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Nonalcoholic fatty liver disease (NAFLD) is becoming an increasingly serious disease worldwide. Unfortunately, no specific drug has been approved to treat NAFLD. Accumulating evidence suggests that lipotoxicity, which is induced by an excess of intracellular triacylglycerols (TAGs), is a potential mechanism underlying the ill-defined progression of NAFLD. Under physiological conditions, a balance is maintained between TAGs and free fatty acids (FFAs) in the liver. TAGs are catabolized to FFAs through neutral lipolysis and/or lipophagy, while FFAs can be anabolized to TAGs through an esterification reaction. However, in the livers of patients with NAFLD, lipophagy appears to fail. Reversing this abnormal state through several lipophagic molecules (mTORC1, AMPK, PLIN, etc.) facilitates NAFLD amelioration; therefore, restoring failed lipophagy may be a highly efficient therapeutic strategy for NAFLD. Here, we outline the lipophagy phases with the relevant important proteins and discuss the roles of lipophagy in the progression of NAFLD. Additionally, the potential candidate drugs with therapeutic value targeting these proteins are discussed to show novel strategies for future treatment of NAFLD., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Acute ischemic stroke is a challenging disease that threatens the life of older people. Dysfunction of brain endothelial cells is reported to be involved in the pathogenesis of acute ischemic stroke. Ramelteon is a novel agonist of melatonin receptor developed for the treatment of insomnia. Recently, the promising protective effect of Ramelteon on brain injury has been widely reported. The present study aims to investigate the protective effect of Ramelteon against free fatty acid (FFA)-induced damages in brain vascular endothelial cells and the underlying mechanism. Firstly, we discovered that Ramelteon administration remarkably reversed the decreased cell viability, increased LDH release, activated oxidative stress, and excessive released inflammatory factors caused by FFAs. Secondly, Ramelteon extensively suppressed the attachment of U937 monocytes to bEnd.3 brain endothelial cells induced by FFAs. In addition, the elevated expression of E-selectin and the reduced expression of KLF2 induced by FFAs were pronouncedly alleviated by Ramelteon. Lastly, silencing of KLF2 abolished the protective effects of Ramelteon against FFA-induced expression of E-selectin and the attachment of U937 monocytes to bEnd.3 brain endothelial cells. In conclusion, Ramelteon mitigated FFA-induced attachment of monocytes to brain vascular endothelial cells by increasing the expression of KLF2 and reducing the expression of E-selectin., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid β-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. Furthermore, flavin adenine dinucleotide (FAD) can enhance the expression and enzyme activity of SCAD. However, whether FAD can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of FAD on pathological cardiac hypertrophy and fibrosis. FAD significantly inhibited PE-induced cardiomyocyte hypertrophy and AngII-induced cardiac fibroblast proliferation. In addition, FAD ameliorated pathological cardiac hypertrophy and fibrosis in SHR. FAD significantly increased the expression and enzyme activity of SCAD. Meanwhile, ATP content was increased, the content of free fatty acids and reactive oxygen species were decreased by FAD in vivo and in vitro. In addition, molecular dynamics simulations were also used to provide insights into the structural stability and dynamic behavior of SCAD. The results demonstrated that FAD may play an important structural role on the SCAD dimer stability and maintenance of substrate catalytic pocket to increase the expression and enzyme activity of SCAD. In conclusion, FAD can inhibit pathological cardiac hypertrophy and fibrosis through activating SCAD, which may be a novel effective treatment for pathological cardiac hypertrophy and fibrosis, thus prevent them from developing into heart failure., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rationale: Depression and anxiety can cause the development of chronic pain. However, the mechanism of chronic pain induced by emotional dysfunction is still unknown. Previously, we demonstrated that the G protein-coupled receptor 40/free fatty acid receptor 1 (GPR40/FFAR1) signaling in the brain is related to regulation of both pain and emotion. In the present study, we proved that the role of GPR40/FFAR1 signaling in the development of chronic pain is induced by emotional dysfunction., Results: Repeated social defeat (SD)-stressed mice showed the impairment of social interaction and anxiety behavior. These mice also caused pain prolongation after paw-incision comparison with non-SD mice. This pain prolongation was markedly continued by infusion of the GPR40/FFAR1 antagonist, GW1100 during SD stress but not non-SD stress. Although, infusion of the GW1100 during SD stress did not cause deterioration of the emotional behavior. Furthermore, GW1100-treated SD-mice showed strong tendency of emotional dysfunction after paw incision., Conclusion: Our findings indicate that the dysfunction of fatty acids-GPR40/FFAR1 signaling in the brain underlying stress condition might be related to the development of chronic pain.

In view of the occurrence of diabetic ketoacidosis associated with the use of sodium-glucose transport protein-2 inhibitors in patients with type 1 diabetes (T1DM) and the relative absence of this complication in patients treated with liraglutide in spite of reductions in insulin doses, we investigated the effect of liraglutide on ketogenesis. Twenty-six patients with inadequately controlled T1DM were randomly divided into 2 groups of 13 patients each. After an overnight fast, patients were injected, subcutaneously, with either liraglutide 1.8 mg or with placebo. They were maintained on their basal insulin infusion and were followed up in our clinical research unit for 5 hours. The patients injected with placebo maintained their glucose and glucagon concentrations without an increase, but there was a significant increase in free fatty acids (FFA), acetoacetate and β-hydoxybutyrate concentrations. In contrast, liraglutide significantly reduced the increase in FFA, and totally prevented the increase in acetoacetate and β-hydroxybutyrate concentrations while suppressing glucagon and ghrelin concentrations. Thus, a single dose of liraglutide is acutely inhibitory to ketogenesis., (© 2017 John Wiley & Sons Ltd.)

Pancreatic β-cell lipotoxicity is a central feature of the pathogenesis of type 2 diabetes. To study the mechanism by which fatty acids cause β-cell death and develop novel approaches to prevent it, a high-throughput screen on the β-cell line INS1 was carried out. The cells were exposed to palmitate to induce cell death and compounds that reversed palmitate-induced cytotoxicity were ascertained. Hits from the screen were analyzed by an increasingly more stringent testing funnel, ending with studies on primary human islets treated with palmitate. MAP4K4 inhibitors, which were not part of the screening libraries but were ascertained by a bioinformatics analysis, and the endocannabinoid anandamide were effective at inhibiting palmitate-induced apoptosis in INS1 cells as well as primary rat and human islets. These targets could serve as the starting point for the development of therapeutics for type 2 diabetes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: This study aimed to investigate whether exogenous hydrogen sulfide (H2S) can protect the RAW264.7 macrophages against the inflammation induced by free fatty acids (FFA) by blunting NLRP3 inflammasome activation via a specific TLR4/NF-κB pathway., Methods: RAW264.7 macrophages were exposed to increasing concentrations of FFA for up to 3 days to induce FFA-induced inflammation. The cells were pretreated with NaHS (a donor of H2S) before exposure to FFA. Cell viability, cell apoptosis, TLR4, NF-κB, NLRP3 inflammasome, IL-1β, IL-18 and cleaved caspase-3 expression were measured by a combination of MTT assay, ELISA, and immunoblotting., Results: H2S attenuated FFA-induced cell apoptosis, and reduced the expression of NLRP3, ASC, pro-caspase-1, caspase-1, IL- 1β, IL-18 and caspase-3. In addition, H2S inhibited the FFA-induced activation of TLR4 and NF-κB. Furthermore, NLRP3 inflammasome activation was regulated by the TLR4 and NF-κB pathway., Conclusion: The present study demonstrated for the first time that H2S appears to suppress FFA-induced macrophage inflammation and apoptosis by inhibiting the TLR4/ NF-κB pathway and its downstream NLRP3 inflammasome activation. Thus H2S might possess potential in the treatment of diseases resulting from FFA overload like insulin resistance and type diabetes., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Puerarin, a type of isoflavone, was shown to have multiple protective effects on myocardial injury. The objective of this study was to investigate the role of puerarin in the progression of lipotoxic cardiomyopathy. Primary cardiomyocytes were isolated from FATP1 transgenic (Tg) mice with lipotoxic cardiomyopathy, and various concentrations of puerarin were used to incubate with the cardiomyocytes. Our results showed low-dose puerarin (≤20 μM) treatment increased the cell viability and decreased the accumulation of free fatty acid (FFA). The data on enzyme-linked immunosorbent assay indicated that 15 μM puerarin treatment greatly increased Na-K-ATPase activity and decreased C-reactive protein secretion, thus suppressing the expression of CD36, a key contributor to the FFA accumulation. Additionally, low-dose puerarin (≤100 mg/kg body weight) administration improved Na-K-ATPase activity. Our data on serum analysis and histological detection in vivo indicated that systemic inflammation, CD36-induced lipid infiltration, and cardiomyocyte apoptosis were markedly alleviated in Tg mice injected with 90 mg/kg dose of puerarin. Finally, the uptake rates of H-palmitate and C-glucose were monitored on ex vivo working hearts that were obtained from wild-type (WT), Tg-control, and Tg-puerarin mice. Compared with WT hearts, Tg hearts displayed a significant decrease in Na/K-ATPase activity and glucose consumption rate and an increase in palmitate uptake rate and FFA accumulation. In Tg-puerarin hearts, Na/K-ATPase activity and glucose consumption rate were significantly rescued, and palmitate uptake and FFA accumulation were sharply suppressed. In conclusion, low-dose puerarin suppressed Na-K-ATPase-mediated CD36 expression and systemic inflammation and alleviated cardiac lipotoxicity in vitro and in vivo.

CD36 is a key transporter involved in fatty acid (FA) uptake and contributes to the accumulation of FA in cardiomyocytes. The objective of this study was to investigate the role of ouabain, a glycoside regulator of Na(+)/K(+)-ATPase, in the regulation of CD36 expression and FA accumulation. FATP1 transgenic (Tg) mice with lipotoxic cardiomyopathy displayed significantly increased cardiac CD36 expression and free fatty acid accumulation. The data on enzyme-linked immunosorbent assay showed that endogenous ouabain was decreased in the serum of Tg mice versus wild-type mice. CD36 expression and free fatty acid accumulation in their primary cardiomyocytes were abated by treatment with 0.15-0.30 μM ouabain. CD36 expression was suppressed by 0.2 μM ouabain treatment, and the suppression was rescued by C-reactive protein. CD36 expression and free fatty acid accumulation in the heart were markedly reduced in Tg mice injected with 30 or 40 ng of ouabain (P < 0.01). Obvious fatty infiltration was found in noninjected Tg mice but not in the mice injected with 40 ng of ouabain. In conclusion, low-dose exogenous ouabain increased Na(+)/K(+)-ATPase activity, suppressed C-reactive protein-mediated CD36 expression, and alleviated murine cardiac lipotoxicity in vitro and in vivo.

Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of lesions ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). The excess influx of fatty acids (FAs) into the liver is recognized as a main cause of simple steatosis formation and progression to NASH. Recently, administration of lactoferrin (LF), a glycoprotein present in milk, was suggested to prevent NAFLD development. However, the effect of LF on the contribution of FA to NAFLD development remains unclear. In this study, the effects of LF on FA mixture (FAm)-induced lipotoxicity using human hepatocarcinoma G2 cells were assessed. FAm significantly decreased cell viability and increased intracellular lipid accumulation, whereas LF significantly recovered cell viability without affecting lipid accumulation. FAm-induced lactic dehydrogenase (LDH) and caspase-3/7 activities were significantly decreased by LF and SP600125, a c-Jun N-terminal kinase (JNK) specific inhibitor. We also found that LF added to FAm-treated cells induced Akt phosphorylation, which contributed to inhibition of JNK signaling pathway-dependent apoptosis. Akt inhibitor VIII, an allosteric Akt inhibitor, significantly attenuated the effect of LF on LDH activity and abrogated the ones on cell viability and caspase-3/7 activity. In summary, the present study has revealed that LF has a protective effect on FAm-induced lipotoxicity in a HepG2 model of NAFLD and identified the activation of the Akt signaling pathway as a possibly major mechanism.

Scope: Resveratrol (RSV), a natural polyphenol, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD); however, its underlying mechanism is unclear. Autophagy was recently identified as a critical protective mechanism during NAFLD development. Therefore, we investigated the role of autophagy in the beneficial effects of RSV on hepatic steatosis., Methods and Results: Via Oil red O staining, triglyceride, and β-hydroxybutyrate detection, we found that RSV decreased palmitate-induced lipid accumulation and stimulated fatty acid β-oxidation in hepatocytes. Based on Western blot assay, confocal microscopy and transmission electron microscopy, we found that RSV induced autophagy in hepatocytes, whereas autophagy inhibition markedly abolished RSV-mediated hepatic steatosis improvement. Moreover, RSV increased cAMP levels and the levels of SIRT1 (sirtuin 1), pPRKA (phosphorylated protein kinase A), and pAMPK (phosphorylated AMP-activated protein kinase), as well as SIRT1 activity in HepG2 cells. Incubation with inhibitors of AC (adenylyl cyclase), PRKA, AMPK, SIRT1, or with AC, PRKA, AMPK, or SIRT1 siRNA abolished RSV-mediated autophagy. Similar results were obtained in mice with hepatic steatosis., Conclusion: RSV improved hepatic steatosis partially by inducing autophagy via the cAMP-PRKA-AMPK-SIRT1 signaling pathway, which provides new evidence regarding RSV's effects on NAFLD treatment., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: To investigate whether docosahexaenoic acid (DHA) could inhibit linoleic acid (LA) induced monocyte chemoattractant protein (MCP)-1 expression in human retinal pigment epithelial (RPE) cells., Methods and Results: ARPE-19 cells were pretreated with DHA and then exposed to LA. The expression of MCP-1 and PPARγ was examined using RT-PCR and Western blot analysis. LA at 10, 25, or 50 μM induced expression of MCP ARPE-19 cells in a dose-dependent manner (p < 0.05). DHA at 50 and 100 μM effectively inhibited LA-induced MCP-1 expression and production (p < 0.05) and NF-κB activation. In addition, the culture medium from LA-stimulated ARPE-19 cells could induce tube formation in choroidal endothelial cells (RF6A), whereas 100 μM DHA inhibited tube formation. DHA at 100 μM increased the expression and activity of PPARγ (p < 0.05). Pretreatment with PPARγ inhibitor (GW9662) abolished the inhibitory effect of DHA (100 μM) on LA-induced IκB degradation, p65 translocation, and MCP-1 expression in ARPE-19 cells (p < 0.05), as well as tube formation in RF6A., Conclusion: DHA reduced LA-induced MCP-1 expression via a PPARγ- and NF-κB-dependent pathway in ARPE-19 cells. These results suggest the molecular mechanisms underlying the beneficial effects of increased consumption of DHA and reduced consumption of LA on age-related macular degeneration., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study aimed to explore the effect of angiotensin (1-7) (Ang (1-7)) on palmitate-induced apoptosis in islet endothelial cells and the mechanism of action. MS-1 cells were treated with palmitate in the presence or absence of Ang (1-7). The percentage of apoptotic cells was determined by DNA fragmentation and flow cytometry. Reactive oxygen species (ROS) production was measured using a Reactive Oxygen Species Assay Kit. Expression of AKT, eNOS, C-Jun N-terminal kinase (JNK), and p38 was detected by western blotting. Compared with palmitate treated group, palmitate-induced apoptosis was decreased in MS-1 cells which were preincubated with Ang (1-7) (P < 0.05). Palmitate decreased the phosphorylation of AKT and eNOS, and Ang (1-7) increased the phosphorylation of these kinases (P < 0.05), with a concomitant reduction in MS-1 cells apoptosis. Ang (1-7) also inhibited the palmitate-induced ROS production and attenuated the apoptosis-related signaling molecule JNK and p38 activation (all P < 0.05). PI3K/AKT, eNOS, p38 MAPK, and JNK inhibitors blocked the antilipoapoptosis of Ang (1-7) (all P < 0.05). Our findings suggest that Ang (1-7) reduces palmitate-induced islet endothelial cells apoptosis. AKT/eNOS/NO signaling and JNK and p38 pathway are involved in the Ang (1-7)-mediated modulation of islet endothelial cells lipoapoptosis.

Non-alcoholic fatty liver disease is associated with inhibited AMP-activated kinase (AMPK) and activation of sterol regulatory element binding protein 1 (SREBP-1). AMPK phosphorylation inhibits SREBP-1, a major transcription factor of de novo lipogenesis, by inhibiting the liver X receptor (LXR) or by direct phosphorylation. Resveratrol, a polyphenol, has regulatory effects on hepatic lipid metabolism as a potent AMPK activator. In this study, we evaluated the anti-steatogenic effects of resveratrol and its derivatives and identified the molecular mechanism in vitro and in vivo. Resveratrol and its derivatives decreased lipid accumulation by free fatty acids (FFA mixture; 0.5 mM, oleic acid:palmitic acid = 2: 1) in H4IIEC3 cells. Synthesized derivatives of resveratrol had lower cytotoxicity than the parental molecule with similar potency. SY-102 suppressed SREBP-1 maturation by T0901317, an LXR agonist, and decreased SRE luciferase activity and the mRNA levels of lipogenic genes. Inhibition of AMPK by pre-treatment with compound C completely blocked the effects of SY-102. To evaluate their efficacy in vivo, mice were fed a high-fat diet for 5 days, and resveratrol or SY-102 was administered orally for the last 2 days. Oral administration of the SY-102 increased AMPK phosphorylation, followed by reduced hepatic triglyceride accumulation to a similar extent as resveratrol. These data demonstrate that SY-102, a synthesized derivative of resveratrol, might provide a promising therapeutic effect against fatty liver disease.

We evaluated the effect of sitagliptin on glycemic control, endogenous insulin secretion, and beta cell function in Japanese patients with type 2 diabetes mellitus (T2DM) receiving a combination of oral antidiabetics and basal insulin analog glargine (basal-supported oral therapy [BOT]). Twenty-one patients showing inadequate glycemic control with BOT were given dipeptidylpeptidase-4 inhibitor (DPP-4I) sitagliptin at 50 mg/day for 12 weeks. Clinical markers of glycemic control, HbA1c, glycated albumin (GA), and 1,5-anhydroglucitol (1,5-AG), were measured before and 4 and 12 weeks after the start of sitagliptin. A 2-hour morning meal test was performed upon enrollment and at 12 weeks, and plasma glucose (PG), serum C-peptide, and plasma intact proinsulin (PI) were measured. HbA1c, GA, and 1,5-AG at 4 and 12 weeks were significantly improved over enrollment levels. The area under the PG concentration curve (AUC-PG) during the meal test at 12 weeks was significantly reduced (from 350 ± 17 mg ･ hr/dL before sitagliptin treatment to 338 ± 21 mg ･ hr/dL [mean ± SE], P < 0.05,); the AUC-C-peptide was unchanged (from 3.4 ± 0.4 ng ･ hr/mL to 3.6 ± 0.5 ng ･ hr/mL). However, both fasting and 2-hour PI/C-peptide ratios at 12 weeks were significantly decreased (from 13.3 ± 2.3 to 11.1 ± 2.0 [P < 0 .05] and from 9.5 ± 1.6 to 5.3 ± 0.9 [P < 0.01], respectively). Adding sitagliptin to BOT in Japanese T2DM patients appears to improve glycemic control without increasing endogenous insulin secretion and to reduce fasting and 2-hour postprandial PI/C-peptide ratios.

The fungal pathogen Fusarium graminearum is the causal agent of Fusarium head blight (FHB); a devastating crop disease resulting in heavy yield losses and grain contamination with mycotoxins. We recently showed that the secreted lipase FGL1, a virulence factor of F. graminearum, targets plant defense-related callose biosynthesis during wheat head infection. This effector-like function is based on a FGL1-mediated release of polyunsaturated free fatty acids (FFA) that can inhibit callose synthase activity. The importance of FGL1 in successful wheat head colonization was demonstrated in FGL1 disruption mutants (Δfgl1), where infection was restricted to directly inoculated spikelets and accompanied by strong callose deposition in the spikelet's phloem. The application of polyunsaturated FFA to Δfgl1-infected spikelets prevented callose deposition in the phloem and partially restored wheat head colonization.   The comparative analysis of 3 wheat cultivars revealed that the level of resistance to FHB correlated with resistance to FFA-dependent inhibition of callose biosynthesis. Therefore, resistance of callose biosynthesis to FFA inhibition might be used as marker and/or direct target in the breeding of FHB-resistant wheat cultivars.

Free fatty acids (FFAs) have been implicated in the pathogenesis of insulin resistance. Reducing plasma FFA concentration in obese and type 2 diabetic (T2DM) subjects improves insulin sensitivity. However, the molecular mechanism by which FFA reduction improves insulin sensitivity in human subjects is not fully understood. In the present study, we tested the hypothesis that pharmacological FFA reduction enhances insulin action by reducing local (muscle) inflammation, leading to improved insulin signalling. Insulin-stimulated total glucose disposal (TGD), plasma FFA species, muscle insulin signalling, IBα protein, c-Jun phosphorylation, inflammatory gene (toll-like receptor 4 and monocyte chemotactic protein 1) expression, and ceramide and diacylglycerol (DAG) content were measured in muscle from a group of obese and T2DM subjects before and after administration of the antilipolytic drug acipimox for 7 days, and the results were compared to lean individuals. We found that obese and T2DM subjects had elevated saturated and unsaturated FFAs in plasma, and acipimox reduced all FFA species. Acipimox-induced reductions in plasma FFAs improved TGD and insulin signalling in obese and T2DM subjects. Acipimox increased IBα protein (an indication of decreased IB kinase-nuclear factor B signalling) in both obese and T2DM subjects, but did not affect c-Jun phosphorylation in any group. Acipimox also decreased inflammatory gene expression, although this reduction only occurred in T2DM subjects. Ceramide and DAG content did not change. To summarize, pharmacological FFA reduction improves insulin signalling in muscle from insulin-resistant subjects. This beneficial effect on insulin action could be related to a decrease in local inflammation. Notably, the improvements in insulin action were more pronounced in T2DM, indicating that these subjects are more susceptible to the toxic effect of FFAs.

Aims: The damage of human brain vascular endothelial cells (HBVECs) is the key pathogenesis of diabetes-associated cerebral vascular complications. The aim of this study was to elucidate the effects of glutathione (GSH) on free fatty acids (FFAs)-induced HBVECs apoptosis, oxidative stress, and the involved possible signaling pathway., Methods: After culturing HBVECs for 72 h with GSH and FFAs, we determined cell proliferation by CCK8, detected apoptosis by caspase-3 and Annexin V-FITC/PI staining, and judged oxygen stress by determining the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP). We investigated whether the Akt pathway was involved in FFAs-induced signaling pathway alteration and whether GSH influenced the above effects., Results: After being cultured in 200 μM FFAs for 72 h, the HBVECs proliferation significantly decreased; HBVECs apoptosis increased; the ROS levels increased; and the HBVECs MMP subsequently decreased. FFAs induced a significant decrease in phosphorylated active Akt. These alterations were obviously prevented when 1 mM GSH was added to culture medium containing FFAs, and the above effects of GSH were blocked by Akt inhibitor., Conclusion: GSH may prevent FFAs-induced HBVECs damage, oxidative stress, and apoptosis through activating the Akt pathway., (© 2013 Blackwell Publishing Ltd.)

Background/aim: We tested the hypothesis that a persistent reduction in free fatty acid (FFA) levels improves cardiac function and systemic insulin sensitivity via a reduction in the myocardial and skeletal muscle adiposities and a modulation in adipokine release., Methods: Study subjects (body mass index 22-30 kg/m(2), 57 ± 3 yr old) underwent magnetic resonance imaging and spectroscopy to measure the cardiac function and the amounts of fat inside and around the myocardium and skeletal muscle, before (n = 10) and after acute (n = 8) and 1 wk (n = 7, one excluded from analysis) lowering of circulating FFA by acipimox. Circulating adipokines (leptin, adiponectin, resistin, TNFα, IL-6, IL-8, plasminogen activator inhibitor-I, macrophage chemoattractant protein-1) were measured., Results: The ejection fraction (62 ± 2 vs. 56 ± 1%, P = 0.0035), cardiac output (6.6 ± 0.3 vs. 5.5 ± 0.2 liters/min, P = 0.0018), and forward work (708 ± 49 vs. 539 ± 44 mm Hg × liters/min, P = 0.018) were significantly lower after 1 wk of FFA lowering. In the six subjects undergoing all sessions, the stroke and end-diastolic volumes were also reduced, insulin sensitivity was increased by 33%, and adiponectinemia was decreased (-26%, P = 0.03). No change in intracellular cardiac and skeletal muscle triglyceride levels was observed. Metabolic changes correlated with the lowering of FFA. The reduction in cardiac function was related with changes in glycemia and insulin sensitivity, whereas the deflection in left ventricular work was correlated with the decline in FFA, lipid, and blood pressure levels., Conclusions: A 1-wk FFA depletion suppressed cardiac function and improved insulin sensitivity. Intracellular triglyceride deposits in the heart and skeletal muscle played no role in the observed changes. Our data show that FFA participate in the physiological regulation of adipokine levels.

Development of a preventive strategy against tubular damage associated with proteinuria is of great importance. Recently, free fatty acid (FFA) toxicities accompanying proteinuria were found to be a main cause of tubular damage, which was aggravated by insufficiency of peroxisome proliferator-activated receptor alpha (PPARα), suggesting the benefit of PPARα activation. However, an earlier study using a murine acute tubular injury model, FFA-overload nephropathy, demonstrated that high-dose treatment of PPARα agonist (0.5% clofibrate diet) aggravated the tubular damage as a consequence of excess serum accumulation of clofibrate metabolites due to decreased kidney elimination. To induce the renoprotective effects of PPARα agonists without drug accumulation, we tried a pretreatment study using low-dose clofibrate (0.1% clofibrate diet) using the same murine model. Low-dose clofibrate pretreatment prevented acute tubular injuries without accumulation of its metabolites. The tubular protective effects appeared to be associated with the counteraction of PPARα deterioration, resulting in the decrease of FFAs influx to the kidney, maintenance of fatty acid oxidation, diminution of intracellular accumulation of undigested FFAs, and attenuation of disease developmental factors including oxidative stress, apoptosis, and NFκB activation. These effects are common to other fibrates and dependent on PPARα function. Interestingly, however, clofibrate pretreatment also exerted PPARα-independent tubular toxicities in PPARα-null mice with FFA-overload nephropathy. The favorable properties of fibrates are evident when PPARα-dependent tubular protective effects outweigh their PPARα-independent tubular toxicities. This delicate balance seems to be easily affected by the drug dose. It will be important to establish the appropriate dosage of fibrates for treatment against kidney disease and to develop a novel PPARα activator that has a steady serum concentration regardless of kidney dysfunction., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study investigated the effects and molecular mechanisms of genistein in improving insulin resistance induced by free fatty acids (FFAs) in HepG2 hepatocytes. A model of insulin resistance in HepG2 cells was established by adding palmitic acid (0.5 mmol/L) to the culture medium and the cells were treated by genistein. Glucose consumption of HepG2 cells was determined by glucose oxidase method. The levels of c-jun N-terminal kinase (JNK) phosphorylation, insulin receptor substrate-1 (IRS-1) Ser307 phosphorylation, JNK, IRS-1, phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 1 (GLUT1) proteins were detected by Western blotting. The results showed that after the treatment with palmitic acid for 24 h, the insulin-stimulated glucose transport in HepG2 cells was inhibited, and the glucose consumption was substantially reduced. Meanwhile, the expressions of IRS-1, PI-3K p85 protein and GLUT1 were obviously reduced, while the levels of JNK phosphorylation and IRS-1 Ser307 phosphorylation and the expression of JNK protein were significantly increased, as compared with cells of normal control. However, the aforementioned indices, which indicated the existence of insulin resistance, were reversed by genistein at 1-4 μmol/L in a dose-dependent manner. It was concluded that insulin resistance induced by FFAs in HepG2 hepatocytes could be improved by genistein. Genistein might reverse FFAs-induced insulin resistance in HepG2 cells by targeting JNK.

Objective: To investigate whether Astragalus membranaceus (AM) can protect endothelium-dependent vasodilatation (EDV) function of aorta from the damage induced by high level of free fatty acid (FFA)., Methods: Ten male SD rats, 8 weeks old and 250-300 g in weight, were sacrificed and thoracic aorta were harvested. Aorta rings incubated in organ baths were divided into three groups, Control group, FFA group and FFA+ AM group. The control group was incubated in 20 mL Krebs-Henseleit solution; the FFA group was incubated in 20 mL KH solution mixed with FFA(800 micromol/L) the FFA + AM group was incubated in 20 mL KH solution mixed with FFA (800 micromol/L) and AM (4 g/L). The relaxation levels of aorta rings response to acetylcholine and sodium nitroprusside were measured, the expression of NF-kappaB and the level of NOx in the organ bath were analyzed by immunohistochemistry., Results: Severe endothelial dysfunction were induced in FFA group (maximal vasorelaxation in response to Ach: 61.1% +/- l6.9% vs. 93.1% +/- 2.7% in control, P < 0.05), while EDV in FFA+AM group was significantly improved by the incubation with AM (P < 0.05). Compared with the control group (104.1 +/- 14.2) micromol/g, NOx levels of FFA group was (83.1 +/- 8.4) micromol/g (P < 0.05), and the treatment of AM increased the levels of NOx (98.8 +/- 10.7) micromol/g (P < 0.05). The control vascular ring had a little NF-kappaB expression in endothelial nucleus, FFA increased the activation of NF-kappaB, while the treatment of AM lower the elevated NF-kappaB level., Conclusion: FFA can directly injure EDV, while AM may ameliorate it, with the possible mechanism related to the signal pathway of NF-kappaB and NO.

During the onset of an acute coronary syndrome, an immediate increase in plasma free fatty acid (FFA) concentrations presents the ischaemic underperfused myocardium with a metabolic challenge to its survival. For the maintenance of contraction and to counter ventricular arrhythmias, parenteral treatment to reduce FFA availability should be available for paramedics when the patient is first seen. The best treatment options are insulin which partly inhibits FFA release from adipose tissue, immediate reduction of adipose tissue lipolysis, perhaps by new analogues of nicotinic acid, or measures to decrease the FFA/molar binding ratio with albumin. None has yet been tested in appropriate clinical trials.

The free fatty acid receptor, GPR40, is implicated in the pathophysiology of type 2 diabetes, and is a new potential drug target for the treatment of type 2 diabetes. Its antagonist is thought to be not only a useful chemical probe for further exploring the function of GPR40 but also a lead structure for drug development. With virtual screening based on a homology model followed by a cell-based calcium mobilization assay, we found that sulfonamides are a new class of small organic antagonists for GPR40. One of the compounds, DC260126, dose-dependently inhibited GPR40-mediated Ca(2+) elevations stimulated by linoleic acid, oleic acid, palmitoleic acid and lauric acid (IC(50): 6.28+/-1.14, 5.96+/-1.12, 7.07+/-1.42, 4.58+/-1.14 microM, respectively), reduced GTP-loading and ERK1/2 phosphorylation stimulated by linoleic acid in GPR40-CHO cells, suppressed palmitic acid potentiated glucose-stimulated insulin secretion, and negatively regulated GPR40 mRNA expression induced by oleic acid in Min6 cells.

The pivotal therapeutic role of myocardial metabolic modulation in ischemic heart disease (IHD) is increasingly recognized. Among the others, inhibitors of free fatty acids (FFA) oxidation have been consistently shown to play an important role in the therapeutic strategy of IHD patients. Additionally, abnormalities of glucose homeostasis are consistently present in patients with IHD, definitely contributing to the progression of the primary disease. If not adequately treated, in most patients glucose metabolism abnormalities will heavily contribute to the occurrence of complications, of whom severe left ventricular dysfunction is at present one of the most frequent and insidious. Apart from a meticulous metabolic control of frank diabetes, special attention should be also paid to insulin resistance, a condition that is generally underdiagnosed as a distinct clinical entity. An important metabolic alteration in diabetic patients is the increase in free fatty acid concentrations and the increased muscular and myocardial free fatty acid uptake and oxidation. The increased uptake and utilization of free fatty acid and the reduced utilization of glucose as source of energy during stress and ischemia are responsible for the increased susceptibility of the diabetic heart to myocardial ischemia and to a greater decrease of myocardial performance for a given amount of ischemia compared to non diabetic hearts. In order to shift cardiac metabolism from FFA to preferential glucose utilization, the use of FFA inhibitors has been advocated. Among FFA inhibitors etomoxir, perhexiline, oxfenicine and trimetazidine have been evaluated. Among them, trimetazidine, specifically a 3-ketoacyl coenzyme A thiolase inhibitor, has been shown to improve overall glucose metabolism in IHD patients with diabetes and left ventricular dysfunction. The observed combined beneficial effects of FFA inhibitors on myocardial ischemia, left ventricular function and glucose metabolism, represent an additional advantage of these drugs, especially when myocardial and glucose metabolism abnormalities coexist. In this paper, the recent literature on the beneficial therapeutic effects of FFA oxidation inhibitors on myocardial ischemia, left ventricular dysfunction and glucose metabolism in patients with ischemic heart disease and abnormalities of carbohydrate metabolism is reviewed and discussed.

Coronary artery disease (CAD) is due to subintimal deposition of atheromatous plaques in large and medium-sized coronary arteries. Different risk factors have been identified such as hypertension, hypercholesterolemia, diabetes and smoking. Both hypertension and diabetes mellitus affect the same major target organs. The common hypertensive/diabetic target is the vascular tree, hence renal function is particularly exposed in these patients and often reduced by vascular injury. Consequently, renal protection is a major concern for patients with CAD and/or diabetes who are facing vascular or abdominal surgery, potential nephrotoxic treatment or contrast agents-induced nephropathy. Ischemia reperfusion injury (IRI) is also a common and important clinical cause of renal disease such as renal transplantation and following shock from any cause. Acute renal failure and chronic renal insufficiency are significant complications associated with prolonged warm ischemia (WI). The WI duration remains the most important factor governing the return of postoperative renal function in surgical procedure in which renal blood flow is interrupted. Beside traditional therapy, metabolic therapy is another approach for the treatment of myocardial ischemia at the cellular level itself, with agents that have the capacity to exert their action on the cell without affecting the hemodynamic condition. Such therapies could also be of major interest in the prevention of renal damage and limitation of long term effect of renal IRI, particularly for patients with reduced functional nephron mass. The absence of hemodynamic effect is useful in situations such as shock.

Alterations of cardiac metabolism can be present in several cardiac syndromes. Heart failure may itself promote metabolic changes such as insulin resistance, in part through neurohumoral activation, and determining an increased utilization of non-carbohydrate substrates for energy production. In fact, fasting blood ketone bodies as well as fat oxidation have been shown to be increased in patients with heart failure. The result is depletion of myocardial ATP, phosphocreatine and creatine kinase with decreased efficiency of mechanical work. A direct approach to manipulate cardiac energy metabolism consists in modifying substrate utilization by the failing heart. To date, the most effective metabolic treatments include several pharmacological agents, such as trimetazidine and perhexiline, that directly inhibit fatty acid oxidation. These agents have been originally adopted to increase the ischemic threshold in patients with effort angina. However, the results of current research is supporting the concept that shifting the energy substrate preference away from fatty acid metabolism and toward glucose metabolism could be an effective adjunctive treatment in patients with heart failure, in terms of left ventricular function and glucose metabolism improvement. In fact, these agents have also been shown to improve overall glucose metabolism in diabetic patients with left ventricular dysfunction. In this paper, the recent literature on the beneficial therapeutic effects of modulation of cardiac metabolic substrates utilization in patients with heart failure is reviewed and discussed.

The possibility of modifying cardiac metabolism by switching the fuel used by the myocardium could become increasingly important. Inhibitors of free fatty acid (FFA) oxidation could have an important role in therapeutic strategy for patients with heart failure, and shifting the energy substrate preference away from FFA metabolism and toward glucose metabolism may be an effective adjunctive treatment. Additionally, abnormalities of glucose homeostasis in patients with heart failure contribute to the progression of the primary disease. If not adequately treated, these abnormalities can contribute to the occurrence of complications, including severe left ventricular dysfunction. Apart from meticulous metabolic control of frank diabetes, special attention should be paid to insulin resistance, a distinct clinical entity. The observed combined beneficial effects of FFA inhibitors on left ventricular function and glucose metabolism represent an additional advantage of these drugs, especially when abnormalities of myocardial and glucose metabolism coexist.

Objective: Free fatty acid inhibition with trimetazidine (TMZ) improves myocardial metabolism and myocardial ischemia in patients with coronary artery disease (CAD). Because of its effect on myocardial glucose utilization TMZ may represent a therapeutic option in diabetic patients with CAD. Aim of the present study was to evaluate whether the metabolic effect of TMZ may improve episodes of myocardial ischemia in diabetic patients with CAD., Research Design and Methods: We assessed the effect of TMZ on 24 h ambulatory ECG monitoring (AEM) in 30 patients (22 males and 8 females, mean (SE) age 67+/-6.5 years) with NIDDM and ischemic cardiomyopathy. Patients were randomized to receive on top of standard therapy either TMZ (20 mg, tds) or placebo (tds) and were evaluated at baseline and after 6 months., Results: Patients randomized to TMZ or placebo were comparable regarding demographic data, distribution of CAD, and glicated haemoglobin levels. TMZ significantly reduced the number of episodes of transient myocardial ischemia (-24% compared to baseline, p<0.01; -27% compared to placebo, p<0.01), and Total Ischemic Burden (-28% compared to baseline, p<0.01; -29% compared to placebo, p<0.01). TMZ also significantly reduced the number of silent episodes of myocardial ischemia (-42% compared to baseline and -39% compared to placebo, p<0.01) and the time of silent myocardial ischemia/24 h (-37% compared to baseline and -35% compared to placebo, p<0.01). No significant changes in heart rate were detected between baseline, placebo and TMZ evaluations., Conclusions: TMZ is effective in reducing silent and symptomatic episodes of transient myocardial ischemia in diabetic patients with CAD on standard anti-anginal therapy.

Elevated plasma FFA cause beta-cell lipotoxicity and impair insulin secretion in nondiabetic subjects predisposed to type 2 diabetes mellitus [T2DM; i.e., with a strong family history of T2DM (FH+)] but not in nondiabetic subjects without a family history of T2DM. To determine whether lowering plasma FFA with acipimox, an antilipolytic nicotinic acid derivative, may enhance insulin secretion, nine FH+ volunteers were admitted twice and received in random order either acipimox or placebo (double-blind) for 48 h. Plasma glucose/insulin/C-peptide concentrations were measured from 0800 to 2400. On day 3, insulin secretion rates (ISRs) were assessed during a +125 mg/dl hyperglycemic clamp. Acipimox reduced 48-h plasma FFA by 36% (P < 0.001) and increased the plasma C-peptide relative to the plasma glucose concentration or DeltaC-peptide/Deltaglucose AUC (+177%, P = 0.02), an index of improved beta-cell function. Acipimox improved insulin sensitivity (M/I) 26.1 +/- 5% (P < 0.04). First- (+19 +/- 6%, P = 0.1) and second-phase (+31 +/- 6%, P = 0.05) ISRs during the hyperglycemic clamp also improved. This was particularly evident when examined relative to the prevailing insulin resistance [1/(M/I)], as both first- and second-phase ISR markedly increased by 29 +/- 7 (P < 0.05) and 41 +/- 8% (P = 0.02). There was an inverse correlation between fasting FFA and first-phase ISR (r2 = 0.31, P < 0.02) and acute (2-4 min) glucose-induced insulin release after acipimox (r2 =0.52, P < 0.04). In this proof-of-concept study in FH+ individuals predisposed to T2DM, a 48-h reduction of plasma FFA improves day-long meal and glucose-stimulated insulin secretion. These results provide additional evidence for the important role that plasma FFA play regarding insulin secretion in FH+ subjects predisposed to T2DM.

Recent studies have evidenced that alterations of cardiac metabolism can be present in several cardiac syndromes. In heart failure, wasting of subcutaneous fat and skeletal muscle is relatively common and suggests an increased utilisation of non-carbohydrate substrates for energy production. In fact, fasting blood ketone bodies as well as fat oxidation during exercise have been shown to be increased in patients with heart failure. This metabolic shift determines a reduction of myocardial oxygen consumption efficiency. A direct approach to manipulate cardiac energy metabolism consists in modifying substrate utilisation by the heart. To date, the most effective metabolic treatments include several pharmacological agents that directly inhibit fatty acid oxidation. Clinical studies have shown that these agents can substantially increase the ischaemic threshold in patients with effort angina. However, the results of current research is also supporting the concept that shifting the energy substrate preference away from fatty acid metabolism and towards glucose metabolism could be an effective adjunctive treatment in patients with heart failure, in terms of left ventricular function and glucose metabolism improvement. In fact, these agents have also been shown to improve overall glucose metabolism in diabetic patients with left ventricular dysfunction. In this paper, the recent literature on the beneficial therapeutic effects of modulation of cardiac metabolic substrates utilisation in patients with heart failure is reviewed and discussed.

We explored the mechanisms by which a 4-month, placebo-controlled pioglitazone treatment (45 mg/day) improves glycemic control in type II diabetic patients (T2D, n=27) using physiological testing (6-h mixed meal) and a triple tracer technique ([6,6-(2)H(2)]glucose infusion, (2)H(2)O and [6-(3)H]glucose ingestion) to measure endogenous glucose production (EGP), gluconeogenesis (GNG), insulin-mediated glucose clearance and beta-cell glucose sensitivity (by c-peptide modeling). Compared to sex/age/weight-matched non-diabetic controls, T2D patients showed inappropriately (for prevailing insulinemia) raised glucose production (1.05[0.53] vs 0.71[0.36]mmol min(-1) kg(ffm)(-1) pM, P=0.03) because of enhanced GNG (73.1+/-2.4 vs 59.5+/-3.6%, P<0.01) persisting throughout the meal, reduced insulin-mediated glucose clearance (6[5] vs 12[13]ml min(-1) kg(ffm)(-1) nM(-1), P<0.005), and impaired beta-cell glucose-sensitivity (27[38] vs 71[37]pmol min(-1) m(-2) mM(-1), P=0.002). Compared to placebo, pioglitazone improved glucose overproduction (P=0.0001), GNG and glucose underutilization (P=0.05) despite lower insulinemia. GNG improvement was quantitatively related to raised adiponectin. beta-cell glucose sensitivity was unchanged. In mild-to-moderate T2D, pioglitazone monotherapy decreased fasting and post-prandial glycemia, principally via inhibition of gluconeogenesis, improved hepatic and peripheral insulin resistance.

Type 2 diabetes is characterized by peripheral insulin resistance, pancreatic beta-cells dysfunction, and decreased beta-cell mass with increased rate of apoptosis. Chronic exposure to high levels of free fatty acids (FFAs) has detrimental effects on beta-cell function and survival. FFAs have adverse effects on mitochondrial function, with a consequent increase in the production of reactive oxygen species. Hepatocyte growth factor (HGF) plays a critical role in promoting beta-cell survival. In the present study, we investigated whether HGF was capable of protecting beta-cells from death induced by prolonged exposure to FFAs. RINm5F cell line was cultured in the presence of FFAs (oleate:palmitate 2:1) for 72 h in order to induce apoptosis. Simultaneous administration of HGF and FFAs significantly suppressed the impaired insulin secretion and FFA-induced apoptosis. Specifically, HGF exerted its protective effect by counteracting: (i) the overproduction of either hydrogen peroxide and superoxide anion, (ii) the reduction of intracellular gamma-glutamylcysteinylglycine level, and (iii) the depolarization of mitochondrial membrane, induced by prolonged FFAs exposure. These effects appear to be mediated by bcl-2 and phosphatidylinositol 3 kinase (PI3K)/Akt pathways. Indeed, HGF increased mRNA and protein expression of bcl-2 downregulated by FFAs-treatment; moreover, pre-treatment with the specific PI3-kinase inhibitor LY294002, significantly abolished the protective effect of HGF. In conclusion, in rat insulin-producing RINm5F cells, HGF exerts its prosurvival effect by counteracting the increased intracellular oxidative stress and, consequently, by inhibiting apoptosis induced by chronic exposure to FFAs.

Background: Metabolic modulators that enhance myocardial glucose metabolism by inhibiting free fatty acid (FFA) metabolism may improve cardiac function in heart failure patients. We studied the effect of acute FFA withdrawal on cardiac function in patients with heart failure caused by idiopathic dilated cardiomyopathy (IDCM)., Methods and Results: Eighteen fasting nondiabetic patients with IDCM (14 men, 4 women, aged 58.8+/-8.0 years, ejection fraction 33+/-8.8%) and 8 matched healthy controls underwent examination of myocardial perfusion and oxidative and FFA metabolism, before and after acute reduction of serum FFA concentrations by acipimox, an inhibitor of lipolysis. Metabolism was monitored by positron emission tomography and [15O]H2O, [11C]acetate, and [11C]palmitate. Left ventricular function and myocardial work were echocardiographically measured, and efficiency of forward work was calculated. Acipimox decreased myocardial FFA uptake by >80% in both groups. Rate-pressure product and myocardial perfusion remained unchanged, whereas stroke volume decreased similarly in both groups. In the healthy controls, reduced cardiac work was accompanied by decreased oxidative metabolism (from 0.071+/-0.019 to 0.055+/-0.016 min(-1), P<0.01). In IDCM patients, cardiac work fell, whereas oxidative metabolism remained unchanged and efficiency fell (from 35.4+/-12.6 to 31.6+/-13.3 mm Hg x L x g(-1), P<0.05)., Conclusions: Acutely decreased serum FFA depresses cardiac work. In healthy hearts, this is accompanied by parallel decrease in oxidative metabolism, and myocardial efficiency is preserved. In failing hearts, FFA depletion did not downregulate oxidative metabolism, and myocardial efficiency deteriorated. Thus, failing hearts are unexpectedly more dependent than healthy hearts on FFA availability. We propose that both glucose and fatty acid oxidation are required for optimal function of the failing heart.

Aims: Metabolic responses to manipulation of the plasma free fatty acid (FFA) concentration were assessed in six healthy men via cross-over design to determine whether FFAs independently influence insulin sensitivity., Methods: Intramyocellular lipid (IMCL) was measured by proton magnetic resonance spectroscopy and insulin sensitivity via frequently sampled intravenous glucose tolerance test (IVGTT) after 67 h of two identical low carbohydrate/high fat (LC) diets which were used to elevate IMCL and plasma FFAs. To uncouple the influence of FFAs and IMCL on insulin sensitivity, FFAs were suppressed 30 min prior to and during IVGTT in one treatment [LC + nicotinic acid (NA)] by NA ingestion., Results: Vastus lateralis IMCL was significantly elevated in LC (13.3 +/- 1.1 x 10(-3)) and LC + NA (13.5 +/- 1.1 x 10(-3)) (P < 0.01 for both), but was not different between conditions (P > 0.05). Plasma FFAs were raised in LC (0.79 +/- 0.08 mmol/l) and LC + NA (0.80 +/- 0.11 mmol/l) (P < 0.01 for both) and were significantly reduced by NA ingestion prior to (0.36 +/- 0.05 mmol/l, P < 0.01) and during IVGTT (P < 0.05) in LC + NA. Despite marked differences in plasma FFA availability, insulin sensitivity and glucose tolerance were not different between LC and LC + NA (P > 0.05 for both)., Conclusions: Plasma FFAs appear to exert no immediate effect on insulin sensitivity/glucose tolerance independent of their action on intracellular lipid moieties. Further research is required to elucidate the duration of FFA suppression required to restore insulin sensitivity following lipid-induced insulin resistance.

There is an urgent medical need for orally effective drugs to replace insulin injections for the treatment of diabetes mellitus. Vanadium complexes with insulin-mimetic activities have recently been proposed as candidates as new antidiabetic drugs. Following in vitro and in vivo studies on a group of bis(3-hydroxy-4-pyronato)oxovanadium(IV) (1) complexes with VO(O4) coordination mode, bis(allixinato)oxovanadium(IV) (3) which contains allixin, a garlic component, was found to be the most potent antidiabetic agent among them. Complex 3 with a high in vitro insulin-mimetic activity in terms of both free fatty acid (FFA)-release inhibitory and glucose-uptake enhancing activities in isolated rat adipocytes exhibited a high hypoglycemic effect in type 1 diabetic model mice by both intraperitoneal injections and oral administrations. Complex 3 is thus proposed to be one of the most effective candidates for antidiabetic therapy.

Objective: Elevation of free fatty acids (FFAs) by the infusion of triglyceride-heparin emulsion infusion (TG-Hep) causes insulin resistance (IR). We examined the effect of insulin sensitizer (rosiglitazone) on FFA-induced IR., Design: Nine obese subjects underwent a 6-h infusion of TG-Hep before and after 6 wk of rosiglitazone (8 mg/d) treatment. Hyperinsulinemic euglycemic clamps were performed during 0-2 and 4-6 h of TG-Hep., Results: After rosiglitazone for 6 wk, fasting FFA concentration fell, but not significantly (489 +/- 63 at 0 wk; 397 +/- 58 micromol/liter at 6 wk; P = 0.16), whereas C-reactive protein (4.26 +/- 0.95 at 0 wk; 2.03 +/- 0.45 microg/ml at 6 wk) and serum amyloid A (17.36 +/- 4.63 at 0 wk; 8.77 +/- 1.63 microg/ml at 6 wk) decreased significantly. At 0 wk, TG-Hep infusion caused a decrease in glucose infusion rate (GIR) from 4.49 +/- 0.95 mg/kg.min to 3.02 +/- 0.59 mg/kg.min (P = 0.018). Rosiglitazone treatment resulted in an increase in baseline GIR to 6.29 +/- 0.81 mg/kg.min (P = 0.03 vs. 0 wk), which decreased to 4.52 +/- 0.53 mg/kg.min (P = 0.001) after 6 h of TG-Hep infusion. The decrease in GIR induced by TG-Hep infusion was similar before and after rosiglitazone therapy [1.47 +/- 0.50 vs. 1.77 0.3 mg/kg.min (28.9 +/- 6.5 vs. 26.4 +/- 3.7%); P = 0.51]. The rise in FFAs and triglycerides after TG-Hep infusion was significantly lower at 6 wk (P = 0.006 for FFAs; P = 0.024 for triglycerides)., Conclusions: We conclude that rosiglitazone: 1) causes a significant increase in GIR; 2) induces a decrease in inflammatory mediators, C-reactive protein, and serum amyloid A; 3) decreases the rise in FFAs and triglycerides after TG-Hep infusion; and 4) does not prevent FFA-induced IR.

Nicotinic acid has, like the Roman God Janus, two faces. One is the vitamin. The other is the broad-spectrum lipid drug. The Canadian pathologist Rudolf Altschul discovered 50 years ago that nicotinic acid in gram doses lowered plasma levels of cholesterol. From the point of view of treatment of the dyslipidaemias that are risk factors for clinical atherosclerosis nicotinic acid is a miracle drug. It lowers the levels of all atherogenic lipoproteins--VLDL and LDL with subclasses as well as Lp(a)--and in addition it raises more than any other drug the levels of the protective HDL lipoproteins. Trials have shown that treatment with nicotinic acid reduces progression of atherosclerosis, and clinical events and mortality from coronary heart disease. The new combination treatment with statin-lowering LDL and nicotinic acid-raising HDL is reviewed. A basic effect of nicotinic acid is the inhibition of fat-mobilizing lipolysis in adipose tissue leading to a lowering of plasma free fatty acids, which has many metabolic implications which are reviewed. The very recent discovery of a nicotinic acid receptor and the finding that the drug stimulates the expression of the ABCA 1 membrane cholesterol transporter have paved the way for exciting and promising new 50 years in the history of nicotinic acid.

Background: Elevated non-esterified fatty acids (NEFA) concentrations in non-pregnant animals have been reported to decrease pancreatic responsiveness. As ovine gestation advances, maternal insulin concentrations fall and NEFA concentrations increase. Experiments were designed to examine if the pregnancy-associated rise in NEFA concentration is associated with a reduced pancreatic sensitivity to glucose in vivo. We investigated the possible relationship of NEFA concentrations in regulating maternal insulin concentrations during ovine pregnancy at three physiological states, non-pregnant, non-lactating (NPNL), 105 and 135 days gestational age (dGA, term 147+/- 3 days)., Methods: The plasma concentrations of insulin, growth hormone (GH) and ovine placental lactogen (oPL) were determined by double antibody radioimmunoassay. Insulin responsiveness to glucose was measured using bolus injection and hyperglycaemic clamp techniques in 15 non-pregnant, non-lactating ewes and in nine pregnant ewes at 105 dGA and near term at 135 dGA. Plasma samples were also collected for hormone determination. In addition to bolus injection glucose and insulin Area Under Curve calculations, the Mean Plasma Glucose Increment, Glucose Infusion Rate and Mean Plasma Insulin Increment and Area Under Curve were determined for the hyperglycaemic clamp procedures. Statistical analysis of data was conducted with Students t-tests, repeated measures ANOVA and 2-way ANOVA., Results: Maternal growth hormone, placental lactogen and NEFA concentrations increased, while basal glucose and insulin concentrations declined with advancing gestation. At 135 dGA following bolus glucose injections, peak insulin concentrations and insulin area under curve (AUC) profiles were significantly reduced in pregnant ewes compared with NPNL control ewes (p < 0.001 and P < 0.001, respectively). In hyperglycaemic clamp studies, while maintaining glucose levels not different from NPNL ewes, pregnant ewes displayed significantly reduced insulin responses and a maintained depressed insulin secretion. In NPNL ewes, 105 and 135 dGA ewes, the Glucose Infusion Rate (GIR) was constant at approximately 5.8 mg glucose/kg/min during the last 40 minutes of the hyperglycaemic clamp and the Mean Plasma Insulin Increment (MPII) was only significantly (p < 0.001) greater in NPNL ewes. Following the clamp, NEFA concentrations were reduced by approximately 60% of pre-clamp levels in all groups, though a blunted and suppressed insulin response was maintained in 105 and 135 dGA ewes., Conclusions: Results suggest that despite an acute suppression of circulating NEFA concentrations during pregnancy, the associated steroids and hormones of pregnancy and possibly NEFA metabolism, may act to maintain a reduced insulin output, thereby sparing glucose for non-insulin dependent placental uptake and ultimately, fetal requirements.

We have recently proposed the existence of some potent vanadyl complexes with blood glucose-lowering activity in experimental diabetic animals based on the results of an in vitro FFA (free fatty acids)-release assay in isolated rat adipocytes treated with epinephrine and evidence of an in vivo blood glucose lowering effect in experimental diabetic animals. However, the FFA assay depends indirectly on the glucose-uptake of vanadyl complexes in adipocytes. It is therefore necessary to develop a more reliable in vitro glucose-uptake assay, in place of the glucose uptake method using radioactive compounds such as (14)C-glucose, to identify insulin-mimetic vanadyl complexes. In the present study, we proposed a combined in vitro assay by using the conventional glucose oxidase method for glucose-uptake and FFA assay in isolated rat adipocytes. Insulin, vanadyl sulfate (VOSO(4)), bis(picolinato)vanadyl (VO(pa)(2)), and bis(6-methylpicolinato)vanadyl (VO(6mpa)(2)) complexes exhibited concentration-dependent uptake of (+)-D-glucose and inhibition of FFA release in the adipocytes treated with epinephrine. Vanadyl complexes were found to accelerate glucose-uptake at lower concentrations than VOSO(4). In vitro high insulin-mimetic activity of VO(pa)(2) and VO(6mpa)(2) were thus indicated by both glucose-uptake and FFA-release, with the insulin-mimetic activity of VO(6mpa)(2) being higher than that of VO(pa)(2), as suggested by the partition coefficient (0.330 for VO(pa)(2) and 0.595 for VO(6mpa)(2)). The proposed assay provides a more reliable method than each single method for the evaluation of in vitro insulin-mimetic activity of compounds.

The synthetic compound NO-1886 is a lipoprotein lipase activator that has been proven to be highly effective in lowering plasma triglycerides and elevating high-density lipoprotein cholesterol. Recently, we found that NO-1886 also had a plasma glucose-reducing action in high-fat/high-sucrose diet-induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 on the morphology of adipocytes, plasma levels of tumor necrosis factor-alpha (TNF-alpha) and free fatty acids (FFA) in miniature pigs fed a high-fat/high-sucrose diet. Our results showed that feeding a high-fat/high-sucrose diet to miniature pigs increased the size of adipocytes, and the plasma levels of TNF-alpha, FFA, and glucose. This diet also induced insulin resistance and impaired the acute insulin response to glucose loading. Supplementing 1% NO-1886 to the high-fat/high-sucrose diet inhibited adipocyte enlargement, and suppressed plasma levels of TNF-alpha, FFA, and glucose. The decrease in plasma TNF-alpha and FFA was simultaneous with the decrease in plasma glucose. We also found an increased whole body glucose clearance and an increased acute insulin response to intravenous glucose loading by NO-1886 supplementation. These data suggest that NO-1886 improves the glucose metabolism in high-fat/high-sucrose diet-induced diabetic minipigs by decreasing fat deposit, and suppressing plasma TNF-alpha and FFA levels. Therefore, NO-1886 is potentially beneficial for the treatment of insulin-resistant syndrome.

DA-11004 is a synthetic, potent NADP-dependent isocitrate dehydrogenase (IDPc) inhibitor where IC50 for IDPc is 1.49 microM. The purpose of this study was to evaluate the effects of DA-11004 on the high fat high sucrose (HF)-induced obesity in male C57BL/6J mice. After completing a 8-week period of experimentation, the mice were sacrificed 1 hr after the last DA-11004 treatment and their blood, liver, and adipose tissues (epididymal and retroperitoneal fat) were collected. There was a significant difference in the pattern of increasing body weight between the HF control and the DA-11004 group. In the DA-11004 (100 mg/kg) treated group the increase in body weight significantly declined and a content of epididymal fat and retroperitoneal fat was also significantly decreased as opposed to the HF control. DA-11004 (100 mg/ kg) inhibited the IDPc activity, and thus, NADPH levels in plasma and the levels of free fatty acid (FFA) or glucose in plasma were less than the levels of the HF control group. In conclusion, DA-11004 inhibited the fatty acid synthesis in adipose tissues via IDPc inhibition, and it decreased the plasma glucose levels and FFA in HF diet-induced obesity of C57BL/6J mice.

The bactericidal potencies of saturated and unsaturated fatty acids (FAs) and monoglycerides (MGs) against Helicobacter pylori were determined following short incubations with freshly harvested cells over a range of pHs. FAs and their derivatives with an equivalent-carbon number of 12 were the most potent: lauric acid had a minimum bactericidal concentration (MBC) at pH 7.4 of 1 mM, myristoleic and linolenic acid were the most potent unsaturated FAs (MBCs of 0.5 mM, pH 7.4), and monolaurin was the most potent MG (MBC 0.5 mM). Potencies of saturated FAs were increased sharply by lowering pH, and a decrease of only 0.5 pH units can cause a change from non-lethal to lethal conditions. Conversely, the bactericidal action of monolaurin was not pH-dependent. The bactericidal potencies of unsaturated FAs increased with degree of unsaturation. When more than one FA or FA plus MGs were present, their combined action was additive. Urea and endogenous urease did not protect H. pylori from the bactericidal action of FAs. These results suggest that H. pylori present in the stomach contents (but not necessarily within the mucus barrier) should be rapidly killed by the millimolar concentrations of FAs and MGs that are produced by pre-intestinal lipase(s) acting on suitable triglycerides such as milk fat.

The lipolytic effects of catecholamines are mediated through members of the beta(2)-adrenergic receptor (BAR-2) family. Previous studies have suggested that genetic variants in the BAR-2 gene may be associated with obesity in some populations. To our knowledge, no studies have directly examined the effects of this polymorphism on circulating nonesterified fatty acid (NEFA) levels. To explore this issue further, a cohort of 604 Caucasian individuals (aged 40-65 yr) was genotyped for a common polymorphism in the BAR-2 gene (Gly16Arg), and the relationships between genotype, body mass index (BMI), NEFA, and lipid levels were examined. Women bearing the Arg16 allele had higher BMI values (P < 0.01) than Gly16Gly women. Women carriers of the Arg16Arg genotype had lower fasting plasma NEFAs (P < 0.01) and greater suppression of NEFAs (P < 0.01) after an oral glucose load than women bearing the Gly16 allele. In multivariate analysis after adjustment for age, sex, and smoking status, the interaction between the BAR-2 genotype and BMI in determining fasting NEFA concentrations was statistically significant (P < 0.05). The availability of objective measures of total energy expenditure in this population permitted the further examination of interactions, particularly that between genotype and physical activity. In the population as a whole, after adjustment for confounding by age, smoking, and BMI, the effect of the Arg16Arg genotype on the suppression of NEFA levels was modified by physical activity level (P for interaction <0.05). These data suggest the existence in this population of a gene-physical activity interaction on NEFA levels.

To test the hypothesis that GH-induced insulin resistance is mediated by an increase in FFA levels we assessed insulin sensitivity after inhibiting the increase in FFA by a nicotine acid derivative, Acipimox, in nine GH-deficient adults receiving GH replacement therapy. The patients received in a double blind fashion either Acipimox (500 mg) or placebo before a 2-h euglycemic (plasma glucose, 5.5 +/- 0.2 mmol/liter) hyperinsulinemic (serum insulin, 28.7 +/- 6.3 mU/liter) clamp in combination with indirect calorimetry and infusion of [3-(3)H]glucose. Acipimox decreased fasting FFA by 88% (P = 0.012) and basal lipid oxidation by 39% (P = 0.015) compared with placebo. In addition, the insulin-stimulated lipid oxidation was 31% (P = 0.0077) lower during Acipimox than during placebo. Acipimox increased insulin-stimulated total glucose uptake by 36% (P = 0.021) compared with placebo, which mainly was due to a 47% (P = 0.015) increase in glucose oxidation. GH induced insulin resistance is partially prevented by inhibition of lipolysis by Acipimox.

To evaluate the effects of acute lowering of FFAs on glucose-induced insulin secretion and GH response to GHRH in polycystic ovary syndrome (PCOS), 27 PCOS subjects (11 lean and 16 obese) and 17 body mass index-matched controls (8 lean and 9 obese) were investigated. Patients underwent an oral glucose tolerance test and a GHRH test before and after administration of the antilipolytic drug acipimox (250 mg orally 3 h and 1 h before the starting of the tests). Blood samples were collected for 2 h after GHRH bolus and for 4 h after the oral glucose tolerance test. Serum concentrations of GH, insulin, glucose, and c-peptide were assayed in each sample, and the results were expressed as area under the curve (AUC). No significant differences were found as to glucose, insulin, and c-peptide AUC before and after acute FFA plasma reduction in any of the investigated groups. Basally, lower GH-AUC was found in lean PCOS compared with body mass index-matched controls and in obese vs. lean controls; no significant differences were found as to the same variable between the two obese groups. The acipimox induced FFA suppression elicited in the four groups a sustained increase in the GH response to its trophic hormone; indeed, the GH-AUC nearly doubled with respect to basal evaluation in all the studied groups. However, the antilipolytic drug was not able to abolish the differences found between lean groups in basal conditions. In conclusion, the presented data confirm that FFAs have a main role in regulating GH secretion at the pituitary level; however, it does not seem that they could explain the GH as well as insulin dysfunction of PCOS.

Reversal of the Na(+)/Ca(2+) exchanger (NCX) occurs during ischemia-reperfusion injury as a result of changes in intracellular pH and sodium concentration. Inhibition of NCXs has been shown to be neuroprotective in vitro. In this study, we evaluated the effects of KB-R7943 (50 microM), a specific inhibitor of the reverse mode of NCX, applied topically onto rat cerebral cortex prior to and during ischemia. Amino acid and free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high-performance liquid chromatography. During a 20-min period of ischemia in control animals, there were significant increases in all amino acids and in all FFAs. Following reperfusion, all FFAs remained significantly elevated. Application of KB-R7943 (50 microM) significantly inhibited effluxes of phosphoethanolamine, but had no effect on glutamate, aspartate, taurine or GABA levels. KB-R7943 also resulted in significant reductions in levels of myristic, docosahexaenoic and arachidonic acid during ischemia and in reperfusion levels of arachidonic and docosahexaenoic acids. These data indicate that inhibition of Na(+)/Ca(2+) exchange likely prevented the activation of phospholipases that usually occurs following an ischemic insult as evidenced by its attenuation of phosphoethanolamine and free fatty acid efflux. The inhibition of phospholipases may be an essential component of the neuroprotective benefits of Na(+)/Ca(2+) exchange inhibitors in ischemia-reperfusion injury and may provide a basis for their possible use in therapeutic strategies for stroke.

The insulin-like action of a novel class of potential insulin-mimetic complexes was investigated in terms of free fatty acid (FFA) release from isolated rat adipocytes. Vanadyl complexes such as VO(ema)2 [(bis(2-ethyl-3-hydroxy-4-pyrone)VO], VO(mpp)2 [bis (3-hydroxy-2-methyl-4(1H)-pyridinone)VO], VO(dmpp)2 [bis(1,2-dimethyl-3-hydroxy-4(1H)-pyridinone)VO] and VO(empp)2 [bis(2-ethyl-3-hydroxy-1-methyl-4(1H)-pyridinone)VO] were tested together with vanadyl sulfate for comparison. The inhibitory effect of the vanadium complexes on FFA release, from rat adipocytes treated with epinephrine, is dependent on concentration and for that reason the results are reported in terms of the IC50 value, the 50% inhibition concentration. The results show that all the complexes have an inhibitory effect on FFA release and that two pyridinone complexes, VO(mpp)2 and VO(empp)2, have a significantly better insulin-mimetic activity than that of vanadyl sulfate.

A new vanadyl complex, bis(5-iodopicolinato)oxovanadium(IV), VO(IPA)2, with a VO(N2O2) coordination mode, was prepared by mixing 5-iodopicolinic acid and VOSO4 at pH 5, with the structure characterized by electronic absorption, IR, and EPR spectra. Introduction of the halogen atom on to the ligand enhanced the in vitro insulinomimetic activity (IC50 = 0.45 mM) compared with that of bis(picolinato)oxovanadium(IV) (IC50 = 0.59 mM). The hyperglycemia of streptozotocin-induced insulin-dependent diabetic rats was normalized when VO(IPA)2 was given by daily intraperitoneal injection. The normoglycemic effect continued for more than 14 days after the end of treatment. To understand the insulinomimetic action of VO(IPA)2, the organ distribution of vanadium and the blood disposition of vanadyl species were investigated. In diabetic rats treated with VO(IPA)2, vanadium was distributed in almost all tissues examined, especially in bone, indicating that the action of vanadium is not peripheral. Vanadyl concentrations in the blood of normal rats given VO(IPA)2 remain significantly higher and longer than those given other complexes because of its slower clearance rate. VO(IPA)2 binds with the membrane of erythrocytes, probably owing to its high hydrophobicity in addition to its binding with serum albumin. The longer residence of vanadyl species shows the higher normoglyceric effects of VO(IPA)2 among three complexes with the VO(N2O2) coordination mode. On the basis of these results, VO(IPA)2 is indicated to be a preferred agent to treat insulin-dependent diabetes mellitus in experimental animals.

Troglitazone is an antidiabetic agent that improves the ability of adipocytes to store triglycerides by enhancing their insulin sensitivity. Although potent in insulin-resistant states, the effect of troglitazone on lipid and glucose turnover in normal animals has not been assessed. Euglycemic clamps were performed as an insulin dose response in normal mongrel dogs (n = 6). Somatostatin was infused without hormone replacement (zero insulin) for 90 min. Insulin was then either portally replaced (1.8 pmol x min(-1) x kg(-1), overreplaced (5.4 pmol x min(-1) x kg(-1)), or overreplaced peripherally to match the systemic levels of the portal overreplacement dose (2.3 pmol x min(-1) x kg(-1)) for 180 min. A total of 600 mg troglitazone was then given orally each day for 3 weeks and continued throughout a second experimental phase, at which point the euglycemic clamps were repeated. In concordance with previous studies, endogenous glucose production (EGP) was similar whether insulin was delivered portally or peripherally, both before and during troglitazone treatment. Although free fatty acids (FFAs) at zero insulin were not affected, there was a leftward shift of the insulin-FFA dose response curve secondary to a suppression of FFA release into plasma. EGP was paradoxically elevated by troglitazone treatment because of an elevation of both gluconeogenesis and glycogenolysis. In conclusion, troglitazone reduced hepatic sensitivity to FFAs. Because EGP is a primary determinant of fasting blood glucose, we hypothesize that a protective mechanism exists in normal animals, preventing hypoglycemia during insulin sensitization with troglitazone.