Funding: H.Y.J. was supported by grants from the Novo Nordisk, the Sigrid Juselius and the EVO Foundations and the Finnish Government Research Fund. P.K.L. was supported by the Academy of Finland (350545), the Sigrid Jusélius Foundation, the Novo Nordisk Foundation (NNF22OC0074397), the Emil Aaltonen Foundation, the Finnish Medical Foundation, the Wilhelm and Else Stockmanns Foundation, the Early Career Investigator Fund of the University of Helsinki and the Finnish Government Research Fund.
Handling Editor: Stefano Romeo
ABSTRACT
Background and Aims
Steatotic liver disease (SLD) associated with insulin resistance (IR) and the metabolic syndrome (‘IR-SLD’) increases the risk of liver disease, type 2 diabetes and cardiovascular disease (CVD). SLD associated with the PNPLA3 I148M variant (‘PNPLA3-SLD’) also predisposes individuals to liver disease but protects against type 2 diabetes and CVD. Although in real life the two causes of SLD commonly co-exist, the opposite effects of ‘IR-SLD’ and ‘PNPLA3-SLD’ on CVD and liver disease suggest their pathogenesis differs.
Methods and Results
This review summarises human data comparing the effects of ‘IR-SLD’ and ‘PNPLA3-SLD’ on the human liver lipidome, hepatic handling of fatty acids, pathways of intrahepatocellular triglyceride synthesis, circulating lipids and lipoproteins and adipose tissue inflammation. We also discuss how steatosis in PNPLA3 I148M carriers leads to defects in mitochondrial function.
Summary
The pathogenesis of SLD caused by insulin resistance (‘IR-SLD’) and PNPLA3 I148M (‘PNPLA3-SLD’) differ.
‘IR-SLD’ predisposes both to CVD, type 2 diabetes and liver disease, while ‘PNPLA3-SLD’ only increases the risk of liver disease. The variant appears to be protective against CVD and type 2 diabetes.
The liver lipidome in ‘IR-SLD’ is characterised by excess saturated fatty acids, triglycerides and ceramides. This results in hypertriglyceridaemia and low HDL cholesterol in the circulation. In ‘PNPLA3-SLD’ excess liver triglycerides are polyunsaturated, and bioactive insulin resistance-inducing lipids are absent. Circulating lipids display an antiatherogenic pattern in ‘PNPLA3-SLD’, especially in insulin-resistant patients.
Substrate excess and increased rates of hepatic de novo lipogenesis (DNL) and adipose tissue lipolysis are features of ‘IR-SLD’ while there is no substrate excess in ‘PNPLA3-SLD’. The rate of adipose tissue lipolysis is unaltered and DNL is subnormal in ‘PNPLA3-SLD’.
Subnormal DNL in ‘PNPLA3-SLD’ reflects impaired TCA flux, which is attributable to an increased hepatic mitochondrial redox state, resulting from increased intrahepatic lipolysis and beta oxidation.
Abbreviations
BMI
body mass index
CVD
cardiovascular disease
DNL
de novo lipogenesis
HOMA-IR
homeostatic model assessment for insulin resistance
1 PNPLA3 I148M Protects Against Cardiovascular Disease CVD and Diabetes
As reviewed by Petta et al. in this journal [1], in 11 longitudinal studies and one case–control study examining the impact of the PNPLA3 I148M variant on the risk of CVD, none found a significant increase, although the variant consistently is associated with hepatic steatosis. In a recent Mendelian randomisation analysis including more than 500,000 subjects, the PNPLA3 variant had opposite effects on CVD and liver disease [2]. This is in marked contrast with abundant data documenting MASLD to be a major risk factor for both CVD and liver disease [3]. MASLD associated with the PNPLA3 I148M variant has also been shown to protect against the development of type 2 diabetes in two large prospective studies, one performed in Chinese [4] and one in Korea [5].
Since insulin resistance predisposes to both CVD and liver disease while the PNPLA3 I148 M variant has opposite effects on these outcomes, it seems plausible that the pathogenesis of SLD depends on the aetiology. For the purpose of this review, we here define insulin resistance-associated SLD (‘IR-SLD’) as a condition where the individual has insulin resistance/the metabolic syndrome and steatosis, and PNPLA3 I148M variant-associated steatosis as ‘PNPLA3-SLD’. Given that the worldwide prevalence of the insulin resistance/metabolic syndrome is at least 25% [6] and that of the PNPLA3 I148M variant is 30%–50% [7], the two conditions commonly co-exist. Furthermore, we have learned from studies such as the Dallas Heart study that adiposity amplifies the risk of steatosis conferred by PNPLA3 I148M [8] (Figure 1).
Liver fat content (measured by proton magnetic resonance spectroscopy) by body mass index and PNPLA3 I148M genotype (II, noncarrier; IM, heterozygous carrier; MM, homozygous carrier) in the Dallas Heart Study. The ability of the PNPLA3 I148M to increase liver fat content was amplified by obesity (p-value for interaction I148M × BMI on liver fat = 4 × 10−5). n denotes the number of subjects. Reproduced with permission from Stender et al. [8].
2 Why Does PNPLA3 I148M Cause Steatosis but Not CVD or Diabetes?
2.1 Unchanged Plasma Insulin in Epidemiological Studies
Insulin resistance is a hallmark of MASLD and a bona fide risk factor for CVD and diabetes [6]. In the Dallas Heart Study discovering the PNPLA3 I148M variant, insulin resistance, as measured by HOMA-IR (essentially the product of plasma insulin and glucose concentrations), was not influenced by the variant [7]. Dissociation between liver fat and insulin resistance in PNPLA3 I148M carriers has subsequently been confirmed in multiple studies in Europeans [9-15], Hispanic Americans and Africans [16], Taiwanese [17] and Chinese [18]. Lack of insulin resistance in PNPLA3 I148M carriers versus noncarriers has been confirmed by direct measurements of in vivo hepatic insulin sensitivity of the liver, muscle and adipose tissue using the euglycaemic hyperinsulinaemic clamp technique combined with glucose tracers [9].
2.2 No Increase in Insulin Resistance-Inducing Bioactive Lipids in the Human Liver
Lack of hepatic insulin resistance in ‘PNPLA3-SLD’ raised the possibility that insulin resistance-inducing lipids in the liver could differ between ‘IR-SLD’ and ‘PNPLA3-SLD’. In the hitherto largest study examining this possibility in the human liver, 125 liver biopsies were collected and divided into two equally sized groups based on median HOMA-IR which were called ‘High HOMA-IR’ and ‘Low HOMA-IR’. In addition, the subjects were divided into two groups based on PNPLA3 I148M carrier status entitled ‘PNPLA3 I148M carriers’ and ‘PNPLA3 noncarriers’ [19]. All groups were similar with respect to age, gender and obesity. The amount of liver fat and the prevalence of MASH were similarly increased in the ‘High HOMA-IR’ and ‘PNPLA3 I148M carrier’ groups as compared to their respective control groups. In the ‘High HOMA-IR’ group, the liver lipidome was characterised by an increase in saturated fatty acids, triglycerides (Figure 2) and insulin resistance-inducing ceramides (Figure 3, panel on the right), compared to the ‘Low HOMA-IR’ group. Ceramides can be synthesised de novo, or derived from sphingomyelin hydrolysis or the salvage pathway (Figure 3, panel on the left). In ‘IR-SLD’, the increase in ceramide synthesis is a consequence of de novo synthesis. This pathway produces dihydroceramides with distinct acyl chain lengths [21]. Ablation of the gene (Cers6) encoding ceramide synthase 6 (CERS6) [22, 23] or the gene encoding ceramide synthase 5 (CER5) which produces C16:0 ceramides resolves high-fat diet-induced obesity, glucose intolerance and insulin resistance in mice. Tissue-specific depletion of Degs1 encoding DES1, which converts dihydroceramides to ceramides, from the whole animal, liver or adipose tissue resolves hepatic steatosis and insulin resistance in mice caused by obesogenic diets [21]. Ceramides are degraded by ceramidase, which is activated via adiponectin binding to its receptors [24]. Adiponectin deficiency may therefore contribute to ceramide accumulation in ‘IR-SLD’ [19, 24]. Circulating levels of ceramides have predicted cardiovascular disease and diabetes independent of classic risk factor in multiple longitudinal studies (see Chaurasia et al. [25] for review).
Differences in hepatic triglycerides between ‘IR-SLD’ and ‘PNPLA3-SLD’ in the human liver based on analysis of 125 liver biopsies. The heatmaps depict the ratio of triglycerides as a function of the number of double bonds (X-axis) and chain length (Y-axis). The red colour denotes an increase and blue a decrease. *p < 0.05, **p < 0.01, ***p < 0.001. On the left, the ratio of absolute triglycerides between subjects with a HOMA-IR above median (3.19) (‘IR-SLD’) are compared to those with a HOMA-IR below or equal to the median HOMA-IR. The subjects in both HOMA-IR groups were similar to age, gender, body mass index and PNPLA3 genotype. On the right carriers of the PNPLA3 I148M variant ('PNPLA3-SLD') are compared to noncarriers. These groups were similar with respect to age, gender, body mass index and HOMA-IR. Reproduced with permission from Luukkonen et al. [19].
Differences in ceramides and dihydroceramides between ‘IR-SLD’ and ‘PNPLA3-SLD’. The panel on the left depicts pathways of ceramide synthesis and degradation and concentrations of dihydroceramides (DihydroCer), hexosylceramides (HexCer) and sphingomyelins (SM) in ‘IR-SLD’ [20]. The panel on the right shows concentrations of ceramides with a d18:1 and d18:2 sphingoid base in ‘IR-SLD’ (third and fourth column from right side of the panel) and ‘PNPLA3-SLD’ (first and second column from the right side of the panel). *p < 0.05, **p < 0.01, ***p < 0.001. The red colour denotes an increase and blue a decrease relative to the respective control groups. Modified with permission from Luukkonen et al. [19].
In ‘PNPLA3 I148M carriers’, the liver was enriched with polyunsaturated triglycerides (Figure 2) with no changes in ceramides (Figure 3) or other lipotoxic intermediates [19]. These changes in the lipidome of PNPLA3 I148M carriers as compared to noncarriers also characterises adipose tissue, which expresses more PNPLA3 protein in humans than the liver [26]. This change in adipose tissue composition is an interesting exception to the rule that adipose tissue reflects dietary intake [27]. Kinetic studies comparing acute handling of a mixed meal labelled with a polyunsaturated fatty acid linoleate (C18:2) and a saturated fatty acid palmitate (16:0) showed the human liver to retain the polyunsaturated linoleate relative to the saturated palmitate (Figure 4). Consistent with these data, VLDL particles in ‘PNPLA3 I148M carriers’ were deficient in polyunsaturated triglycerides [28].
The PNPLA3 I148M causes retention of polyunsaturated fatty acids compared to saturated fatty acids in the human liver. This leads to a deficiency of polyunsaturated fatty acids in VLDL-triglycerides (TG). This study compared the handling of labelled polyunsaturated fatty acids to saturated fatty acids administered in a mixed meal between 14 subjects lacking the PNPLA3 I148M variant and 12 subjects homozygous for the variant. Reproduced with permission from Luukkonen et al. [28].
Regarding the mechanism underlying the accumulation of polyunsaturated triglycerides, Johnson SM et al. recently showed in ATGL −/− HeLa cells that wild-type PNPLA3 protein degrades polyunsaturated triglycerides in an ATGL-independent manner [29]. The ability of PNPLA3 to hydrolyse polyunsaturated triglycerides was abolished when either the catalytic serine in PNPLA3 was mutated into alanine or the PNPLA3 I148M mutation was introduced [29]. Further studies using primary mouse hepatocytes lacking PNPLA3 showed PNPLA3 to facilitate the flux of polyunsaturated fatty acids from triglycerides to secreted phospholipids. Expression of the PNPLA3 I148M variant in mouse hepatocytes was shown to impair polyunsaturated fatty acid mobilisation from triglycerides to phospholipids [29]. In keeping with these data, Mitsche et al. found very long-chain polyunsaturated fatty acids to be enriched in triglycerides and depleted in phospholipids in hepatic lipid droplets in mice where PNPLA3 had been inactivated by substituting the catalytic serine with alanine [30]. However, it is important to emphasise that the mechanism underlying steatosis may be distinct from this alteration in lipid composition [31].
2.3 No Inflammation in Adipose Tissue
Inflammation in adipose tissue is a well-established feature of insulin resistance in both mice and humans [32]. Its features include accumulation of ‘crown-like structures’, that is, macrophages surrounding necrotic adipocytes [33, 34] and ceramides [20, 34], upregulation of pro-inflammatory markers such as MCP-1, TNFalpha and IL-6 [35] and adiponectin deficiency [36]. These changes are observed in subjects with MASLD compared to age-, weight- and gender-matched subjects without MASLD [34]. Several adipokines have been causally related to both steatosis, cardiovascular disease and diabetes [37, 38]. However, adipose tissue inflammation, determined from gene expression of the macrophage marker CD-68, MCP-1, TNFalpha and adiponectin, in biopsies from 92 subjects, does not seem to characterise the PNPLA3 I148M variant carriers as compared to noncarriers [39].
2.4 Antiatherogenic Changes in Serum Lipids and Lipoproteins
In ‘IR-SLD’, impaired inhibition of hepatic VLDL production by insulin leads to atherogenic dyslipidaemia, which is characterised by hypertriglyceridaemia, a low concentration of HDL cholesterol and predominance of small dense LDL particles [40]. Thus, steatosis and hypertriglyceridaemia are closely linked in ‘IR-SLD’. In contrast, steatosis caused by the PNPLA3 I148M variant is not characterised by hypertriglyceridaemia [14, 41, 42] or a low HDL cholesterol concentration [14, 42]. In contrast, the PNPLA3 I148M carriers compared to noncarriers are characterised by antiatherogenic changes, the magnitude of which is particularly strong in carriers who are insulin resistant [14] (Figure 5). Consistently, a meta-analysis of 63 studies including 81 003 subjects demonstrated that serum triglycerides were significantly lower in PNPLA3 carriers than in noncarriers [41]. The meta-analysis confirmed that triglyceride concentrations were especially reduced in PNPLA3 I148M carriers who were obese or had type 2 diabetes [41].
Impact of ‘IR-SLD’ and ‘PNPLA3-SLD’ on concentrations of circulating lipoproteins. The panel on the left compares subjects with HOMA-IR above the median to those with HOMA-IR below or equal to the median. All subjects were lacking the PNPLA3 I148M variant. The middle panel compares heterozygous to homozygous carriers of the PNPLA I148M variant and the panel on the right homozygous PNPLA I148M carriers to those lacking the variant. Colour coding represents the fold change in mean concentrations between the groups. The brighter the red colour, the greater the increase; the darker the blue colour, the greater the decrease in the lipoprotein concentration between the groups. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; L, large; LDL, low-density lipoprotein; M, medium; S, small; VLDL, very low-density lipoprotein; XL, very large; XXL, extremely large. Reproduced with permission from Luukkonen and Qadri et al. [14].
2.5 No Increase in Coagulation Factor Activities
Hepatocytes or sinusoidal endothelial cells are major sites of production of coagulation factors. Coagulation factor activities such as FVII [43-45], FVIII [43, 45-47], FIX [47], FXI [47], FXII [47], FXIII [43], von Willebrand factor [43, 48] and fibrinogen [43, 45, 48] are all increased, and prothrombin time and activated partial thromboplastin time are shortened [43] in obese insulin-resistant subjects with MASLD compared to leaner, more insulin-sensitive subjects. In carriers of the PNPLA3 I148M compared to age-, gender- and BMI-matched noncarriers, activities of coagulation factors FVII, FVIII, FIX, FXI, FXII and FXIII, as well as prothrombin time, activated partial thromboplastin time and activities of fibrinogen and VWF are unchanged [43]. These data imply that higher coagulation factor activities are consequences of insulin resistance rather than hepatic steatosis and that the PNPLA3 I148M does not increase the risk of thrombosis.
3 Lack of Substrate Excess, Unchanged Adipose Tissue Lipolysis and Low DNL in ‘PNPLA3-SLD’
Insulin resistance is characterised by circulating substrate surplus [42] including glucose, glycolytic intermediates, amino acids [49, 50], free fatty acids [51, 52] and lipoproteins [53]. Hepatic DNL consumes excess products of glycolysis and amino acid catabolism and is increased in ‘IR-SLD’ [42, 54]. Free fatty acids reflect accelerated adipose tissue lipolysis in ‘IR-SLD’ [42, 51, 52]. In ‘PNPLA3-SLD’, there is no insulin resistance and no substrate surplus [42]. The rate of adipose tissue lipolysis is unchanged, and hepatic DNL is subnormal in PNPLA3-I148M carriers compared to noncarriers [42, 55].
Regarding the subnormal rate of DNL in ‘PNPLA3-SLD’, the main regulators of DNL are glucose, insulin and substrate availability originating from the tricarboxylic acid (TCA) cycle (citrate). Since plasma glucose and insulin concentrations are unchanged in ‘PNPLA3-SLD’, the low DNL may reflect impaired production of citrate by the TCA cycle (vide infra). These normal or subnormal rates of fatty acid synthesis in the liver in ‘PNPLA3-SLD’ thus markedly contrast with the accelerated rates of these pathways in ‘IR-SLD’.
4 How Does ‘PNPLA3-SLD’ Influence Mitochondrial Function? (Figure 6)
The effect of PNPLA3 I148M in the human liver. After an overnight fast, homozygous PNPLA3 I148M carriers are characterised by increased intrahepatic lipolysis, resulting in increased fatty acid supply in hepatic mitochondria, where they undergo beta oxidation into acetyl CoA molecules. Beta oxidation increases the hepatic mitochondrial redox state (i.e., [NADH]/[NAD+] ratio), which in turn inhibits the mitochondrial TCA cycle, favouring the entry of mitochondrial acetyl CoA into the ketogenic pathway. Consistently, exogenous fatty acids from a mixed meal are also preferentially channelled towards ketogenesis in I148M carriers. The TCA cycle is an important regulator of hepatic de novo lipogenesis (DNL), as it provides the rate-limiting substrate, citrate. Consistent with a significant decrease in the mitochondrial TCA cycle flux that provides citrate for DNL, PNPLA3 I148M carriers have markedly decreased rates of hepatic DNL. Since hepatic DNL and fatty acid oxidation are reciprocally regulated, this decrease in DNL could further stimulate the entry of fatty acids into mitochondria, thereby forming a vicious circle leading to mitochondrial dysfunction. Reproduced with permission from Luukkonen et al. [56].
The TCA cycle in hepatic mitochondria plays a key role in DNL by converting sugar- and amino acid-derived acetyl CoA into citrate, which is the rate-limiting substrate in DNL [57]. Hepatic mitochondria are also the site of the breakdown of fatty acids via beta oxidation into acetyl CoA molecules [58]. These acetyl CoA molecules can either undergo terminal oxidation in the mitochondrial TCA cycle or, in the case of a defective TCA cycle, enter the ketogenic pathway (Figure 6) [58]. Thus, investigation of hepatic DNL, mitochondrial TCA cycle flux and ketogenesis can provide important insights into mitochondrial (dys)function in the human liver.
After an overnight fast, homozygous PNPLA3 I148M carriers are characterised by markedly lower rates of hepatic DNL than noncarriers [55, 56]. Since DNL is dependent on citrate supply from the mitochondrial TCA cycle and since other main regulators of DNL, such as glucose, insulin and substrate availability, are unchanged in I148M carriers (vide supra), the decrease in DNL in I148M carriers could be explained by the decreased hepatic mitochondrial TCA cycle flux. Indeed, PNPLA3 I148M carriers have markedly lower rates of hepatic mitochondrial TCA cycle flux compared to noncarriers, as determined by a triple stable isotope PINTA method [56].
It is well established that hepatic DNL and fatty acid oxidation/ketogenesis are reciprocally regulated [58]. Thus, the decrease in DNL in I148M carriers might be expected to lead to a reciprocal increase in hepatic fatty acid oxidation/ketogenesis. Indeed, PNPLA3 I148M carriers are characterised by increased hepatic ketogenesis from both endogenous and exogenous (i.e., dietary) fatty acids [56]. The PNPLA3 I148M carriers also have a marked increase in plasma beta-hydroxybutyrate/acetoacetate ratio, a bona fide marker of hepatic mitochondrial redox state (i.e., [NADH]/[NAD+] ratio) [59] that inhibits the TCA cycle [59, 60] and associates with the severity of MASLD [42, 61]. This is consistent with increased hepatic mitochondrial reductive stress secondary to excessive beta oxidation of fatty acids.
Where do the excess fatty acids undergoing beta oxidation in PNPLA3 I148M originate from? While it is clear that the PNPLA3 I148M causes hepatic lipid accumulation during hyperinsulinaemic–hyperglycaemic states that sustain its gene and protein expression, it has also been repeatedly shown to associate with a greater loss of intrahepatic triglycerides (i.e., intrahepatic lipolysis) during relatively hypoinsulinaemic–hypoglycaemic states, such as a ketogenic diet [56, 62].
Collectively, these results show that homozygous PNPLA3 I148M carriers have marked alterations in intrahepatic lipid processing that include (1) increased intrahepatic lipolysis during hypoenergetic states, (2) excessive beta oxidation of fatty acids, (3) mitochondrial reductive stress and (4) hepatic mitochondrial dysfunction, a hallmark of progressive liver disease [63-65]. The hepatic mitochondrial dysfunction in I148M variant carriers is manifested as markedly reduced hepatic DNL and preferential partitioning of carbons towards ketogenesis.
5 Conclusions
The pathogenesis of ‘IR-SLD’ and ‘PNPLA3-SLD’ is summarised in Figure 7. ‘IR-SLD’ is essentially a condition that reflects the consequences of overeating and physical inactivity. Tissues are bathing in substrate excess, including sugars, glycolytic intermediates, amino acids and fatty acids. This surplus accelerates lipolysis and de novo lipogenesis. DNL produces exclusively saturated fatty acids [68], which may, in addition to ingestion of excess saturated fat [69-72] explain why triglycerides in the liver contain an excess of saturated fatty acids. Saturated fat induces the formation of ceramides, which induce hepatic insulin resistance and could explain why subjects with ‘IR-SLD’ are at increased risk of both diabetes, CVD and liver disease [25]. ‘PNPLA3-SLD’ lacks all features of insulin resistance and thus, there is no adipose tissue inflammation, no hyperinsulinaemia or hyperglycaemia, no changes in coagulation factor activities and no increase in the risk of diabetes or CVD. Indeed, the PNPLA3-I148M remodels hepatic triglycerides to become enriched with polyunsaturated fatty acids, and there is consequently no change in the rate of ceramide synthesis [19]. Lack of adipose tissue inflammation maintains serum adiponectin levels, and ceramide degradation remains normal. Serum lipids and lipoproteins display an opposite change to those in ‘IR-SLD’ [14].
Summary of the pathophysiology of ‘IR-SLD’ (panel on the left) and ‘PNPLA3-SLD’ (panel on the right). In ‘IR-SLD’, overeating and inactivity cause insulin resistance in adipose tissue, leading to accelerated lipolysis and a deficiency of circulating adiponectin. Both of the latter increase liver fat content [36, 66]. Circulating substrate excess and possibly hyperinsulinaemia stimulate de novo lipogenesis [54]. Liver TGs are predominantly saturated in ‘IR-SLD’ [19, 67]. Insulin resistance is accompanied by hyperglycaemia, hyperinsulinaemia, increased production of coagulation factors, and atherogenic dyslipidaemia, which increases the risk of atherothrombotic vascular disease. AT, adipose tissue; DNL, de novo lipogenesis; fS, fasting serum; HDL, high-density lipoprotein; N, normal; PUFA, polyunsaturated fatty acid; SAFA, saturated fatty acid; TG, triglyceride. In ‘PNPLA3-SLD’, there are no features of insulin resistance. The PNPLA3 I148M variant acts in the liver to induce both steatosis [31] and a change in the composition of liver triglycerides in a polyunsaturated direction [19, 29].
Although ‘IR-SLD’ and ‘PNPLA3-SLD’ differ markedly in their pathogenesis, both conditions frequently coexist, and the presence of obesity/insulin resistance markedly accentuates the actions of the PNPLA3-I148M variant [8]. However, subjects who are carrying the PNPLA3 I148M variant and are insulin resistant are more likely to develop severe liver disease and less likely to develop diabetes and cardiovascular disease [1]. The latter has obvious implications for the follow-up of SLD patients in the clinic. In addition, the differences in pathogenesis may also have implications for the treatment of liver disease. Patients with the PNPLA3 I148M can be predicted not to benefit from inhibitors of acetyl-CoA carboxylase or fatty acid synthase, as inhibition of an already subnormal DNL can be predicted to increase CPT-1, beta oxidation of fatty acids and the mitochondrial redox state, leading to inhibition of mitochondrial oxidation [56].
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