Phosphatidylinositol 4-kinase IIIβ mediates contraction-induced GLUT4 translocation and shows its anti-diabetic action in cardiomyocytes

In the diabetic heart, long-chain fatty acid (LCFA) uptake is increased at the expense of glucose uptake. This metabolic shift ultimately leads to insulin resistance and a reduced cardiac function. Therefore, signaling kinases that mediate glucose uptake without simultaneously stimulating LCFA uptake could be considered attractive anti-diabetic targets. Phosphatidylinositol-4-kinase-IIIβ (PI4KIIIβ) is a lipid kinase downstream of protein kinase D1 (PKD1) that mediates Golgi-to-plasma membrane vesicular trafficking in HeLa-cells. In this study, we evaluated whether PI4KIIIβ is involved in myocellular GLUT4 translocation induced by contraction or oligomycin (an F1F0-ATP synthase inhibitor that activates contraction-like signaling). Pharmacological targeting, with compound MI14, or genetic silencing of PI4KIIIβ inhibited contraction/oligomycin-stimulated GLUT4 translocation and glucose uptake in cardiomyocytes but did not affect CD36 translocation nor LCFA uptake. Addition of the PI4KIIIβ enzymatic reaction product phosphatidylinositol-4-phosphate restored oligomycin-stimulated glucose uptake in the presence of MI14. PI4KIIIβ activation by PKD1 involves Ser294 phosphorylation and altered its localization with unchanged enzymatic activity. Adenoviral PI4KIIIβ overexpression stimulated glucose uptake, but did not activate hypertrophic signaling, indicating that unlike PKD1, PI4KIIIβ is selectively involved in GLUT4 translocation. Finally, PI4KIIIβ overexpression prevented insulin resistance and contractile dysfunction in lipid-overexposed cardiomyocytes. Together, our studies identify PI4KIIIβ as positive and selective regulator of GLUT4 translocation in response to contraction-like signaling, suggesting PI4KIIIβ as a promising target to rescue defective glucose uptake in diabetics.

Abstract 233: Metabolic Trapping Effects of ACS1 for Fatty Acid Uptake and Intracellular Trafficking are Different in Hearts of Males and Females

Disruption of cardiomyocyte lipid metabolism has been observed in the responses to both pathogenic and nutrient stresses in human patients and animal models, with distinct sex differences. This investigation examined cardiac acyl CoA synthetase-1 (ACS)-mediated, LCFA uptake and trafficking with hearts of male and female mice. Isolated mouse hearts with cardiac specific overexpression of ACS1 (MHC-ACS) and non-transgenic littermates (NTG) were perfused with buffer containing 13C palmitate, 10 mM glucose, and 1 mM lactate. Dynamic 13C NMR elucidated the two phases of LCFA incorporation into triacylglycerol (TAG). The time constant (τ) of the initial exponential phase, corresponding to LCFA trans-sarcolemmal uptake, was lower, indicating faster LCFA uptake and esterification in male ACS hearts compared to NTG males (ACS male τ = 1.59±0.67 min vs NTG male τ = 2.85±0.90 min, P<0.05). Consistent with greater metabolic trapping, acyl CoA content was 470% higher in male ACS hearts (NTG male 57.5±9.4 vs ACS male 327.4±42 pmol/mg protein. P<0.001). Despite no differences in ACS content, NTG females displayed faster uptake than NTG males at a rate similar to ACS males, which was reversed by ovariectomy (OVX) indicating an estrogen dependent response (NTG female τ = 1.56±0.46 min, P<0.05 vs NTG OVX female τ = 3.02±0.77 min, P<0.05). Despite prior reports of ACS localization distal from the sarcolemma, ACS level and female gender both independently accelerated LCFA uptake without affecting TAG content or turnover. The data are consistent with metabolic trapping to facilitate LCFA uptake due to ACS activity. ACS also leads to higher total ceramide in hearts, with specific elevation of C18 and C22 in both genders, C24 in males, and C20 in females (male P<0.05; female P<0.01). ACS overexpression induced lower content of the cardiac fatty acid transporter (FATP) isoform, FATP6, indicating cooperative regulation of LCFA uptake between membrane transport proteins and intracellular acyl chain activation (P<0.05 male; P<0.001 female and female OVX). These results demonstrate that perturbation of LCFA metabolism though facilitated metabolic trapping by ACS1 affects sarcolemma transporter expression and induces the conversion of the CoA activated LCFA to ceramide.

Calcium signaling recruits substrate transporters GLUT4 and CD36 to the sarcolemma without increasing cardiac substrate uptake

Activation of AMP-activated protein kinase (AMPK) in cardiomyocytes induces translocation of glucose transporter GLUT4 and long-chain fatty acid (LCFA) transporter CD36 from endosomal stores to the sarcolemma to enhance glucose and LCFA uptake, respectively. Ca2+/calmodulin-activated kinase kinase-β (CaMKKβ) has been positioned directly upstream of AMPK. However, it is unknown whether acute increases in [Ca2+]i stimulate translocation of GLUT4 and CD36 and uptake of glucose and LCFA or whether Ca2+ signaling converges with AMPK signaling to exert these actions. Therefore, we studied the interplay between Ca2+ and AMPK signaling in regulation of cardiomyocyte substrate uptake. Exposure of primary cardiomyocytes to inhibitors or activators of Ca2+ signaling affected neither AMPK-Thr172 phosphorylation nor basal and AMPK-mediated glucose and LCFA uptake. Despite their lack of an effect on substrate uptake, Ca2+ signaling activators induced GLUT4 and CD36 translocation. In contrast, AMPK activators stimulated GLUT4/CD36 translocation as well as glucose/LCFA uptake. When cardiomyocytes were cotreated with Ca2+ signaling and AMPK activators, Ca2+ signaling activators further enhanced AMPK-induced glucose/LCFA uptake. In conclusion, Ca2+ signaling shows no involvement in AMPK-induced GLUT4/CD36 translocation and substrate uptake but elicits transporter translocation via a separate pathway requiring CaMKKβ/CaMKs. Ca2+-induced transporter translocation by itself appears to be ineffective to increase substrate uptake but requires additional AMPK activation to effectuate transporter translocation into increased substrate uptake. Ca2+-induced transporter translocation might be crucial under excessive cardiac stress conditions that require supraphysiological energy demands. Alternatively, Ca2+ signaling might prepare the heart for substrate uptake during physiological contraction by inducing transporter translocation.

Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes

The liver expresses high levels of two proteins with high affinity for long-chain fatty acids (LCFAs): liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2). Real-time confocal microscopy of cultured primary hepatocytes from gene-ablated (L-FABP, SCP-2/SCP-x, and L-FABP/SCP-2/SCP-x null) mice showed that the loss of L-FABP reduced cellular uptake of 12- N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid (a fluorescent-saturated LCFA analog) by ∼50%. Importantly, nuclear targeting of the LCFA was enhanced when L-FABP was upregulated (SCP-2/SCP-x null) but was significantly reduced when L-FABP was ablated (L-FABP null), thus impacting LCFA nuclear targeting. These effects were not associated with a net decrease in expression of key membrane proteins involved in LCFA or glucose transport. Since hepatic LCFA uptake and metabolism are closely linked to glucose uptake, the effect of glucose on L-FABP-mediated LCFA uptake and nuclear targeting was examined. Increasing concentrations of glucose decreased cellular LCFA uptake and even more extensively decreased LCFA nuclear targeting. Loss of L-FABP exacerbated the decrease in LCFA nuclear targeting, while loss of SCP-2 reduced the glucose effect, resulting in enhanced LCFA nuclear targeting compared with control. Simply, ablation of L-FABP decreases LCFA uptake and even more extensively decreases its nuclear targeting.

Insulin- and leptin-regulated fatty acid uptake plays a key causal role in hepatic steatosis in mice with intact leptin signaling but not in ob/ob or db/db mice

Hepatic steatosis results from several processes. To assess their relative roles, hepatocellular long-chain fatty acid (LCFA) uptake was assayed in hepatocytes from C57BL/6J control mice, mice with steatosis from a high-fat diet (HFD) or 10%, 14%, or 18% ethanol (EtOH) in drinking water [functioning leptin-signaling groups (FLSGs)], and ob/ob and db/db mice. Vmax for uptake was increased vs. controls ( P < 0.001) and correlated significantly with liver weight and triglycerides (TGs) in all FLSG mice but was minimally or not increased in ob/ob and db/db mice, in which liver weights and TGs greatly exceeded projections from regressions in FLSG animals. Coefficients of determination ( R2) for these FLSG regressions suggest that increased LCFA uptake accounts for ∼80% of the increase in hepatic TGs within these groups, but increased lipogenic gene expression data suggest that enhanced LCFA synthesis is the major contributor in ob/ob and db/db. Got2, Cd36, Slc27a2, and Slc27a5 gene expression ratios were significantly upregulated in the EtOH groups, correlating with sterol regulatory element binding protein 1c ( SREBP1c) and Vmax, but only Cd36 expression was increased in HFD, ob/ob, and db/db mice. Comparison of Vmax with serum insulin and leptin suggests that both hormones contribute to upregulation of uptake in the FLSG animals. Thus, increased LCFA uptake, reflecting SREBP1c-mediated upregulation of four distinct transporters, is the dominant cause of steatosis in EtOH-fed mice. In ob/ob and db/db mice, increased LCFA synthesis appears more important. In FLSG animals, insulin upregulates hepatocellular LCFA uptake. Leptin appears to upregulate LCFA uptake or to be essential for full expression of upregulation by insulin.

FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase

Fatty acid transport protein (FATP)2, a member of the FATP family of fatty acid uptake mediators, has independently been identified as a hepatic peroxisomal very long-chain acyl-CoA synthetase (VLACS). Here we address whether FATP2 is 1) a peroxisomal enzyme, 2) a plasma membrane-associated long-chain fatty acid (LCFA) transporter, or 3) a multifunctional protein. We found that, in mouse livers, only a minor fraction of FATP2 localizes to peroxisomes, where it contributes to approximately half of the peroxisomal VLACS activity. However, total hepatic (V)LACS activity was not significantly affected by loss of FATP2, while LCFA uptake was reduced by 40%, indicating a more prominent role in hepatic LCFA uptake. This suggests FATP2 as a potential target for a therapeutic intervention of hepatosteatosis. Adeno-associated virus 8-based short hairpin RNA expression vectors were used to achieve liver-specific FATP2 knockdown, which significantly reduced hepatosteatosis in the face of continued high-fat feeding, concomitant with improvements in liver physiology, fasting glucose, and insulin levels. Based on our findings, we propose a model in which FATP2 is a multifunctional protein that shows subcellular localization-dependent activity and is a major contributor to peroxisomal (V)LACS activity and hepatic fatty acid uptake, suggesting FATP2 as a potential novel target for the treatment of nonalcoholic fatty liver disease.

Differential regulation of cardiac glucose and fatty acid uptake by endosomal pH and actin filaments

Insulin and contraction stimulate both cardiac glucose and long-chain fatty acid (LCFA) uptake via translocation of the substrate transporters GLUT4 and CD36, respectively, from intracellular compartments to the sarcolemma. Little is known about the role of vesicular trafficking elements in insulin- and contraction-stimulated glucose and LCFA uptake in the heart, especially whether certain trafficking elements are specifically involved in GLUT4 versus CD36 translocation. Therefore, we studied the role of coat proteins, actin- and microtubule-filaments and endosomal pH on glucose and LCFA uptake into primary cardiomyocytes under basal conditions and during stimulation with insulin or oligomycin (contraction-like AMP-activated protein kinase activator). Inhibition of coat protein targeting to Golgi/endosomes decreased insulin/oligomycin-stimulated glucose (−42%/−51%) and LCFA (−39%/−68%) uptake. Actin disruption decreased insulin/oligomycin-stimulated glucose uptake (−41%/−75%), while not affecting LCFA uptake. Microtubule disruption did not affect substrate uptake under any condition. Endosomal alkalinization increased basal sarcolemmal CD36 (2-fold), but not GLUT4, content, and concomitantly decreased basal intracellular membrane GLUT4 and CD36 content (−60% and −62%, respectively), indicating successful CD36 translocation and incomplete GLUT4 translocation. Additionally, endosomal alkalinization elevated basal LCFA uptake (1.4-fold) in a nonadditive manner to insulin/oligomycin, and decreased insulin/oligomycin-stimulated glucose uptake (−32%/−68%). In conclusion, 1) CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process depending on coat proteins, and 2) GLUT4 and CD36 trafficking are differentially dependent on endosomal pH and actin filaments. The latter conclusion suggests novel strategies to alter cardiac substrate preference as part of metabolic modulation therapy.

Development and Validation of a High-Throughput Screening Assay for Human Long-Chain Fatty Acid Transport Proteins 4 and 5

Dietary long-chain fatty acid (LCFA) uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). Six subtypes of this transporter are differentially expressed throughout the human and rodent body. To facilitate drugs discovery against FATP subtypes, the authors used mammalian cell lines stably expressing the recombinant human FATP4 and 5 and developed a high-throughput screening (HTS) assay using a 96-well fluorometric imaging plate reader (FLIPR). LCFA uptake signal-to-background ratios were between 3- and 5-fold. Two 4-aryl-dihydropyrimidinones, j3 and j5, produced inhibition of FATP4 with a half-maximal inhibitory concentration (IC50) of 0.21 and 0.63 µM, respectively, and displayed approximately 100-fold selectivity over FATP5. The US Drug Collection library was screened against the FATP5. A hit rate of around 0.4% was observed with a Z′ factor of 0.6 ± 0.2. Two confirmed hits are bile acids, chenodiol and ursodiol with an IC50 of 2.4 and 0.22 µM, respectively. To increase throughput, a single time point measurement in a 384-well format was developed using the Analyst HT, and the results are comparable with the 96-well format. In conclusion, the FATP4 and 5 cell-based fluorescence assays are suitable for a primary drug screen, whereas differentiated cell lines are useful for a secondary drug screen.

Etomoxir-induced partial carnitine palmitoyltransferase-I (CPT-I) inhibition in vivo does not alter cardiac long-chain fatty acid uptake and oxidation rates

Although CPT-I (carnitine palmitoyltransferase-I) is generally regarded to present a major rate-controlling site in mitochondrial β-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall LCFA (long-chain fatty acid) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg of body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity, were not altered in the hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and oxidation, and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. It is likely that sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases.