KEY FIGURE

Highlights

• Hepatic de novo lipogenesis (DNL), FFA flux, and dietary fat contribute to steatosis.

• Hepatic DNL is promoted by complex mechanisms driven by insulin and substrates.

• Divergent insulin signaling and zonation characterize hepatic insulin resistance.

• DNL targeting offers a modifiable MASLD therapy.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of conditions ranging from simple steatotic liver disease (SLD) to metabolic dysfunction-associated steatohepatitis (MASH), progressive fibrosis, and ultimately cirrhosis [1-3]. MASLD is associated with an increased risk of cardiovascular (CV) events, chronic kidney disease, hepatic and extrahepatic malignancies, and liver-related outcomes, including liver failure and hepatocellular carcinoma [1,3,4]. Quantitative assessments of liver fat for the diagnosis of SLD can be performed using various clinically available diagnostic tools: ≥5% of hepatocytes containing fat, as assessed by light microscopy; ≥5% proton density fat fraction (PDFF) on magnetic resonance imaging (MRI); or >5.56% fat content on proton magnetic resonance spectroscopy (¹H-MRS) [5,6].

Overall, the global prevalence of MASLD has been steadily increasing, currently reaching approximately 40%, and it is projected to exceed 55% by 2040 [7,8]. In patients with type 2 diabetes mellitus (T2DM), more than 65% are reported to have coexisting MASLD. Moreover, among these individuals, approximately 41% have MASH and around 15% present with advanced fibrosis (stage ≥F3) [7].

Given that insulin resistance (IR) is widely recognized as the key underlying mechanism of metabolic syndrome, it is reasonable to consider IR as a central factor in the pathogenesis of MASLD, which can be viewed as the hepatic manifestation of metabolic syndrome [2,9-12]. However, the precise mechanisms linking IR to sustained hepatic lipid accumulation remain a subject of ongoing controversy. Macronutrients, especially carbohydrates, and insulin concentrations in the portal circulation may be up to 10 times higher than those in the systemic circulation [13]. It remains to be further clarified to what extent insulin’s direct regulation of hepatic lipid metabolism, including de novo lipogenesis (DNL), is preserved or impaired during IR, and how other intrahepatic and extrahepatic factors, such as elevated circulating free fatty acids (FFAs), further contribute to MASLD [12]. This mechanistic gap limits the development of targeted therapies to effectively control hepatic steatosis and improve insulin sensitivity at the same time. Thus, gaining a more specific and detailed understanding of insulin action in the liver—particularly its mechanistic links with MASLD—could further contribute to the development of effective strategies for the prevention and treatment of MASLD. In this review, we aim to focus on hepatic IR and steatosis with a particular emphasis on DNL, highlighting their mechanisms and implications in the context of MASLD.

OVERVIEW OF INSULIN ACTIONS IN THE LIVER

Insulin signaling pathways

All of insulin’s actions are mediated by its receptor, the insulin receptor (INSR), a cell surface protein that signals through multiple pathways involving protein and lipid phosphorylation, activation of small G-protein molecular switches, regulation of trafficking events, and control of a network of enzymes and transcription factors that together define insulin’s unique actions [10]. In the liver, insulin regulates glycogenesis, gluconeogenesis, and lipogenesis by activating INSR and downstream signaling pathways (Fig. 1) [2,9-12,14]. During signaling in the liver, insulin binds to its receptor and is internalized into liver cells. There, about 50% is degraded by insulinase and lysosomal enzymes [15]. Hyperinsulinemia resulting from decreased clearance can potentiate or even cause IR through classic homologous desensitization pathways [10,16,17]. Several studies have consistently shown reduced hepatic insulin clearance in subjects with MASLD [14]. Hepatic insulin clearance may be influenced not only by the hepatic expression of INSR but also by the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which promotes insulin clearance. Upon phosphorylation by INSR, CEACAM1 facilitates receptor-mediated insulin endocytosis and degradation in hepatocytes [14]. Mice lacking hepatic CEACAM1 exhibit hepatic steatosis, inflammation, peripheral hyperinsulinemia, and IR [18].

The INSR is a glycosylated, disulfide-linked α2β2 tetramer that belongs to a subfamily of receptor tyrosine kinases [10]; insulin binding to the INSR triggers autophosphorylation of β-subunits (Fig. 1). Insulin-stimulated autophosphorylation of its receptor recruits several proteins for phosphorylation to initiate signaling pathways [2]. Insulin receptor substrate 1 (IRS1) and IRS2 among IRS1–6 are the main adaptor proteins in the liver, activating phosphatidylinositol-3 kinase (PI3K) [9,11,15,19]. IRS2 gene expression is downregulated by insulin, so its mRNA expression rises during fasting (low insulin) and decreases after eating (high insulin) [20]. In contrast, IRS1 gene expression is not suppressed by insulin and stays stable in both fasting and fed states. Therefore, IRS2 mainly functions during fasting and immediately post-meal, regulating gluconeogenesis via phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), while IRS1 primarily acts in the fed state to promote lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) [9,11,15,20,21].

Class IA PI3K, consisting of the p85 regulatory subunit and p110 catalytic subunit, is activated by the two Src homology 2 (SH2) domains in the regulatory subunit interacting with tyrosine- phosphorylated IRS proteins [10,11]. These phosphorylated IRS proteins serve as docking sites for the PI3K p85 subunit, and this docking then leads to the activation of the p110 catalytic subunit [9]. PI3 kinase catalyzes the formation of the lipid second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) from phosphatidylinositol-4,5-triphosphate (PIP2). PIP3 binds to the pleckstrin homology (PH) domains of target proteins, altering activity or subcellular localization, as in the activation of 3-phosphoinositide-dependent protein kinase 1 (PDK1). This pathway can be terminated by phosphoinositide phosphatases, such as phosphatase and tension homologue deleted on chromosome 10 (PTEN) and SH2 domain-containing inositol 5ʹ-phosphatase 2 (Fig. 2) [2,9-11,22]. Hepatocyte-specific PTEN knockout (KO) (LPTENKO) mice exhibited improved glucose tolerance and enhanced systemic insulin sensitivity, but also developed massive hepatomegaly and MASH with triglyceride (TG) accumulation, accompanied by elevated expression of lipogenic genes. Additionally, enhanced hepatocarcinogenesis was observed in the LPTENKO mice [9,23,24].

PDK1 activates AKT (named for its discovery as the oncogene responsible for thymoma in Ak mice) and atypical protein kinases Cs (aPKCs; aPKCζ and aPKCι/λ) [11,25,26]. Phosphorylation of both Thr308/Thr309 and Ser473 (AKT1)/Ser474 (AKT2) is required for maximal activity of AKT. The Ser473/Ser474 phosphorylation stabilizes Thr308/Thr309 phosphorylation, leading to the activation of AKT kinase ability [11,12]. While PDK1 phosphorylates AKT at Thr308/Thr309 and PKCζ on Thr410, phosphorylation of Ser473/Ser474 is facilitated primarily by the mechanistic target of rapamycin complex 2 (mTORC2) [11,12]. Activation of AKT leads to phosphorylation of downstream substrates such as glycogen synthase kinase 3β (GSK3β), forkhead box protein O1 (FoxO1), Bcl-2-associated death promoter, phosphodiesterase 3B, and tuberous sclerosis complex 2 (TSC2) [11,27]. Insulin-stimulated AKT activates mTORC1 signaling by phosphorylating and inhibiting TSC2 which forms a complex with the scaffolding protein TSC1 that negatively controls the small guanosine triphosphatase Ras homolog enriched in brain (RHEB), a key regulator of the mTORC1 [10-12]. While GTP-loaded RHEB activates mTORC1, TSC negatively regulates RHEB and mTORC1 signaling [12,27]. There are three isoforms: AKT1, AKT2, and AKT3 [11,15]. Although AKT1 and AKT2 are prominently expressed in various tissues, including classical insulin-sensitive tissues like the liver and muscle, AKT3 expression is restricted to the testes, brain, lung, and fat. Some variability in AKT actions may reflect the presence of three AKT isoforms [12]. Although AKT1 impacts cell survival and growth, AKT2 appears to play a more prominent role in the liver [10-12]. Thus, AKT2-deficient mice show IR, mild glucose intolerance, and mild growth deficiency [15]. A study showed that insulin-induced AKT2 activity, but not AKT1 activity, was significantly decreased in the livers of obese insulin-resistant rats as compared with that in the livers of lean rats [28].

AKT activation promotes glycogen synthesis [11]. Hormonal activation of glycogen synthase (GYS) involves both allosteric interaction with glucose-6-phosphate (G6P) and dephosphorylation promoted by kinase inhibition including GSK3β, protein kinase A, and adenosine monophosphate-activated protein kinase (AMPK) and phosphatase activation (primarily protein phosphatase 1 [PP1]) (Fig. 1) [10]. How insulin regulates GYS is still not fully understood, and many mechanisms remain to be elucidated. AKT inhibits gluconeogenesis, which is regulated by FoxO1 and peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC1α) through the control of PEPCK and G6Pase [15]. When glucagon and glucocorticoids are elevated in the fasting state, expression of PGC1α is increased [29,30]. PGC1α binds and activates FoxO1, leading to activation of gluconeogenic gene expression. FoxO1 activation is suppressed by AKT-mediated phosphorylation, and AKT functions to specifically disrupt the FOXO1-PGC1α interaction [2,10,17,29]. A constitutively active FoxO1 mutation induces hypertriglyceridemia in mouse models, highlighting the additional significance of the AKT-FoxO1 axis in maintaining lipid homeostasis under normal physiological conditions [12,31]. In mice, concomitant deletion of FoxO1 in an AKT-deficient liver is not sufficient to drive DNL but restores insulin sensitivity and glucose tolerance, suggesting there are other pathways downstream of AKT that contribute to DNL [12,32].

Maintaining glucose homeostasis requires quick insulin signaling that is rapidly reversed when insulin dissociates. Protein tyrosine phosphatases (PTPs) dephosphorylate the INSR and its substrates, and some PTPs involved in this process are upregulated in insulin-resistant states [10].

Insulin-mediated SREBP1c activation and lipogenesis

The liver synthesizes several lipids, including TGs, diacylglycerols (DAGs) and sterols, which are synthesized at the level of the endoplasmic reticulum (ER) by esterification of FFAs derived from (1) adipose tissue lipolysis, (2) dietary chylomicrons taken up from the circulation, or (3) synthesized through hepatic DNL and then stored within lipid droplets (LDs) [33]. TG synthesis begins with the entry of FAs into the ER where they are converted to acyl-coenzyme A (CoA) and used for synthesis of TG by diacylglycerol acyltransferase 1 (DGAT1) and 2 (DGAT2). Insulin stimulates lipogenesis through activation of INSR-IRS-PI3KAKT- mTORC1-SREBP1c pathway, in which SREBP1c is an insulin- regulated transcription factor and upregulates various lipogenic genes, including genes encoding acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), adenosine triphosphate (ATP) citrate lyase (ACLY), and genes encoding the enzymes of the FA elongase complex [2,9,15,34]. Insulin increases Srebp1c mRNA and is also involved in the posttranslational activation of the SREBP1c protein [17]. SREBPs are activated by binding of the SREBP cleavage-activating protein (SCAP), causing a conformational change in SCAP to initiate site-1 cleavage of SREBPs, which releases them from Golgi membrane and allows their translocation to the nucleus. Insulin-induced gene 1 (INSIG-1) and INSIG-2 can bind SCAP, preventing the SCAP-dependent cleavage of SREBPs [12,35]. In a state of over-nutrition, such as obesity or IR, circulating insulin concentrations are positively associated with hepatic SREBP1c levels [2,34].

Insulin promotes SREBP1c cleavage via AKT and mTORC1. SREBP1c phosphorylation by AKT enhances SCAP-SREBP1c trafficking to the Golgi by increasing its affinity for coat protein complex II proteins, while mTORC1 and p70 S6-kinase (S6K) also support SREBP1c processing [36]. Additionally, AKT reduces Insig-2a mRNA expression, ultimately facilitating the release of the SREBP1c/SCAP from the ER [2,34,37]. In addition, mTORC1 is required for insulin-mediated induction of SREBP1c mRNA [36]. On the contrary, the inhibition of mTORC1 is required for the fasting-induced activation of PPARα and the activation of hepatic ketogenesis [38]. Moreover, suggesting a more complex pathway, selective inhibition of mTORC1 signaling in mice, through deletion of the RagC/D guanosine triphosphatase–activating protein folliculin (FLCN), promotes activation of transcription factor E3 (TFE3) in the liver without affecting canonical mTORC1 targets and protects against MASLD and MASH. TFE3 in the liver both induces a catabolic (lysosomal and oxidative) gene program and simultaneously suppresses DNL by suppressing proteolytic processing and activation of SREBP1c and by interacting with SREBP1c on chromatin [39].

However, stimulation of mTORC1 and SREBP1c is not sufficient to drive postprandial lipogenesis in the absence of AKT2 [2]. While inhibition of mTORC1 can repress hepatic DNL in the normal state, it does not do so when AKT is hyperactivated. In summary, although mTORC1 can sense abundant nutrients and drive hepatic DNL, AKT plays more crucial roles [2,40].

Mouse models with liver-specific deficient in insulin signaling (INSR-deficient, IRS1/IRS2 double-KO, PDK1-KO, and AKT2 KO) demonstrate impaired glucose tolerance (IGT), impaired suppression of hepatic glucose production (HGP), and a failure of induction of lipogenic genes, highlighting the importance of IRS1/2-AK2-FoxO1 pathways in hepatic insulin action [2,9,12,15]. Although additional deletion of FoxO1 in liver-specific INSR KO mice restored glucose tolerance and enabled normal suppression of gluconeogenic gene expression after feeding or systemic insulin administration, the induction of lipogenic gene expression by feeding remained suppressed [9,32,41]. This suggests that hepatic insulin signaling pathways other than the INSR-AKT-FoxO1 axis are required for SREBP1c induction and DNL in vivo [9]. In addition, in the absence of both AKT and FoxO1, mice adapt appropriately to both the fasted and fed states, and insulin suppresses HGP normally, but DNL is not normalized [41,42]. Interestingly, simultaneous deletion of FOXO1 and TSC1 from livers lacking AKT completely restored lipogenesis in vivo, independent of increased Insig2a expression, indicating that both inhibition of FoxO1 and mTORC1 activation by insulin are required and sufficient to induce DNL and to regulate lipid metabolism by insulin in liver in vivo [40,41]. These data suggest that a major role of hepatic AKT is to restrain the activity of FoxO1 and that in the absence of FoxO1, AKT is largely dispensable for insulin- and nutrient-mediated regulation of hepatic metabolism in vivo [32,42]. Additionally, insulin signaling can increase hepatic lipogenesis via atypical PKCs (e.g., PKC-ζ and PKC-λ), which involve SREBP1c and other pathways not related to AKT [43].

The PI3K-AKT-mTORC1 pathway also promotes protein synthesis and cell proliferation by phosphorylating of S6K1 and eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1).

Hepatic insulin action summary

Insulin signaling in the liver tightly coordinates glucose and lipid metabolism through distinct but interlinked molecular cascades involving IRS proteins, AKT, FoxO1, SREBP1c, and mTORC1. These pathways are also temporally regulated by feeding state, with IRS-2 crucial for gluconeogenesis suppression after fasting and IRS-1 supporting lipogenesis in the fed state.

HEPATIC INSULIN RESISTANCE AND STEATOSIS

Hepatic insulin resistance in the context of hepatic steatosis

In clinical studies, hepatic IR is typically demonstrated by the impaired ability of insulin to suppress HGP, as shown in hyperinsulinemic-euglycemic clamp studies [2,9,14,20]. In many cases, it reflects defective insulin signal transduction, primarily driven by chronic systemic inflammation and elevated FFA levels in obesity [15]. In relation to hepatic IR, an interesting study has provided compelling evidence of a direct and causal relationship between hepatic IR and intrahepatic fat accumulation [44], showing that 3 days of high-fat diet (HFD) feeding selectively induced hepatic fat accumulation and hepatic IR, without affecting peripheral fat depots or causing peripheral IR [44]. These changes occurred independently of alterations in visceral fat mass or portal vein FA concentrations. Mechanistically, the fat-induced hepatic IR appears to be mediated by activation of PKC-ε and/or c-Jun N-terminal kinase 1, leading to impaired tyrosine phosphorylation of IRS1 and IRS2. This disruption in insulin signaling compromises insulin’s ability to activate GYS and increases the contribution of gluconeogenesis to endogenous glucose production (Fig. 2) [44]. Under conditions of lipogenic substrate overload to the liver and hyperinsulinemia, resultant hepatic steatosis leads to elevated levels of lipid intermediates such as DAG and ceramides [2,9]. Classic and novel PKCs are activated by DAG, whereas atypical isoforms (aPKCζ and aPKCι/λ) are activated through the IRS/ PI3K pathway, as reviewed in detail elsewhere [25,26]. Elevated DAG levels activate PKCε, which inhibits INSR kinase activity by phosphorylating INSR at Thr1160 [2,14]. Multiple studies have demonstrated that membrane-bound DAG accumulation, but not DAG localized in cytoplasmic LDs, plays a key role in hepatic IR by activating protein PKCε [9,14,33]. In parallel, ceramides—lipids composed of sphingosine and FAs—also increase with excess FFA influx (Fig. 2). Ceramides can activate PKCζ or protein phosphatase 2A (PP2A), thereby inhibiting AKT activation by either phosphorylating the PH domain of AKT at a Thr34 or Ser34 residue, or by dephosphorylating AKT, respectively [2,14,25,26,45]. Moreover, ceramides also interact with FFAs, inflammatory cytokines, and glucocorticoids, further contributing to hepatic IR. Excess ceramides may accumulate in mitochondria, enhancing reactive oxygen species production and aggravating IR [2]. Elevated FFAs can also trigger inflammation by activating Toll-like receptor 4 [15].

Selective or total hepatic insulin resistance

As previously mentioned, under normal physiological conditions, insulin acts in the liver to suppress gluconeogenesis and promote DNL [9]. In line with this, liver-specific KO of INSR, dual IRS1/2, or AKT2 in mice exhibits both increased gluconeogenesis and decreased lipogenesis, a phenotype referred to as ‘total IR’ [17,41,46-48]. A paradox, as seen in patients with metabolic dysfunction such as T2DM, obesity, and MASLD, is the coexistence of hyperglycemia (due to impaired insulin action on glucose metabolism) and hepatic steatosis (due to preserved or exaggerated insulin action on lipid metabolism). This phenomenon has been termed ‘selective IR’ [2,9,15,17,49]. As increased hepatic DNL in obese people with MASLD has been reported to be inversely correlated with hepatic and whole-body insulin sensitivity and directly correlated with the plasma insulin levels [50], it is possible that selectively enhanced hepatic insulin signaling resulting from hyperinsulinemia in cases of MASLD might directly promote hepatic DNL. In this model, insulin fails to suppress gluconeogenesis via PEPCK, while simultaneously stimulating SREBP1c-mediated lipogenesis [9]. Evidence suggests that hepatic zonation and differential expression of IRS isoforms may underlie this mechanism (see the following sections) [2,9,15].

In addition, there are two insulin signaling pathways in the liver that diverge after AKT and before mTORC1 [12], the latter of which is also important for lipogenesis, but not for the inhibition of gluconeogenesis. Studies have shown that activation of mTORC1 and its downstream target S6K1 is markedly elevated in the liver of diet-induced obesity models [51]. Insulin-induced activation of mTOR and S6K1 is associated with increased inhibitory phosphorylation of IRS-1 at Ser636/Ser639 and impaired AKT activation [27,51]. In addition, insulin also regulates SREBP1c activation through AKT-mTORC1-independent pathways, such as WD repeat domain 6 (WDR6) and the CREB/ATF bZIP transcription factor (CREBZF; also known as Zhangfei), which was identified as a basic region-leucine zipper (bZIP) transcription factor of the activating transcription factor (ATF)/cAMP response element-binding protein (CREB) gene family. Interestingly, CREBZF promotes hepatic lipogenesis and adipose inflammation via insulin signaling, linking it to IR and steatosis; its inhibition may offer therapeutic benefits (Fig. 2) [2,37,52,53]. Additionally, mTORC1 phosphorylates and blocks the nuclear entry of the phosphatidic acid phosphatase lipin1, which has an inhibitory effect on nuclear SREBP protein levels [27,54]. Torin 1, an mTORC1 kinase inhibitor that inhibits both S6K and 4E-BP1 phosphorylation, causes a redistribution of cytoplasmic lipin1 to the nucleus and suppresses SREBP target gene expression—effects that are not observed with rapamycin, which does not inhibit 4E-BP1 phosphorylation consistently [54]. Considering the differences in the effects of rapamycin and Torin1 on the regulation of lipin1 and SREBP, it is clear that further investigation into the differential potencies of various mTORC1 inhibitors on cellular and systemic lipid and glucose homeostasis is needed. Moreover, it has been suggested that substrates for DNL are produced via glucokinase (GCK) even earlier than the activation of SREBP1c following insulin stimulation [55]. These findings suggest the presence of multiple alternative pathways beyond the classical AKT-mediated signaling. Several other factors, such as amino acids or tumor necrosis factor α (TNFα), are also implicated in the activation of mTORC1 and the promotion of IR [2,51]. It seems likely that both the zonation-dependent mechanism and multiple layers of divergent signaling mechanisms may contribute to the development of selective hepatic IR (Fig. 2) [20,56].

Conserved aPKC activation in diabetic liver is noteworthy, as aPKC mediates insulin and feeding effects on levels and activation of SREBP1c, which regulates levels of multiple enzymes engaged in lipid synthesis [43]. Notably, in contrast to AKT, the activation of aPKC by insulin is fully intact in livers of IRS1-KO mice with tissue-specific differences [57,58]. Atypical PKC activation by insulin was found to be fully or nearly fully maintained in the livers of Goto-Kakazaki diabetic rats and ob/obdiabetic mice, whereas AKT activation was markedly compromised in these diabetic livers [57]. In support of a critical mediatory role of aPKCs, in both models, inhibition of hepatic aPKC by adenovirally mediated expression of kinase-inactive aPKC markedly diminished diet/insulin-dependent activation of hepatic SREBP1c and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and concomitantly improved hepatosteatosis, hypertriglyceridemia, hyperinsulinemia, and hyperglycemia. Thus, in obesity, conserved hepatic aPKC-dependent activation of SREBP1c and NF-κB contributes importantly to the development of hepatic lipogenesis, hyperlipidemia, and systemic IR [57,59]. However, the precise mechanisms underlying the divergent signaling responses of AKT and aPKC downstream of IRS signaling in the insulin-resistant liver remain incompletely understood and warrant further investigation.

Lipid metabolism in hepatic steatosis

Liver TG is synthesized from fatty acyl-CoA, whose hepatocellular concentration is determined by the balance between FFA input (from circulating FFAs and DNL) and utilization through lipid synthesis and oxidation (Fig. 3A) [33]. Tracer studies in animals and humans have estimated that approximately one-fourth to one-third of labeled FFAs are extracted by the liver from perfusing blood and are subsequently retained within the organ [60-64]. The rate of hepatic removal of FFAs was similar in fed and fasted rats. And, about two-thirds of the tracer activity is recovered in hepatic TG, while the remainder is in the phospholipid fraction [64]. Apparently, under normal conditions, the liver extracts small amounts of FFAs from blood with low FFA concentrations (e.g., after glucose loading or insulin treatment) and larger amounts when concentrations are higher (e.g., during fasting or after epinephrine treatment). However, the percentage of hepatic FFA uptake does not change. The liver extracts more FFAs when plasma levels are high and less when they are low, indicating a homeostatic role in this aspect of fat metabolism [64].

Under insulin-resistant conditions, the increased rate of lipolysis due to adipose tissue IR raises the plasma concentrations of FFAs and glycerol, which stimulate gluconeogenic flux by providing glycerol as the substrate and FFA for activation of gluconeogenesis in the liver [14]. Although FFAs do not provide net carbon to gluconeogenesis, they indirectly fuel the gluconeogenic flux by providing, through their hepatic β-oxidation, the energy needed to drive gluconeogenesis as well as mitochondrial acetyl-CoA, an allosteric activator of the gluconeogenic pyruvate carboxylase [14,65]. Patients with MASLD have an upregulation of gluconeogenesis from glycerol rather than from alanine or lactate but similar rates of glycogenolysis [14,66]. This switch from lactate to glycerol as a substrate for gluconeogenesis indicates a failure to meet excessive metabolic challenges. Liver fat accumulation, coupled with reduced metabolic adaptability, may lead to a vicious pathogenic cycle contributing to the comorbidities of MASLD.

However, in a study that examined whether chronic mild hyperglycemia and hyperinsulinemia affect hepatic FFA uptake in patients with IGT, researchers found that absolute hepatic FFA uptake and fractional extraction of circulating FFA by the liver were reduced in IGT subjects compared with healthy controls. This reduction may result from intrahepatic substrate competition between FFAs and glucose/lactate [61].

The TG–FA composition and sources contributing to plasma very low-density lipoprotein (VLDL)-TG have been shown to closely reflect hepatic TG content. Therefore, the characteristics of VLDL-TG are commonly used to assess hepatic TG fluxes (Fig. 3) [60,67]. In well-fasted, lean subjects, FFAs derived from adipose tissue stores are the primary source of FAs used by the liver for VLDL-TG synthesis, while DNL typically represents about 5%–10% of these sources, and dietary contribution in the fasting state is usually minor [68]. Patients with MASLD show a marked increase in hepatic DNL and elevated nocturnal plasma FFA levels [60,69,70]. In individuals with MASLD and obesity, hepatic TG is reported to derive approximately 59% from circulating FFAs, 26% from DNL, and 15% from dietary sources [67]. In a well-designed study by Smith et al. [50], the contribution of hepatic DNL to intrahepatic TG palmitate was 11%, 19%, and 38% in the lean, obese, and obese-MASLD groups, respectively. Hepatic DNL was inversely correlated with hepatic and whole-body insulin sensitivity, but directly correlated with 24-hour plasma glucose and insulin concentrations. Furthermore, non-obese participants who achieved a 10% weight loss showed a 35% decrease DNL in lipoprotein-TG and a 50% reduction in intrahepatic TG [50]. In contrast, a recent study showed that the proportional contribution of FFAs to VLDL-TG was greater following weight loss, and the rates at which plasma FFAs were recycled into VLDL-TG that was secreted were also greater (Fig. 3B) [60]. The combined effect of excessive mobilization of FFAs from adipose tissues and upregulation of DNL further exacerbates the accumulation of liver TGs. Relative increase in IRS1 or IRS2 is associated with increase in liver-specific FA transport proteins 2 and 5, and increased lipid storage [13,71]. Another study showed that CD36 protein levels in the liver are significantly elevated during diet-induced obesity, and these elevated levels correlate with increased hepatic TG storage and secretion [72].

Hepatic de novo lipogenesis

Implications of hepatic de novo lipogenesis in MASLD

Elevated hepatic DNL is a key abnormality in MASLD, with overfeeding carbohydrates significantly raising liver fat, while caloric restriction reduces it (Fig. 3) [13,50,60,67,73]. Lambert et al. [69] showed that individuals with high liver fat (H-LF) have a 3.5-fold greater contribution of FAs from DNL to VLDL-TG than those with low liver fat (L-LF), while the portion of TG palmitate from diet was similar in both groups (about 5% in each). As a percentage of TG palmitate, DNL was 2-fold higher in subjects with H-LF and this level was independently associated with the level of intrahepatic TG [69]. Even higher proportions of DNL-derived palmitate (43%) were recorded in patients suspected of having MASH and fibrosis, suggesting that progressive disease may be associated with higher DNL [74].

In a recent elegant study by Lambert et al. [60], participants were stratified at baseline into L-LF or H-LF groups by liver fat measured by ¹H-MRS. Following a 6-month intervention designed to reduce energy intake and improve food quality and composition by reduction of intake of simple sugars and increasing consumption of whole foods, both groups achieved comparable and substantial reductions in body weight of approximately 10%. Notably, weight loss resulted in a significant 75.6% relative reduction in intrahepatic TG in the H-LF group, leading to normalization of hepatic TG levels (<5.6%) in the majority of these participants (Fig. 3B) [60]. In the study, multiple stable isotope tracers were used to assess the contribution of FFAs, DNL, and dietary fat to VLDL-TG palmitate. After the 6 months of dietary intervention and marked weight reduction, VLDL-TG concentrations fell by 38% because of a 67% reduction in the contribution from DNL, whereas the contributions from FFAs and dietary fat to VLDL-TG remained unchanged compared to baseline values. Reduced DNL was significantly associated with loss of intrahepatic TG [60]. Given the strong effect of energy restriction to reduce FA synthesis, the results support a principal role of reduced DNL to lead to greater utilization of plasma FFAs for VLDL-TG synthesis and secretion, further leading to reductions in liver fat. These results underscore the pathological, yet highly modifiable, role of DNL in MASLD development.

Hepatic de novo lipogenesis pathways

Hepatic DNL is a process by which lipids are endogenously synthesized from dietary sources, usually carbohydrates, or stored energy depots [13,75-77]. DNL consists of three main steps: FA synthesis, elongation/desaturation, and TG assembly (Fig. 4). Dietary carbohydrates like starch and sucrose break down into glucose and fructose, which are metabolized to acetyl-CoA for the tricarboxylic acid cycle. Fructose has emerged as a potent activator of carbohydrate response element-binding protein (ChREBP), which is particularly relevant in the context of MASLD, given the global rise in fructose consumption that parallels the increasing prevalence of MASLD [78- 82]. Fructose enhances its own metabolism by upregulating ketohexokinase (KHK), which is elevated in MASLD; diets high in both fat and fructose worsen MASLD, but fructose alone more strongly induces DNL enzymes like ACC1, FASN, and stearoyl-CoA desaturase 1 (SCD1) compared to HFDs [13]. Fructose metabolism rapidly reduces hepatic ATP through KHK-driven phosphorylation, which is much faster than glucose phosphorylation. This rapid ATP reduction increases uric acid production, which promotes hepatic steatosis through generation of mitochondrial oxidative stress. Furthermore, the prolonged ATP reduction, even after peak fructose phosphorylation within 5 minutes, may result from fructose-induced DNL, which inhibits mitochondrial β-oxidation. Patients with MASLD often show reduced hepatic ATP [13]. Fructose is metabolized in the liver via KHK to form fructose 1-phosphate (F1P), which bypasses the regulatory phosphofructokinase-1 step in glycolysis. This leads to an unregulated supply of three-carbon intermediates that promote DNL. F1P also activates GCK independently of insulin, enhancing hepatic glucose uptake and metabolism. Additionally, fructose can stimulate pyruvate kinase transcription, further accelerating glycolysis and favoring DNL from both fructose and glucose. Thus, the combination of glucose and fructose—as in sucrose—synergistically enhances hepatic DNL [76].

When energy is abundant, citrate accumulates, exits the mitochondria, and is converted back to acetyl-CoA by ACLY in the cytoplasm, initiating FA synthesis (Fig. 4). It is relevant to highlight that the higher availability of glucose in the liver, which accelerates DNL, also favors cholesterologenesis under insulin stimulation [12,76]. Citrate also activates ACC, which produces malonyl-CoA, the key building block for the enzyme FASN. FASN extends the carbon chain, forming palmitate, the primary FA product. High malonyl-CoA levels inhibit carnitine palmitoyltransferase 1a (CPT1a), limiting mitochondrial FA oxidation. Palmitate (C16:0) is the main FA made in the body and can be elongated by elongase of long chain fatty acids family 6 (ELOVL6) to stearate (C18:0) or longer FAs. Both can be desaturated by SCD1 to produce monounsaturated fats. These FAs are esterified to form TG. Glycerol-3-phosphate (G3P) acyltransferase (GPAT) initiates TG assembly by adding an acyl-CoA to G3P, followed by 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) and lipin1 actions to generate DAG. DGAT completes the process by adding the third FA to form TG (Fig. 4). Notably, unsaturated fats like oleate are favored in this step. Importantly, DAG buildup, not TG, is linked to hepatic IR via PKC activation. Supporting this, patients with MASLD have higher liver DAG levels, but mice overexpressing DGATs accumulate TG without developing IR, highlighting the distinct metabolic roles of these lipids [13].

Molecular regulation of hepatic de novo lipogenesis

DNL is minimal during fasting but increases after a meal as glucose and insulin levels rise, activating insulin signaling via PI3K, AKT, aPKC, mTORC, DNA-dependent protein kinase, and phosphatases [34,83]. A high-carbohydrate diet further amplifies this response, promoting greater FA and fat synthesis [34]. Thus, insulin activates specific kinases and phosphatases that can modify transcription factors such as upstream stimulatory factor (USF), SREBP1c, ChREBP, and liver X receptor (LXR), or regulate the expression of these transcription factors (Fig. 4) [34,83]. Additionally, specific glucose metabolites that rise after a meal may influence the function or localization of certain transcription factors [34]. For detailed interactions and complex regulatory mechanisms among these transcription factors, readers are encouraged to consult more in-depth review articles [34]. In particular, two major transcription factors have been studied much [2,10,84]: SREBP1c, which is controlled by both insulin signaling and insulin-independent pathways [2,20,75], and ChREBP, which is activated by carbohydrate uptake [9,34,78,83].

Transgenic mice overexpressing nuclear form of SREBP1c in adipose tissue (adipocyte-specific SREBP1c) showed impaired white fat differentiation, hypertrophic brown fat, increased preadipocyte factor-1 (Pref-1) and TNFα, severe IR with very high insulin levels, diabetes unresponsive to insulin, hepatic steatosis, and hypertriglyceridemia [84]. This secondary MASLD model caused by increased adipose tissue lipolysis is associated with decreased ChREBP-mediated lipogenesis in the liver and results in total hepatic and whole-body IR, followed by oxidative stress as well as portal and lobular inflammation [84]. This is in line with the results from some studies in that hepatic esterification of FAs into TG, unlike DNL, depends on substrate delivery rather than insulin action [84-86].

In a mouse model with hepatocyte-specific SREBP1c overexpression, SREBP1c targets specific genes and key enzymes in DNL and lipid metabolism to be upregulated with a characteristic liver-specific IR without peripheral IR in skeletal muscle [84]. This hepatocyte-specific SREBP1c mice developed SLD with a considerably increased visceral adipose tissue. Their liver-specific IR was associated with increased C18:1-DAG content and PKCε translocation [84]. Compared to control mice, hepatocyte-specific and adipocyte-specific SREBP1c overexpressed mice had larger livers and showed greater accumulation of LDs, as confirmed by 87% and 191% higher hepatic lipid content. Histological analysis revealed portal and lobular inflammation without hepatocellular ballooning or fibrosis in the adipocyte-specific SREBP1c model [84]. Of note, IR in these two different models with MASLD can result not from impaired mitochondrial function but from increased accumulation of specific DAG [84]. Actually, impaired mitochondrial function (i.e., decreased oxidative capacity and/or increased oxidative stress) did not precede IR. Thus, the study indicates that mitochondrial function initially increases in a compensatory manner, but after eventually reaches a limit, beyond which mitochondrial dysfunction seems to contribute to the progression to MASH and hepatic fibrosis [14,66].

As a negative feedback regulation, unsaturated FAs were shown to decrease the nuclear content of SREBP1, but not SREBP2, in cultured human embryonic kidney-293 cells [87]. The potency of unsaturated FAs increased with increasing chain length and degree of unsaturation. However, the saturated FAs palmitate and stearate were not effective. In line with this finding, a study showed that dietary polyunsaturated FAs coordinately suppressed transcription of a group of hepatic genes, with a 4-fold decrease in SREBP1c protein in hepatic nuclear protein extracts [88]. Downregulation occurred at two levels: the expression of SREBP1a and SREBP1c genes were markedly reduced, and the proteolytic processing of these SREBPs was also inhibited [87,88].

Progression from simple steatosis to MASH may depend on secondary hits, including ER stress and inflammation [89,90]. Elevated ER stress and inflammatory pathways involving TNFα also drive the progression by inducing caspase-2 (Casp2), which activates SREBP1/2 via SCAP-independent cleavage by site-1 protease (S1P) [90]. A study showed that ER-stress-mediated SREBP1/2 activation, TG and cholesterol accumulation, as well as MASH progression, all depend on Casp2 [91]. Thus, ER stress might contribute to the development of increased hepatic DNL in hepatic IR, at least in the advanced stages of MASLD/MASH [91]. Importantly, Casp2 activates SREBP1 and 2 through a hitherto undescribed mechanism that is not subject to feedback inhibition by sterols or unsaturated FAs, which inhibit normal SCAP-dependent SREBP activation [87]. These findings explain how ER stress and over-nutrition overcome the normal homeostatic controls that safeguard multicellular animals against excessive lipogenesis. Additionally, that the FLCN:mTORC1:TFE3 arm is dominant in the anabolic regulation of SREBP1c and DNL. However, the loss of TSC1 may promote protein anabolism through activation of the mTORC1:S6K arm but suppress lipid anabolism through feedback inhibition of the FLCN:mTORC1:TFE3 arm [39,92].

ChREBP, which is a member of the Mondo family of transcription factors, senses carbohydrates and activates genes involved in glycolysis, DNL, and lipid trafficking [2,34,78]. Under high-carbohydrate conditions, ChREBP is activated through dephosphorylation, allowing its translocation into the nucleus, where it binds to its response elements. Additionally, metabolic intermediates such as xylulose 5-phosphate, G6P, fructose-2,6-bisphosphate, and acetyl-CoA are additional potential activators of ChREBP translocation and activity [2,34,78,93]. In addition to the canonical form of ChREBP (renamed ChREBPα), a short but highly active isoform (177 amino acids shorter and 20-fold more active than ChREBPα), termed ChREBPβ, was discovered [14,78]. Well-accepted targets of ChREBP include not only enzymes in glucose metabolism, such as liver-type pyruvate kinase (L-PK), G6Pase catalytic subunit, glucose transporter 4, G3P dehydrogenase, and GCK regulatory protein, but also lipogenic enzymes, such as ACLY, FASN, ACC, and SCD [34,78]. Transient overexpression of ChREBP in the liver results in hepatic steatosis without IR through induction of the lipogenic enzyme SCD1, suggesting that increased ChREBP can dissociate hepatic steatosis from IR [94]. This result also demonstrates that hepatic steatosis is not necessarily accompanied by IR in DNL in the liver and can be initiated by factors other than insulin signaling [34]. In this regard, ChREBP expression in liver biopsies from patients with MASH was observed to be two-fold higher when the histologic steatosis score was greater than 50% compared to those with less than 50%. However, it decreased in the presence of severe IR [34,94]. Glucose can induce translocation of GCK from the nucleus to cytoplasm to facilitate synthesis of G6P. And, multiple factors influencing the level or activity of GCK, followed by ‘carbon push’ to DNL, could partially account for insulin-independent hepatic DNL in the hyperglycemia condition (Fig. 4) [95].

ChREBPβ is transcriptionally induced by ChREBPα and amplifies the glucose response in the liver and adipose tissue [78,86]. Unlike ChREBPα, hepatic ChREBPβ is strongly induced by high-fructose feeding in mice, accompanied by increased expression of glycolytic (Pklr, Gpi1, and Eno1), fructolytic (Aldob, Khk, and Dak), and lipogenic (Fasn, Acly, and Acaca) genes [86]. Furthermore, fructose activates ChREBP and induces G6Pase gene expression and enzyme activity in the absence of FoxO1a, indicating that carbohydrate-induced activation of ChREBP and G6Pase dominates over the suppressive effects of insulin to enhance glucose production [86]. Importantly, ChREBP induces the expression of fibroblast growth factor 21 (FGF21) as a hepatokine in response to glucose and fructose [76]. FGF21, acting via a liver-brain axis, suppresses sugar intake and regulates energy homeostasis. This ChREBP-FGF21 pathway requires PPARα and serves as a protective feedback loop against carbohydrate overconsumption and metabolic disease [14]. By co-regulating the expression of FGF21, ChREBP mediates liver to brain dialogue and contributes to the regulation of sugar intake and energy expenditure [14,96].

In addition, unsuppressed glucose production is an important causal factor of hepatic DNL. ChREBP plays an important role in sensing glucose (Fig. 4). Of note, hepatic ChREBP activation is not specific to fructose ingestion. Any carbohydrate that can increase the intrahepatic hexose- and triose-phosphate pool can likely activate hepatic ChREBP [86]. Therefore, G6P is naturally one of the primary activators of ChREBP [2,78]. Mice with hepatocyte-specific ChREBP deletion did not develop carbohydrate-induced hepatosteatosis in spite of IGT. Mice with ChREBP overexpression showed significant accumulation of hepatic TG levels, with reduced G6P and glycogen concentrations. These findings concur with the roles of ChREBP in redirecting glucose toward FA synthesis and support that glucose-driven DNL makes significant contributions to IR-induced liver lipid deposition [2]. In IR, it is difficult to distinguish the contribution of lipid substrate supply from the role of insulin in regulating DNL. Taken together, these findings demonstrate that ChREBP-driven hepatic steatosis can be dissociated from IR [94]. ChREBP and SREBP1c respond to different kinds of nutrient signals, and the synergistic function of ChREBP and SREBP1c allows the liver to maintain lipogenesis under different nutrient conditions [2,86].

Studies suggest that IRS1 and IRS2 have distinct roles in regulating hepatic lipid metabolism [20,30,97]. Decreased IRS2 expression but preserved IRS1 levels in the liver under hyperinsulinemia, HFD conditions, or in human MASLD [20,97], is associated with increased hepatic SREBP1c and DNL, whereas IRS1 deletion reduces DNL and TG content without altering SREBP1c levels. These findings indicate that IRS2 may be more important for suppressing DNL [2]. However, insulin-driven SREBP1c under IR condition is likely independent of IRS1, IRS2, or both, implying that additional pathways may be involved: in HFD models, levels of SREBP1c increased after refeeding, regardless of the absence of IRS1, or of both IRS1 and IRS2 [2,20]. Additional mechanisms, such as hepatic zonation, substrate-driven pathways, inflammatory mediators, and interplay with other tissues and/or transcriptional pathways, may be explainable factors [2,9,20,86]. In a liver-specific IRS KO mouse model study, liver-specific IRS2 KO (LIRS2KO) mice, but not liver-specific IRS1 KO (LIRS1KO) mice, developed hepatic steatosis on an HFD, while LIRS1KO mice were protected from steatosis [20]. Additionally, DNL is markedly decreased in LIRS1KO mice but not in LIRS2KO mice. Neither mTORC1 nor SREBP1c is necessary or sufficient for insulin-related DNL, but AKT is crucial for this. The partial independence of mTORC1 or SREBP1c also implies that insulin/IRS may also likely function via alternative pathways, e.g., via CREBZF or WDR6, although the detailed mechanisms still need to be elucidated (Fig. 2) [37,52]. Thus, the contributions of other factors should be taken into consideration [2]. Moreover, hepatic glucose and its derivative G6P could increase GCK and GYS2 activity independently of insulin and subsequently promote DNL through substrate supply [2,98].

Collectively, hepatic DNL, driven by increased macronutrient influx, plays a major role in the early development of hepatic steatosis and contributes to the onset of selective hepatic IR. While this mechanism is important in the initial stages, the progression of MASLD ultimately depends on the development of total hepatic IR and systemic IR [84].

Insulin-dependent and insulin-independent pathways in the metabolic paradox

Several insulin-dependent and insulin-independent pathways are implicated in the paradoxical coexistence of increased hepatic glucose output and enhanced hepatic lipogenesis in MASLD [14,34,43,56,86]. mTORC1, which lies downstream of AKT, mediates SREBP1c induction despite the impaired signaling during insulin-resistant conditions. Because mTORC1 can function independently of AKT, it does not phosphorylate and inhibit FoxO1, as AKT normally does, and thus promotes lipogenic gene transcription while gluconeogenic gene transcription is still active [56]. In addition, insulin signaling can increase hepatic lipogenesis via atypical PKCs, which involve SREBP1c and other pathways not related to AKT [43].

Additionally, substrate-driven pathways as insulin-independent mechanisms contribute to elevated DNL and enhanced esterification of FFAs into hepatic TG [34,70,85,86]. Notably, Ter Horst et al. [70] demonstrated that glucose-stimulated, insulin-mediated DNL was not elevated in insulin-resistant steatotic livers, calling into question the notion that increased hepatic lipogenesis in human MASLD is primarily driven by pathway-selective insulin action. In contrast, glucose-stimulated hepatic DNL was significantly higher in control subjects with greater insulin sensitivity. Moreover, fructose—a substrate that enters hepatocytes independently of insulin—robustly induced gene expression of the active ChREBPβ isoform and stimulated hepatic lipogenesis in obese individuals regardless of MASLD status [70,86].

Supporting this perspective, monosaccharides have been shown to activate ChREBP and other key lipogenic transcription factors—including SREBP1c, LXR, and PPARγ—via insulin-independent mechanisms [34,70,83,86,99]. Thus, in individuals with IR, an increase in hepatic insulin action may not be necessary to explain the upregulated DNL; rather, it is likely driven by an excess supply of lipogenic substrates—including carbohydrates such as fructose as well as amino acids, lactate, and citrate—that fuel hepatic DNL [70,86]. Although increased DNL is a feature of human MASLD, it does not necessarily lead to IR. Likewise, hepatic IR develops in some, but not all, animal models of DNL-mediated MASLD. However, augmented FA flux to the liver and/or peripheral IR may lead to so-called ‘secondary MASLD’ and total hepatic IR [84]. Luukkonen et al. [100]. showed that 3 weeks of a hypercaloric diet rich in saturated FAs induced the highest increase in hepatic TG (+55%) compared to the diets high in unsaturated FAs (+15%) or simple sugars (+33%). The high saturated FA diet increased lipolysis, adipose IR, and ceramide levels [33,100]. Many studies suggested that hepatic esterification of FAs into TG, unlike DNL, depends on substrate delivery but not insulin action [84-86]. Additionally, nutrient-derived ROS may augment hepatic steatosis through insulin-independent activation of the PI3K pathway [101]. Insulin-independent regulation of FA esterification has also been reported: a metabolic flux study reported that the rate of FA esterification into hepatic TG was dependent on the plasma FA infusion rates, but independent of changes in the plasma insulin concentrations or hepatic insulin signaling [85].

Hepatic zonation and insulin resistance

The concept of hepatic zonation of insulin signaling components can also help explain the paradoxical coexistence of increased hepatic glucose output and increased hepatic lipogenesis in MASLD [15,20]. As summarized in Table 1, the liver exhibits spatial metabolic compartmentalization, with periportal (PP) hepatocytes predominantly responsible for gluconeogenesis, while perivenous (PV) hepatocytes are specialized for lipogenesis and glycolysis. While PP hepatocytes are suppressed by insulin signaling, PV hepatocytes are stimulated by insulin signaling [20,102,103]. Among the core insulin signaling mediators—including INSR, IRS1, IRS2, AKT1, and AKT2—IRS1 shows marked zonal expression, with approximately 2-fold higher levels in PV compared to PP regions [9,20]. In hyperinsulinemic states such as obesity or MASLD, transcriptional downregulation of IRS2 occurs, particularly in PP hepatocytes [20]. This leads to impaired insulin signaling in gluconeogenic zones, resulting in insufficient suppression of HGP and thus contributing to systemic hyperglycemia. In contrast, PV hepatocytes retain or even upregulate IRS1 expression, preserving insulin sensitivity in this zone and enhancing DNL, thereby promoting hepatic lipid accumulation [20]. The reason why LIRS1KO mice show ‘total IR’ with hyperglycemia and suppression of steatosis, whereas LIRS2KO mice show ‘selective IR’ with hyperglycemia and steatosis may be attributable to the differential distribution and alterations of Irs1 and Irs2 expressions in the liver [15,20]. In humans with MASLD, hepatic IRS2 expression is significantly reduced and correlates with elevated expression of gluconeogenic enzymes, while lipogenic genes such as FASN remain highly expressed and positively correlate with IRS1 levels [97,104]. The enrichment of enzymes responsible for generating some specific FA and their metabolites in a PV distribution in MASH provides further evidence to implicate lipid mediators in liver injury [74,105].

Recent studies also implicate the Wnt/β-catenin signaling pathway in establishing the zonated expression of IRS1. Specifically, the β-catenin/T-cell factor 4 transcriptional complex directly binds to the IRS1 promoter, suggesting a molecular mechanism by which hepatic architecture regulates insulin signal distribution and contributes to metabolic dysregulation in MASLD [106].

INSULIN RESISTANCE AND VERY LOW-DENSITY LIPOPROTEIN SECRETION

The availability of lipids within the lumen of the ER of hepatocytes may dictate the amount of apolipoprotein B (ApoB) eventually secreted. In addition, in the absence of adequate core lipids, partially ER-translocated ApoB is exposed to the cytosol and subjected to degradation [107]. Normal activity of microsomal triglyceride transfer protein (MTP) is required for the assembly of ApoB lipoproteins [107]. Under physiological conditions, insulin represses ApoB synthesis, MTP expression, and VLDL-TG and ApoB secretion via the INSR-IRS-PI3K pathway and related mechanisms, such as protein tyrosine phosphatase 1B (PTP1B) and PTEN [2,107,108]. Most de novo synthesized TG is stored in cytosolic LDs and a smaller portion is secreted in the form of VLDL (Fig. 4) [108,109]. TG synthesis in ER and further processing are detailed elsewhere [109]. Increased FFA flux, as observed in IR states, has been suggested to increase intracellular availability of TG and indirectly stimulate the assembly and secretion of VLDL particles [12,107,109]. Upon being taken up by hepatocytes, FFA must be activated for further processing and utilization. Long-chain FAs undergo thioesterification to form fatty acyl-CoA, a process facilitated by the acyl-CoA synthetase long chain family member 1 (ACSL1, 3,4,5, and 6) [12]. Among them, ACSL5 overexpression in cells increases FFA uptake and flux into TG synthesis (Fig. 4) [104]. Additionally, ACSL5 mRNA expression is induced upon insulin stimulation and SREBP1c activation, suggesting this isoform contributes to the anabolic FA uptake cascade [12,104, 110]. FAs from different sources are processed differently in the liver (e.g., a portion of FFAs is immediately re-secreted in VLDL, while de novo FAs appear to enter a liver storage pool before secretion, etc.). And, the FA sources contributing to store hepatic TG are reflected in the fractional sources found in plasma VLDL-TG [60]. Many studies have shown that insulin- resistant conditions are accompanied by a substantial increase in MTP and apolipoprotein C3 (ApoCIII) expression and the production of hepatic VLDL-ApoB and VLDL-TG [2, 9,108,111]. Additionally, IR-associated PKC cascades can increase the binding of an insulin-sensitive 110-kDa RNA-binding protein (p110) to the 5ʹ UTR of the ApoB mRNA, increasing ApoB translation [2]. The increase in VLDL-TG production under IR conditions appears to result from decreased sensitivity to the inhibitory effects of insulin on VLDL secretion (Fig. 2) [2,107]. Supporting the notion that enhanced DNL contributes to increased VLDL-TG in MASLD, elevated ChREBP activity promotes the transcription of Mttp and other DNL-related genes, while LXRα may facilitate VLDL particle enlargement [2]. In addition, increased FFA flux to the liver and upregulation of SREBP1c and chronic stimulation of hepatic DNL in turn enhance intracellular availability of TG and drive VLDL assembly and secretion [107]. The IR-induced increase in VLDL-TG secretion may help to alleviate lipid overload; however, once hepatic fat content exceeds a certain level (e.g., intrahepatic TG >10%), VLDL secretion reaches a plateau [111-113]. Furthermore, overweight males with MASLD exhibit lower insulin-mediated suppression of VLDL particle size compared to those without MASLD [111]. Thus, under insulin-resistant conditions, increased VLDL secretion does not significantly contribute to the improvement of MASLD. On the contrary, the resulting increase in VLDL and changes in its size may have greater clinical relevance by raising the risk of CV diseases. Additionally, increased consumption of sugar-sweetened beverages (SSBs) and the resultant increase in DNL lead to a significant shift in low-density lipoprotein particle distribution toward smaller, more atherogenic particles associated with CV diseases [80,114].

CLINICAL IMPLICATIONS

Sustained weight loss remains a cornerstone in managing MASLD [1,5]. Evidence suggests that in adults with MASLD and overweight/obesity, dietary and behavioral therapy induced weight loss should aim at a sustained reduction of ≥5% to reduce liver fat, but at least 7%–10% to improve liver inflammation and ≥10% to improve fibrosis [3,33]. Recent studies demonstrated that diet therapy aimed at reducing total calorie and simple sugars achieved approximately 10% weight loss with a 75% reduction in hepatic TG, with normalization of liver fat in most individuals with high baseline levels. This reduction in hepatic TG was mainly driven by suppressed hepatic DNL, rather than changes in FFA or dietary fat flux [60]. DNL is an efficient biochemical pathway and plays a key role in explaining the metabolic benefits of low-carbohydrate diets, including improved insulin sensitivity, lipid profiles, and reduced hepatic steatosis in individuals with obesity, diabetes, or MASLD [76,115-117]. Because DNL is highly responsive to reductions in energy intake, this pathway represents the most highly modifiable biochemical contributor to MASLD [60]. Therefore, DNL is a valuable parameter for interpreting long-term metabolic data, even when weight loss is similar across diets.

Free sugar is defined as sugar added by the manufacturer, cook, or consumer, plus sugar found in honey, syrup, or fruit juice. It is often added as a sweetener to food and drink in the form of sucrose (table sugar) or high-fructose corn syrup (55% fructose), which are commonly used in SSBs [118]. In the United States, it is recommended to avoid added sugars before the age of 2 years and to limit added sugar to less than 10% of calorie intake per day starting at 2 years of age [119]. In Europe, recommendations similarly call for restricting free sugars—especially SSBs—to no more than 10% of total energy intake [120]. The UK’s Scientific Advisory Committee on Nutrition [121] recommend limiting free sugar intake to less than 5% of overall energy intake. Excessive fructose intake is defined as more than 50 grams per day, based on population studies suggesting that obesity rates are greater than 10% when mean intake increases beyond this amount [122,123].

Bariatric surgery offers substantial therapeutic benefits in patients with MASLD and obesity unresponsive to lifestyle changes as fully reviewed by Liu et al. [124]. Our bariatric surgery cohort also showed that approximately 25% weight reductions at 6 months after sleeve gastrectomy led to 70% reduction in MRI-PDFF [125]. Verrastro et al. [126] conducted the first large randomized clinical trial (the BRAVES study) including adults with biopsy-confirmed MASH and obesity. The primary outcome was MASH resolution without worsening fibrosis at 1 year. In the intention-to-treat analysis, significantly more patients in the Roux-en-Y gastric bypass (56%) and sleeve gastrectomy (57%) groups achieved the primary outcome compared to lifestyle modification alone (16%) [126]. In a long-term follow-up study of patients with MASH who underwent bariatric surgery, the resolution of MASH in liver samples was observed from 84% of patients 5 years later. The reduction of fibrosis is progressive, beginning during the first year and continuing through 5 years [4,127].

The clinical efficacy of pharmacologic agents for at-risk MASH, particularly their effects on MASH resolution and fibrosis inhibition, is only briefly summarized here. Readers are encouraged to consult recent literature for in-depth reviews [128]. Pharmacotherapy is rapidly evolving. Glucagon-like peptide-1 receptor agonist (GLP1RA) alone [129] or GLP1RA-based combination agents, including dual (e.g., tirzepatide and survodutide) and triple (e.g., retatrutide) agonists, now demonstrate histological improvements in MASH and fibrosis with weight loss approaching that of bariatric surgery [3,130-133]. Among insulin sensitizers, pioglitazone shows some effect on MASH but variable effect on fibrosis [134]. However, its combination with other agents is expected to yield beneficial effects [135]. The pan-PPAR agonist lanifibranor demonstrated significant MASH resolution and fibrosis improvement in clinical trials, though it was associated with weight gain [3,136]. Importantly, resmetirom, a selective thyroid hormone receptor-β agonist, became the first U.S. Food and Drug Administration-approved drug for non-cirrhotic MASH with F2–F3 fibrosis. It has shown histologic improvement in MASH and liver fibrosis, as well as improvement of non-invasive biomarkers, with an acceptable safety profile [3,4]. Resmetirom treatment increased hepatic fat oxidation and decreased LDL-cholesterol concentrations (–16.3%) without affecting body weight [137]. FGF21-based therapeutics, efruxifermin and pegozafermin showed promising efficacy in phase 2 trials, with significant improvements in fibrosis and MASH resolution compared to placebo [138-140].

In addition, drugs targeting the DNL pathways, such as citrate-isocitrate carrier inhibitors [75], ACLY inhibitors [13,141], ACC inhibitors (alone or in combination with DGAT inhibitors) [105,142,143], FASN inhibitors [144], and adiponectin receptor 1/2 agonists have shown preclinical and/or clinical promises in reducing fibrosis and MASH progression, raising high expectations for their future clinical efficacy [145]. Notably, bempedoic acid (BA) works as an ACLY inhibitor, which is clinically used as a plasma cholesterol-lowering drug. BA treatment also reduced TG and total cholesterol content in liver LDs. BA is a prodrug that requires activation by a liver-specific enzyme, very long-chain ACSL1. It effectively lowers LDL-cholesterol and reduces CV risk without muscle-related side effects. BA is a particularly important therapeutic agent, as it simultaneously inhibits cholesterol synthesis and DNL, making it potentially effective for treating MASH while also improving major CV outcomes [146-148].

Finally, even for drugs that have shown efficacy for MASH and fibrosis, longer-term efficacy data are still needed. Ultimately, adherence to lifestyle modification and weight loss must remain the cornerstone of treatment, and this cannot be overemphasized.

CONCLUSIONS

In MASLD, hepatic steatosis results from elevated FFA flux, enhanced DNL, and dietary fat. This occurs within a complex context involving various types of IR, substrate overflow, diverse combinations of insulin signaling pathways, and other contributing factors. In addition to weight reduction therapies, including lifestyle modification, bariatric surgery, and emerging therapeutics, targeting hepatic DNL—the most modifiable contributor—offers a promising strategy to reduce liver fat and improve insulin sensitivity in MASLD.

NOTES

CONFLICTS OF INTEREST

Dae Ho Lee has been an international editorial board of the Diabetes & Metabolism Journal since 2023. He was not involved in the review process of this article. Otherwise, there was no conflict of interest.

FUNDING

This study was supported by grants from Gachon University(GCU-202309080001 and GCU-20240928000, to Dae Ho Lee).

ACKNOWLEDGMENTS

None

Fig. 1.

Insulin signaling pathways regulating hepatic lipid and glucose metabolism in the normal insulin-sensitive state. Insulin signaling in the liver orchestrates glucose and lipid metabolism through INSR-mediated activation of IRS1/2, PI3K, AKT, and mTORC1. IRS2 primarily regulates gluconeogenesis via FoxO1 during fasting, while IRS1 promotes lipogenesis via SREBP1c in the fed state. AKT phosphorylates FoxO1, suppressing gluconeogenic genes, and activates mTORC1 to induce SREBP1c and lipogenic genes. While mTORC1 can drive DNL, AKT is essential; deletion of FoxO1 rescues glucose regulation but not lipogenesis in AKT-deficient livers. Atypical PKCs also contribute to insulin-induced lipogenesis. Pathways are represented as follows: orange- colored nodes and pathways indicate an increase by insulin action, while bright green ones indicate a decrease. Red lines indicate increased pathways, while blue lines represent decreased pathways, with arrows (→) marking progression pathways and blunt arrows (⊣) denoting inhibitory pathways. *Abbreviations: GP, glycogen phosphorylase.

Fig. 2.

Integrated hepatic insulin signaling and metabolic pathways under insulin resistant conditions such as MASLD. Hepatic IR, a hallmark of MASLD, is characterized by impaired insulin suppression of HGP, primarily driven by excess FFAs, DAGs, and ceramides. These lipid metabolites activate PKCs and inhibit INSR–IRS–AKT signaling. A key paradox in MASLD is selective hepatic IR: insulin fails to suppress gluconeogenesis but still promotes lipogenesis via mTORC1, SREBP-1c, and aPKCs. These pathways also feature multiple divergent insulin signaling components. DNL is highly active in MASLD, driven by hyperinsulinemia and carbohydrate-induced ChREBP activation. In IR states, a failure of insulin to facilitate degradation of ApoB as well as increased levels of FFAs and increased lipogenesis stimulate VLDL secretion. Nodes: lines and pathway regulation are presented similarly to the Fig. 1, except that pink-colored nodes and pathways indicate an increase by insulin action. *Abbreviations: GLUT2, glucose transporter 2; MRC, mitochondrial respiratory chain; mtProtein, mitochondrial ribosomal protein; NRF1, nuclear respiratory factor 1; SIRT1, sirtuin 1; and TFAM, mitochondrial transcription factor A.

Fig. 3.

Lipid flux related to hepatic TG accumulation in MASLD and changes in lipid flux through the liver following weight reduction by dietary intervention in obese subjects with low and high liver fat. (A) FA sources contributing to hepatic and VLDLTG: Donnelly et al. [67]. reported that in patients with MASLD, hepatic TG originates primarily from FFAs, followed by DNL and dietary fat. DNL was elevated even in the fasting state and showed no diurnal variation. The figure highlights the significant roles of peripheral lipolysis and hepatic DNL in MASLD pathogenesis and underscores the need to consider all four pathways—FFAs, DNL, dietary spillover, and chylomicron remnants—in understanding hepatic fat accumulation. Three major boxed pathways indicate the metabolic routes from FFAs to intrahepatic TG and VLDL-TG. (B) Summary of changes in the contribution of the three major lipid fluxes to hepatic VLDL-TG palmitate following dietary intervention in a study conducted by Lambert et al. [60]. In the study, overweight adults with H-LF (hepatic TG ≥ 5.6%) or L-LF (hepatic TG < 5.6%) received dietary counseling for six months. For additional information, please refer to the literature [60,67].

Fig. 4.

Hepatic DNL pathways. Elevated hepatic DNL, driven by carbohydrate overfeeding—especially fructose—plays a central role in MASLD pathogenesis by promoting TG accumulation. Fructose activates ChREBP, upregulates DNL enzymes, and increases palmitate synthesis, contributing significantly to VLDL-TG. Excessive fructose intake leads gut microbiota to produce acetate, which is converted to acetyl-CoA in the liver by ACSS2. In MASLD, DNL-derived FAs constitute a larger fraction of VLDLTG. The ER and Golgi microsome-associated lumenal lipid droplets (MALD) represent a metabolically active TG pool that serves as the precursor for hepatic VLDL production. *Abbreviations: ACSLs, long-chain fatty acyl-CoA synthetases; ACSS2, acetyl- CoA synthetase 2; AGPAT, 1-acylglycerol-3-phosphate acyltransferase; ALDOB, aldolase B; ApoB100, apolipoprotein B100; AQPs, aquaglyceroporins; DHAP, dihydroxyacetone phosphate; GA, glyceraldehyde; GA3P, glyceraldehyde-3-phosphate; GPD1/2, glycerol-3-phosphate dehydrogenase 1 and 2; LPA, lysophosphatidic acid; MCTs: monocarboxylate transporters; OAA, oxaloacetate; PA, phosphatidic acid; and TPI, triose topoisomerase.

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About this article

Truong XT, Lee DH. Hepatic Insulin Resistance and Steatosis in Metabolic Dysfunction-Associated Steatotic Liver Disease: New Insights into Mechanisms and Clinical Implications. Diabetes Metab J. 2025;49(5):964-986.

Received: Jul 23, 2025; Accepted: Aug 21, 2025

DOI: https://doi.org/10.4093/dmj.2025.0644.

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