Leptin in nonalcoholic fatty liver disease: A narrative review

    Leptin in nonalcoholic fatty liver disease: a narrative review Stergios A. Polyzos, Jannis Kountouras, Christos S. Mantzoros PII: DOI: Reference:

S0026-0495(14)00306-0 doi: 10.1016/j.metabol.2014.10.012 YMETA 53107

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Metabolism

Please cite this article as: Polyzos Stergios A., Kountouras Jannis, Mantzoros Christos S., Leptin in nonalcoholic fatty liver disease: a narrative review, Metabolism (2014), doi: 10.1016/j.metabol.2014.10.012

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ACCEPTED MANUSCRIPT Leptin in nonalcoholic fatty liver disease: a narrative review

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Stergios A. Polyzos,1,2 Jannis Kountouras,1 Christos S. Mantzoros2

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Second Medical Clinic, Department of Medicine, Aristotle University of Thessaloniki,

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Ippokration Hospital, Thessaloniki, Greece 2

Division of Endocrinology, Diabetes and Metabolism, Department of Internal Medicine,

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Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

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Corresponding author:

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Running title: Leptin and the liver

Stergios A. Polyzos, MD, MSc, PhD Endocrinologist

13 Simou Lianidi, 551 34 Thessaloniki, Greece

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Tel./Fax: +302310424710

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E-mail: [email protected]

Word count: 6906 (text); 184 (abstract); references: 162

Funding: No sources of financial support for this study.

Disclosure statement: SAP and JK: No conflict of interest; CSM has served as consultant for Astra Zeneca and had received research support through his Institution from Amgen.

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ACCEPTED MANUSCRIPT Abbreviation list AMPK, 5'-adenosine monophosphate–activated protein kinase; BMI, body mass index; CHB, chronic hepatitis B; CHC, chronic hepatitis C; ChREBP, carbohydrate response element

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binding protein; Fox, forkhead box protein; HCC, hepatocellular carcinoma; HSCs, hepatic stellate cells; IR, insulin resistance; IRS, insulin receptor substrate; JAK, Janus kinase; LepR, leptin receptor; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of

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rapamycin; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score; NASH,

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nonalcoholic steatohepatitis; PI3K, phosphatidylinositol-3 kinase; PNPLA, patatin-like phospholipase domain-containing; SH2-B, src homology 2 domain-containing adapter protein

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B; SHIP, src homology 2-domain containing inositol 5'-phosphatase; SNP, single nucleotide polymorphism; SOCS, suppressor of cytokine signaling; SREBP, sterol regulatory element-

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binding protein; SS, simple steatosis; STAT, signal transducer and activator of transcription; SVR, sustained virological response; T2DM, type 2 diabetes mellitus; TGF, transforming

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growth factor; TNF, tumor necrosis factor.

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ACCEPTED MANUSCRIPT Abstract Leptin, the first described adipokine, interplays with hepatic metabolism. The aim of this review was to summarize available data on the association between leptin and

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nonalcoholic fatty liver disease (NAFLD). Leptin has a potential dual action on NAFLD experimental models, exerting a possible anti-steatotic, but also a proinflammatory and profibrogenic action. Observational clinical studies have shown higher or similar leptin levels

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between simple steatosis or nonalcoholic steatohepatitis (NASH) compared with controls.

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Interventional studies showed that circulating leptin diminishes together with body mass index after successful weight loss following lifestyle modifications or bariatric surgery.

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Studies providing evidence for the effect of other medications on leptin levels in NAFLD populations are limited and of low power. Data from small studies claim that recombinant

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leptin administration had a possibly beneficial effect on steatosis, but not fibrosis, in NAFLD patients with hypoleptinemia. Although the aforementioned dual leptin action has not yet been validated in humans, leptin administration in NAFLD patients with normoleptinemia or hyperleptinemia is discouraged. Further well-controlled studies in cautiously selected

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populations are needed to elucidate whether leptin has any prognostic and therapeutic role in

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NAFLD patients.

Key-words: insulin resistance; leptin; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis; leptin resistance; steatosis

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ACCEPTED MANUSCRIPT 1. Introduction Nonalcoholic fatty liver disease (NAFLD) is regarded as the hepatic component of metabolic or insulin resistance (IR) syndrome, increasing recently in parallel with the

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epidemics of obesity and type 2 diabetes mellitus (T2DM) [1,2]. NAFLD is now a global public health problem, with a prevalence of 10-46% in the general US population and of 635% in the rest of the world (median 20%) [3]. It is the most common cause of chronic liver

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disease in US adults. NAFLD ranges from nonalcoholic simple steatosis (SS) to nonalcoholic

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steatohepatitis (NASH) characterized by steatosis, inflammation and/or fibrosis. Apart from its systemic consequences concerning IR, metabolic complications, cardiovascular and/or

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chronic kidney disease, NASH may lead to subacute liver failure, liver cirrhosis and/or hepatocellular carcinoma [3]. Although NAFLD is a field of intensive research, the

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mechanisms underlying its development remain to be elucidated [4]. However, IR and adipokines contribute to the pathogenesis of SS and the progression to NASH and NASHrelated cirrhosis, as reviewed elsewhere [1]. Leptin, the first described adipokine [5], is an adipocyte-secreted hormone playing a

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crucial role in the regulation of energy homeostasis, and in metabolic, reproductive and neuroendocrine functions [6-8]. There are also emerging roles of leptin, including cognition,

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immune function and related autoimmune disorders, as well as bone metabolism [9]. Leptin is secreted proportionally to the amount of white adipose mass; circulating leptin levels reflect primarily the body energy stores and secondarily acute changes in caloric intake [10]. The importance of leptin could be derived from animal and human pathophysiology. Mice homozygous for mutations in the obese (ob) gene (ob/ob), which prevent leptin production or lead to secretion of an inactive leptin molecule, exhibit hyperphagia, IR, early-onset obesity, diabetes and several neuroendocrine abnormalities, which are improved by exogenous leptin administration [6,9]. Similarly, leptin replacement is highly effective for many metabolic derangements observed in congenital or acquired lipodystrophy patients, who have low leptin levels (hypoleptinemia) associated with a variety of metabolic dysfunctions, including IR, T2DM, dyslipidemia and possibly fatty liver [11]. Notably, both the US Food and Drug

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ACCEPTED MANUSCRIPT Administration (FDA) and the Japanese Pharmaceuticals and Medical Devices Agency have recently approved recombinant human methionyl leptin (metreleptin) replacement for the treatment of congenital generalized lipodystrophy on the basis of uncontrolled, non-

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randomized, open-label studies [12-14].

On the other hand, most patients with garden-variety obesity and associated comorbidities, including T2DM and NAFLD, have hyperleptinemia, presumably because of

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leptin resistance or tolerance, thereby leptin action is impaired. Despite the initial

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expectations, recombinant leptin administration in these patients has not been associated with significant weight loss or significant reduction in metabolic complications [6,9,15].

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The potential pathogenetic links and therapeutic role of leptin in NAFLD, if any, are attracting significant interest, but remain poorly elucidated. This interest for the possible

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association between leptin and NAFLD derives mainly from the large percentage of individuals with garden-variety obesity, and therefore hyperleptinemia, usually observed in NAFLD patients [3]. Experimental data provide early evidence for a potential adverse role of leptin treatment in non-lipodystrophic patients with NAFLD [6]. The aim of this review is to

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summarize evidence on the association between leptin and NAFLD with a special focus on data from human studies. A deeper insight into the interplay between leptin and NAFLD may

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lead to the design of new clinical trials aiming at elucidating the role of leptin in NAFLD, a disease which currently represents an unmet medical need [16].

2. Literature search We performed a computerized literature search using the PubMed electronic database, not limited by publication time. Studies of any design providing data for leptin, and NAFLD and/or SS and/or NASH were eligible for this review, with a special focus on clinical studies. Studies were excluded, if: they were exclusively related to diseases or conditions other than NAFLD; they had considerable patients’ overlap; and they were seriously methodologically flawed (PRISMA and MOOSE reporting guidelines were used to assess the quality of randomized controlled and observational studies, respectively). Reviews, editorials

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ACCEPTED MANUSCRIPT and letters to the editor were also generally excluded; however, limited reviews and letters were included, if they reported important information (reviews) or raw data (letters). Studies that provided comparative leptin data between NAFLD and other liver diseases were also

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included. Although clinical studies providing data on biopsy-proven NAFLD populations were preferable, in specific issues with inadequate data, non-biopsy-proven studies were included. The search followed a strategy as proposed elsewhere [17], and it is described in

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detail in supplementary material, together with search results and the selection of the studies.

3. Molecular pathways of leptin action in the liver

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Leptin acts through specific leptin receptors (LepR), which belong to the cytokine receptor class I family. Six isoforms of leptin receptor have currently been identified in rats

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(LepRa to LepRf), which are products of alternative RNA splicing of LEPR gene dividing into three classes, according to structural differences: long, short and soluble isoforms [18]. In humans only 4 (LepRa–LepRd) alternative spliced isoforms have been identified. Among LepR isoforms, only the long isoform LepRb can fully transduce activation signaling into the

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target cells, thereby mediating most of the biological effects of leptin. The soluble isoform (sLepR; previously soluble Ob receptor [sOb-R]) regulates serum leptin levels and serves as a

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carrier protein delivering leptin to its membrane receptors [18]. In hepatic cell, leptin acts mainly by activating the Janus kinase (JAK)2/ signal

transducer and activator of transcription (STAT)3 pathway. Specifically, leptin binds LepRb resulting in autophosphorylation and activation of JAK2, which leads to phosphorylation of highly conserved tyrosine residues (Tyr) located in the intracellular domain of the LepRb (Tyr985, Tyr1077 and Tyr1138) [19,20]. Phosphorylated Tyr985 mediates docking with src homology 2-domain containing inositol 5'-phosphatase (SHIP)-2 and subsequent activation of mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) pathway. Phosphorylated Tyr1077 mediates STAT5 activation. Phosphorylated Tyr1138 mediates both STAT3 and STAT5 activation. Subsequently, STAT3 activation leads to increased transcription and expression of suppressors of cytokine signaling (SOCS)-3, which

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ACCEPTED MANUSCRIPT acts as a feedback inhibitor molecule by attenuating LepRb signaling, partly by binding Tyr985 [19,20]. LepRb signaling also results in the activation of phosphatidylinositol-3 kinase

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(PI3K)/ Akt/ mammalian target of rapamycin (mTOR). Normally, leptin improves insulin sensitivity via PI3K in the liver, by suppressing hepatic glucose production [21]. Other pathways, including the 5'-adenosine monophosphate–activated protein kinase (AMPK) and

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forkhead box protein (Fox)O1, have been proposed to contribute to leptin signaling [9],

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though their specific hepatic role is not fully elucidated.

There is evidence for an overlap between hepatic leptin and insulin signaling with

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potential implications in NAFLD [22] (Figure 1). The src homology 2 domain-containing adapter protein B (SH2)-B interacts with both JAK2 and insulin receptor substrate (IRS)-1

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and IRS-2 and promotes IRS-1/2 mediated activation of PI3K in response to leptin [23]. SH2B loss-of-function mutation leads to significant metabolic defects, including hyperleptinemia, hyperinsulinemia and hepatic steatosis [24,25]. Furthermore, SOCS-3, which protects overactivation of leptin signaling under normal conditions, impairs leptin

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signaling when overexpressed, thereby playing a crucial role in leptin resistance [26]. Apart from its activation through leptin signaling itself, SOCS-3 expression can be also induced by

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insulin [27]. Reversely, overexpression of SOCS-1 and SOCS-3 in the liver causes IR, whereas their inhibition improves IR and hepatic steatosis [28]. There is also evidence that elective deletion of SOCS-3 in mice improves both IR and leptin resistance [29]. Therefore, decreasing expression of SOCS proteins in the liver offers a potential approach to prevent and/or treat hepatic steatosis and other components of IR syndrome.

4. Leptin in NAFLD: Evidence from experimental studies Leptin seems to exert a dual action on NAFLD experimental models; it may protect from hepatic steatosis, at least at the initial stages of the disease, but it may act as an inflammatory and fibrogenic factor, when the disease persists or progresses (Figure 2).

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ACCEPTED MANUSCRIPT 4.1. Leptin in hepatic steatosis Hepatic steatosis can be induced by both abnormal lipid and glucose metabolism [1]. An important role of leptin is to confine the storage of triglycerides to the adipocytes, while

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limiting triglyceride storage in non-adipose tissues, including the liver, thus protecting them from lipotoxicity and lipoapoptosis [30]. Leptin prevents hepatic steatosis in animal models by affecting both lipid and glucose metabolism. Under normal conditions, leptin suppresses

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hepatic glucose production and hepatic lipogenesis, thereby providing an insulin sensitizing

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and anti-steatotic effect [6].

Chronic central leptin infusion reduces hepatic lipogenic gene expression and

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decreases triglyceride content by stimulating hepatic sympathetic activity in animals; this function requires PI3K signaling, since impairment of leptin-mediated PI3K signaling results

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in hepatic steatosis without inducing obesity. More specifically, leptin inhibits hepatic de novo lipogenesis, whereas it stimulates fatty acid oxidation [31,32], thereby reducing lipid content in isolated livers [33] (Figure 2). Paradigms establishing the association between leptin and hepatic steatosis are the ob/ob mice and fa/fa Zucker rats: the former lack leptin,

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whereas the latter harbor a loss of function mutation in the LepR; both develop hepatic steatosis, together with IR, obesity and diabetes [34,35]. Leptin administration or liver-

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specific overexpression of LepRb, respectively, improve or prevent hepatic steatosis in both models [32,34]. Both fa/fa rats and ob/ob mice exhibit increased sensitivity to further pathogenetic “hits” (i.e., endotoxinemia), thus quickly progressing from SS to NASH [36]. Apart from the above mentioned genetically modified animal models, anti-steatotic leptin action is also observed in other animal models: in non-obese mice with uncontrolled type 1 diabetes, leptin treatment reduces both lipogenic and cholesterologenic transcription factors and enzymes, normalizes the levels of hepatic intermediary metabolites and decreases plasma and tissue lipids, in contrast to insulin monotherapy [37]. Similarly, leptin treatment corrects both plasma hyperlipidemia and hepatic steatosis in high-sucrose fed rats, similarly to chowfed controls, indicating that hepatic leptin action remains intact in these animals [38]. Notably, leptin treatment maintains its hepatic triglyceride-depleting and oxidative effect,

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ACCEPTED MANUSCRIPT even in the co-treatment with insulin in rats; thus, under normal conditions, leptin might augment the suppressive effect of insulin on hepatic very low density lipoprotein production by decreasing liver triglycerides and stimulating oxidative metabolism [39]. Nevertheless, the

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anti-steatotic effect of leptin is not observed in all animal models. Leptin treatment decreases hepatic triglyceride content in lean, but not in diet-induced obese, rats, indicating that leptin resistance seen in obesity may impair the anti-steatotic leptin action [40]. Similarly, leptin

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treatment decreases hepatic triglyceride content in young, but not old, rats. Notably, basal

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leptin levels in old rats were 25-fold higher compared with young rats, suggesting severe leptin resistance; although leptin treatment doubled circulating leptin levels, leptin failed to

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reduce hepatic triglyceride content [41]. Summarizing the aforementioned data, the antisteatotic effect of leptin may be observed in the initial stages of the disease, but when the

steatotic leptin action.

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NAFLD and/or obesity progress, the presence of leptin resistance may impair the anti-

Regarding glucose metabolism, persistent hyperglycemia (glucotoxicity) and subsequent IR play a crucial role in the pathogenesis of hepatic steatosis by affecting both

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adipose tissue and hepatic metabolism [1]. IR impairs the suppression of hormone-sensitive lipase in adipocytes, thereby increasing lipolysis and fatty acid flow from adipose tissue to the

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liver. Besides, persistently elevated glucose and hyperinsulinemia stimulate hepatic de novo lipogenesis by upregulating hepatic lipogenic transcription factors, such as sterol regulatory element-binding protein (SREBP)-1c and carbohydrate response element binding protein (ChREBP). Thus, the activity of lipogenic enzymes, such as glucokinase, fatty acid synthase, and acetyl-coenzyme A carboxylase, is enhanced. Leptin treatment decreases hepatic glucose production and normalizes fasting glucose, independently of food intake, body weight and insulin levels (Figure 2) [42]. Leptin decreases hepatic glycogenolysis [32,43,44], whereas its effect on hepatic gluconeogenesis remains conflicting: some authors reported that leptin decreases [42], others that it increases [43], while others that it has no effect on [44] hepatic gluconeogenesis. Therefore, it seems that the effect of leptin on hepatic glucose production is mainly due to the reduction in glycogenolysis.

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ACCEPTED MANUSCRIPT Apart from its direct hepatic effect, leptin may affect hepatic glucose metabolism indirectly, through its central action. Leptin infusion into the intra-cerebral ventricle in mice with type 1 diabetes duplicates the glucagon-suppressing action of the peripherally induced

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hyperleptinemia [45] In LepR-deficient Koletsky rats, adenovirally-induced expression of leptin receptors in the area of the hypothalamic arcuate nucleus improves peripheral insulin sensitivity via enhanced suppression of hepatic glucose production, but no change in insulin-

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stimulated glucose uptake or disposal [21]. This effect is associated with increased insulin

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signal transduction via PI3K in the liver and with reduced hepatic expression of the gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate kinase. Moreover, the

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beneficial effects of hypothalamic leptin signaling on hepatic insulin sensitivity are blocked by selective hepatic vagotomy, implying that hypothalamic leptin action on the liver is

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mediated via the hepatic branch of the vagus nerve [21]. Interestingly, the hypothalamic leptin action on glucose metabolism is exerted independently of its effects on energy balance and seems to be mediated by pro-opiomelaninocortin-expressing neurons, which may regulate glucose homeostasis [46].

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It should be also highlighted that apart for genetic factors, which generally convey a degree of susceptibility, external factors, especially diet and exercise, also play a significant

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role. For example, methionine restriction prevented the progression of hepatic steatosis and diminished inflammatory markers in ob/ob mice [47]. Moreover, physical inactivity leads to hepatic steatosis in male Otsuka Long-Evans Tokushima fatty (OLETF) rats, whereas regular exercise prevents hepatic steatosis by improving hepatic lipid metabolism [48].

4.2. Leptin in hepatic inflammation and fibrosis It has been hypothesized that adipokines, including leptin, may have a bidirectional role in IR and NAFLD; most alterations of adipokines during adipose tissue expansion are compensatory and aim to provide beneficial effects, but they may simultaneously provoke harmful effects [49]. In this regard, it has been proposed that leptin increases with increasing fat mass as a compensatory mechanism to limit the expansion of fat mass and preserve insulin

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ACCEPTED MANUSCRIPT sensitivity, thereby exerting an anti-steatotic effect on the liver. However, a failure of leptin to compensate for an increase of IR and steatosis beyond a certain point (i.e., when adipose tissue continues to expand) may ultimately lead to harmful effects of leptin, by acting as

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proinflammatory and profibrogenic adipokine [49]. In this regard, leptin may interact with other contributors to NAFLD progression; for example, regucalcin, which is considered to prevent the progression of NAFLD, downregulates leptin in both hepatic and adipose tissue

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[50].

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Although the liver does not produce leptin under normal conditions, activated hepatic stellate cells (HSCs) may produce leptin in vivo, thus triggering or enhancing fibrogenesis.

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Either high fat diet or chronic toxic liver injury did not upregulate collagen-I or provoke hepatic fibrosis in ob/ob mice, in contrast to their genetic controls; however, leptin

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administration reverted decreased fibrogenesis in ob/ob mice [51,52]. Similarly, thioacetamide- or xenobiotics-induced liver fibrosis was almost completely prevented in Zucker fa/fa rats; in parallel, induction of transforming growth factor (TGF)-β1 and activation of HSCs were abolished [53,54]. Furthermore, it has been proposed that activated HSCs

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express LepRb, whose activation leads to increased expression of proinflammatory and proangiogenic cytokines and growth factors, such as angiopoietin-1 and vascular endothelial

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growth factor, which affect hepatic inflammation and fibrosis [55]. Moreover, leptin may induce hepatic fibrogenesis in activated HSCs by upregulating collagen α1 [56], stimulating the production of tissue inhibitor of metalloproteinase-1 [57] and repressing matrix metalloproteinase-1 gene expression [58]. Additionally, leptin facilitates proliferation and prevents apoptosis of HSCs [59,60], and it modulates all features of the activated phenotype of HSCs in a profibrogenic manner (myofibroblastic phenotype) [61] (Figure 2). Likewise, leptin increases β-catenin levels in cultured HSCs and the β-catenin pathway contributes to leptin regulation of SREBP-1c expression in HSCs and leptin-induced liver fibrosis in mice; the effect of leptin-induced STAT3 pathway on SREBP-1c expression in HSCs may contribute to leptin-produced liver fibrosis [62]. Once activated, HSCs contribute to further leptin expression [63], thereby possibly establishing a vicious cycle [22]. Notably, curcumin,

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ACCEPTED MANUSCRIPT an antioxidant, was shown to interrupt the activation of leptin signaling, leading to HSC inactivation [64,65]. Data regarding the role of leptin on hepatic fibrosis via an effect on Kupffer or

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sinusoidal endothelial cells are currently limited. Both Kupffer and sinusoidal endothelial cells express LepRb, through which leptin upregulates the expression of matrix remodelling genes, including TGF-β1 [54] (Figure 2). Leptin may promote hepatic fibrogenesis through

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upregulation of TGF-β and connective tissue growth factor in isolated Kupffer cells; based on

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this finding a Kupffer cells - HSCs cross-talk has been proposed for hepatic fibrosis [66]. Furthermore, leptin action in macrophages of the steatotic liver triggers peroxynitrite-

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mediated oxidative stress, thereby activating Kupffer cells; leptin-mediated protein radical formation, tyrosine nitration and activation of Kupffer cells are induced by peroxynitrite

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formation [67]. In this regard, higher levels of oxidative stress-induced leptin mediated CD8+CD57+ T cells might play a significant role in NASH development. Importantly, upregulation of CD14 (an endotoxin receptor recognizing bacterial lipopolysaccharide) in Kupffer cells resulted in NASH progression in high-fed diet-induced

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steatosis mice, but not in chow-fed-control mice [68]. When recombinant leptin was administered in chow-fed mice, an upregulation of CD14 in Kupffer cell was similarly

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observed, resulting in hepatic inflammation and fibrosis without steatosis (Figure 2). On the contrary, recombinant leptin administration to ob/ob mice did not upregulate CD14 or induce inflammation and fibrosis, despite severe steatosis [68]; these results demonstrated that leptin deficiency may lead to hepatic steatosis, that leptin treatment may be protective against the progression of SS to NASH, and also that leptin in excess may favor hepatic inflammation and fibrosis. In line with the aforementioned data, it was previously speculated that leptin under normal conditions may prevent liver steatosis, while its effect on quiescent HSCs, Kupffer and sinusoidal cells is minimal. However, prolonged hyperleptinemia, commonly observed in leptin tolerance states (i.e. garden obesity), may ultimately result in overexpression of SOCS3 and in activation of HSCs, Kupffer and sinusoidal cells. Overexpressed SOCS-3 may

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ACCEPTED MANUSCRIPT aggravate both leptin tolerance and IR in hepatocytes and outweigh the primarily beneficial effect of leptin on hepatocytes. Activated HSCs, Kupffer and sinusoidal cells, which seem to

cascade, advancing progression of SS to NASH [22].

4.3. Leptin, NAFLD and hepatocellular carcinoma

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display minimal or no leptin tolerance, may trigger the proinflammatory and profibrogenic

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There is experimental evidence indicating that leptin acts as a tumor promoter in

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NASH-related hepatocellular carcinoma (HCC). Liver hyperplasia was evident at an earlier stage of NAFLD and HCC was observed in higher rates in ob/ob mice; metabolic aberrations,

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rather than cirrhosis, initiated the hepatic neoplastic process [69]. Other authors showed that HCC developed in the control, but not in Zucker fa/fa rats [70]; hepatic neovascularization

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and the expression of vascular endothelial growth factor, a potent angiogenic factor, increased only in the control rats, in parallel with fibrogenesis and carcinogenesis, implying leptin signaling as a prerequisite for the development of HCC via augmented angiogenesis and vascular invasiveness [70]. In vitro, leptin exerts a mitogenic and anti-apoptotic effect [71],

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and enhances the migration and invasiveness of HCC cells [72]. In this regard, leptin seems to have opposite effects than adiponectin on HCC [73]. Other authors showed that in vitro leptin

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upregulates telomerase reverse transcriptase expression, a known mediator of cellular immortalization, in HCC tissues [74]. They also showed that leptin affected HCC progression and invasion through its interaction with cytokines and matrix metalloproteinases in the tumorigenic microenvironment [74]. However, other authors have shown an opposite effect of leptin. Leptin seems to inhibit HCC cell growth in vitro via a p38-MAPK-dependent signaling pathway. Moreover, leptin administration induced a significant reduction in tumor size and improved survival rate in athymic nude mice transplanted with Hep3B cells, an effect possibly mediated by induction of natural killer cell proliferation and activation [75]. There is no solid explanation for this discrepancy; distinct leptin action in ob/ob mice or fa/fa rats, in which leptin is lacking or its

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ACCEPTED MANUSCRIPT signaling is impaired, compared with other animal models without impaired leptin signaling

5. Leptin in NAFLD: Evidence from clinical studies

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cannot be excluded.

Although experimental studies provide strong evidence for the anti-steatotic, but also inflammatory and fibrogenic role of leptin in NAFLD models, relevant data from clinical

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studies in NAFLD patients are inconclusive. They derived mainly from cross-sectional

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studies, while prospective cohort studies are limited. These studies may provide limited clues for interplay between leptin and NAFLD, but currently no causative relationship could be

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established.

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5.1. Data from observational studies assessing circulating leptin and LepR Data derived form clinical studies evaluating circulating leptin and/or LepR in biopsy-proven NAFLD patients [76-116] are summarized in Supplementary Table 1. Some authors reported higher leptin levels in NAFLD patients [77,85-87,96,101,107,110], whereas

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others reported similar levels in NAFLD and controls [81,82,100]. Likewise, some authors reported higher leptin levels in NASH patients than controls [78,91,101,102,106,113-116],

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whereas others similar between groups [76,79,83,89,92,103,104,108,111]. Notably, no study showed lower leptin levels in NAFLD or NASH patients than controls, or in NASH than SS patients (Supplementary Table 1); in one study, leptin was lower in NAFLD patients than controls of significantly higher body mass index (BMI), but not compared to lean controls [84]. Comparisons between SS and NASH patients provided consistently similar leptin levels [81,82,85,87,92,98,100,101,103,105,108,116]. Importantly, similar leptin between NAFLD patients and controls was observed in a study, when liver biopsy was not performed in controls, but higher leptin in NAFLD compared to biopsy-proven controls (healthy donors for liver transplantation) [79]. The results of the studies without liver biopsies in controls should be cautiously interpreted, mainly because NAFLD patients may have been included within controls (false negative results of non-invasive estimation).

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ACCEPTED MANUSCRIPT Notably, leptin levels were escalading from controls to SS and NASH in men, but not in women in a study [80], implying a potential gender-specific effect in NAFLD. Generally, higher

circulating

leptin

was

observed

in

women

than

men

with

NAFLD

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[77,78,96,98,103,117], as consistently observed in other populations. This possibly reflects the higher subcutaneous adipose mass in the former, but some authors reported higher leptin levels in women even after correction for differences in body fat composition [118]. Leptin

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levels decline significantly after menopause; based on this observation, it has been

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hypothesized that estrogen increases leptin levels, while androgens suppress them [118], but further data are required to clarify the interplay between leptin and sex steroids.

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There are also comparative data for specific histologic end-points. Regarding NAFLD activity score (NAS), the latest scoring system for NAFLD [119], leptin levels were

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increasing by increasing NAS in two studies with pediatric NAFLD populations [90,93]. Cross-sectional data regarding hepatic steatosis, inflammation and fibrosis are controversial. Leptin levels were higher in: more severe steatosis grade in some [90,96], but not all [79,94,97], studies; more severe inflammation grade in some [78,81,116], but not all

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[79,88,90] studies; more advanced fibrosis stage in some [81,90,97,105,116], but not all [78,79,88,102,103], studies. Of note, no study showed lower leptin levels in less severe

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steatosis, inflammation or fibrosis. In line with the aforementioned experimental data showing upregulation of CD14 as a mechanism mediating the effect of leptin on the progression to NASH [68], higher serum leptin and hepatic CD14 mRNA levels were observed in NASH patients (n=8) compared with BMI-matched controls (n=6); however, these findings need validation by a large-scale study [68]. Regarding sLepR, lower levels were reported in NAFLD patients than controls; furthermore, an inverse association between sLepR and circulating leptin was shown in the same study [96]. Although the authors proposed that this association may be counteracting to sLepR downregulation [96], given that sLepR is a carrier for leptin, this inverse association might amplify any positive or adverse consequences of increasing leptin. Likewise, others reported lower circulating LepRb levels in NAFLD patients than controls; LepRb levels were

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ACCEPTED MANUSCRIPT also inversely correlated with leptin levels [110]. However, other investigators reported no association of sLepR levels with NAS, hepatic steatosis, inflammation or fibrosis in children with NAFLD [90]. Finally, in a single cohort of morbidly obese patients undergoing bariatric

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surgery, sLepR levels were positively correlated with the stage of hepatic fibrosis, whereas the ratio of leptin to sLepR (free leptin index) was inversely associated to hepatic steatosis [117].

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There are controversial data comparing leptin levels between patients with NAFLD

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and chronic viral hepatitis. Some authors reported higher leptin levels in NAFLD patients than those with chronic hepatitis C (CHC) or chronic hepatitis B (CHB) [77,97]; However,

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others have reported similar leptin levels between NAFLD and CHC or CHB patients [76,86]. Of note, apart from the stage of fibrosis and the viral genotype, the extent of hepatic steatosis

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might affect the likelihood of sustained virological response (SVR) in CHC patients; leptin seems to contribute to the pathogenesis of steatosis, and increased serum leptin concentration may be an independent predictor of non-SVR. No study has evaluated leptin levels in patients with pure NASH-related cirrhosis.

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Based on studies in cirrhosis of other or mixed etiology, leptin levels were increased [120,121] in most, but not all [122], studies. The decrease in hepatic and/or renal leptin

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clearance may result in increased leptin levels, whereas cachexia due to nutritional and/or metabolic aberrations may result in low leptin levels. Therefore, leptin levels largely depend on the studied population. Given these data, further studies in pure NASH human populations are needed to clarify whether leptin may play a role in the pathogenesis of cirrhosis and portal hypertension or it is only a bystander. Experimental and clinical studies provide evidence for a potential association between NAFLD and HCC, as is reviewed elsewhere in detail [123,124]. Circulating leptin levels were shown to increase in either cirrhotic or non-cirrhotic patients with HCC of mixed etiology [125]. HCC may arise in NASH patients even in the absence of cirrhosis [126], which makes the need for prognostic indices of paramount importance. Nevertheless, clinical data linking leptin levels in pure NAFLD population and HCC do not currently exist.

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ACCEPTED MANUSCRIPT Therefore, it is essential to determine whether adipokine (including leptin) dysregulation may be prognostic for HCC in patients with NASH, thereby improving the screening and prevention of NASH-related HCC.

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The cross-sectional nature of the aforementioned clinical studies and the heterogeneity between them render any conclusion uncertain. Their controversial results might be partly attributed to differences in populations, including age (some studies refer to

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pediatric NAFLD), race, BMI (some studies refer to severely obese patients, whereas others

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to normal-weighted individuals), co-morbidity (i.e., some studies have included T2DM and liver cirrhosis patients, whereas others did not), staging and grading of the disease. Another

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important issue that adds to heterogeneity is that NASH, even if graded with the same histological system, includes patients with different duration of the disease (which remains

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largely unknown) and different disease severity, thereby being at different risk for advanced disease. Furthermore, BMI may have affected circulating leptin, thereby leptin being higher in NAFLD or NASH patients when they have higher BMI than controls (Supplementary Table 1); however, this is not a constant observation; there are studies in which leptin was

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higher in NAFLD than controls, despite similar BMI between groups [78,80,85,93,115,116]. Finally, the effect of leptin on the liver may be autocrine or paracrine and, therefore, the

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levels of circulating leptin may not reflect its hepatic or adipose tissue levels nor the effect of leptin on hepatocytes, HSC, sinusoidal cells, and Kupffer cells. Larger studies with homogenous populations and carefully matched controls are of importance.

5.2. Data from observational studies assessing leptin and LepR expression or polymorphisms Data on leptin (LEP) and leptin receptor (LEPR) gene expression and single nucleotide polymorphisms (SNPs) in NAFLD populations are summarized in Table 1. Generally, there are limited data that are further restricted by the different tissues on which genetic analysis was performed. Moreover, all existing studies are observational, thereby having limited value to show a cause-effect relationship. Especially for SNP studies, they

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ACCEPTED MANUSCRIPT usually require large sample size to achieve statistical power; however it is not usually feasible to perform liver biopsies in studies with large populations, because this is time and resource consuming and may raise ethical considerations.

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Regarding LEP expression, two studies have shown no LEP expression in hepatic tissue [79,89]. LEP expression was similar in NAFLD and controls in peripheral blood leukocytes [79] and visceral adipose tissue [127] in two studies with small sample size. In

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another study, leptin staining was higher in NAFLD than controls and correlated with serum

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leptin levels [107]; however this study was based on immunohistochemistry, thereby the source of hepatic leptin was of unknown origin. In a network analysis study, LEP-regulated

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genes were associated with hepatic steatosis [128]. Notably, carriers of the G allele of patatinlike phospholipase domain-containing 3 (PNPLA3) SNP rs738409 showed lower peripheral

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LEP expression, together with higher rates of hepatic steatosis and smaller adipocytes compared with CC genotype [129]. The findings of this study, although observational, seem to be important, since this PNPLA3 SNP has been strongly associated with liver fat accumulation and susceptibility towards more aggressive disease; in a meta-analysis, GG, as

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compared with CC homozygous, showed 73% higher hepatic fat content, 3.2-fold greater risk for higher necroinflammatory scores and 3.2-fold greater risk for developing fibrosis

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[130]. Whether this PNPLA3 SNP exerts its effect partly via affecting LEP expression, or lower LEP expression in G carriers is an epiphenomenon, remains to be elucidated. Regarding LEP SNPs, lower rates of A and higher of G allele in position -2548 of

LEP gene in NAFLD than controls was shown only in women. Nevertheless, whether this finding has a pathogenetic and/or clinical implication remains to be shown [131]. Regarding LEPR expression, two studies have shown similar hepatic LEPRa and/or LEPRb in NASH and SS or obese controls [89,92]. However, higher LEPRb expression was shown in NASH patient with than without fibrosis; LEPRb expression was further correlated with TGF-β expression, a factor playing a crucial role in fibrosis [89]. Hepatic leptin staining, on the other hand, was higher in NAFLD than controls [107], but the methodology was different in this study.

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ACCEPTED MANUSCRIPT LEPR SNPs have been investigated more than LEP SNPs. A positive association between specific SNPs has been observed in some [110,132-134], but not all [135], studies (Table 1). Remarkably, no study to-date has evaluated the same SNP; therefore, any

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conclusion for their pathogenetic role or any potential clinical importance should be cautiously interpreted, until validation by other observational and, more importantly, by prospective cohort studies. It should be also highlighted that a meta-analysis of observational

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studies did not show an overall association between LEPR SNPs and overweightness [136];

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although obesity and NAFLD are closely related, this meta-analysis did not exclude an

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association between LEPR SNPs and NAFLD, independently from obesity.

5.3. Data from longitudinal studies assessing the natural course of NAFLD

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Evidence from longitudinal cohort studies providing data for leptin changes when NAFLD progresses or regresses overtime may more effectively focus on the pathophysiologic interplay between leptin and NAFLD, but is limited, mainly because of the need for repeat liver biopsies, which usually result in high drop-out rate.

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In an early study, circulating leptin was not significantly different between patients who had fibrosis progression (n=10) and those who did not (n=29) after a mean follow up of

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28 months (range 12–86) [81]. BMI also remained essentially unchanged during the followup. However, a second liver biopsy was performed in 39 of the 88 initially recruited patients [81]. In a 3-year prospective study with paired-biopsies, serum leptin levels decreased in the total cohort at the end of the study, whereas BMI remained essentially the same. Notably, at the end of the study, leptin decreased more in patients with stable or improved disease compared with those with worsening disease; however leptin change could not predict the disease progression or fibrosis independently from BMI change, which followed a similar pattern [137]. A given advantage of this study is the low drop-out rate (2 of 54). In another 7year prospective study, individuals without NAFLD at baseline (n=147) who developed NAFLD at 7 years (n=28) had higher baseline leptin levels, as well as BMI and waist circumference. However, among individuals with NAFLD at baseline (n=66), leptin levels

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ACCEPTED MANUSCRIPT were similar in those with or without disease remission [138]. A limitation of this study was its non-invasive nature (NAFLD was not biopsy-proven). Further prospective studies with

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paired-biopsies and long-term follow-up are needed.

5.4. Interventional studies assessing the effect of diet and exercise on leptin in NAFLD patients

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Circulating leptin levels decrease in parallel with BMI after successful weight loss

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after bariatric surgery [139,140] or exercise alone [141] or interdisciplinary interventions [142-146] (Table 2). Leptin levels decreased only if substantial weight loss was achieved,

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whereas increased in those who did not receive intervention [146]. Importantly, changes in circulating leptin were correlated with changes in both aspartate and alanine transaminase in

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children with or without ultrasound-proven NAFLD [146]. In one study, a decrease in circulating leptin was accompanied by a decrease in hepatic LepR expression, whereas hepatic LEP expression was unaffected by weight loss [139]. The combination of diet and exercise for three months decreased circulating leptin and BMI more than diet alone [145]. In

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another prospective study, the combination of aerobic and resistance exercise for 12 weeks decreased leptin levels in NAFLD patients more than aerobic exercise alone, although BMI

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was similarly affected [141]. In a cross-sectional study, fewer NAFLD patients were engaged in resistance training compared to controls, whereas the rates of those engaged in aerobic exercise were similar [147]. In the same study, men who engaged in exercise at least once weekly had lower leptin levels than those engaged less than once weekly, changes that were generally in parallel with BMI changes [147]. However, NAFLD was biopsy-confirmed in only two of these studies [139,140], whereas paired biopsies were performed in only one [139]. All, except for one [146], studies are further limited by the small sample size and relative short duration of interventions. Therefore, large-scale prospective studies with paired biopsies are required to investigate whether changes in leptin achieved after weight loss are associated with changes in hepatic histology and may affect NAFLD progression, or leptin is only a gauge of weight loss.

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ACCEPTED MANUSCRIPT 5.5. Interventional studies assessing the effect of medications on leptin in NAFLD patients There are only sporadic data on the effect of various therapeutic interventions on circulating leptin levels in NAFLD patients, which are summarized in Table 3; all these

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studies are characterized by small sample size; some of them are further limited by the absence of a control group and/or the high drop-out rates. Notably, none of these studies had serum leptin as the main end-point.

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Regarding anti-diabetic medications, circulating leptin decreased equally after

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metformin was added vs. not added to lifestyle modification [148]. Leptin levels were increased (in parallel with BMI) after pioglitazone treatment in one study [149], without

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being associated with histological changes. However, other authors reported that they remained unaffected after pioglitazone [150] or rosiglitazone [151,152] treatment. It should

studies [150,152].

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be highlighted that leptin levels remained unaffected despite increase in BMI in two of these

Anti-lipidemic medications, including atorvastatin [153], ezetimibe [154] and probucol [155], had no effect on circulating leptin levels. Regarding other medications

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investigated in NAFLD patients, ursodeoxycholic acid with or without vitamin E did not affect leptin levels or BMI, despite an improvement in hepatic steatosis [156]. On the other

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hand, short-term melatonin administration resulted in an increase in leptin levels, but it is unknown whether it was accompanied by a histological improvement, because hepatic biopsies were not repeated [114]. Finally, cysteamine administration in overweight children with NAFLD did not affect leptin levels in the total cohort, but it decreased leptin in a small subgroup of responders to treatment [157]. To the best of our knowledge, there are no data for the effect of other medications investigated for NAFLD on leptin levels, including older and newer anti-obesity drugs (orlistat, sibutramine, rimonabant, phentermine/topiramate, lorcaserin), other anti-diabetic medications (including glucagon-like peptide-1 analogues, dipeptidyl peptidase-4 inhibitors), other statins (including simvastatin, rosuvastatin and pitavastatin), fibrates, omega-3 fatty acids, anti-hypertensive, and anti-oxidative medications. Similarly to weight loss studies,

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ACCEPTED MANUSCRIPT investigating the potential association between post-treatment changes in leptin and changes in hepatic histology is of paramount importance in clinical terms.

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5.6. Interventional studies assessing the effect of recombinant leptin on hepatic imaging or histology

To the best of our knowledge, no study has to-date investigated or plans to investigate

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the effects of recombinant leptin administration on NAFLD patients with normal or high

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leptin levels. For this reason, evidence could be indirectly derived from studies with patients with lipodystrophy and NAFLD [13,158-161], which are summarized in Table 4. The main

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characteristic of all these studies is the lack of control group and the small sample size, which was under or equal to 10 in all but one study, and was further decreased by drop-outs or lack

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of availability of data, in some cases [161]. Generally, metreleptin treatment decreased hepatic volume and steatosis, but had no effect on inflammation and fibrosis. In one case report, no change in hepatic histology was reported after 24 months of metreleptin treatment [160]. This discrepancy might be attributed, at least partly, to low compliance reported in this

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case, or the coexistence of autoimmune hepatitis reported in patients with acquired generalized lipodystrophy. Clinical results are generally similar to those shown by Imago et

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al. in an experimental model of NASH [68], indicating that recombinant leptin administration in patients with lipodystrophy and NAFLD may possibly improve steatosis, but not fibrosis.

6. Future perspectives Despite the initial enthusiasm which followed leptin discovery and the expectations for a drug targeting obesity and related disorders, 20-year of research have provided evidence for limited use of recombinant leptin in clinical practice, i.e., in patients with leptin deficiency or lipoatrophy. Mainly experimental studies indicate that leptin administration in NAFLD patients with normoleptinemia or hyperleptinemia might have adverse effect on liver fibrosis, thereby possibly worsening the prognosis of liver disease. There is no ongoing study evaluating the effect of recombinant leptin administration in NAFLD patients with

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ACCEPTED MANUSCRIPT normoleptinemia or hyperleptinemia. On the contrary, NAFLD patients with hypoleptinemia might possibly benefit from leptin replacement, although this remains to be fully proven. Importantly, the frequency of NAFLD patients with hypoleptinemia remains largely

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unknown, but is expected to represent only a small minority. Currently, there are two ongoing clinical trials investigating the effect of metreleptin administration on hepatic histology of NAFLD patients with hypoleptinemia. In the first study (single-group, prospective, open-

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label), metreleptin (0.1 mg/kg/day) is expected to be administered for one year to 10 adult

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males with NASH and hypoleptinemia, but without lipodystrophy (NCT00596934). In the second study (single-group, prospective, open-label), metreleptin (0.1 mg/kg/day) is expected

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to be administered for one year to 20 patients with SS or NASH and lipodystrophy (NCT01679197). Whether these open-label studies could provide proof of concept is doubtful

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given the lack of control groups.

Recently, a new leptin analogue (7i) was shown to exert beneficial effect on body weight and hepatic steatosis in a mouse model of diet-induced obesity [162]. In this regard, novel leptin analogues preserving the anti-steatotic, but lacking the fibrogenic leptin action

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would be of paramount importance. Thus, leptin might exert its beneficial effect on steatosis by acting on hepatocytes without acting on HSCs, Kupffer or sinusoidal endothelial cells.

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Another issue for future research is a potential dose-related difference in leptin actions on various tissues, including liver cells. According to this speculation, leptin may exert hepatoprotective action, when it is subtly increased, by acting mainly on hepatocytes, whereas it may exert an inflammatory and fibrogenic action, when it is further increased, by acting on HSCs, Kupffer, and sinusoidal cells. In this respect, it should be investigated whether hepatocytes, HSCs, Kupffer, and sinusoidal cells have distinct degrees of leptin resistance, which may also explain the potential dual leptin action. It would be also proposed that pharmaceutical trials targeting NAFLD histology include circulating leptin and/or hepatic leptin expression in their secondary end-points, which would help to clarify any association between histological endpoints and changes in leptin.

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ACCEPTED MANUSCRIPT Moreover, early evidence for pathogenetic relationship between leptin and irisin in NAFLD warrants further research on this potential pathogenetic triangle. We have recently shown that irisin tends to follow a pattern similar to leptin in histological endpoints of

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patients with NAFLD [116]. It would be important to clarify whether both irisin and leptin have a common denominator or they may affect each other with certain pathophysiologic and potentially therapeutic implications.

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Finally, well-designed prospective cohort studies evaluating the prognostic value of

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leptin during the natural course of NAFLD would be of value.

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7. Conclusions

Leptin seems to display a potential dual action on NAFLD experimental models

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exerting anti-steatotic, but also proinflammatory, profibrogenic, and possibly oncogenic actions. On the other hand, relative evidence is not as clear in human studies, partly because of poorly controlled studies and poor understanding of central and peripheral leptin actions. In this regard, data from observational clinical studies are conflicting. Leptin levels have been

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shown to be higher or similar in SS or NASH compared with controls, whereas virtually no study reported higher levels in the controls. Furthermore, clinical data on leptin levels in

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NASH-related cirrhosis or HCC are scarce. Interventional studies showed that circulating leptin levels decrease together with BMI after successful weight loss, following exercise, interdisciplinary interventions or bariatric surgery. Studies providing evidence for the effect of medications on leptin levels in NAFLD populations are limited and of low power. Although experimental data should be cautiously extrapolated to humans, given the anatomical, biochemical and physiological differences between animals and humans, they do not warrant leptin administration in NAFLD patients with normoleptinemia or hyperleptinemia. On the other hand, recombinant leptin administration in lipodystrophic NAFLD patients with hypoleptinemia resulted in generally beneficial effects on steatosis, although not on fibrosis, in uncontrolled studies of limited size, power and thus significance. Nevertheless, further studies are needed to elucidate the long-term efficacy and safety of

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ACCEPTED MANUSCRIPT recombinant leptin on hepatic parameters, especially fibrosis, in patients with NAFLD and

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relative hypoleptinemia.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. SOCS3 as a potential suppressor of both hepatic leptin and insulin signaling. In hepatocytes, leptin acts mainly by activating the JAK2/STAT3 pathway; STAT3 dimers

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translocate into the nucleus and affect the transcription of SOCS3, which acts as a negative feedback, protecting overactivation of leptin signaling under normal conditions. However, when SOCS3 is overexpressed, it impairs leptin signaling, thereby inducing leptin resistance.

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SOCS3 expression may also be induced by insulin. Reversely, overexpression of SOCS3 in

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the hepatocyte causes insulin resistance by impairing IRS action. As a consequence, elective deletion of SOCS3 in mice improved both IR and leptin resistance. Other leptin pathways are

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omitted in this figure, so as to be focused on the SOCS3.

Abbreviations: IRS, insulin receptor substrate; JAK, janus kinase; LepR, leptin receptor; Rec,

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receptor; SOCS, suppressors of cytokine signaling; STAT, signal transducer and activator of transcription

Figure 2. A hypothesis on the dual role of leptin in the pathogenesis of nonalcoholic fatty

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liver disease. By acting on hepatocytes, leptin affects both lipid and glucose metabolism; it inhibits hepatic de novo lipogenesis, whereas it stimulates FFAs oxidation, thereby decreasing

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hepatic triglyceride content, lipotoxicosis and lipoapoptosis. Furthermore, leptin suppresses hepatic glycogenolysis and increases gluconeogenesis, thereby decreasing hepatic glucose production and glucotoxicosis. Thus, leptin seems to have an anti-steatotic action. On the other hand, by acting on SECs and Kupffer cells, leptin upregulates TGF-β1, as well as other matrix remodelling enzymes. Moreover, leptin upregulates CD14 in Kupffer cells, which renders them susceptible to other stimuli, like LPS, and increases oxidative stress. By acting on HSCs, leptin contributes to their activation; activated HSCs produces TGF-β1, angiopoietin-1, VEGF and collagen-I, all of which contribute to enhance inflammation and hepatic fibrosis. Finally, activated HSCs seem to produce leptin themselves, which stimulates HSCs proliferation and prevents their apoptosis, thereby establishing a vicious cycle. Nevertheless, this hypothesis has not been validated in humans yet.

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ACCEPTED MANUSCRIPT Abbreviations: FFA, free fatty acid; HSC, hepatic stellate cell; LepR, leptin receptor; LPS, lipopolysaccharide; SEC, sinusoidal endothelial cell; TGF, transforming growth factor;

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VEGF, vascular endothelial growth factor

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ACCEPTED MANUSCRIPT Funding: No sources of financial support for this study.

Disclosure statement: SAP and JK: No conflict of interest; CSM has served as consultant for

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Astra Zeneca and had received research support through his Institution from Amgen.

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Table 1. Main findings of clinical observational studies evaluating LEP or LEPR gene expression, and LEP or LEPR gene SNPs in NAFLD populations. Method (SNP, Liver Reference* Design Tissue Populations (n) Main findings when needed) biopsy LEP expression Hepatic and 1) No LEP expression in the hepatic tissue. Chalasani, CrossNASH (5) and SS PCR peripheral blood Yes 2) Similar LEP expression between groups in 2003 [79] sectional patients (5) leukocytes leukocytes. Obese patients with Baranova, CrossVisceral adipose Similar LEP expression between groups of low and PCR NAFLD with (11) Partly (21) 2006 [127] sectional tissue high IR. and without (10) IR PCR and NASH patients (55) 1) No LEP expression in the hepatic tissue. Cayon, 2006 Crossimmunohistoche Hepatic tissue and SS or obese Yes 2) Leptin staining was higher in NASH with fibrosis [89] sectional mistry controls (35) than SS. Hepatic and Obese NASH patients Network analysis showed an association between Baranova, CrossMicroarrays visceral adipose (27), obese (7) and Yes leptin-TNFα regulated genes and steatosis in hepatic 2007 [128] sectional tissue lean (6) controls tissue, but not adipose tissue. Obese children and Santoro, CrossSubcutaneous Carriers of the G allele of PNPLA3 SNP rs738409 PCR adolescents with and Partly (n=6) 2010 [129] sectional adipose tissue showed lower LEP expression. without NAFLD (18) 1) Higher LEP staining in NAFLD than controls. Xu, 2011 CrossImmunohistoch NAFLD patients (30) Hepatic tissue Yes 2) Positive correlation between serum and hepatic [107] sectional emistry and controls (30) leptin. LEP SNPs Zhou, 2010 CrossPCR (position Peripheral blood NAFLD patients (50) Lower rates of A and higher of G allele in NAFLD No [131] sectional 2548) leukocytes and controls (50) women (but not men) than controls LEPR expression 1) Similar LEPRb expression between groups. PCR and NASH patients (55) 2) Higher LEPRb expression in NASH patient with Cayon, 2006 Crossimmunohistoche Hepatic tissue and SS or obese Yes than without fibrosis. [89] sectional mistry controls (35) 3) LEPRb expression was positively correlated with TGF-β expression. Le, 2007 CrossNASH (21) and SS Similar hepatic LEPRa and LEPRb between SS and PCR Hepatic tissue Yes [92] sectional (10) patients NASH Xu, 2011 CrossImmunohistoch Hepatic tissue NAFLD patients (30) Yes Higher LEPR staining in NAFLD than controls.

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sectional

emistry

and controls (30)

Chen, 2006 [135]

Crosssectional

PCR (Lys109Arg)

Peripheral blood leukocytes

Lu, 2008 [132]

Crosssectional

PCR (G3057A)

Peripheral blood leukocytes

Aller, 2011 [133]

Crosssectional

PCR (Lys656Asn)

Peripheral blood leukocytes

NAFLD patients (76)

Yes

Swellam, 2012 [110]

Crosssectional

PCR (rs6700896)

Peripheral blood leukocytes

Obese NAFLD (90) and lean controls (30)

No

Higher rates of NAFLD in the carriers of mutant allele.

Partly (not in controls)

1) Higher rates of G allele of both SNPs in NASH than controls. 2) Higher rates of G allele of rs1137100, but not rs1137101 in SS than controls. 3) Similar rates rates of G allele of both SNPs in SS and NASH. 4) The presence of G allele of rs1137100, but not rs1137101, was inversely associated with fibrosis (F≥2), but not steatosis or inflammation. 5) The combination of rs1137100 and PNPLA3 SNP rs738409 further increases the risk for NAFLD. 6) Race differences were observed.

No genotype difference between patients and controls

RI

No

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PCR (rs1137100 and rs1137101)

Peripheral blood leukocytes

Patients with NASH (111), SS (33) and controls (144)

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Zain, 2013 [134]

1) Higher rates of A and lower of G alleles in T2DM with NAFLD than NGT. 2) Similar rates of alleles between T2DM with and without NAFLD. 1) Lys656Asn/Asn656Asn had higher BMI, fat mass, glucose and HOMA-IR, but similar leptin, than Lys656Lys genotype. 2) No difference in steatosis, inflammation and fibrosis between different genotypes.

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NAFLD patients (n=117) and controls (n=63) T2DM patients with (104) or without (112) NAFLD, and NTG (108)

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[107] LEPR SNPs

(*) References are presented, first, in gene expression or SNP order and, second, in publication order. Abbreviations: F, fibrosis stage; HOMA-IR, homeostasis model of assessment IR; IR, insulin resistance; LEP, leptin gene; LEPR, leptin receptor gene; NAFLD, nonalcoholic fatty liver disease; NASH, steatohepatitis; NGT, normal glucose tolerance; PCR; polymerase chain reaction; PNPLA3, patatin-like phospholipase domain-containing 3; SNP, single nucleotide polymorphisms; SS, simple steatosis; T2DM, type 2 diabetes mellitus; TGF, transforming growth factor; TNF, tumor necrosis factor.

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Change in histology

Additional information

Yes

12

Decreased

Decrease in steatosis, but not inflammation or fibrosis

1) BMI decreased. 2) Paired biopsy in 60%. 3) Hepatic leptin expression unaffected. 4) Hepatic LepR expression decreased.

Yes

18

Decreased similarly in SS and NASH

No repeat biopsy

1) BMI decreased. 2) Drop-out 32%.

12

Decreased more after combined aerobic + resistance training

No biopsy was performed

BMI decreased equally between groups.

No

12

Decreased only if substantial weight loss/ Increased in control group

No biopsy was performed

BMI changes resembles leptin changes.

Obese children without NAFLD (22)

No

12

Decreased only in NAFLD group

No biopsy was performed

BMI decreased in both groups.

No prospective control group

No

12

Decreased only in children who lost weight

No biopsy was performed

BMI changes resembles leptin changes.

Obese adolesents without NAFLD

No

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Decreased in both groups

No biopsy was performed

BMI decreased in both groups.

Cohort

Bariatric surgery

Morbidly obese with NAFLD (30)

No control group

Felipo, 2013 [140]

Cohort

Bariatric surgery

Morbidly obese with SS (29) or NASH (18)

No prospective control group

Cohort with comparator arm

Aerobic ± resistance training

Obese adolescents with NAFLD (28)

Obese adolescents without NAFLD (30)

Diet + exercise + psychological support Diet + exercise + psychological support Diet + exercise + psychological support Diet + exercise +

Obese children with and without NAFLD (130)

Obese children with and without NAFLD/ no intervention (50)

ED

Moschen, 2009 [139]

Cohort with comparator arm

Campos, 2012 [142]

Cohort with comparator arm

Reinehr, 2012 [143]

Cohort

Sanches, 2014 [144]

Cohort with comparator

Obese children with NAFLD (18) Obese children with and without NAFLD (16) Obese adolesents with

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Reinehr, 2009 [146]

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Interdisciplinary interventions

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Exercise De Piano, 2012 [141]

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Table 2. The impact of various interventions targeting weight loss on serum leptin levels in clinical studies with NAFLD patients. Durati BiopsyIntervention Control on Change in serum Reference* Design Patients (n) proven (dose) intervention (n) (month leptin NAFLD s) Bariatric surgery

No

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NAFLD (33)

(46)

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arm

Cohort with Obese men with Decreased more BMI decreased more Diet + Obese men with No biopsy was comparator NAFLD/ Diet alone No 3 after combined after combined diet + exercise NAFLD (na) performed arm (na) diet + exercise exercise (*) References are presented, first, in type of intervention order and, second, in publication order. Abbreviations: BMI, body mass index; na, not available; LepR, leptin receptor; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; SS, simple steatosis.

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Table 3. The impact of pharmaceutical interventions on serum leptin levels in clinical studies with NAFLD patients. Durati BiopsyIntervention Control on Reference* Design Patients (n) proven (dose) intervention (n) (month NAFLD s) Anti-diabetic medications Obese adults Nar, 2009 Randomized, Metformin Lifestyle with T2DM and No 6 [148] open label (1700 mg/d) intervention (15) NAFLD (19)

Change in histology

Additional information

Decrease in both groups

No biopsy was performed

BMI decreased in both groups.

12

Unchanged

Decrease in steatosis, inflammation and fibrosis

1) Leptin did not correlate with histological improvement. 2) BMI increased.

Yes

12

Increase in pioglitazone group

Yes

12

Unchanged

Yes

6

Unchanged

Histological results were not reported

Weight was reported unchanged.

Decrease in steatosis, but not inflammation or fibrosis

1) Leptin changes were not correlated with steatosis changes. 2) BMI unchanged.

Open label

Pioglitazone (30 mg/d)

NASH (18)

No control group

Aithal, 2008 [149]

Double-blind RCT

Pioglitazone (30 mg/d)

Non-diabetic NASH (31)

Placebo (30)

Ratziu, 2008 [152]

Double-blind RCT

Rosiglitazone (8 mg/d)

NASH (32)

Placebo (31)

Rosiglitazone (4 mg/d)

NAFLD (27)

No prospective control group

NASH with hyperlipidemia (31)

Hyogo, 2008 [153]

Open label

Atrovastatin (10 mg/d)

Park, 2011 [154]

Open label

Ezetimibe (10 mg/d)

Ishitobi, 2013 [155]

Open label

Probucol (500 mg)

PT

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SaryuszWolska, 2011 Open label [151] Anti-lipidemic medications

Yes

ED

Lutchman, 2006 [150]

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Change in serum leptin

No control group

Yes

24

Unchanged

NAFLD (45)

No control group

Yes

24

Unchanged

NASH with hyperlipidemia (26)

No control group

Yes

12

Unchanged

No decrease in steatosis, inflammation or fibrosis Decrease in steatosis, but not inflammation or fibrosis

Decrease in steatosis, and inflammation, but not fibrosis No decrease in steatosis, inflammation or fibrosis

Weight increased in pioglitazone group. Weight increased in rosiglitazone group.

BMI unchanged.

BMI unchanged.

Other medications

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Cysteamine (0.6-2.0 g/d)

Children with NAFLD (13)

No control group

Gonciarz, 2013 [114]

Open label

Melatonin (10 mg/d)

Overweight with NASH (16)

No prospective control group

Yes

No repeat biopsy

Increased

No repeat biopsy

PT

Open label

6

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Dohil, 2011 [157]

1) Totally unchanged 2) Decreased in responders (n=7)

Yes

1

1) 11 children completed the study. 2) ALT was normalized or reduced >50% in 7/11 children. 3) BMI unchanged. 1) Decrease in HOMAIR and liver function tests. 2) BMI change not reported.

1) UCDA (12-14 Decrease in steatosis; mg/kg/d)/ Placebo Balmer, 2009 inflammation and Open label NASH (14) (14) Yes 24 Unchanged BMI unchanged. [156] fibrosis are not 2) Placebo/ Placebo reported (13) (*) References are presented, first, in medication order and, second, in publication order. Abbreviations: ALT, alanine transferase; BMI, body mass index; na, not available; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; RCT, randomized controlled trial; T2DM, diabetes mellitus type 2; UDCA, ursodeoxycholic acid.

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UCDA (12-14 mg/kg/d)/ Vitamin E (800/d)

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Table 4. The impact of recombinant leptin replacement on hepatic histology or MR imaging in clinical studies with patients with inherited or acquired lipodystrophy. BiopsyIntervention Control Duration Reference* Design Patients (n) proven Change in histology or MR imaging Additional information (dose) intervention (n) (months) NAFLD Metreleptin Inherited or Oral, 2002 Decrease in hepatic volume associated Open label (0.015-0.06 acquired No control group No 4 No liver biopsy. [13] with decrease in liver function tests. mg/kg/day) lipodystrophy (9) 1) Decrease in hepatic volume. Inherited or Metreleptin 2) Decrease in incidence of NASH. Repeat biopsy in 100% Javor, 2005 acquired Open label (0.015-0.06 No control group Yes 6.6 ± 1.4 3) Improvement of steatosis and (n=10). [158] lipodystrophy and mg/kg/day) ballooning. NAFLD (10) 4) No effect on fibrosis 1) Decrease in hepatic volume. 1) Repeat biopsy in both Acquired Metreleptin 36 (patient 2) Resolution of NAFLD (patient #1). patients. Park, 2008 generalized Case report (0.015-0.08 No control group Yes #1) & 14 3) Deterioration of hepatic 2) Worsening of pre-existing [159] lipodystrophy and mg/kg/day) (patient #2) inflammation (patient #2; possible coproteinuria (patient #2; died T1DM (2) existence of autoimmune hepatitis). of renal failure). Acquired Metreleptin Kamran, 2012 generalized Case report (0.06 No control group Yes 60 Unchanged (month 24) Repeat biopsy (at month 24) [160] lipodystrophy and mg/kg/day) NASH (1) 1) Decrease in incidence of NASH. Inherited or Metreleptin 2) Improvement in steatosis, ballooning Repeat biopsy in 54% Safar Zadeh, acquired Open label (0.06-0.24 No control group Yes 25.8 ± 3.7 and NAS. (n=27). 2013 [161] ** lipodystrophy and mg/kg/day) 3) No effect on lobular or portal NAFLD (50) inflammation, and fibrosis. (*) References are presented in publication order. (**) The Safar Zadeh et al. study includes longer-term data of 6/10 patients previously described by Javor et al. study. Abbreviations: BMI, body mass index; MR, magnetic resonance; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; T1DM, diabetes mellitus type 1; RCT, randomized controlled trial.

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Figure 1

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Figure 2

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