23 kg/m2,n =9).10 patients with newly-onset type 2 diabetes free from ketosis (OB-DM:BMI ≥ 25 kg/m2,n =10) were enlisted as control.Detailed assessments of medical history and symptoms of hyperglycemia were performed.The islet cell antibody (ICA),insulin autoantibody (IAA),anti-glutamic acid decarboxylase antibody (GAD-Ab),fasting plasma glucose,serum insulin,C-peptide and free fat acids concentrations were measured.All of the subjects underwent oral and intravenous glucose tolerance tests,euglycemic-hyperinsulinemia and hyperglycemia clamp test,to evaluate the insulin secretion and insulin sensitivity respectively.Insulin sensitivity was determined by glucose disposal rate (GDR) of steady state during euglycemic clamp and acute insulin secretion was calculated by insulin area under curve(AUCins 0-10 min) during IVGTT.Maximal insulin secretion was determined by glucose infusion rate (GIR) and serum insulin concentration of steady state during hyperglycemic clamp test.Results Age,sex,duration of diabetes were matched among groups.A family history of diabetes was strongly associated with those patients with obesity,compared with lean ketosis prone diabetes(16/22 vs 1/9).GDR was (4.91 ± 1.82) mg · kg 1 · min-1 in subjects with OB-KPD,being lower than that in Lean-KPD patients[(6.26 ± 1.89) mg · kg 1 · min-1] and OB-DM group[(6.78-± 1.69) mg · kg 1 · min-1,P＜0.01].Serum insulin and C-peptide in OB-KPD patients were higher than Lean-KPD patients.Area under the insulin curve [AUCins0-10min (183.86 ± 31.1) mIU/L] and GIR[(2.65 ±1.53) mg · kg-1 · min-1] in OB-KPD patients were lower than those in OB-DM group[(697.06-± 231.9) mIU/L,(6.53 ± 2.21)mg · kg 1 · min-1,P＜0.0 1],but slightly higher than the Lean-KPD group [AUCins0 10min (92.1 ±29.8) mUU/L,GIR (2.55 ± 1.49) mg · kg 1 · min-1,P＜0.05].Glucose disposal rate (GDR) was strongly associated with casual plasma glucose (r =-0.502,P＜0.01),HbA1C(r =-0.553,P＜0.0 1) and FFA eoneentrations (r=-0.504,P＜0.01) on admission.Conclusions Insulin resistance and β-cell dysfunction coexist in all KPD patients.OB-KPD patients exhibit more severe insulin resistance,while Lean-KPD patients have lower insulin secretion.KPD patients had severe hyperglycemia,hypertriglyceridemia,and high plasma FFA levels on admission,suggesting that hyperglycemia and elevated FFA levels could result in serious insulin resistance,β-cell dysfunction,and diabetic ketosis in patients with KPD. Key words: Diabetes mellitus ; Ketosis ; β-cell function

Abstract C29: Free fatty acid induced epithelial to mesenchymal transition (EMT) in hepatocellular carcinoma cells

Abstract Background: The incidence and mortality rates of hepatocellular carcinoma (HCC) have steadily increased in the United States over the past few decades. From an epidemiological perspective, it has been observed that HCC related mortality rates are significantly higher in obese patients indicating a possible correlation between obesity and metastatic progression of HCC. At the physiological level, obesity manifests an elevation in free fatty acid levels (FFA) levels which is thought to be detrimental for liver cells. While most previous studies have focused on cytotoxic effects of elevated FFA, very few studies have investigated their potential role in progression of liver cancer. In a previous study, we found reduced expression of desmoplakin (DSP) in FFA treated HepG2 cells, suggesting a loss of cell adhesion – one of the features of cells undergoing metastatic progression via EMT. Hence, we hypothesized that elevated FFA levels possess the ability to induce EMT in HCC cells. Methodology and Results: We studied the in vitro effects of FFA on HepG2 and Hep3B cells – two human HCC cell lines of different etiologies. We performed all experiments using FFA conjugated to BSA (bovine serum albumin), and used BSA as treatment control. We first evaluated the overall impact of FFA treatment on cell adhesion. We performed a dispase-based dissociation assay and found significant loss of cell adhesion in FFA treated cells. Next, using scratch wound-healing assay and modified Boyden's chamber assay, we found that FFA significantly enhanced the migration and invasiveness of HCC cells. We further confirmed the occurrence of EMT by measuring the change in expression levels of various markers (Keratin 8/18, Vimentin, E-cadherin) in FFA treated cells using Western blots. Additionally, we screened the change in expression levels of several transcription factors that have been described as inducers of EMT using qRT-PCR. Our studies indicated enhanced expression of a number of EMT-related transcription factors including SNAIL, TWIST, ZEB1 and FOXC2, suggesting a potential transcriptional regulatory mechanism by which elevated FFA could induce EMT. Furthermore, we evaluated the possibility of a direct role of DSP loss in inducing EMT. Using siRNA mediated DSP knock-down we found enhanced expression of EMT markers in both HepG2 and Hep3B cells. Conclusions and future perspectives: Our studies provide evidence that elevated FFA levels may be involved in metastatic progression of HCC cells through transcriptional activation of EMT pathways. Future in vivo studies based on mouse xenograft model will provide additional support to confirm the effects of elevated FFA and DSP loss on enhancing aggressiveness and metastatic potential of HCC. These results may impact HCC therapeutic intervention and drug development, as well as future studies on obesity as a risk factor for cancer tumorigenesis and progression. Citation Format: Aritro Nath, Christina Chan. Free fatty acid induced epithelial to mesenchymal transition (EMT) in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C29.

Role of plasminogen activator inhibitor and free fatty acids in diagnosis of insulin resistance in patients with myocardial infarction

Purpose: To access the insulin resistance markers dynamics in ST-segment elevation patients with MI with presented and non-presented T2DM in acute and post-acute rehabilitation periods. Methods: 95 patients with myocardial infarction and 60 patients with MI and associated T2DM were included in the study; the control group consisted of 30 healthy subjects. The concentration of serum FFA, glucose, C-peptide and insulin were measured on the 1st and 12th days of the study. MI was diagnosed according to the 2007 National Scientific Cardiology Society. The study design was approved by the Institutional Review Board. The insulin resistance level was estimated using a mathematical model incorporating fasting serum insulin and glucose measurement with the QUICKI calculation. The results were statistically processed using non-parametric criteria; differences were considered to be statistically significant in p < 0.05. Results: Blood glucose level increased in the Group 1 and on the 1st day after MI onset. An elevation of glucose level was noticed in the Group 2 during the whole follow-up period, whereas in the Group 1 blood glucose level decreased on the 12th day. Postprandial glucose level increased in the Group 2 in comparison with the control values on the 12th day from the MI onset. In contrast, fasting serum insulin and C-peptide level tended to increase in both groups on the 1st and 12th days from the MI onset. Postprandial insulin level raised equally in both groups compared to the control group. In the Group 1, the QUICKI indices were 0,316±0.005 and 0,319±0.005 on the 1st and 12th days after the MI onset, respectively, and they were interpreted as breakpoint values between severe and moderate IR. In the Group 2, the QUICKI indices were 0,296±0.009 and 0,300±0.005, that verified a pronounced tissue insulin resistance. FFA levels in the Group 1 and in the Group 2 exceeded the control group values. PAI-1 concentration was higher in the Group 1 and higher in the Group 2 compared to the control group on the 1st day. In addition, PAI-1 concentration significantly reduced in both groups on the 12th day. Postprandial insulin positively correlated to FFA levels (R=0,47, p=0.02) and QUICKI index negatively correlated to FFA levels (R=-0,229, p=0,006). Moreover, a negative correlation between PAI-1 concentration and QUICKI index was reported in Group 2 and in Group 1 (R= -0,77, p=0,005; R= -0,47, p=0,005, respectively). Conclusion: Elevated FFA and PAI-1 levels are significant indicators of impaired insulin sensitivity under the conditions of acute MI compared to the postprandial glycemia and insulinemia.

Lipocalin prostaglandin D synthase and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism in vivo.

Mice lacking Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) have unexpectedly normal glucose tolerance and mild insulin resistance. Mice lacking PPARγ2 were found to have elevated levels of Lipocalin prostaglandin D synthase (L-PGDS) expression in BAT and subcutaneous white adipose tissue (WAT). To determine if induction of L-PGDS was compensating for a lack of PPARγ2, we crossed L-PGDS KO mice to PPARγ2 KO mice to generate Double Knock Out mice (DKO). Using DKO mice we demonstrated a requirement of L-PGDS for maintenance of subcutaneous WAT (scWAT) function. In scWAT, DKO mice had reduced expression of thermogenic genes, the de novo lipogenic program and the lipases ATGL and HSL. Despite the reduction in markers of lipolysis in scWAT, DKO mice had a normal metabolic rate and elevated serum FFA levels compared to L-PGDS KO alone. Analysis of intra-abdominal white adipose tissue (epididymal WAT) showed elevated expression of mRNA and protein markers of lipolysis in DKO mice, suggesting that DKO mice may become more reliant on intra-abdominal WAT to supply lipid for oxidation. This switch in depot utilisation from subcutaneous to epididymal white adipose tissue was associated with a worsening of whole organism metabolic function, with DKO mice being glucose intolerant, and having elevated serum triglyceride levels compared to any other genotype. Overall, L-PGDS and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism.

Cyclosporine A and palmitic acid treatment synergistically induce cytotoxicity in HepG2 cells

Immunosuppressant cyclosporine A (CsA) treatment can cause severe side effects. Patients taking immunosuppressant after organ transplantation often display hyperlipidemia and obesity. Elevated levels of free fatty acids have been linked to the etiology of metabolic syndromes, nonalcoholic fatty liver and steatohepatitis. The contribution of free fatty acids to CsA-induced toxicity is not known. In this study we explored the effect of palmitic acid on CsA-induced toxicity in HepG2 cells. CsA by itself at therapeutic exposure levels did not induce detectible cytotoxicity in HepG2 cells. Co-treatment of palmitic acid and CsA resulted in a dose dependent increase in cytotoxicity, suggesting that fatty acid could sensitize cells to CsA-induced cytotoxicity at the therapeutic doses of CsA. A synergized induction of caspase-3/7 activity was also observed, indicating that apoptosis may contribute to the cytotoxicity. We demonstrated that CsA reduced cellular oxygen consumption which was further exacerbated by palmitic acid, implicating that impaired mitochondrial respiration might be an underlying mechanism for the enhanced toxicity. Inhibition of c-Jun N-terminal kinase (JNK) attenuated palmitic acid and CsA induced toxicity, suggesting that JNK activation plays an important role in mediating the enhanced palmitic acid/CsA-induced toxicity. Our data suggest that elevated FFA levels, especially saturated FFA such as palmitic acid, may be predisposing factors for CsA toxicity, and patients with underlying diseases that would elevate free fatty acids may be susceptible to CsA-induced toxicity. Furthermore, hyperlipidemia/obesity resulting from immunosuppressive therapy may aggravate CsA-induced toxicity and worsen the outcome in transplant patients.