Encyclopedia
Please note this is a comparison between Version 1 by Majid Mufaqam Syed-Abdul and Version 2 by Lindsay Dong.
Metabolic-associated steatotic liver disease (MASLD) is a cluster of pathological conditions primarily developed due to the accumulation of ectopic fat in the hepatocytes. During the severe form of the disease, i.e., metabolic-associated steatohepatitis (MASH), accumulated lipids promote lipotoxicity, resulting in cellular inflammation, oxidative stress, and hepatocellular ballooning. If left untreated, the advanced form of the disease progresses to fibrosis of the tissue, resulting in irreversible hepatic cirrhosis or the development of hepatocellular carcinoma. Although numerous mechanisms have been identified as significant contributors to the development and advancement of MASLD, altered lipid metabolism continues to stand out as a major factor contributing to the disease.
- MASLD
- MASH
- cholesterol
- DNL
- fatty acid oxidation
1. Metabolic-Associated Steatotic Liver Disease
Metabolic-associated steatotic liver disease (MASLD) is a recent nomenclature that has been approved to broaden the diagnostic criteria and to avoid stigmatization for a previously known condition called nonalcoholic fatty liver disease (NAFLD) [1], which encompasses a spectrum of liver conditions, ranging from simple fatty liver (hepatic steatosis), which can be detected through imaging or histological methods, to MASH, which involves inflammation and can lead to more severe liver damage [2]. Currently, characteristics indicative of MASLD have been observed in nearly a quarter of the global population [3][4][3,4], with these prevalence rates showing an upward trend [5]. This escalating occurrence of MASLD closely mirrors the concurrent rise in obesity and constitutes a significant contributing factor to the expanding burden of chronic hepatic diseases on a worldwide scale [3][4][5][6][7][8][9][10][3,4,5,6,7,8,9,10]. As anticipated, in concurrence with the rising prevalence of MASLD, there has been a significant 2.0–2.5-fold increase in the incidence of MASH in recent years [4][11][4,11] which was robustly linked to liver-related morbidity [12][13][12,13]. Additionally, it is important to highlight that projections indicate that MASH is nearing the position of becoming the second most prevalent causative factor necessitating liver transplantation [14].
2. Pathophysiological Changes at the Molecular Level in MASLD
Serological studies and numerous genomic studies performed on hepatocytes sourced from patients diagnosed with MASLD and individuals undergoing bariatric surgery consistently reveal a noticeable increase in several biochemical parameters [15] and the upregulation of key enzymes integral to the de novo lipogenesis (DNL) pathway [16][17][18][19][20][21][16,17,18,19,20,21]. As a master regulator of the DNL pathway, sterol regulatory element-binding protein 1c (SREBP1c), primarily activated by insulin, exhibited a significant increase in MASLD patients compared to those without MASLD [17][20][22][23][24][25][17,20,22,23,24,25], underscoring its central role in governing this metabolic process. Contrary to these findings, single-cell RNA sequencing (scRNA-seq) combined with computational network analyses to explore lipid signatures in mice with MASLD showed that, despite its traditional role as a driver of lipid synthesis, high SREBP1 expression is not predictive of hepatic lipid accumulation in non-alcoholic fatty liver disease (NAFLD); instead, the study identifies the constitutive androstane receptor (CAR) as a key player in regulating functional modules associated with cholesterol homeostasis, bile acid metabolism, fatty acid metabolism, and estrogen response, demonstrating its correlation with steatohepatitis in human livers [26]. Notably, among the subsequent enzymes regulated by SREBP1c, the isoforms of acetyl-CoA carboxylase (ACC) exhibited a remarkable increase, i.e., a more than eight-fold increase in expression in MASLD patients compared to those with normal liver profiles [16][17][19][16,17,19]. In addition to an increase in DNL enzyme expression, patients with MASLD also exhibited altered expression of FA binding protein (FABP), FA transport protein (FATP) [16][17][21][27][28][16,17,21,27,28], and CD36 [16][21][28][16,21,28]—genes responsible for FA uptake. Moreover, genes associated with triacylglycerols (TAGs) synthesis, including diacylglycerol o-acyltransferase 2 (DGAT2) [19] and microsomal triglyceride transfer protein (MTTP) [16][17][29][16,17,29], along with genes impacting very-low-density lipoprotein (VLDL) kinetics, exemplified by apolipoprotein B100 (apoB100) [16][17][29][16,17,29], exhibited increased expression levels. Furthermore, genes related to the oxidation of FAs, including peroxisome proliferator-activated receptor gamma (PPAR-γ) [27] and carnitine palmitoyltransferase 1 (CPT1) [16][27][16,27], were upregulated, while PPAR-γ coactivator 1α (PGC-1α) was downregulated [30]. It is worth noting that Moore et al. reported decreased expression of FA oxidation genes in MASLD patients [31]. MASH patients, in comparison with MASLD patients, exhibited lower expression of peroxisome proliferator-activated receptor α (PPAR-α), MTTP, and apoB100, but no changes were observed in SREBP1c, FASN, DGAT1 and 2, FABP and FATP, and CD36 [32][33][34][32,33,34].3. Lipid Synthesis in MASLD
3.1. Fatty Acids
As shown in Figure 1, FA synthesis is a complex biochemical process responsible for the synthesis of FAs, utilizing glycerol and carbon molecules. When FAs are generated from non-lipid sources, notably carbohydrates, this metabolic pathway is termed DNL [35][36][37,38]. Serving as the master regulator, SREBP1c controls the activation of enzymes of the DNL pathway, collectively governing the complex process of FA synthesis at the molecular level through their specific functions. The activation of SREBP-1c has been demonstrated to be regulated by insulin concentrations, with higher insulin levels leading to increased SREBP-1c activation [22]. At the molecular level, crucial enzymes involved in FA synthesis include ACC, which catalyzes acetyl-CoA carboxylation, a pivotal step in FA synthesis. FASN plays an orchestrating role in the intricate assembly of FAs, ensuring the formation of these essential molecules. SCD-1 is responsible for facilitating desaturation reactions, crucial for modifying FA chains to confer specific properties.Figure 1. Fatty acid synthesis and regulation in the development of MASLD. Legend: This figure illustrates the intricate process of Fatty Acid (FA) synthesis and its regulation. The synthesis of FAs involves a complex biochemical pathway, primarily driven by the activities of key enzymes such as ACC; FASN; SCD-1; and elongases (ELOV). The master regulator SREBP1c, influenced by insulin levels, governs the activation of enzymes in de novo lipogenesis (DNL). Additionally, ChREBP serves as a regulator activated during postprandial states and hyperglycemia, impacting gene transcription and enzymatic activity in the DNL pathway. The red dot (●) represents changes altered during MASLD pathogenesis. Figure abbreviations: ACC—acetyl-CoA carboxylase; CE—cholesterol ester; ELOVL5—elongases 5; FASN—fatty acid synthase; SCD-1—stearoyl-CoA desaturase-1; TCA—tricarboxylic cycle; and TAG—triacylglycerols.
The onset of MASLD is perhaps driven by insulin resistance, triggered by overnutrition, caloric surplus, or physiological changes [37][38][39][40][41][41,42,43,44,45]. This condition leads to hyperinsulinemia, which in turn results in an elevation in SREBP1c expression and the consequential upregulation of essential enzymes within the pathway of DNL (ACC, FASN, SCD-1). These observations were consistent in animal models as well as in in vitro studies, providing valuable insights into the mechanisms underlying MASLD [42][43][44][45][46][47][48][49][50][51][52][46,47,48,49,50,51,52,53,54,55,56]. Carbohydrate response element-binding protein (ChREBP) serves as another regulator of hepatic DNL. It is primarily activated during a postprandial state and hyperglycemia [53][54][55][57,58,59] and has been reported to increase gene transcription or directly enhance enzymatic activity [54][56][57][58][58,60,61,62]. In contrast, ChREBP downregulation in mice liver, known for its primary role in maintaining glucose and lipid homeostasis [59][60][61][62][63,64,65,66], significantly mitigated steatosis induced by high-carbohydrate feeding. Furthermore, with ChREBP downregulation, endogenous glucose production increased, FA oxidation decreased, and insulin resistance increased [63][67]. Intriguingly, the insulin resistance state reversed in a mouse model of diabetes and insulin resistance (i.e., ob/ob mice) when ChREBP was downregulated [54][64][58,68]. Although inhibiting ChREBP decreases DNL caused by fructose and elevates TAG levels [65][69], it has been linked to adverse effects such as fructose intolerance, gastrointestinal distress including diarrhea and irritable bowel syndrome, as well as cholesterol-induced liver damage [65][66][67][68][69,70,71,72].
Saturated vs. Unsaturated FAs in MASLD
Both the mono-(MUFA) and poly-unsaturated FAs (PUFA) have been studied briefly in relation to MASLD pathophysiology. Observational studies reported a significant relationship between low PUFA concentrations and MASLD presence. As reviewed by Yan et al., in a meta-analysis of 18 studies involving 1424 patients, PUFA supplementation (omega-3 in particular) resulted in lower hepatic enzyme levels and fat accumulation compared to control groups [69][97]. Similarly, impaired expression and activity of FADS1, a key desaturase in lipid metabolism, has been shown to contribute to altered FA profiles in MASH, leading to an imbalance in the n-6:n-3 ratio and impacting the synthesis of pro-inflammatory lipid-signaling molecules [70][98]. Others have also utilized the n-6:n-3 ratio of long-chain PUFA which is an indication of increased synthesis and decreased oxidation/secretion of FAs, and reported a relationship between the n-6/n3 ratio and steatosis [71][99].
At the molecular level, when the FASN and SCD expression at the level of mRNA and protein were considered, a concurrent increase was observed [72][105], suggesting that once the FAs are made via DNL, these FAs are desaturated to MUFA, resulting in a strong correlation between DNL (percent) and MUFA (percent of total FAs) but not between percent DNL and percent SFA [73][106]. Similarly, Knebel et al. found a simultaneous increase in DNL (measured indirectly using multiple DNL calculators) and percent MUFA in C57BL6 mice.
3.2. Triacylglycerol and Diacylglycerol
As illustrated in Figure 2, three distinct sources of FAs DNL from dietary carbohydrates, non-esterified FAs (NEFA) released from adipose tissue, and FAs obtained from the diet, undergo a sequence of enzymatic conversions, resulting in the formation of fatty acyl-CoA molecules. Subsequently, fatty acyl-CoA undergoes a series of enzymatic reactions, with glycerol-3-phosphate acyltransferase (GPAT) facilitating their conjugation to glycerol-3-phosphate, leading to the synthesis of lysophosphatidate. Next, another fatty acyl-CoA is incorporated into the process through the enzymatic activity of 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT), resulting in the formation of phosphatidate. The generated phosphatidate molecules, in turn, undergo enzymatic modification via phosphohydrolase-1 (PPH-1), resulting in the production of diglycerides (DAG). It is noteworthy that DAGs can also be derived from monoglycerides (MAG), with monoacylglycerol acyltransferase (MGAT) playing a pivotal role, although this specific pathway is not depicted in Figure 2.Figure 2. Triacylglycerol and diacylglycerol synthesis in MASLD. Legend: This figure illustrates the general metabolism of triacylglycerol (TAG) and diacylglycerol (DAG) synthesis. Three distinct sources of fatty acids (FAs) undergo enzymatic conversions, leading to the formation of fatty acyl-CoA. Notably, in MASLD, elevated DAG levels contribute to lipotoxicity, exacerbating fat accumulation, oxidative stress, inflammation, and insulin resistance. Mechanistically, DAG inhibits insulin receptor tyrosine kinase via PKC-ε activation, underscoring its significance in MASLD pathogenesis. Increased triacylglycerol (TAG) levels signify increased hepatic TAG accumulation, marking a crucial stage in MASLD progression. The red dot (●) represents changes altered during MASLD pathogenesis. Figure abbreviations: AGPAT—1-acylglycerol-3-phosphate-O-acyltransferase; CD36—cluster of differentiation 36; CE—cholesterol ester; DGAT—diacylglycerol acyltransferase enzyme; DNL—de novo lipogenesis; FA—fatty acids; GPAT—glycerol-3-phosphate; Pi—phosphate; NEFA—nonesterified fatty acids; PDAT—Phospholipid: diacylglycerol acyltransferase; PPH-1—phosphohydrolase–1; TAG—triacylglycerols; and VLDL—very-low-density lipoprotein.
In addition to TAG, the intermediate product [56][74][60,116] DAG has been identified as a contributing factor to MASLD development and progression primarily due to its lipotoxic properties [75][117]. Additionally, increased levels of hepatic DAG have been associated with key characteristics of MASLD i.e., fat accumulation, oxidative stress, inflammation, and insulin resistance [75][117]. At the molecular level, DAG has been shown to inhibit the tyrosine kinase activity of the insulin receptor [76][118] via activation of protein kinase C-ε (PKC-ε) [77][119] in the liver [56][78][60,120], which, at the intracellular level, impairs the downstream signaling cascade of insulin, resulting in insulin resistance [79][121].
3.3. Cholesterol
Depicted in Figure 3 is the synthesis of cholesterol. Catalyzed by acetyl-CoA acetyltransferases (ACAT), two molecules of acetyl-CoA transform into acetoacetyl-CoA. Later, another molecule of acetyl-CoA is further added to acetoacetyl-CoA to form a molecule that plays a central role in the cholesterol synthesis pathway known as β-hydroxy β-methylglutaryl-CoA (HMG-CoA). In the presence of the HMG-CoA reductase enzyme (HMGCR), HMG-CoA undergoes a series of reactions to eventually produce mevalonic acid. Mevalonic acid is then converted into squalene and eventually to cholesterol [23][80][23,126]. The metabolic fate of cholesterol includes the synthesis of bile acid, which is transported through an ATP-binding cassette sub-family G member 5/8 (ABCG5/8). Additionally, cholesterol can be esterified to produce CE in the presence of an enzyme called acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2), also termed SOAT2. Lastly, cholesterols can also be incorporated into very-low-density lipoprotein (VLDL) particles for secretion or can be stored in the form of a lipid droplet for later utilization [80][126]. In the context of MASLD pathophysiology, numerous studies have reported elevated levels of total cholesterol and low-density lipoprotein cholesterol (LDLc) concentrations in plasma. Conversely, reduced concentrations of high-density lipoprotein cholesterol (HDLc) have been observed in patients with MASLD when compared to those without MASLD [4][81][82][83][84][85][4,109,110,111,112,127].
Figure 3. Cholesterol synthesis and implications in MASLD. Legend: This figure outlines cholesterol synthesis, with acetyl-CoA acetyltransferases initiating the pathway and HMG-CoA reductase progressing to cholesterol. In MASLD, elevated total cholesterol and LDLc, along with reduced HDLc, are observed. SREBP-2, a master regulator, shows increased expression, contributing to heightened cholesterol synthesis. Enhanced HMGCR activity in MASLD and MASH exacerbates cholesterol production, impacting liver function. Altered cholesterol metabolism plays a crucial role in MASLD progression, with potential implications for inflammation and fibrosis within liver cells. The red dot (●) represents changes altered during MASLD pathogenesis. Figure abbreviations: ABCG8—ATP-binding cassette sub-family G member 8; ACAT—acetyl-CoA acetyltransferases; DNL—de novo lipogenesis; HMGCR—β-Hydroxy β-methylglutaryl-CoA reductase; HMGCS—β-Hydroxy β-methylglutaryl-CoA synthase; LDL-R—low-density lipoprotein receptor; NCEH1—neutral cholesterol ester hydrolase 1; NEFA—nonesterified fatty acids; and SOAT2—sterol O-acyltransferase 2.
3.4. Ceramides
Ceramides, a lipid class within the sphingolipid family, have recently garnered attention due to their lipotoxic properties and their integral role in cell membrane composition [75][86][117,142]. As shown in Figure 4, ceramides, vital lipid molecules for cell membrane integrity, are synthesized in the endoplasmic reticulum and Golgi apparatus [87][143]. The process involves condensing a long-chain fatty acid with serine, catalyzed by serine palmitoyltransferase (SPT) to form 3-ketodihydrosphinganine. Subsequent enzymatic steps lead to dihydroceramide, which is converted into ceramide through desaturation by dihydroceramide desaturase [88][144]. Ceramides are involved in skin barrier function, apoptosis regulation, and lipid metabolism [89][90][145,146]. Ceramides produce metabolic byproducts, including sphingosine, which regulates cell growth and apoptosis; ceramide-1-phosphate (C1P) involved in cell signaling and inflammation; sphingomyelin, which is crucial for cell membrane integrity; and sphingosine-1-phosphate (S1P), a signaling molecule influencing cell migration and immune responses [87][143].
Figure 4. Ceramide metabolism in MASLD. Legend: This figure outlines ceramide synthesis and impacts, emphasizing crucial roles in cell membrane integrity, skin barrier function, apoptosis, and lipid metabolism. Elevated ceramide levels in metabolic disorders, cardiovascular diseases, and MASLD are linked to increased lipid peroxidation and reduced mitochondrial respiration in MASH. Ceramides contribute significantly to hepatic steatosis and insulin resistance by activating PKCζ and PP2A, impairing AKT translocation, suppressing β-oxidation, and activating the NLRP3 inflammasome. The red dot (●) represents changes altered during MASLD pathogenesis. Figure abbreviations: 3-KDSR—3-ketodihydrosphinganine reductase; C1P—ceramide-1-phosphate; Cer—ceramides; CerDase—ceramidase; CerS—ceramide synthase; DES—dihydroceramide desaturase; S1P—sphingosine-1-phosphate; SK—sphingosine kinase; SMase—sphingomyelinase; SMS—sphingomyelin synthase; and SPT—serine palmitoyltransferase.
4. Lipid oxidation in MASLD
FA Oxidation
As illustrated in Figure 5, besides the FAs originating from DNL, dietary intake, and NEFA, additional sources of FAs stem from TAG catabolism and CE hydrolysis. These FAs are subject to cellular oxidation to meet energy demands [91][162]. Carnitine palmitoyltransferase 1 (CPT1) plays a pivotal role as an outer-membrane mitochondrial enzyme, facilitating FA uptake and their subsequent oxidation through the ß-oxidation pathway [92][93][94][95][163,164,165,166]. Typically, each FA undergoes oxidation, hydration, oxidation, and thyolisis, respectively—four primary steps of ß-oxidation, with two carbons being eliminated from FA during each cycle [96][97][167,168]. The final three steps occur within a protein assembly known as mitochondrial trifunctional protein (MTP) [98][99][100][169,170,171].
Figure 5. Fatty acid oxidation in MASLD. Legend: Illustration of diverse fatty acid sources contributing to cellular oxidation in non-alcoholic fatty liver disease (MASLD). Carnitine palmitoyltransferase 1 (CPT1), a pivotal outer-membrane mitochondrial enzyme, facilitates fatty acid uptake and oxidation through the ß-oxidation pathway. Malonyl-CoA, an intermediate in de novo lipogenesis (DNL), inhibits CPT1, preventing the entry of fatty acids from plasma non-esterified fatty acids (NEFA), diet, triglyceride (TAG) catabolism, and cholesterol ester (CE) hydrolysis into mitochondria for oxidation. This dysregulation results in hepatic lipid accumulation, fostering the progression of liver steatosis and metabolic dysfunction. The red dot (●) represents changes altered during MASLD pathogenesis. Figure abbreviations: 2C—two carbons; ATGL—Adipose triglyceride lipase; CD36—cluster of differentiation 36; CE—cholesterol esters; CPT1—carnitine palmitoyltransferase 1; DGAT—diacylglycerol acyltransferase enzyme; DNL—de novo lipogenesis; FA—fatty acids; HSL—hormone-sensitive lipase; LDL-R—low-density lipoprotein receptor; MTP—mitochondrial trifunctional protein; NCEH—neutral cholesterol ester hydrolase; NEFA—non-esterified fatty acids; SOAT2—sterol O-acyltransferase 2; and TAG—triacylglycerols.
5. Lipid Flux and Storage in MASLD
As illustrated in Figure 6, FAs appearing in the circulation stemming from all three sources (i.e., DNL, adipose tissue, referred to as NEFA, and dietary fats) primarily undergo two metabolic fates. They are either subjected to oxidation for energy production (to be elaborated upon later) or used in the biosynthesis of lipids (i.e., TAG, CER, and PL) using glycerol 3-phosphate or esterification of cholesterols utilizing cholesterol derived from exogenous or endogenous sources [101][40]. Depending on the body’s needs or the presence of pathological conditions, synthesized lipids are either secreted into circulation through the VLDL particles [80][102][103][114,126,139] or they are stored as lipid droplets in the liver. While the former mechanism appears to be predominantly activated by DGAT 1, i.e., the incorporation of fats into the lipoprotein particles in conjunction with apoB100 [102][104][105][114,192,193]; the later fate (storage via incorporation of fats into lipid droplets) appears to be driven by the activation of DGAT2 and is subsequently employed by lipolysis. In healthy males, the in vivo administration of insulin during an euglycemic hyperinsulinemic clamp resulted in decreased NEFA concentrations and reduced synthesis and secretion of VLDL [106][194].
Figure 6. Lipid flux in MASLD. Legend: This figure illustrates the intricate fate of fatty acids (FAs). FAs from de novo lipogenesis (DNL), non-esterified FAs (NEFA), and dietary fats undergo oxidation for energy or are utilized in lipid biosynthesis (triglycerides [TAG], ceramides [CER], and phospholipids [PL]). While DGAT1 activation drives FA incorporation into lipoprotein particles (VLDL) for circulation, DGAT2 activation leads to storage in hepatic lipid droplets. Insulin resistance in MASLD diminishes insulin-induced suppression of VLDL synthesis, contributing to elevated VLDL secretion and subsequent lipid dysregulation. The red dot (●) represents changes altered during MASLD pathogenesis. Figure abbreviations: APOB100—apolipoprotein B100; ATGL—adipose triglyceride lipase; CE—cholesterol esters; DAG—diacylglycerols; DGAT—diacylglycerol acyltransferase enzyme; DNL—de novo lipogenesis; FA—fatty acids; MTTP—microsomal triglyceride transfer protein; NEFA—nonesterified fatty acids; PL—phospholipids; TAG—triacylglycerols; and VLDL—very-low-density lipoprotein.
6. Interplay of Cholesterol Metabolism and DNL in MASLD
Regarding the correlation between cholesterol levels and the progression of MASLD, elevated lipid levels are frequently observed in MASLD patients [4][81][82][83][84][4,109,110,111,112]. In a lipidomic analysis of liver tissue obtained from healthy individuals, MASLD patients, and MASH patients, the concentrations of liver-CE were not significantly different between all groups. Regardless, in another study, FC concentrations were reported to be higher in patients with MASH only [107][100]. However, for total cholesterol levels, both MASLD and MASH patients exhibited higher concentrations with no differences in HDLc and LDLc, suggesting greater cholesterol content in VLDL and IDL remnant particles [107][100]. In another study, total cholesterol levels exhibited no distinctions; nonetheless, LDLc was markedly elevated in both groups of patients (i.e., MASLD and MASH) when compared to their healthy counterparts. Simultaneously, HDLc was lower only in the MASH group [108][132]. When gene analysis was performed, genes involved with cholesterol synthesis (i.e., HMGCR increased), clearance (LDL-R reduced), and those involved in the synthesis of bile (CYP 7A1/27A reduced) or transport (ABCG1/8 reduced) were altered, which may have resulted in higher FC in MASH patients.
SREBP2 serves as a vital regulator in cholesterol pathways, essential for generating an endogenous sterol ligand activating SREBP1c [24]. Intriguingly, the concomitant increase in TAG and cholesterols observed in patients with MASH, along with overexpression of the key master regulator, SREBP-2, clearly indicates a potential interplay between both the pathways of FA synthesis (i.e., the DNL) and pathways involving the synthesis of cholesterols [24][109][24,129]. Acetoacetyl-CoA has been proposed as a potential link connecting these two pathways [110][205]. This occurs because, within the DNL pathway, acetyl-CoA combines with malonyl-CoA, resulting in the formation of an acetoacetyl-CoA molecule (also referred to as ß-ketoacyl-ACP). Conversely, in the cholesterol pathway, two acetyl-CoA molecules give rise to acetoacetyl-CoA. Therefore, it is likely that the acetoacetyl-CoA molecule generated during lipogenesis by the FASN enzyme could potentially be routed toward the cholesterol synthesis pathway [110][205]. Additionally, the positive associations reported between the DNL percent and plasma total cholesterol and plasma LDLc provide additional evidence for this notion [111][206].
