PET imaging of glucose and fatty acid metabolism for NAFLD patients
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The aim of the paper by Tang and colleagues was to investigate the association between NAFLD and myocardial glucose uptake to see if alterations in 18F-FDG uptake could be an indicator of cardiac dysfunction in NAFLD individuals. They retrospectively assessed 18F-FDG PET imaging data of a total of 201 subjects with NAFLD and 542 without NAFLD who were imaged over the years from December 1, 2011 to November 30, 2017. The liver (Figure 1B) in addition to adipose tissue is a major source of lipoproteins (Figure 1C), which are a major substrate for cardiac ATP production. NAFLD is a disease with an extensive amount of fat in the liver. The diagnosis of fatty liver disease was confirmed by CT where disease was indicated if liver attenuation was at least 1 Hounsfield Unit (HU) less than the spleen and the attenuation ratio of liver to spleen was less than 1.0. It was found that myocardial 18F-FDG uptake was significantly lower in individuals with NAFLD compared with those without fatty liver. The authors also demonstrated that in NAFLD patients the glucose uptake was inversely proportional to the increase in left ventricular (LV) filling pressure, suggesting that NAFLD individuals with lower myocardial 18F-FDG uptake are more likely to have high risk of impaired diastolic heart function. The reasons for alterations of the biochemical processes of myocardial glucose uptake in patients with NAFLD is not clear, but probably is the result of several complex biochemical processes involving genetic factors, atherosclerosis, inflammatory cytokines, insulin resistance, and other alterations in biochemistry of glucose and fatty acid metabolism.6,7
The process of glucose and fatty acid metabolism converts chemical energy into mechanical energy that is regulated by hemodynamic factors, neurohumoral factors, and oxygen availability.8,9 It involves translocation of molecular substrates into the cell and mitochondria, the facilitated diffusion of molecular species, enzymatic processes in metabolic pathways, and genetic regulation of enzyme production, where many things can go wrong. In the heart, energy production in the form of ATP involves the metabolism of 70% to 80% fatty acids and 20% to 30% glucose (glucose, lactate, glycogen).10 The ATP produced is used in several chemical reactions involving sequestering of substrates into the cell, transport of ions (such as Ca++) in and out of internal vesicles and ions (such as Na+, K+) in and out of myocytes during nerve impulse propagation, breaking down of metabolic substrates (chemical synthesis), and the production of mechanical translation between actin and myosin fibers (muscle contraction). The heart turns over its ATP pool every 10 seconds.10 Metabolism of carbohydrates produces 4 kcal/gram of energy, whereas metabolism of fatty acids produces 9 kcal/gram. The use of fatty acids as a substrate generates the greater number of ATP, but it comes at the expense of a greater oxygen requirement than the use of glucose. Because of the many chemical processes and transport of molecular species, the conversion of chemical energy into mechanical energy is only approximately 20% efficient.10
Abnormal structure and function of liver cells in NADFL patients may provide insight into cardiac cellular abnormalities. The liver (Figure 1B) is the central organ for handling lipids including storage of glycerols and fats in its hepatocytes, manufacturing triglycerides and cholesterol, synthesizing glycogen, and producing bile from cholesterol. It is also involved in making proteins and blood clotting factors. Another important organ closely related to the liver for manufacturing and storage of energetic substrates is adipose tissue whose main role is to store energy in the form of lipids, where there is a constant flux of free fatty acids entering and leaving its cellular adipocytes. Free fatty acids are liberated from lipoproteins (Figure 1C) by lipoprotein lipase (LPL) and enter the adipocyte, where they are reassembled into triglycerides by esterifying it onto glycerol. The net direction of this flux is controlled by insulin and leptin and only when insulin is low can free fatty acids leave adipose tissue.
It is recognized that cardiac cells differ in many respects to liver cells. Cardiac cells need continuous energy supply but have poor capacity to synthesize and store energy substrates, whereas liver cells can synthesize, store, and release energy substrates. However, they have similarities and understanding irregularities in liver cells of NAFLD patients may give insight into potential defects in cardiac cells. In NAFLD patients, an increase in hepatic levels of triacylglycerol (TG), diacylglycerol (DG), cholesteryl ester (CE), and free cholesterol and a change in the percent of polyunsaturated fatty acid (PUFA) in these lipids has been observed. As indicated in an excellent review by Peng and colleagues,14 this may be due to mitochondrial abnormalities found in NAFLD patients indicating metabolic alterations. These abnormalities are seen as abnormal morphological changes, leaky mitochondrial membranes, accumulation of structural and enzymatic proteins, and oxidative stress. Mitochondrial molecular components of cardiolipin, ubiquinone, and acyl-carnitine play an important role in mitochondrial function of oxidative phosphorylation. It is suggested that cardiolipin and ubiquinone levels increase in NAFLD to enhance mitochondrial respiration, thus reducing steatosis (abnormal retention of lipids). On the other hand, acyl-carnitine involved in the carnitine shuttle (Figure 3B) increases with NAFLD indicative of mitochondrial stress, mitochondrial dysfunction, and impaired fatty acid oxidation. Modulation of action of enzymatic proteins through allosteric regulation may also change the mix of metabolic substrates with the disease. For example, methylmalonate semialdehyde dehydrogenase and propionyl-CoA carboxylase are involved in the metabolism of succinyl-CoA (component of the tricarboxylic acid cycle). It is known that these enzymes, which are decreased in mitochondria of obese individuals, could also be decreased in NAFLD patients. In addition, there is evidence that increased dihydroceramide and dihexosylceramides with NAFLD may increase metabolism of ceramides and contribute to metabolic disorders in the disease.14 Ceramides (composed of sphingosine and a fatty acid) are found in high concentrations within the cell membrane providing supportive function and also cellular signaling, including regulating differentiation, proliferation, and programmed cell death.
Decreased glucose metabolism may be a potential indication of cardiac lipotoxicity in NAFLD patients. Cardiac lipotoxicity (Wan and Rodrigues,8 Schulze et al.15) is the excessive accumulation of intramyocellular fatty acids and their metabolites, and is a main contributor to the pathophysiology of insulin resistance and dysfunctioning of heart muscle.16 Fatty acids are delivered to the heart by (1) lipolysis in adipose tissue releasing fatty acid into the plasma, (2) LPL mediated breakdown of TG-rich lipoproteins from the liver (VLDL) and gut (chylomicron), and (3) endogenous triglyceride (TG) breakdown within the heart. The paper of Tang and colleagues in this issue found higher levels of fatty acids in the blood of NAFLD patients. Arterial fatty acid concentration is the primary determination of the rate of myocardial fatty acid uptake and oxidation, and intramyocardial triacylglycerol (TAG) content.12 The transport of fatty acids into the cell is facilitated by FAT/CD transporters. Both muscle contraction and insulin (Figure 3A) stimulate the translocation of FAT/CD36 to the cell membrane, which is mediated by 5’ adenosine monophosphate-activated protein kinase (AMPK). This translocation occurs independently of the insulin-induced translocation mediated by phosphatidylinositol 3–kinase (PI3K).16 It has been shown that increased CD36 abundance in the cell membrane and an increased plasma fatty acid concentration not only can lead to an excessive increase in fatty acid uptake and oxidation but also leads to an increased rate of fatty acid esterification into TAG and increased concentrations of fatty acid metabolites of diacylglycerols (DAG) and ceramides. These intermediates of TAG, DAG, and ceramides are potentially harmful intracellular components that can create insulin resistance by reducing the insulin-induced GLUT4 translocation to the sarcolemma and lowering the rate of glucose uptake.16
There are many feedback controls between glucose and fatty acid metabolism. For one, the oxidation of pyruvate and the activity of pyruvate dehydrogenase (PDH) in the heart are decreased by elevated rates of fatty acid β-oxidation; whereas, pyruvate oxidation is enhanced by suppression of fatty acid β-oxidation. The enzyme AMPK is particularly important as a fuel sensor that plays a big role in cellular energy homeostasis to increases fatty acid uptake and increase β-oxidation during times of increased energy demand or decrease fatty acid uptake and β-oxidation in times of low demand.12 For example, through the AMPK-ACC-MCD axis the concentration of malonyl-CoA depends on the balance between acetyl-CoA carboxylase (ACC) and malonyl-CoA decarboxylase (MCD). Other enzymes involved in the fatty acid β-oxidation are sensitive to feedback inhibition through allosteric control by the products of enzymatic reactions, including FADH2 and NADH. These enzymes are also under a high degree of transcriptional control by peroxisome proliferator-activated receptors (PPARs) that regulate the expression of genes (Figure 3C). For one, the nuclear receptor PPAR\( \alpha \) is a major transcriptional regulator of fatty acid metabolism whereby overexpression of PPAR\( \alpha \) results in increase in cardiac fatty acid uptake, fatty acid β-oxidation, and lipid overload; and under expression of PPAR\( \alpha \) results in decreased expression of fatty acid β-oxidation and a parallel increase in glucose oxidation.
There are several other biological components involved in controlling the homeostasis of energy production. For example, phospholipids in cell membranes are involved in signaling to activate metabolic processes. In the membrane of the mitochondrion there are uncoupling proteins that are transport proteins providing an alternate means for the re-entry of protons from the inter-membrane space to the mitochondrion matrix that is not coupled to synthesis of ATP. These help maintain concentration and electrostatic balance between the inner mitochondrial membrane and the space between the inner and outer mitochondrial membrane. In disease these concentrations can become out of balance. It has been shown that enzymatic activities of mitochondrial complex I and II are significantly reduced in 24 week-old spontaneously hypertensive rats (SHR).17 Recently, increased levels of branched-chain amino acid (BCAA) have also been reported in animal models of heart failure.18 The end products of catabolism of BCAAs can enter the Krebs cycle either for oxidative decarboxylation or anaplerosis (the formation of intermediates of a metabolic pathway).10 BCAAs, especially leucine, increase mammalian target of rapamycin (mTOR) activity and thereby promote protein synthesis, cellular metabolism, and cell growth.19
Advances in PET metabolic imaging provides a necessary tool to further our understanding of various forms of cardiovascular disease and potentially improve the care of cardiac patients.20 In particular, PET imaging of glucose metabolism,21 fatty acid metabolism,22 and oxygen utilization23,24 provides information about metabolic shifts related to cardiac function,25 making PET an outstanding tool to measure cardiac metabolic changes in patients with NAFLD. There have been some imaging studies investigating liver metabolism in NAFLD patients, one using 11C-palmitate26 and one using 18F-FDG and 18F-FTHA27 and another study of NAFLD patients using 18F-FDG to image vascular inflamation,2 but no studies to evaluate the disease relationship to cardiac metabolism other than the study presented in this issue of the Journal by Tang and colleagues using 18F-FDG. Using other metabolic imaging tracers to measure myocardial fatty acid metabolism would help to better measure more precisely and understand the reason for the metabolic shifts in this particular disease.
Most of our understanding of shifts in metabolic substrates in the heart has been elicited from PET imaging studies in heart failure.10,11,28–32 Using 15O-labeled water and 11C-labeled acetate, palmitate, and glucose tracers, one can assess simultaneously myocardial blood flow, energy expenditure, and fatty acid and glucose metabolism.28 Studies have shown decreased fatty acid utilization and increased myocardial glucose metabolism in patients with idiopathic dilated cardiomyopathy.28 On the other hand, patients with congestive heart failure, myocardial uptake of a radiolabeled fatty acid analog (18F-FTHA) was greater and radiolabeled deoxyglucose (18F-FDG) uptake was less than controls.31 When patients in class III HF were treated with carvediol, myocardial uptake of 18F-FTHA decreased and 18F-FDG uptake was unchanged.32 The discrepancy among these investigations may be attributable to the severity of HF, where in the early stages there is a normal (or slightly elevated) rate of fatty acid oxidation, with a downregulation in advanced stage of HF.10 In all of our imaging studies33,34 glucose metabolism was consistently elevated in the hypertensive SHRs compared to the normotensive WKY controls. When fed, the fatty acid metabolism in the SHRs was less than with the WKY controls.33 Whereas with fasting, there was an observed increase (though not significant) in fatty acid metabolism in the SHR model compared with WKY controls.34 Metabolic changes appear to precede mechanical changes of LVH progression in the SHR model.
A recent paper in this journal by Manabe and colleagues35 gives an extensive review of possible PET cardiac tracers to evaluate cardiovascular disease by targeting myocardial perfusion, metabolism, innervation, and inflammation. Another interesting paper by Li and colleagues36 highlights efforts to develop tracers that target mitochondrion, which plays a fundamental role in cellular processes ranging from metabolism to apoptosis. Although perfusion is important, PET imaging of cardiac metabolism in heart failure continues to be the main focus in the development of new tracers.37 Another important application is the targeting of the development of fibrosis, especially important in hypertensive cardiac myopathy. Potentially using 11C-martinostat, which binds with high affinity to class 1 and 2 histone deacetylases (HDACs),38 would help to facilitate the development of novel anti-fibrotic therapies to reduce fibrosis in many heart failure patients.
A paper by Wu and colleagues39 provides insights into the design of PET fatty acid tracers. [11C]-palmitate is a 16-carbon fatty acid commonly used for measuring myocardial fatty acid metabolism in PET studies,40 however, it is degraded rapidly within 2 minutes by complete β-oxidation in cardiac tissue.41 Therefore, effort has been made to develop tracers that are trapped in the cell by insertion of an S-atom or substitution with a methyl group preventing oxidative degradation.42 An example of a β-methyl modified fatty acid labeled with 11C is 1-[11C]- β-R,S-methylheptadecanoic acid ([11C]-BMHA). [11C]-BMHA has high retention of activity in the myocardium and excellent imaging properties.41 Examples of fatty acids with an insertion of a S-atom include thia fatty acids labeled with 18F: 14-18F-fluoro-6-thia-heptadecanoic acid ([18F]-FTHA),42 18-[18F]fluoro-4-thia-oleate ([18F]-FTO), 16-[18F]fluoro-4-thia-palmitate ([18F]-FTP). These 18F-labeled tracers demonstrate excellent properties as myocardial PET tracers.39,41
Metabolic therapies that stimulate myocardial carbohydrate oxidation and inhibit myocardial fatty acid oxidation can improve cardiac performance and prevent or reverse the progression of LV dysfunction and remodeling.10,11 Specifically, such compounds as metformin21 and dichloroacetate (DCA)43 can improve glucose metabolism. Metformin acts by enhancing insulin sensitivity via the AMPK pathway. Studies have shown that metformin also attenuates cardiac fibrosis by inhibiting collagen synthesis.44 Moreover, studies in rats treated with dichloroacetate (DCA) showed increased glucose oxidation by attenuating increase in energy reserves, activation of the pentose phosphate pathway, and reduced oxidative stress improving cardiac function and animal survival.21 DCA has been used since its discovery in 1969 to treat lactic acidosis complications of congenital mitochondrial diseases and diabetes. It stimulates PDH and carbohydrate oxidation by inhibiting pyruvate dehydrogenase (PDH) kinase. Another drug (rapamycin) used for the prevention of transplant rejection inhibits mammalian target of rapamycin (mTOR) in SHRs, reducing cardiac hypertrophy but does not reduce blood pressure.45 Heart failure with preserved ejection fraction (HFpEF) is frequently accompanied by left ventricle hypertrophy and myocardial fibrosis. Histone deacetylases (HDACs) are a class of enzymes that cause conformation changes in the 3D architecture of DNA, modifying its transcription. HDAC inhibition with ITF2357 (givinostat) decreased hypertrophy and fibrosis and improved diastolic function in HFpEF patients.46 Other possible therapies for lipotoxicity in hearts of NAFLD patients can be learned from the diabetic heart. Wan and Rodrigues8 report on how the protein “ensemble” (heparanase-VEGF-LPL) cooperates in the diabetic heart to switch to predominantly use of fatty acid for energy; however, increased accumulation of fatty acid can trigger cell death by eliciting messengers such as ceramides. Understanding the interplay between heparanase, vascular endothelial growth factors (VEGFs), and LPL might help develop therapies for restoring metabolic equilibrium in cardiac lipotoxicity.
NAFLD is a serious disease with significant prognosis for cardiac death. The disease demonstrates abnormal structure and function in the liver cells that could potentially provide some insight into the same potential maladaptation of cardiac cells. The decrease in myocardial glucose uptake in NAFLD patients is a possible indicator of lipotoxicity in cardiac cells. Most PET studies reported in the literature involve using 18F-FDG to assess cardiac glucose metabolism. However, the complex mechanisms of cardiac metabolism can only be appreciated using multiple imaging probes. PET imaging of metabolic processes has been predominately aimed at studying heart failure; however, PET imaging of cardiac fatty acid and glucose metabolism and perfusion in NAFLD patients would significantly help to direct therapeutic intervention and to prolong life for these patients. Dynamic cardiac PET can significantly help in the management of NAFLD by improving the diagnosis of cardiac disease.
Grant T. Gullberg receives financial compensation outside the submitted work from Spectrum Dynamics Medical, Inc. for consultancy and Solving Dynamics, Inc. for work performed on a small business grant from NIH and NSF. The other authors have no conflict of interest.
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