Non-alcoholic fatty liver disease (NAFLD): a multi-system disease 1 influenced by ageing and sex, and affected by adipose tissue and intestinal 2 function.

In recent years, a wealth of factors are associated with increased risk of developing non- 36 alcoholic fatty liver disease (NAFLD) and NAFLD is now thought to increase the risk of 37 multiple extra-hepatic diseases. The aim of this review is firstly to focus on the role of ageing 38 and sex as key, poorly understood risk factors in the development and progression of 39 NAFLD. Secondly, we aim to discuss the roles of white adipose tissue (WAT) and intestinal 40 dysfunction, as producers of extra-hepatic factors known to further contribute to the 41 pathogenesis of NAFLD. Finally, we aim to summarise the role of NAFLD as a multi-system 42 disease affecting other organ systems beyond the liver. Both increased age and male sex 43 increase the risk of NAFLD and this may be partly driven by alterations in the distribution 44 and function of WAT. Similarly, changes in gut microbiota (GM) composition and intestinal 45 function with ageing and chronic overnutrition are likely to contribute to the development of 46 NAFLD both directly (i.e. by affecting hepatic function) and indirectly via exacerbating 47 WAT dysfunction. Consequently, the presence of NAFLD significantly increases the risk of 48 various extra-hepatic diseases including cardiovascular disease, type 2 diabetes mellitus, 49 chronic kidney disease and certain extra-hepatic cancers. Thus changes in WAT and 50 intestinal function with ageing and chronic overnutrition contribute to the development of 51 NAFLD - a multi-system disease that subsequently contributes to the development of other 52 chronic cardiometabolic diseases. 53 thought to contribute to an increased risk of NAFLD both directly (via inducing hepatic 535 mitochondrial function, inflammation and steatosis) and indirectly through detrimentally impacting 536 WAT function (impairing WAT expansion, metabolic flexibility and increasing the production of pro- 537 inflammatory cytokines). The increased production of inflammatory cytokines is thought to lead to a 538 state of chronic low-grade inflammation which is likely to further disrupt the function of tight 539 junction-associated proteins – thus forming a viscous cycle of worsening metabolic dysfunction and 540 NAFLD disease severity. Trimethylamine N-oxide,

Introduction effectively expand and store lipid, rather than the obesity per se, is a pivotal factor in the relationship 134 between increased adiposity and NAFLD risk. 135 The distribution of WAT is known to differ significantly between sexes, changes with increasing age 136 and has been hypothesised to be partly responsible for the increased prevalence of NAFLD in men 137 and older age groups, particularly post-menopausal women (Figure 1) (38)(39)(40) . Whilst the mechanisms 138 regulating the distribution of WAT remain largely elusive, evidence indicates that ageing and male 139 sex are associated with a restricted capacity to effectively expand so-called "metabolically protective" 140 SAT depots (41) . Whilst pre-menopausal women typically have greater total adiposity, men tend to 141 accumulate greater amounts of VAT with ageing and pre-menopausal women accumulate gluteal 142 femoral SAT which is associated with a lower risk of metabolic disease and NAFLD (42) . In both men 143 and women, older age (i.e. post-menopausal women and men > 50 years) is associated with a 144 reduction in the capacity of SAT to expand and an increase in VAT (43)(44)(45) . The limited capacity of 145 SAT to store TAG in men and with increasing age is likely to re-direct lipid accumulation ectopically 146 in non-adipose tissues, including the liver, leading to lipotoxicity, a chronic local and systemic pro-147 inflammatory environment and eventually NAFLD development (46) . The importance of effective SAT 148 expansion can be seen in individuals with certain genetic or acquired lipodystrophies that are 149 characterised by the complete or partial absence of SAT (47) . In spite of their often lean appearance, 150 these individuals appear to exhibit much higher rates of NAFLD/NASH progression and other 151 cardiometabolic complications than would be expected based on their BMI alone (31,47) . Given this, it 152 is likely that differences in WAT distribution between sexes and changes occurring with increasing 153 age are both important in the increased risk of NAFLD associated with ageing and with male sex. 154 155

Adipose tissue dysfunction and NAFLD 156
White adipose tissue is composed of mature unilocular adipocyte fraction and a stromal vascular 157 fraction, comprised of numerous cell types such as vascular, mesenchymal and immune cells. At a 158 cellular level, WAT expansion can be mediated by an enlargement of individual adipocytes 159 (hypertrophy), an increase in the number of adipocytes (hyperplasia) or a mixture of both. Adipocyte 160 hypertrophy, rather than hyperplasia, is more closely associated with WAT dysfunction and metabolic 161 disease (48) . Factors including hypoxia, low-grade chronic inflammation (i.e. metaflammation) and 162 improper extracellular matrix remodelling are thought to limit adipocyte differentiation and the healthy 163 expansion of adipose tissue (hyperplasia) (49,50) . This limit can result in adipocyte hypertrophy, 164 dysfunction, stress and eventually death (51,52) . In this context, WAT dysfunction refers to a reduction 165 in the tissues ability to effectively sense and respond to dynamic changes in nutrient availability (i.e. 166 metabolic inflexibility) and can coexist with adipose insulin resistance (IR) and metaflammation. 167 Specifically, this dysfunction is thought to affect WAT metabolism and in particular its ability to handle 168 lipids and increase the lipolytic rate of WAT due to a reduction in tissue insulin sensitivity, increasing 169 the flux of non-esterified fatty acids (NEFAs) to the liver and consequently increasing the risk of 170 NAFLD (31,(53)(54)(55) . 171 Accompanying the changes in the distribution of WAT, ageing is associated with a marked reduction 172 in insulin, lipolytic and NEFA responsiveness in WAT. This metabolic inflexibility may underly the 173 known association between ageing and increased risk of NAFLD (43)(44)(45) . The reduction in SAT with 174 ageing in both men and women may in part be driven by a reduction in the adipogenic potential of 175 progenitor cells and the accumulation of senescent adipocytes in aged WAT. Preadipocytes isolated 176 from peripheral SAT in elderly individuals were found to have a reduced rate of replication compared 177 to those isolated from younger individuals (56) . Additionally, ageing is associated with an accumulation 178 of senescent adipocyte-derived stem cells within SAT which lack the ability to differentiate into 179 adipocytes in response to metabolic stress, consequently affecting the tissue's capacity to store TAG 180 (57) . Through their senescence-associated secretory phenotype, senescent adipocyte progenitor cells 181 within WAT are also likely to contribute to WAT inflammation and subsequent metabolic 182 complications (58,59) . 183 In addition to ageing, there are also sexually dimorphic differences in WAT function whereby WAT in 184 females is generally more insulin sensitive, more lipogenic and less susceptible to inflammation than 185 WAT from males. This phenomenon is also strongly associated with differences in sex hormone 186 concentrations (60,61) . Menopause appears to associate with a preferential increase in VAT (rather than 187 SAT) in both obese and non-obese women (62-65) , further supporting a role for sex hormones, such as 188 estrogen, in regulating the beneficial distribution and function of WAT. Circulating concentrations of 189 estrogen decrease markedly after menopause which is thought to lead to the redistribution of lipids into 190 VAT and the liver which, in combination with overnutrition, increases the risk of VAT accumulation 191 and NAFLD in post-menopausal women (66) . Pre-clinical studies utilising ovariectomised murine 192 models also support a causative relationship between reduced estrogen production, increased VAT mass 193 and the development of NASH (42,(66)(67)(68) . Whilst an in-depth discussion of the role of estrogen within 194 WAT is beyond the scope of this review (see other relevant reviews (42,69,70) ), it is thought that the 195 increased expression of estrogen receptor alpha (ERα) in the gluteal femoral SAT of premenopausal 196 women promotes lipoprotein lipase activity and accumulation of TAG in adipocytes within this depot 197 (71) . Thus, it is likely that differences in WAT function (partly driven by differences in sex hormone 198 concentrations and the expression of functional target receptors) is an important factor underlying the 199 observed differences in NAFLD risk between men and women. Furthermore, changes in WAT with 200 ageing are likely to exacerbate WAT dysfunction associated with a state of chronic energy surplus and 201 are likely to have an important role in the increased risk of NAFLD associated with older age. 202 203

Adipokines and NAFLD 204
White adipose tissue is an endocrine tissue capable of secreting a wide range of adipokines which have 205 various roles in the regulation of whole-body energy homeostasis and inter-organ communication (72) . 206 The aberrant production of these adipokines has been linked to multiple obesity-related metabolic 207 diseases. Amongst these adipokines, leptin and adiponectin are predominately produced by adipocytes. 208 In addition to its well-established role in regulating appetite and energy homeostasis (49,73) , leptin exerts 209 a dual action on hepatic function and NAFLD severity. Recent meta-analyses including an analysis of 210 over 30 studies indicated that circulating concentrations of leptin are elevated in patients with NAFLD 211 compared to healthy controls and supports a positive relationship between leptin and NAFLD (74) . As 212 recently highlighted (75,76) , under normoleptinemia conditions, leptin is thought to suppress hepatic 213 glucose production and hepatic lipogenesis thus providing an insulin sensitising anti-steatotic effect. 214 Conversely, in the context of chronic hyperleptinemia as is common in obesity, a state of leptin 215 resistance can result, which may also contribute to the NASH phenotype. It is suggested that in the liver, 216 high concentrations of leptin can increase the expression of matrix remodelling enzymes via interacting 217 with leptin receptors on Kupffer and sinusoidal endothelial cells, in turn activating hepatic stellate cells 218 (HSCs), and possibly contributing to liver fibrosis (77) . 219 Sexual dimorphism has also been reported for leptin expression (78) . Despite their lower risk of NAFLD, 220 circulating concentrations of leptin are higher in pre-menopausal women compared to age-matched men 221 and higher leptin levels are thought to be driven by both greater adiposity and an increased production 222 rate of leptin per unit mass of WAT in women compared to men (79) . In both men and women, circulating 223 concentrations of leptin are thought to gradually decline with ageing, with reductions being most 224 noticeable in women compared to men whilst appearing to be independent of menopausal status (79,80) . 225 Despite these findings, it is currently unknown whether differences in circulating concentrations of 226 leptin between sexes and age groups have an impact on the risk of NAFLD. 227 Similar to leptin, a wealth of studies indicate that the circulating concentrations of adiponectin, the most 228 systemically abundant adipokine, are altered in patients with NAFLD (as reviewed in (81) ). Adiponectin 229 is a hepatoprotective adipokine that has well-established anti-inflammatory (82-84) and insulin sensitising 230 effects (85) both systemically and within the liver. Meta-analysis indicates that adiponectin 231 concentrations are significantly lower in patients with NAFLD compared to healthy controls, 232 furthermore, NASH is associated with lower adiponectin when compared to simple steatosis (86) . 233 Conversely, adiponectin concentrations are thought to increase in patients with NAFLD-cirrhosis 234 potentially due to a reduction in the hepatic clearance of adiponectin and/or an increase in its production 235 as a result of the tissue repair process associated with NAFLD-cirrhosis (87)(88)(89) . Along with its well-236 established role in promoting hepatic insulin sensitivity (90,91) , evidence indicates that adiponectin also 237 has antifibrogenic effects via inhibiting the proliferation of HSCs (92) . Whilst the role of adiponectin in 238 ageing remains uncertain, it is thought that circulating concentrations of adiponectin are paradoxically 239 increased in older age and are positively associated with physical disability and mortality in elderly 240 individuals (93) . Furthermore, some evidence suggests that the association between adiponectin and 241 ageing may be modified by sex (94) . In addition to leptin and adiponectin, a wealth of other studies have 242 demonstrated that numerous other adipokines may be involved in the development and progression of 243 NAFLD (Table 1). It should be noted that there is a substantial amount of conflicting evidence 244 regarding the changes in circulating concentrations of other adipokines in the context of NAFLD and 245 little is known about the potential pathological role of these adipokines in NAFLD (Table 1) A plethora of studies have revealed that GM dysbiosis is associated with and is a contributing factor 281 to NAFLD (112)(113)(114)(115) . The dominating phyla within human GM are Bacteroidetes and Firmicutes with a 282 significant inter-individual variation in the GM at lower taxonomical levels (116,117) . Previous evidence 283 indicates that the relative abundance of Bacteroidetes is lower in patients with NASH compared to 284 those with hepatic steatosis and healthy controls (117) . More recently, Bacteroides abundance was 285 found to be significantly increased in patients with NASH and the abundance of Ruminococcus was 286 increased in patients with liver fibrosis (118) . As recently reviewed (113) , this shifting in GM in relation 287 to NAFLD severity is supported by numerous other studies. Indeed, the presence of bacteria 288 belonging to the Proteobacteria phylum was increased significantly in patients with ≥F3 when 289 compared to patients with F0-F2 liver fibrosis ( Table 2) (119) . Emerging evidence also supports a 290 strong link between the GM and NAFLD-cirrhosis indicating that the composition of the GM may be 291 a useful tool for the identification and staging of NAFLD. Utilising a unique twin and family study 292 design, one study identified a specific GM signature that had a robust diagnostic accuracy, with an 293 area under the reciever operating characteristic of 0.92, for the detection of NAFLD-cirrhosis (120) . 294 Further work demonstrated the robustness and potential universal applicability of this microbiome 295 signature of NAFLD-cirrhosis in two independent cohorts across geographically and culturally 296 distinct populations (121) . However, given the impact of host genetics and environmental factors on the 297 composition of GM (122) , it is unlikely that a single GM signature will be able to distinguish between 298 NAFLD phenotypes at an individual level. 299 Luca Miele and colleagues were the first to identify that patients with NAFLD generally have 300 increased intestinal permeability and alterations in intestinal tight junction integrity (observed as a 301 reduction in zonula occludens-1 within intestinal crypt cells), compared to healthy subjects (123) . 302 Recent meta-analysis found that 39.1% of NAFLD patients had evidence of increased intestinal 303 permeability compared to 6.8% of healthy controls (OR 5.08, 95% CI 1.98 -13.05) (124) . Furthermore, 304 subgroup analysis indicated that there was a higher incidence of increased intestinal permeability in 305 patients with NASH compared to patients with simple steatosis (124) . It is generally well-accepted that 306 the increased intestinal permeability commonly seen in NAFLD facilitates the translocation of GM-307 derived metabolites and bacterial products (such as lipopolysaccharides (LPS) and ethanol) which 308 may in turn contribute to metaflammation and the pathogenesis of NAFLD (125) . 309 In addition to altered GM and intestinal permeability, the abundance of GM-dependent metabolites is 310 thought to be altered in NAFLD, many of which may be detected in stool samples and may offer a 311 tool for the assessment of disease severity. For example, work comparing the abundance of distinct 312 stool metabolites in patients with NAFLD-cirrhosis vs healthy subjects revealed 17 metabolites 313 which, in combination, were able to accurately detect the presence of NAFLD-cirrhosis (AUROC 314 0.91, 95% CI 0.89 -0.93) (121) . Thus, evidence is accumulating to suggest that accumulation of certain 315 microbial species, changes in intestinal function and increased intestinal permeability, are likely to 316 contribute not only to the pathogenesis of NAFLD but also to increased liver disease severity. Further 317 studies are required to elucidate the potential role of non-bacterial species within the GM on the 318 development and progression of NAFLD. 319

Intestinal dysfunction, dysbiosis and links with WAT function in NAFLD 320
Associated with WAT dysfunction are changes in intestinal function and GM dysbiosis, which have 321 also been proposed to be key factors contributing to NAFLD. Receiving around 70% of its blood supply 322 from intestinal vascularisation, the liver is constantly exposed to the metabolic products, toxins and 323 nutrients produced by the GM (126) . It has been suggested that when in a dysbiotic state, GM may 324 contribute to the development and progression of NAFLD via a range of pathways; including changes 325 in dietary energy harvest (127,128) , alterations in short-chain fatty acid (SCFA) production (particularly 326 butyrate) (129, 130) , increased bacterial lipopolysaccharide (LPS) translocation (125,131) , alternations in bile 327 acid profiles (132) and increased endogenous ethanol production (133) . Indeed, the potential effects of these 328 factors on hepatic function and NAFLD have been discussed in various recent reviews (110, 112-114, 125, 134) , 329 furthermore, alterations in appetite-regulating gut hormones are also likely to have an important role in 330 the development and progression of NAFLD, as recently reviewd (135)(136)(137) . 331 Disruptions in intestinal permeability associated with obesity and NAFLD are likely to be accompanied 332 by a reduction in the integrity of intestinal tight junctions (123,138) . Increased intestinal permeability in 333 the presence of GM dysbiosis is thought to facilitate the translocation of bacterial products including 334 pro-inflammatory endotoxins such as LPS. Circulating concentrations of LPS were found to be 335 significantly higher in patients with NAFLD compared to healthy controls (139,140) and have been shown 336 to be positively associated with the expression of pro-inflammatory genes within both VAT and SAT 337 in individuals with obesity (141) . This is supported by evidence from pre-clinical murine studies 338 indicating that increased LPS may directly contribute to WAT inflammation and increase the release of 339 WAT-derived pro-inflammatory cytokines (142) . Accompanying these findings, various other studies 340 have proposed additional mechanisms by which changes in intestinal function and GM dysbiosis may 341 impact NAFLD development both directly and in-directly via detrimentally impacting WAT function 342 (Table 3 and Figure 2). 343 Evidence also suggests that the composition of the GM and intestinal function can differ between sexes 344 and such differences may partly explain differences in the risk of metabolic disease between sexes (143-345 145) . Similarly, changes in GM composition and intestinal function are strongly associated with ageing 346 and are likely to contribute to the increased risk of NAFLD associated with older age both directly and 347 indirectly via exacerbating WAT dysfunction (146)(147)(148) . Similar to obesity, ageing is also associated with 348 disruptions in intestinal permeability subsequently facilitating the translocation of bacterial products 349 such as LPS which are known to contribute to both hepatic and WAT dysfunction (Figure 2) (149,150) . 350 Collectively, existing studies demonstrate the existence of a gut-WAT axis which, in addition to the 351 well-established gut-liver axis, may indirectly contribute to NAFLD pathogenesis. Furthermore,   (154) . We also showed in this work that GDF-15 may be an important factor contributing to 375 the increased risk of liver fibrosis associated with T2DM, and that HbA1c levels explained ~30% of 376 the variance in GDF-15 concentrations (154) . However, further work is required to fully elucidate the 377 role of GDF-15 in the development and progression of NAFLD in patients with T2DM. 378 The estimated global prevalence of NAFLD among patients with T2DM is 55.5% (95% CI: 47.3-379 63.7%) with prevalence estimates varying between geographical regions (155) . This study also found 380 that the estimated global prevalence of NASH and advanced fibrosis in patients with T2DM was 381 37.3% (95% CI: 24.7-50.0%) and 4.8% (95% CI: 0.0-17.5%) respectively (155) . The presence of 382 T2DM is also an important risk factor for the faster progression of NAFLD towards NASH, cirrhosis 383 or HCC (152,156,157) . Patients with NAFLD and coexisting T2DM are thought to have between a 2 and 384 6 fold increased risk of developing advanced fibrosis compared to patients with only NAFLD (152) . In 385 addition to T2DM, the presence of MetS is also recognised as an important NAFLD risk factor. The 386 presence of MetS in patients with NAFLD but without diabetes, is associated with more severe 387 NAFLD compared to patients without MetS (158) . Furthermore, this study suggested that a higher 388 number of MetS features was associated with a greater probability of NASH, with 70% of patients 389 diagnosed with NASH having three or more features of MetS. The presence of MetS has also recently 390 been shown to be associated with progression to advanced fibrosis in patients with NAFLD (159) . 391 These findings support those of others which also show that NAFLD severity is positively associated 392 with the presence of MetS features, particularly the level of hypertension, hyperglycaemia and 393 hypertriglyceridemia (160) . 394 It is important to highlight that the link between T2DM, MetS and NAFLD is complex and bi-395 directional. Evidence from a recent large meta-analysis of over 500,000 individuals found that 396 NAFLD was associated with a ~2.2-fold increased risk of incident diabetes independently of age, sex, 397 adiposity and other common metabolic risk factors (161) . Interestingly, in this study, the risk of incident 398 diabetes was found to increase in relation to the underlying severity of NAFLD with a particularly 399 noticeable increase in risk according to the severity of liver fibrosis (n = 5 studies; random-effects HR 400 3.42, 95% CI 2.3-5.1) (161) . These findings support other evidence from meta-analyses and 401 observational studies which demonstrate that individuals with NAFLD had a higher risk for incident 402 T2DM than individuals without NAFLD (8,162) . Evidence collated from eight studies with a median 403 follow-up period of 4.5 years indicated that NAFLD was associated with an increased risk of incident 404 MetS with a pooled relative risk of 3.2 (95% CI: 3.1-3.4) when NAFLD was diagnosed via 405 ultrasonography (163) . Collectively, this evidence suggests that a vicious cycle of worsening disease 406 states is likely to exist between T2DM, MetS and NAFLD (152) . 407 408

NAFLD and cardiovascular disease (CVD) 409
Evidence indicates that NAFLD is an important risk factor for various extra-hepatic diseases and the 410 detrimental relationship between T2DM and NAFLD likely exacerbates this risk. Furthermore, given 411 the strong associations with NAFLD and other cardiometabolic risk factors, including central obesity, 412 atherogenic dyslipidaemia, and hypertension, it is no surprise that NAFLD is also associated with an 413 increased risk of CVD (6, 164) . Recent evidence suggests that CVD is one of the most important causes 414 of death among people with NAFLD (165) , and patients with NAFLD are more likely to experience 415 CVD-related death than a liver-related death (26,164,166) . Recent meta-analysis incorporating a total of 416 16 observational studies and over 34,000 individuals with a median follow-up of ~7 years, concluded 417 that NAFLD conferred an odds ratio of 1.6 for fatal and/or non-fatal CVD events (random-effects OR 418 of 1.6, 95% CI: 1.3-2.1) (167) . This is consistent with findings from others that suggest that the risk of 419 incident CVD events increases further with greater severity of NAFLD even after adjusting for other 420 established CVD risk factors (13) . Emerging data also supports the evolving notion that sex is an 421 important modifier of NAFLD outcomes and suggest that the occurrence and prevalence of CVD 422 related events and mortality are likely to differ between sexes. One study found that in ~108,000 423 individuals with NAFLD, cardiovascular events were 2 times higher in women compared to men (OR 424 2.1, 95% CI: 1.7-2.7) (168) . Women also had higher cardiovascular mortality with advancing age 425 starting at age 42 years further highlighting the importance of both age and sex as important risk 426 factors for both NAFLD and CVD (168) . 427

NAFLD and Chronic kidney disease (CKD) 428
The risk of CKD is also increased in patients with NAFLD. Chronic kidney disease is a complex, 429 progressive chronic condition that is defined by an abnormality in either the structure and/or function 430 of the kidneys for ≥3 months with serious implications for health (7,169) . Evidence from three meta-431 analyses demonstrates a higher incidence of CKD in patients with NAFLD (170)(171)(172) . The first of these 432 studies, which included 33 observational (20 cross-sectional and 13 longitudinal) studies concluded 433 that NAFLD was associated with a 2-fold increased prevalence of CKD (random-effects odds ratio 434 2.1, 95% CI: 1.7-2.7) and that NAFLD was associated with a nearly 80% increased risk of incident 435 CKD (random-effects HR 1.8, 95% CI: 1.7-2.0) (7,170) . Similarly, the second more recent meta-436 analysis confirmed that NAFLD was associated with a ~40% increase in the long-term risk of incident 437 CKD (random-effects HR 1.4, 95% CI 1.2-1.5) (171) . Most recently, findings from a large updated 438 meta-analysis indicate that NAFLD was significantly associated with a ~1.45 fold increased long-term 439 risk of incident CKD and this association was independent of age, sex and conventional CKD risk 440 factors (172) . Interestingly, these studies also support an association between increased NAFLD 441 severity (particularly the presence of advanced fibrosis) and increased risk of CKD (170)(171)(172) . Another 442 large database study in Germany also supports a strong link between NAFLD and increased risk of 443 CKD that is independent of age, sex and the presence of additional cardiometabolic risk factors such 444 as diabetes, obesity and hypertension (173) . 445

NAFLD and non-hepatic cancers 447
In addition to increasing the risk of HCC, recent evidence suggests that NAFLD may also increase the 448 risk of various non-hepatic cancers. Findings from a recent large population-based cohort study 449 concluded that, compared to healthy controls, patients with biopsy-confirmed NAFLD had 450 significantly increased overall cancer incidence over a median 13.8 years follow-up period (adjusted 451 HR 1.3, 95% CI: 1.2-1.4) (174) . Whilst this increase was mostly driven by a higher HCC incidence, the 452 presence of NAFLD was also associated with modestly increased rates of melanoma, pancreatic, and 453 kidney/bladder cancers (174) . In support of these findings, recent meta-analysis of 10 cohort studies (> 454 180,000 individuals, 24.8% with NAFLD) found that NAFLD was significantly associated with a 455 nearly 1.5-fold to two-fold increased risk of developing gastrointestinal cancers (oesophagus, 456 stomach, pancreas or colorectal cancers) independently of confounding factors such as age, sex, 457 obesity, diabetes and smoking status (175) . There is currently very limited data on the severity of 458 NAFLD (particularly the severity of liver fibrosis) and the risk of developing extra-hepatic cancers. 459 One recent study found that more severe NAFLD was associated with significantly increased overall 460 mortality with most of the excess mortality observed being driven by extrahepatic cancer and liver 461 cirrhosis (176) . Whilst it is reasonable to assume that the risk of developing extra-hepatic cancers is 462 increased in relation to NAFLD severity, further large prospective studies are needed to confirm this 463 link. Such studies should account for the potential modifying effect of important genetic variants, age, 464 sex, and obesity along with other NAFLD-associated comorbidities when considering the relationship 465 between NAFLD severity and risk of specific extra-hepatic cancers. This latter consideration is 466 particularly important since it is not yet clear whether NAFLD is associated with an increased risk of 467 certain extra-hepatic cancers simply as a consequence of shared metabolic risk factors or whether 468 NAFLD itself directly contributes to an increased risk of developing extrahepatic cancers (175) . 469 470

Conclusion 471
The risk of developing NAFLD differs between sexes, changes with age and is likely to be modulated 472 by complex interactions between genetic and environmental factors. Differences in WAT mass, its 473 distribution (VAT vs SAT) and functionality (metabolic & endocrine), are likely to be key drivers of 474 hepatic steatosis and NAFLD development. Similarly, differences in the regional distribution and 475 function of WAT between men and women and between age groups are likely to contribute to the 476 increased risk of NAFLD progression associated with sex and age. The development of GM dysbiosis 477 and intestinal dysfunction is likely to contribute to NAFLD both directly and indirectly via the 478 exacerbation of WAT inflammation and dysfunction through a range of GM-derived factors.

CONFLICTS OF INTEREST 493
The authors declare no conflict of interest. 494

AUTHORSHIP 495
The authors had sole responsibility for all aspects of preparation of this paper. Sex and age are key factors that modify the risk of NAFLD and NAFLD progression. NAFLD risk is 512 lower in younger women compared to younger men whereas the risk of NAFLD is similar in older 513 men and women (i.e. post menopausal). Younger women have an increased capacity to preferential 514 expand gluteal femoral SAT consequently protecting them from NAFLD. Age-associated changes in 515 WAT leads to the redistribution of WAT which is typically characterised by a marked reduction in 516 SAT and increased central metabolically-unfavourable VAT which may partly explain the increased 517 risk of NAFLD associated with ageing in both men and women. WAT distribution is different 518 between men and women, is heavily influenced by ageing and is strongly associated with NAFLD are thought to contribute to NAFLD both directly (i.e. by directly impacting hepatic function) and 527 indirectly through detrimentally influencing WAT function. As highlighted on the left -intestinal 528 eubiosis and healthy gut function (such as that typically found in young individuals) promotes 529 intestinal barrier integrity and homeostasis whilst restricting the production and dissemination of 530 metabolically detrimental factors (such as LPS and endogenous ethanol) into circulation, the liver and 531 WAT. Conversely, as highlighted on the right, intestinal dysbiosis (such as that often associated with 532 older age) leads to alterations in various GM-derived factors/metabolites that impair the function of 533 tight junction-associated proteins located within the intestinal epithelium. Consequently, these 534 changes are thought to contribute to an increased risk of NAFLD both directly (via inducing hepatic 535 mitochondrial function, inflammation and steatosis) and indirectly through detrimentally impacting 536 WAT function (impairing WAT expansion, metabolic flexibility and increasing the production of pro-537 inflammatory cytokines). The increased production of inflammatory cytokines is thought to lead to a 538 state of chronic low-grade inflammation which is likely to further disrupt the function of tight 539 junction-associated proteins -thus forming a viscous cycle of worsening metabolic dysfunction and 540 NAFLD disease severity. Largely unknown, potentially induces hepatic mitochondrial dysfunction and promotes hepatic steatosis (190,191) Adipsin Decreased (192) , No association (193) , Increased (194) Largely unknown, low concentrations may impact hepatic function via a reduction in insulin production (195) Chemerin Increased (193,196,197) Largely unknown, increased concentrations potentially protective via the suppression of pro-inflammatory cytokines (198) Apelin Not associated (199) ,  Increased inflammation and decreased insulin sensitivity (131,206,207) . Increased liver inflammation via the activation of hepatic macrophages and platelets (205) . Profibrogenic via the activation of HSCs (208) .

Butyrate
Decreased (212) Decreased production is thought to contribute to increased inflammation and decreased fatty acid oxidation (213,214) .
Decreased production is thought to contribute to hepatic mitochondrial dysfunction (215) , increased hepatic steatosis and inflammation (216) .
Decreased production is thought to contribute to increased hepatic steatosis and inflammation (222) .