How does ectopic fat accumulation in liver (hepatic steatosis) cause insulin resistance and affect cardio-metabolic risk factors?

Although obesity is strongly associated with hepatic steatosis, body fat accumulation is not a sine qua non for developing NAFLD. Patients with lipodystrophy commonly develop hepatic steatosis, strongly suggesting that it is not body fat mass per se that is important, but it is adipose tissue function that is key in the pathogenesis of NAFLD⁽ Reference Jornayvaz and Shulman ^{16 )}. Specifically, increased fatty acid fluxes from the adipose tissue pool throughout the day, increase the availability of long chain fatty acyl-coenzyme A (acyl-CoA) in the liver, particularly in physically inactive individuals⁽ Reference Byrne ^{11 )}. It is likely that fatty acid fluxes from the adipose tissue pool to the liver will be decreased if individuals are engaging in high levels of physical activity, since high levels of aerobic physical activity increase the demand for skeletal and cardiac muscle long chain fatty acid (LCFA) oxidation and for generation of ATP to enable muscle contraction. High levels of muscle mitochondrial β oxidation of LCFA may provide an important buffer to protect the liver from an excessive supply of LCFA that may overwhelm the liver's capacity to export fatty acids (as VLDL, or conjugated to cholesterol derivatives in bile).

With an increasingly sedentary population, increased concentrations of postprandial fatty acids promote lipid synthesis. Within adipose tissue and liver parenchymal cells, fatty acyl-CoA are esterified with glycerol 3-phosphate (derived from glycolysis) to form monoacylglycerol, diacylglycerol (DAG) and TAG (Fig. 1(a)). In insulin sensitive tissues, lipid synthesis may increase production of intermediates such as DAG, di-palmitoyl phosphatidic acid (PA) and other lipid products such as ceramides. Increased production of these lipid products may be very important in causing ‘resistance’ in the hepatic insulin signalling pathway⁽ Reference Samuel and Shulman ^{17 )} (Fig. 1(b)). In liver, ceramides are another lipid product which utilise LCFA⁽ Reference Watt, Barnett and Bruce ^{18 )}. Ceramides are a family of waxy lipid molecules that are found in high concentrations within cell membranes and are one of the component molecules within the lipid bilayer. Ceramides can accumulate in cells via three main routes: the hydrolysis of the membrane phospholipid sphingomyelin, which is coordinated by the enzyme sphingomyelinase; de novo synthesis from LCFA such as palmitate and serine; and a salvage pathway that utilises sphingosine and forms ceramide⁽ Reference Yang, Badeanlou and Bielawski ^{19 ,} Reference Kolesnick and Kronke ^{20 )}. Although in the past it was thought that ceramide was simply a structural molecule, it is now evident that increases in membrane ceramide have the potential to insulin resistance (see review⁽ Reference Lipina and Hundal ^{21 )}; Fig. 2).

Fig. 1.

(colour online) Synthesis of lipid intermediates and TAG in adipose (a) and liver (b) tissue: links with insulin resistance and inflammation. Adipose tissue ‘inflammation’, generation of long chain fatty acids (LCFA) and the potential role of lipid intermediates (ceramide, di-palmitoyl phosphatidic (di-PPA) and diacylglycerol (DAG)) in hepatic insulin resistance. Liver (b): (1) Acyl-coenzyme A (acyl-CoA): glycerol 3 phosphate acyl transferase (GPAT) catalyses the formation of lysophatidic acid (LPA) the rate limiting step in TAG synthesis. (2) Comparative Gene Identification-58 (CGI-58) is an activator of adipose TAG lipase (ATGL) that converts TAG to DAG. (3) Membrane-associated DAG activates protein kinase Cε (PKCε) membrane translocation to inhibit the insulin receptor kinase. DAG can also be released from membrane lipids by the action of phospholipase C.

Fig. 2.

(colour online) Mechanisms linking hepatic long chain fatty acids with insulin resistance. An increase in caveolar ceramide content causes activation of atypical protein kinase C isoforms (aPKCλ/ζ) and promotes the association of aPKCλ/ζ and Akt (Akt repressed state)⁽ Reference McKeigue, Shah and Marmot ^{1 )}. (Atypical PKCλ, along with aPKCζ (PRKCz), belong to the atypical sub-group of the PKC family (aPKCs).) The formation of intracellular ceramide from serine/palmitate or from sphingosine, leads to the direct activation of protein phosphatase 2, causing the dephosphorylation and inactivation of Akt with consequent decreased insulin signalling. (NB: aPKCλ/ι (also known as PRKCi) is referred to as aPKCλ and aPKC zeta (also known as PRKCz) is referred to as aPKCζ).

Increased concentrations of fatty acids that are not utilised in oxidative metabolism pathways for energy production during catabolism (e.g. skeletal muscle activity in the fasted state) are used in lipogenesis. De novo lipogenesis generates TAG and phospholipids from glycerol 3-phosphate and LCFA (see Fig. 2). Acyl-CoA:glycerol-sn-3-phosphate acyltransferase catalyses the acylation of sn-glycerol-3-phosphate with acyl-CoA to generate lysophosphatidic acid (LPA). LPA is thought to be the rate-controlling step in TAG synthesis. Subsequently, the enzymes acyl-CoA:1-acylglycerol-sn-3-phosphate acyltransferase, PA phosphatase and DAG:acyl-CoA acyltransferase generate PA, DAG and TAG. In the liver, TAG is either deposited in intracellular vacuoles or exported in VLDL particles. LPA and PA require translocation through the cytosol for TAG synthesis at the endoplasmic reticulum if they are not synthesised in the endoplasmic reticulum⁽ Reference Coleman and Lee ^{22 )}. DAG can be hydrolysed to monoacylglycerol by hormone-sensitive lipase and subsequently to glycerol by monoglyceride lipase. These reactions release fatty acids. Glycerol can then be used as a substrate for gluconeogenesis (Fig. 1) but cannot be re-esterified with LCFA to form adipose tissue TAG.

The production of DAG has been suggested as a cause of hepatic insulin resistance and the conversion from TAG to DAG is mediated by adipose TAG lipase. Comparative Gene Identification-58 is an activator of adipose TAG lipase and DAG activates protein kinase Cε membrane translocation to inhibit the insulin receptor kinase and decrease insulin signalling⁽ Reference Cantley, Yoshimura and Camporez ^{23 )}. The importance of intracellular compartmental localisation of DAG in causing lipotoxicity and hepatic insulin resistance has recently been recognised. For example, analysis of the cellular localisation of DAG revealed that DAG increased in the membrane fraction of high fat-fed mice, leading to protein kinase Cε activation and hepatic insulin resistance. In Comparative Gene Identification-58 anti-sense oligonucleotide knock down-treated mice, DAG increased in lipid droplets or lipid-associated endoplasmic reticulum, rather than in the membrane. This location of accumulation of DAG prevented protein kinase Cε translocation to the plasma membrane and thereby the induction of insulin resistance⁽ Reference Cantley, Yoshimura and Camporez ^{23 )}. Taken together, these results explain the disassociation of hepatic steatosis and DAG accumulation from hepatic insulin resistance in Comparative Gene Identification-58 antisense oligonucleotide-treated mice. Phospholipase C can release DAG from membrane lipids. Presently, whether DAG derived from another pathway (i.e. the phospholipase C pathway and from lipid droplets) can lead to protein kinase Cε activation and hepatic insulin resistance remains to be determined⁽ Reference Jornayvaz and Shulman ^{16 )}.

It has been known for a long time that PPARγ agonists are effective drugs for lowering plasma glucose concentrations and PPARγ is a key transcription factor involved in adipogenesis. The PPARγ agonist or thiazolidienedione group of drugs, mainly act in adipose tissue (where PPARγ expression is high). Thiazolidienediones increase GLUT4 translocation to the plasma membrane and thereby facilitate adipose tissue glucose uptake. This class of compound has shown to be effective in some patients with NAFLD⁽ Reference Bhatia, Curzen and Calder ^{15 )}, although it is highly unlikely that this class of drug will ever be licensed for NAFLD because of unacceptable side effects such as increased fracture risk⁽ Reference Montagnani and Gonnelli ^{24 )}, oedema and cardiac failure⁽ Reference Loke, Kwok and Singh ^{25 )}, weight gain and possibly an increase of bladder cancer⁽ Reference Bosetti, Rosato and Buniato ^{26 )}.

Recently, there have been further studies that have added insight as to the role of PPARγ in affecting insulin signalling. PPARγ activity is regulated by nuclear co-repressor and the hepatokine fibroblast growth factor 21. It has been shown that nuclear co-repressor prevents PPARγ activity by facilitating cyclin-dependent kinase 5 binding⁽ Reference Li, Fan and Xu ^{27 )}. Cyclin-dependent kinase 5 phosphorylates PPARγ Ser²⁷³ thereby blocking PPARγ transcriptional activity. Thus nuclear co-repressor may play a role in causing insulin resistance.

The Ser/Thr protein kinase mammalian target of rapamycin (mTOR) is also important in regulating insulin signalling. In combination with other molecules, mTOR forms two complexes; mTOR complex 1 (mTORC1) and mTORC2. mTORC1 contains the protein regulatory associated protein of TOR, whereas mTORC2 contains the protein rictor (rapamycin insensitive companion of TOR). Both complexes are capable of regulating insulin sensitivity (Fig. 3). AMP-activated protein kinase negatively regulates mTORC1 in response to low cellular energy AMP-activated protein kinase and genetically modified mice with defective mTOR signalling in the liver are glucose intolerant, hyperglycaemic, hyperinsulinaemic and display decreased glycogen content indicating that hepatic mTOR plays a major role in glucose homoeostasis⁽ Reference Cornu, Albert and Hall ^{28 )}. AMP-activated protein kinase inhibits mTORC1 signalling and thereby prevents the negative feedback loop that down regulates insulin signalling (Fig. 3). Overexpression of a dominant negative form of regulatory associated protein of TOR in the liver of insulin-resistant mice improved glucose tolerance and restored insulin sensitivity by reducing the negative feedback loop⁽ Reference Koketsu, Sakoda and Fujishiro ^{29 )}. mTORC1 inhibits insulin signalling via the effects of its substrate S6 kinase. These findings suggest that inappropriate mTORC1 activity in the liver contributes to glucose intolerance, at least in part. In contrast, mTORC2 has a positive effect on glucose uptake and glucose tolerance⁽ Reference Laplante and Sabatini ^{30 )}. Lipid synthesis in the liver may also cause insulin resistance via disruption of mTORC2 signalling⁽ Reference Zhang, Wendel and Keogh ^{31 )}. These authors showed that overexpression of acyl-CoA:glycerol 3 phosphate acyl transferase that catalyses the formation of LPA and which is the rate limiting step in TAG synthesis (Fig. 1), suppresses insulin signalling and insulin-mediated suppression of hepatic gluconeogenesis. They showed that mTOR-rictor binding was decreased resulting in diminished mTORC2 phosphorylation of Akt-Ser⁴⁷³ and Akt-Thr³⁰⁸. To determine which lipid intermediate was responsible for inactivating mTORC2, the investigators overexpressed glycerol-sn-3-phosphate acyltransferase 1, acyl-CoA: 1-acylglycerol-sn-3-phosphate acyltransferase or lipin to increase the cellular content of LPA, PA, or DAG, respectively. The inhibition of mTOR/rictor binding and mTORC2 activity coincided with the levels of PA and DAG species that contained 16:0, the preferred substrate of glycerol-sn-3-phosphate acyltransferase 1. Furthermore, di-16:0-PA strongly inhibited mTORC2 activity and disassociated mTOR/rictor in vitro (Fig. 2). Taken together, these data reveal a signalling pathway by which PA synthesised via the glycerol-3-phosphate pathway inhibits mTORC2 activity by decreasing the association of rictor and mTOR, thereby down-regulating insulin action and potentially causing insulin resistance⁽ Reference Zhang, Wendel and Keogh ^{31 )}. In support of these findings, to assess the role of hepatic mTORC2, liver-specific rictor knockout mice have been generated⁽ Reference Hagiwara, Cornu and Cybulski ^{32 )}. Chow fed liver-specific rictor knockout mice displayed loss of Akt-Ser⁴⁷³ phosphorylation and reduced glucokinase and sterol regulatory response element binding protein 1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis. These liver-specific defects resulted in systemic hyperglycaemia, hyperinsulinaemia and hypolipidaemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates both hepatic glucose and lipid metabolism via insulin-induced Akt signalling to control whole-body metabolic homoeostasis. Thus, these data demonstrate a potential important link between nutrient excess, TAG synthesis and hepatic insulin resistance (Fig. 3).

Fig. 3.

(colour online) The effects of exercise/physical activity and nutrient excess on mammalian target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2) regulation of insulin sensitivity. The Ser/Thr protein kinase mammalian target of rapamycin (mTOR) is also important in regulating insulin signalling. In combination with other molecules, mTOR forms two complexes; mTORC1 and mTORC2. mTORC1 contains the protein raptor (regulatory associated protein of TOR), whereas mTORC2 contains the protein rictor (rapamycin insensitive companion of TOR). Both complexes are capable of regulating insulin sensitivity. DAG, diacylglycerol; IRS, insulin receptor substrate; PA, phosphatidic acid.

Ectopic fat, hepatic steatosis and non-alcoholic fatty liver disease progression

NAFLD is not a single disease and NAFLD describes a spectrum of fat-related liver conditions. The spectrum ranges from simple fatty liver (steatosis) to more severe steatosis coupled with marked inflammation, termed non-alcoholic steatohepatitis which is often progressive with development of fibrosis (40–50%) liver cirrhosis (15–17%), liver failure (3%) and potentially hepatocellular carcinoma. Current estimates are that 40% of people with NAFLD develop non-alcoholic steatohepatitis⁽ Reference Ekstedt, Franzen and Mathiesen ^{33 )} and there is increased incidence of coronary (10·8%), cerebrovascular (37·3%) and peripheral (24·5%) vascular disease in individuals with NAFLD⁽ Reference Targher, Bertolini and Padovani ^{34 )}.

NAFLD is defined by the accumulation of liver fat >5% per liver weight, in the presence of <10 g daily alcohol consumption. The characteristic histology of NAFLD resembles that of alcohol-induced liver injury, but occurs in people who consume minimal alcohol. The reported prevalence of NAFLD is 2–44% in the general European population (including obese children) and 42·6–69·5% in people with type 2 diabetes⁽ Reference Blachier, Leleu and Peck-Radosavljevic ^{35 )}. In routine clinical practice, most cases of fatty liver disease are attributable to alcohol excess; however, fatty liver disease can also occur in association with a wide range of toxins, drugs and diseases, such as morbid obesity, cachexia, type 2 diabetes, hyperlipidaemia and after jejunoileal bypass surgery. As important risk factors for NAFLD such as obesity and type 2 diabetes are increasing in prevalence, it is likely that there will be a marked increase in prevalent NAFLD. For people who are free from disease initially, the development of NAFLD will have important implications not just for the patients themselves but also for health care providers, responsible for patients with chronic liver disease, type 2 diabetes and CVD⁽ Reference Ahmed, Byrne, Byrne and Wild ^{36 )}.

Energy expenditure, physical activity, ectopic fat and non-alcoholic fatty liver disease

The benefits of high levels of physical activity in association with lower risk of mortality have been known for over 50 years⁽ Reference Morris, Heady and Raffle ^{37 )}. Over 20 years ago it was shown that a one s.d. increase in free-living energy expenditure (1200·808 kJ/d (287 kcal/d)) was associated with a 32% lower risk of mortality after adjusting for age, sex, race, study site, weight, height, percentage body fat and sleep duration⁽ Reference Manini, Everhart and Patel ^{38 )}. Changes in lifestyle that promote weight loss, or increase physical activity, to induce a net negative energy balance, can be very effective in treating NAFLD⁽ Reference Harrison and Day ^{39 ,} Reference Rodriguez, Torres and Harrison ^{40 )}. Although the most effective strategy for treating NAFLD with lifestyle change is unclear, dietary energy restriction can be very effective in decreasing liver fat⁽ Reference Harrison and Day ^{39 ,} Reference Targher and Byrne ^{41 )}, and 80% decreases in liver fat have been recorded over relatively short periods of time (e.g. 3 months)⁽ Reference Rodriguez, Torres and Harrison ^{40 )}. When energy restriction is severe (2510·4–3347·2 kJ/d (600–800 kcal/d)), marked decreases in liver fat are often noted⁽ Reference Harrison and Day ^{39 ,} Reference Targher and Byrne ^{41 )}. That said, most of the evidence of benefit comes from trials of energy restriction that have tested the intervention in relatively small numbers of individuals, and most of the durations of intervention have often been very short (e.g. 8 weeks)⁽ Reference Lim, Hollingsworth and Aribisala ^{42 )}, but are on average approximately 6 months. In combination with an improvement in liver fat, energy restriction producing weight loss, is also very effective ameliorating many of the features of MetS, such as increased serum TAG, and glucose concentrations and decreased serum HDL-cholesterol concentration and increased blood pressure⁽ Reference Targher and Byrne ^{41 )}.

Increases in physical activity energy expenditure tend to induce a net negative energy balance, although it is unlikely that all the benefit of physical activity (or exercise), is mediated by inducing this change in energy balance. It is presently uncertain as to the best form of physical activity energy expenditure (or exercise) to decrease ectopic fat accumulation in visceral organs such as the liver. Current evidence suggests high intensity activity may have a particularly beneficial effect to decrease hepatic fat and possibly also has a beneficial effect in decreasing risk of progression to liver fibrosis⁽ Reference Hallsworth, Fattakhova and Hollingsworth ^{43 ,} Reference Kistler, Brunt and Clark ^{44 )}. For example, vigorous physical activity at a metabolic equivalent of task (or measure of physical activity energy expenditure) equivalent of greater than or equal to six was associated with decreased odds for having non-alcoholic steatohepatitis (OR 0·65, 95% CI 0·43, 0·98) and also a decreased risk of progression to fibrosis⁽ Reference Kistler, Brunt and Clark ^{44 )}. In addition, interesting recent data suggest that resistance training may be particularly beneficial in decreasing hepatic fat content⁽ Reference Hallsworth, Fattakhova and Hollingsworth ^{43 )}. These authors showed that in nineteen patients with NAFLD, there was a significant 13% decrease in hepatic steatosis, as measured by proton magnetic resonance spectroscopy, after 8 weeks of resistance training (comprising eight different exercises on three non-consecutive days of the week for 45–60 min duration). There were also improvements in systemic lipid oxidation, glucose tolerance and insulin sensitivity. More importantly, these exercise-induced beneficial effects were noted to be independent of weight loss.

However, further evidence is needed before recommending physiologically stressful resistance training for this group of patients, not least because adherence to such a strenuous programme is likely to be low, and many patients with NAFLD have existing co-morbidities, such as CVD. Nevertheless, given that many people with NAFLD have obesity and features of MetS, and there is evidence of a benefit of resistance training in people with MetS⁽ Reference Strasser, Siebert and Schobersberger ^{45 ,} Reference Strasser and Schobersberger ^{46 )}, it is plausible that there could be a substantial benefit in NAFLD. A meta-analysis of thirteen randomised controlled trials testing the effect of resistance training on MetS features found that this intervention improved most features of MetS⁽ Reference Strasser, Siebert and Schobersberger ^{45 )}. Since adipose tissue secretes many adipokines and inflammatory cytokines (e.g. adiponectin, leptin, resistin, TNF-α, IL-6 and plasminogen activator inhibitor 1) and resistance training has been shown to produce a beneficial effect on these bioactive molecules⁽ Reference Strasser and Schobersberger ^{46 )}, it is plausible that resistance training may have a benefit in NAFLD. Table 1 summarises the benefits of lifestyle interventions on ectopic fat and related cardio-metabolic risk factors.

Table 1.

Summary of benefits of lifestyle intervention on ectopic fat (liver) and related cardio-metabolic risk factors

MRS, magnetic resonance spectroscopy, US, ultrasound; NAFLD, non-alcoholic fatty liver disease; HDL-C, HDL-cholesterol.