Search

Decavanadate is a polyoxometalate (POMs) that has shown extensive biological activities, including antidiabetic and anticancer activity. Importantly, vanadium-based compounds as well as antidiabetic biguanide drugs, such as metformin, have shown to exert therapeutic effects in melanoma. A combination of these agents, the metformin-decavanadate complex, was also recognized for its antidiabetic effects and recently described as a better treatment than the monotherapy with metformin enabling lower dosage in rodent models of diabetes. Herein, we compare the effects of decavanadate and metformin-decavanadate on Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from rabbit skeletal muscles and on cell signaling events and viability in human melanoma cells. We show that unlike the decavanadate-mediated non-competitive mechanism, metformin-decavanadate inhibits Ca2+-ATPase by a mixed-type competitive–non-competitive inhibition with an IC50 value about 6 times higher (87 μM) than the previously described for decavanadate (15 μM). We also found that both decavanadate and metformin-decavanadate exert antiproliferative effects on melanoma cells at 10 times lower concentrations than monomeric vanadate. Western blot analysis revealed that both, decavanadate and metformin-decavanadate increased phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine protein kinase AKT signaling proteins upon 24 h drug exposure, suggesting that the anti-proliferative activities of these compounds act independent of growth-factor signaling pathways., This work was funded by the Spanish Ministry of Science, Innovation and Universities through grant RTI2018-094629-B-I00 to WL, and Fundação para a Ciência e a Tecnologia (FCT) (PTDC/MED-ONC/4167/2020 “ENDURING”) to BIF.

Context: Increased circulating free fatty acids (FFAs) have been proposed to contribute to insulin resistance in obesity. Short-term studies have investigated the effects of acipimox, an inhibitor of hormone-sensitive lipase, on glucose homeostasis, but longer-term studies have not been performed. Objective: To test the hypothesis that long-term treatment with acipimox would reduce FFA and improve insulin sensitivity among nondiabetic, insulin-resistant, obese subjects. Design, Setting, Patients, and Intervention: At an academic medical center, 39 obese men and women were randomized to acipimox 250 mg thrice-daily vs identical placebo for 6 months. Main Outcome Measures: Plasma lipids, insulin sensitivity, adiponectin, and mitochondrial function via assessment of the rate of post-exercise phosphocreatine recovery on 31P-magnetic resonance spectroscopy as well as muscle mitochondrial density and relevant muscle gene expression. Results: Fasting glucose decreased significantly in acipimox-treated individuals (effect size, −6 mg/dL; P = .02), in parallel with trends for reduced fasting insulin (effect size, −6.8 μU/mL; P = .07) and HOMA-IR (effect size, −1.96; P = .06), and significantly increased adiponectin (effect size, +668 ng/mL; P = .02). Acipimox did not affect insulin-stimulated glucose uptake, as assessed by euglycemic, hyperinsulinemic clamp. Effects on muscle mitochondrial function and density and on relevant gene expression were not seen. Conclusion: These data shed light on the long-term effects of FFA reduction on insulin sensitivity, other metabolic parameters, and muscle mitochondrial function in obesity. Reduced FFA achieved by acipimox improved fasting measures of glucose homeostasis, lipids, and adiponectin but had no effect on mitochondrial function, mitochondrial density, or muscle insulin sensitivity.

Nutrient deprivation or starvation frequently correlates with amino acid limitation. Amino acid starvation initiates a signal transduction cascade starting with the activation of the kinase GCN2 (general control non-derepressible 2) phosphorylation of eIF2 (eukaryotic initiation factor 2), global protein synthesis reduction and increased ATF4 (activating transcription factor 4). ATF4 modulates a wide spectrum of genes involved in the adaptation to dietary stress. The hormone FGF21 (fibroblast growth factor 21) is induced during fasting in liver and its expression induces a metabolic state that mimics long-term fasting. Thus FGF21 is critical for the induction of hepatic fat oxidation, ketogenesis and gluconeogenesis, metabolic processes which are essential for the adaptive metabolic response to starvation. In the present study, we have shown that FGF21 is induced by amino acid deprivation in both mouse liver and cultured HepG2 cells. We have identified the human FGF21 gene as a target gene for ATF4 and we have localized two conserved ATF4-binding sequences in the 5′ regulatory region of the human FGF21 gene, which are responsible for the ATF4-dependent transcriptional activation of this gene. These results add FGF21 gene induction to the transcriptional programme initiated by increased levels of ATF4 and offer a new mechanism for the induction of the FGF21 gene expression under nutrient deprivation.

HMGCS2 (hydroxymethylglutaryl CoA synthase 2), the gene that regulates ketone body production, is barely expressed in cultured cell lines. In this study, we restored HMGCS2 expression and activity in HepG2 cells, thus showing that the wild type enzyme can induce fatty acid β-oxidation (FAO) and ketogenesis, whereas a catalytically inactive mutant C166A did not generate either process. Peroxisome proliferator-activated receptor (PPAR) α expression also induces fatty acid β-oxidation and endogenous HMGCS2 expression. Interestingly, PPARα-mediated induction was abolished when HMGCS2 expression was down-regulated by RNAi. These results indicate that HMGCS2 expression is both sufficient and necessary to the control of fatty acid oxidation in these cells. Next, we examined the expression pattern of several PPARα target genes in this now “ketogenic” HepG2 cell line. FGF21 (fibroblast growth factor 21) expression was specifically induced by HMGCS2 activity or by the inclusion of the oxidized form of ketone bodies (acetoacetate) in the culture medium. This effect was blunted by SirT1 (sirtuin 1) RNAi, so we propose a SirT1-dependent mechanism for FGF21 induction by acetoacetate. These data suggest a novel feed-forward mechanism by which HMGCS2 could regulate adaptive metabolic responses during fasting. This mechanism could be physiologically relevant, because fasting-mediated induction of liver FGF21 was dependent on SirT1 activity in vivo.

Acetoacetyl-CoA synthetase (AACS) is a cytoplasmic enzyme that activates the ketone body acetoacetate for lipogenesis. Aacs is expressed in liver, thus this tissue produces and consumes ketone bodies. To understand this apparent paradox we studied the expression of Aacs in liver during the transition from fasting to feeding, and characterized the kinetic parameters of the human enzyme. We show that Aacs liver expression is down-regulated by fasting and only up-regulated after 5 h of refeeding, while the levels of circulating ketone bodies returned to basal levels within 20 min of food availability. Since the human enzyme has a high affinity for the ketone body acetoacetate (KM = 37.6 μM), these results indicates that AACS utilizes ketones during periods of feeding. Human AACS was also characterized as an acetylated enzyme in vivo, where lysine 633 (from a P4XGK domain) plays a critical role in the catalytic activity but not in the acetylation state of the protein.

FSP27 [cell death-inducing DFFA-like effector c (CIDEC) in humans] is a protein associated with lipid droplets that downregulates the fatty acid oxidation (FAO) rate when it is overexpressed. However, little is known about its physiological role in liver. Here, we show that fasting regulates liver expression of Fsp27 in a time-dependent manner. Thus, during the initial stages of fasting, a maximal induction of 800-fold was achieved, whereas during the later phase of fasting, Fsp27 expression decreased. The early response to fasting can be explained by a canonical PKA-CREB-CRTC2 signaling pathway because: i) CIDEC expression was induced by forskolin, ii) Fsp27 promoter activity was increased by CREB, and iii) Fsp27 expression was upregulated in the liver of Sirt1 knockout animals. Interestingly, pharmacological (etomoxir) or genetic (Hmgcs2 interference) inhibition of the FAO rate increases the in vivo expression of Fsp27 during fasting. Similarly, CIDEC expression was upregulated in HepG2 cells by either etomoxir or HMGCS2 interference. Our data indicate that there is a kinetic mechanism of autoregulation between short- and long-term fasting, by which free FAs delivered to the liver during early fasting are accumulated/exported by FSP27/CIDEC, whereas over longer periods of fasting, they are degraded in the mitochondria through the carnitine palmitoyl transferase system.

Lipogenic gene expression in liver is repressed in mice upon leucine deprivation. The hormone fi broblast growth factor 21 (FGF21), which is critical to the adaptive metabolic response to starvation, is also induced under amino acid deprivation. Upon leucine deprivation, we found that FGF21 is needed to repress expression of lipogenic genes in liver and white adipose tissue, and stimulate phosphorylation of hormone-sensitive lipase in white adipose tissue. The increased expression of Ucp1 in brown adipose tissue under these circumstances is also impaired in FGF21- defi cient mice. Our results demonstrate the important role of FGF21 in the regulation of lipid metabolism during amino acid starvation. ¿De Sousa-Coelho, A. L., J. Relat, E. Hondares, A. Pérez-Martí, F. Ribas, F. Villarroya, P. F. Marrero, and D. Haro. FGF21 mediates the lipid metabolism response to amino acid starvation.