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The Effects of Hypoglycemic Agents on Non-alcoholic Fatty Liver Disease: Focused on Sodium-Glucose Cotransporter 2 Inhibitors and Glucagon-Like Peptide-1 Receptor Agonists

The only known, effective intervention for non-alcoholic fatty liver disease (NAFLD) is weight loss, and there is no approved pharmacotherapy. Recently, new hypoglycemic agents, such as sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 receptor agonists (GLP-1RAs), and their effects on NAFLD have received substantial interest. Herein, we review the currently available human studies regarding the effects of SGLT2 inhibitors and GLP-1RAs on NAFLD/non-alcoholic steatohepatitis in patients with type 2 diabetes mellitus, and we describe the possible mechanisms explaining the positive effects of these agents on NAFLD.


INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease worldwide, is prevalent in patients with type 2 diabetes mellitus (T2DM). 1,2 NAFLD progresses to steatohepatitis, fibrosis, and cirrhosis. 3 In addition, those with T2DM and NAFLD show a poor hepatic prognosis and have increased risk of cardiovascular disease. 4,5 The only known, effective treatment for NAFLD is weight loss, and there is no approved pharmacotherapy. Because NAFLD and T2DM share pathophysiologic features, such as insulin resistance, hypoglycemic agents, especially pioglitazone, have been evaluated for effectiveness in NAFLD. 6 There is substantial growing interest in the effects of new hypoglycemic agents, such as sodium-glucose cotransporter 2 NAFLD and non-alcoholic steatohepatitis (NASH). 7,8 To date, the effects of SGLT2 inhibitors and GLP-1RAs on hepatic steatosis or fibrosis have been largely investigated with the following information: NAFLD biochemical markers such as alanine aminotransferase (ALT), serum fibrotic markers such as ferritin, and radiologic findings evaluating hepatic fat content (ultrasonography, computed tomography [CT], magnetic resonance imaging [MRI], and transient elastography [so-called FibroScan]). Histologic evaluation has been relatively scarce until now.
The high prevalence and impact on public health of NAFLD and T2DM indicate the need for investigation of the effects of new hypoglycemic agents on NAFLD in patients with T2DM. In this review, we focus on recent human studies regarding the effects of SGLT2 inhibitors and GLP-1RAs on NAFLD/NASH in patients with T2DM, and we describe possible mechanisms to explain the positive effects of these agents on NAFLD.

SGLT2 INHIBITORS
Several studies have reported that SGLT2 inhibitors can inhibit the development of NAFLD/NASH and improve histological hepatic steatosis or steatohepatitis in experimental animal models with T2DM. [9][10][11] On the other hand, published articles on the effects of SGLT2 inhibitors on NAFLD/NASH in human were scarce until 2017, and the majority of human studies was conducted in Japan and recently published in 2018. Many reports regarding the improvement in liver enzymes after treatment with SGLT2 inhibitors have been published, whereas only a few studies have attempted to evaluate the role of SGLT2 inhibitors on liver inflammation and fibrosis histologically in T2DM patients with biopsyconfirmed NAFLD or NASH.

Dapagliflozin
In a randomized controlled trial (RCT) by Shimizu et al. 7 , 57 patients with NAFLD and T2DM were divided into two groups, dapagliflozin 5 mg/day group or the standard treatment without SGLT2 inhibitors group, and treated for 24 weeks. The diagnosis of NAFLD was based on persistent elevation of ALT above the normal limit and fatty liver on ultrasonography. Transient elastography (FibroScan) was performed to assess liver steatosis and fibro-sis. In week 24, only the dapagliflozin group demonstrated a significant decrease in the following: steatosis from FibroScan, serum ALT, γ-glutamyl transpeptidase (γGT) level, and visceral fat mass.
In addition, 14 patients with significant liver fibrosis underwent significantly decreased liver stiffness after dapagliflozin treatment.
This was the first study to show the effect of dapagliflozin on hepatic fibrosis using FibroScan.
One large retrospective study using a Canadian diabetes register investigated the effects of dapagliflozin and canagliflozin (SGLT2 duction than patients in the DPP4 inhibitors group. In addition, the proportion of patients with ALT normalization after treatment was significantly higher in the dapagliflozin group, and the result was consistent even after adjustments for weight loss.

Empagliflozin
The recently published EMPA-REG OUTCOME trial, a study including pooled data from four 24-week placebo-controlled trials, and a trial of empagliflozin vs. glimepiride over 104 weeks investigated the effect of empagliflozin on liver enzymes in patients with T2DM. 16 The adjusted mean difference in change of ALT was −3. A prospective, nonrandomized, single-arm study by Inoue et al. 19 evaluated the effect of 12 months of treatment with canagliflozin 100 mg on biochemical markers, body composition measured by bioelectrical impedance analysis, and MRI-derived hepatic fat fraction (HFF). HFF decreased from a baseline of 17.6% ± 7.5% to 12.1% ± 6.1% after 12 months. In addition, liver enzymes and type IV collagen level were reduced.
In a study by Seko et al. 20 , 10 T2DM patients with biopsy-con-firmed NASH (hepatic fibrosis stage 1-3) received canagliflozin 100 mg for 12 weeks. The primary end point was change in serum ALT level. The change in ALT from baseline to week 12 was −23.9 IU/L. There were also significant improvements in AST and fibrosis-4 index. A different tendency in ALT-change between the stage 1 and stage 2/3 groups was found; the degree of ALT-lowering was greater in the stage 1 group, representing the early stages of NASH, than in the stage 2/3 group.
A 24-week trial by Cusi et al. 21 showed that treatment with canagliflozin 300 mg decreased intrahepatic triglyceride measured by proton-magnetic resonance spectroscopy compared to placebo (-6.9% vs. -3.8%, respectively), and the reduction was strongly correlated with extent of weight loss. T2DM. The authors found that ipragliflozin showed equivalent beneficial effects with pioglitazone on NAFLD during the 24-week trial period. Ipragliflozin ameliorated hepatic steatosis, which was evaluated using the liver-to-spleen ratio on CT, and reduced serum aminotransferase level. Additionally, a case report described the amelioration of liver biopsy-proven histological findings in a patient with NASH and T2DM after administration of ipragliflozin. 25

Weight-or visceral fat-dependent effects (related to reduction of body weight or visceral fat)
Glycosuria by SGLT2 inhibitors is associated with weight loss, mainly fat mass loss. 14 According to a previous meta-analysis, weight loss above 5% improved hepatic steatosis, and losses above 7% also improved histological features of NAFLD. 26 Weight loss and improvement of insulin resistance by SGLT2 inhibitors may ameliorate liver steatosis. 11,18 SGLT2 inhibitors can improve insulin resistance and lipotoxicity and thereby decrease hepatocyte apoptosis and inflammation.

Improvement of glucose control
SGLT2 inhibitors promote glycosuria, which decreases blood glucose level and ameliorates insulin resistance. Glycemic control downregulates carbohydrate responsive element-binding protein, a transcription factor responsible for activating fatty acid synthesis. 27 Amelioration of insulin resistance by glucose control results in downregulation of sterol regulatory element-binding protein 1c, a transcription factor related to lipid production and blockage of de novo lipogenesis. 28

Weight-and glucose-independent effects
Several studies showed that improvement of steatosis was largely independent of changes in body weight, visceral fat mass, or glycated hemoglobin. 7,16,23 SGLT2 inhibitors seem to have a beneficial effect on NAFLD/NASH through weight-and glucose-independent mechanisms, such as reduction in inflammatory markers, decrease in oxidative stress, decrease in lipogenesis, and increase in oxidation of free fatty acids (FFAs). 29

Inhibition of de novo lipogenesis in the liver
Previous studies using a rat/mice model of NAFLD/NASH with diabetes demonstrated that both decrease of de novo lipogenesis and increase of fatty acid β-oxidation play a role in improvement of hepatic steatosis by SGLT2 inhibitors. 9,23 Glucagon effect In addition to its effects on glucose metabolism, glucagon is also known to contribute to lipid metabolism. Glucagon action is important for multiple pathways regulating lipid metabolism and leading to decreased lipogenesis. 30 Decreased glucagon action was associated with development of fatty liver, and exogenous glucagon administration reduced fatty liver in human and animal studies. 31,32 In addition, improvement in glucagon-to-insulin ratio (GI ratio) or use of SGLT2 inhibitors can ultimately increase ketogenesis in the liver. 33 Ketogenesis can protect against steatosis. 34 The above-mentioned studies by Bajaj et al. 12 and Choi et al. 15 showed that SGLT2 inhibitors were associated with considerably greater reduction in ALT than incretin therapies, independent of weight and glycated hemoglobin change. SGLT2 inhibitors induce increased plasma glucagon level; although the precise mechanism is yet known, glycosuria's glucose lowering effect may contribute.
Hansen et al. 35 and Ferrannini et al. 36 explained that the opposing effects of SGLT2 inhibitors and incretin agents on the GI ratio may explain the different impacts of the two drugs on NAFLD. SGLT2 inhibitors cause glucagon stimulation, leading to an increase in GI ratio, whereas incretin agents give rise to glucagon suppression and insulin stimulation, resulting in a decrease in GI ratio. In our previous study, we also observed that a lower GI ratio was significantly associated with NAFLD in patients with T2DM. 37 Therefore, these contrasting glucagon effects may be considered as a possible mechanism in the differential hepatic effects of SGLT2 inhibitors and incretin agents. Future randomized prospective studies are needed to investigate the mechanistic link regarding GI ratio and NAFLD in patients with NAFLD and T2DM.

GLP-1R AGONISTS
Accumulating data support the beneficial effect of GLP-1RAs on NAFLD. 38 In contrast, negative findings have been reported. In an RCT by Tang et al. 47 , liraglutide treatment showed no significant change on liver fat (magnetic resonance spectroscopy-PDFF, liver volume, and total liver fat index). In the LEAD-2 sub-study, liver-to-spleen attenuation ratio on CT was assessed after combination treatment with liraglutide and metformin. 48 There was a non-significant trend

Exenatide
In a study by Kenny et al. 44 , eight T2DM patients with biopsyconfirmed NAFLD were treated with 5-10 μg exenatide for 28 weeks. Liver histology was evaluated using NAFLD activity score, and there was no overall significant improvement in liver histology.
However, three of eight subjects met the primary end point of improved liver histology.
Sixty newly diagnosed T2DM patients with obesity, NAFLD, and elevated liver enzymes were included in the study by Shao et al. 49 The patients were randomly divided into two groups: the "exenatide group" treated with both exenatide and insulin glargine and the "intensive insulin group" treated with insulin glargine and aspart for 12 weeks. Following treatment, the levels of ALT, AST, and γGT in the exenatide group were significantly lower than the levels in the intensive insulin group. The ALT, AST, and γGT changes correlated with mean body weight changes. Moreover, the resolution rate of fatty liver was significantly higher in the exenatide group (93.3%) than in the intensive insulin group (66.7%).

Dulaglutide
Seko et al. 50 retrospectively evaluated 15 T2DM patients with biopsy-confirmed NAFLD who received once-weekly dulaglutide 0.75 mg for 12 weeks. FibroScan and body composition were evaluated before and after treatment in only five patients. Transaminase activities were significantly decreased after dulaglutide treatment.
In addition, liver stiffness measured by FibroScan and total body fat mass also decreased after treatment.

Weight loss and glycemic control
Certainly, GLP-1RAs cause significant weight loss in patients with T2DM. 52 Studies that demonstrated a significant decrease in liver fat content after treatment with GLP-1RAs also showed a significant correlation among weight reduction, glycemic control, and decreased liver fat. 39,53,54 In the Lira-NAFLD study, the reduction in liver fat content obtained after treatment with liraglutide for 6 months was significantly correlated with body weight reduction, even after multivariate adjustment. 46 Similar results were also obtained in the 26-week exenatide treatment study by Dutour et al. 53

Direct action on hepatic cells
A previous in vitro study suggested that GLP-1RAs may act directly on human hepatocytes via a G-protein-coupled receptor, resulting in a direct effect on reduction of hepatic steatosis. 54 However, there are different opinions regarding the presence of GLP-1RAs in human hepatocytes, and this has not yet been determined.

CONFLICTS OF INTEREST
The authors declare no conflict of interest.