1. Introduction
Metabolic dysfunction-associated steatotic liver disease (MASLD), previously termed nonalcoholic fatty liver disease (NAFLD), is defined as the presence of hepatic steatosis in conjunction with at least one cardiometabolic risk factor (obesity, hypertension, type 2 diabetes, dyslipidemia) [
1]. Approximately 1/4 of people with hepatic steatosis progress to metabolic dysfunction-associated steatohepatitis (MASH), which is characterized by hepatocellular ballooning and lobular necroinflammation, and an increased risk of fibrosis, cirrhosis, hepatic decompensation, hepatocellular carcinoma, and all-cause mortality [
2]. The global burden of MASLD is increasing at an alarming rate, with global prevalence reaching up to 32.4% [
3,
4].
A retrospective analysis of patients with MASLD showed that cardiovascular disease (CVD) (38.3%) was the leading cause of death in MASLD patients [
5], indicating that CVD was the most crucial determinant of mortality in MASLD patients. Considering that the development of CVD determines the prognosis of MASLD patients, the treatments for MASLD should improve cardiometabolic risk factors such as obesity and dyslipidemia, in addition to improving liver function [
6]. The metabolic syndrome is significantly associated with the development of MASLD and each component of metabolic syndrome is also associated with the development of MASLD [
7]. Briefly, obesity, type 2 diabetes, hypertension, and dyslipidemia such as high triglyceride (TG) and low high-density lipoprotein cholesterol (HDL-C) levels induce the development of MASLD [
7]. Obesity, especially visceral obesity, is the most common and well-documented risk factor for MASLD. In the meta-analysis, the pooled odds ratios (ORs) for the formation of MASLD in waist circumference and body mass index (BMI) were 2.34 (95%CI [confidence interval], 1.83 to 3.00), and 2.85 (95%CI, 1.60 to 5.08), respectively [
8].
Sodium-glucose cotransporter 2 inhibitors (SGLT2is) are reversible inhibitors of SGLT2, leading to reduction in renal glucose reabsorption and decrease in plasma glucose, in an insulin-independent manner [
9,
10,
11]. This property of SGLT2is is beneficial to reduce visceral fat as well as body weight [
11]. A recent meta-analysis provided evidence that SGLT2is significantly reduced visceral adipose tissue, subcutaneous adipose tissue, and ectopic liver fat, in addition to body weight, especially in young type 2 diabetic patients with MASLD and high BMI [
12].
SGLT2is have multifactorial beneficial properties for MASLD. Liver X receptor (LXR), the potential glucose sensor, comprises two isotypes, LXRα and LXRβ [
13]. They function as nuclear receptors with crucial roles in lipid metabolism [
14,
15,
16]. LXRα is predominantly found in metabolically active tissues such as the liver [
17,
18]. An increase in LXR expression has been demonstrated to correlate with the worsening of MASH [
19,
20]. Bile acids (BAs) are well known to facilitate the absorption of dietary fat and fat-soluble molecules. The presence of metabolic disorders such as MASLD has been reported to be closely related to abnormal levels of BAs in the blood and fecal metabolites of such patients [
21]. Thus, the gut microbiota interacting with BAs and altering BA metabolism are critical in the pathogenesis of such diseases. One of the SGLT2is, dapagliflozin, decreased hepatic lipid contents via inhibiting the expression of LXRα and downstream lipogenesis genes [
22]. Dapagliflozin downregulated LXRα expression through increasing LXRα degradation. Furthermore, dapagliflozin reversed gut dysbiosis induced by BA disruption and altered gut microbiota profile to reduce intestinal lipids absorption [
22].
Hepatic fibrosis is a severe pathological change in the progression of MASH and is characterized by the activation of hepatic stellate cells (HSCs) [
23]. MicroRNAs (miRNAs) are small non-coding RNAs, approximately 21 nucleotides in length, which participate in disease progression [
24,
25]. miR-34a-5p was reported to promote the development of MASLD [
26]. The inhibition of miR-34a-5p prevented HSCs activation by regulating the transforming growth factor-β (TGF-β) signaling pathway [
27]. GREM2 was identified as a direct target of miR-34a-5p [
27]. Empagliflozin ameliorated MASLD-associated fibrosis by downregulating miR-34a-5p and targeting GREM2 to inhibit the TGFβ pathway in HSCs [
27].
SGLT2is improve obesity and diabetes, endoplasmic reticulum (ER) stress, oxidative stress, and inflammation, which is beneficial for MASLD [
28]. However, the evidence on the effects of SGLT2is, especially the long-term effects, on the progression of MASLD in Asian populations with poorly controlled type 2 diabetes is very limited. Here, we investigated the long-term (>3 years) effect of SGLT2is on glucose/lipid metabolism and the markers for hepatic steatosis and hepatic fibrosis in Japanese patients with type 2 diabetes.
4. Discussion
The present study demonstrates that 3-year SGLT2i treatment significantly reduced TC and LDL-C, which disagrees with the results of previous meta-analyses [
34,
35,
36,
37]. All meta-analyses showed that SGLT2is significantly increased LDL-C, and three of four meta-analyses showed a significant increase in TC by SGLT2is. Even in patients without statins, significant increases in TC and LDL-C were not observed in our study. The inclusion of a relatively large number of short-term RCTs and patients of different races in such meta-analyses can explain a different result between ours and meta-analyses. Our previous study showed that SGLT2is did not show a significant effect on LDL-C and TG at 1, 2, 3, and 6 months after the start of SGLT2is [
38,
39], suggesting that a long-term treatment is required to evaluate the effects of SGLT2is on serum lipids. However, a significant reduction in LDL-C and TC in our study might have been induced by the concomitant use of statins.
Our study showed that SGLT2is significantly increased HDL-C, which was observed in all meta-analyses [
34,
35,
36,
37]. A significant increase in HDL-C was observed in both patients with and without statins, suggesting that an elevation in HDL can be caused by SGLT2is alone. We also showed that SGLT2is significantly reduced TG, which was observed in three of four meta-analyses [
35,
36,
37]. Epidemiologic and clinical trials have shown that elevated TG and low levels of HDL-C are independent risk factors for coronary heart disease. Therefore, the 2001 National Cholesterol Education Program Adult Treatment Panel III (ATP III) guidelines placed more emphasis on the importance of managing high TG and low HDL-C by identifying non-HDL-C (LDL-C + very low-density lipoprotein (VLDL)-C) [
40]. An effect of SGLT2is on non-HDL-C has not been sufficiently studied, and only the present study and one meta-analysis reported an effect of SGLT2is on non-HDL-C. The meta-analysis showed that SGLT2is had a significant increase in non-HDL-C by 0.09 mmol/L (95% CI, 0.06 to 0.12;
p < 0.00001) [
37], which disagrees with our result that SGLT2is significantly reduced non-HDL-C. This difference may be due to the inclusion of a relatively large number of short-term RCTs and patients of different races in the meta-analysis. Most studies had shown that SGLT2is reduced TG [
35,
36,
37], and TG is included in non-HDL-C. SGLT2is reduce TG, by reducing TG-rich lipoprotein which is included in non-HDL-C. Therefore, our result may be more reasonable. However, a significant decrease in TG and non-HDL-C were not observed in patients without statins. The effect observed in the study on lipid profiles is likely to be due to statin use rather than SGLT2is. Imada, T. et al. investigated the effect of combination of SGLT2is with statins by a retrospective analysis [
41]. Multivariate regression analyses showed that the change in LDL-C by SGLT2is depended on statin use. Such a synergistic effect with statins was more characteristic for SGLT2is. Three-year SGLT2is treatment significantly reduced body weight and HbA1c, suggesting that long-term SGLT2is treatment improved insulin resistance. Beneficial changes in serum lipids may be induced by an improvement in insulin resistance by 3-year SGLT2is treatment. Changes in TG and non-HDL-C were significantly and positively correlated with changes in HbA1c, supporting our hypothesis.
SGLT2is decrease blood glucose not by increasing insulin secretion, but by reducing renal glucose reabsorption. Such change in glucose and insulin relatively increases the secretion of glucagon, activating hormone-sensitive lipase (HSL) in adipose tissue [
42]. Accumulated TG is hydrolyzed to free fatty acids (FFAs) by activated HSL in adipose tissue, which reduces adipose tissue size, resulting in an improvement in insulin resistance due to reduced inflammatory cytokines and increased adiponectin [
43]. FFAs released from adipose tissue may be promptly used by skeletal muscles and liver because SGLT2is shift the energy metabolism towards FA utilization by the alteration of the glucose–FA cycle [
44]. An increase in adiponectin levels has beneficial effects on glucose and lipid metabolism by activation of adenosine monophosphate-activated protein kinase (AMPK) [
45]. SGLT2is have been reported to activate AMPK and inactivate acetyl-CoA carboxylase (ACC) which regulates FA synthesis [
46]. SGLT2is reduce FA accumulation in the liver, reducing hepatic TG re-synthesis from FA. VLDL, the main component of non-HDL-C other than LDL, is produced in the liver. High TG observed in insulin resistance is induced by hepatic over-production of VLDL and reduced VLDL degradation by impaired lipoprotein lipase [
47]. High VLDL causes an increase in LDL as the metabolite. VLDL is composed of cholesterol, re-synthesized TG, and apoproteins [
47]. Statins reduce hepatic cholesterol synthesis and SGLT2is decrease hepatic TG synthesis, reducing VLDL production in the liver, which may contribute to an improvement in TG, LDL-C, and non-HDL-C.
The evidence on effects of SGLT2is on MASLD is very limited. In our previous study, SGLT2is significantly improved serum transaminase at 3 and 6 months after SGLT2is initiation in type 2 diabetic patients [
38,
39]. We also discovered that the FIB-4 index was significantly improved by the 12-month SGLT2is treatment in a high-risk group [
48]. We also reported that the FIB-4 index significantly decreased in the SGLT2is group but not in the pioglitazone group at 96 weeks [
49]. However, APRI significantly decreased in both groups. SGLT2is reduced body weight by 3.2 kg; however, pioglitazone increased body weight by 1.7 kg.
Our study showed that 3-year SGLT2is treatment significantly reduced HSI as the marker for hepatic steatosis and APRI as the marker for hepatic fibrosis, and a significant reduction in the FIB-4 index in high-risk group was also observed. Changes in both HSI and APRI were significantly and positively correlated with changes in body weight, BMI, and HbA1c, suggesting that weight loss and an improvement of insulin resistance by 3-year SGLT2is treatment may improve hepatic steatosis and fibrosis. Change in HSI was significantly and negatively correlated with change in HDL-C and change in GGT was significantly and positively correlated with changes in TG and non-HDL-C, suggesting that an improvement in serum lipids by SGLT2is may also favorably influence on the progression of MASLD. The multiple regression analysis to detect a determinant of change in HSI showed that changes in BMI and HbA1c were a significant determinant of reduction in HSI. However, a significant determinant of reduction APRI was not detected. This suggests that 3-year SGLT2is treatment is more strongly associated with an improvement in hepatic steatosis.
In the study (
n = 32) by Ito et al., the liver-to-spleen attenuation ratio increased at 5 years after the start of ipragliflozin (an SGLT2is) [
50]. Ipragliflozin significantly improved serum aminotransferase and HbA1c over 5 years. Significant reductions in body weight and visceral fat area were sustained throughout the 5 years. Ipragliflozin significantly reduced serum ferritin and the FIB-4 index as the markers for hepatic fibrosis. Six Japanese patients with MASLD and type 2 diabetes were treated for the long term with canagliflozin [
51]. GLP-1RAs were added to two patients after 3 years due to histological worsening. Histological improvement was obtained in half of the patients at 5 years. An improvement in the scores for steatosis, lobular inflammation, ballooning, and fibrosis stage was observed in 67% of patients at 5 years. Homeostasis model assessment of insulin resistance (HOMA-IR) and serum ferritin significantly decreased at 5 years. One-hundred nine patients who had taken SGLT2is for more than 3 years were investigated [
52]. Improvements in body weight, liver transaminases, and the FIB-4 index were obtained at 3 years of SGLT2is treatment. In the intermediate-risk and high-risk groups (≥1.3 FIB-4 index), the FIB-4 index significantly decreased.
MASLD is strongly associated with obesity, insulin resistance/hyperinsulinemia, type 2 diabetes, and dyslipidemia [
53]. Therefore, SGLT2is and GLP-1RAs which have weight-losing effects, and metformin and pioglitazone which have the effect of improving insulin resistance, have been suggested to be promising therapeutic drugs for MASLD. In this study, 3-year SGLT2i treatment did not improve HSI and APRI in insulin-treated patients; however, SGLT2is significantly reduced both HSI and APRI in insulin-naïve patients, suggesting that exogeneous insulin as well as endogenous hyperinsulinemia may adversely affect MASDL and may attenuate the beneficial effects of SGLT2is for MASLD.
GLP-1RAs hold substantial therapeutic promise for managing diabetes, MASLD, and obesity [
54,
55,
56,
57]. Their mechanisms include augmenting insulin secretion, suppressing glucagon release, and modulating appetite and peripheral insulin sensitivity. The systematic review and meta-analysis using 13 randomized controlled trials (RCTs) which evaluated the impact of GLP-1RAs on visceral adipose tissue and subcutaneous adipose tissue in individuals with diabetes and MASLD or obesity showed that GLP-1RA treatment significantly reduced visceral adipose tissue (−0.55; 95% CI, −0.90 to −0.19), subcutaneous adipose tissue (−0.59; 95% CI, −0.99 to −0.19), body weight (−1.07; 95% CI, −1.67 to −0.47), and BMI (−1.10; 95% CI, −1.74 to −0.47) compared to controls [
58]. GLP-1RA treatment improved fasting blood glucose, postprandial glucose, HbA1c, HOMA-IR, and FIB-4. In another systematic review and meta-analysis of 16 RCTs that used liraglutide, exenatide, dulaglutide, or semaglutide in the treatment of MASDL or MASH, as measured by liver biopsy or imaging techniques, the effect of GLP-1RAs on histologic resolution of MASH with no worsening of liver fibrosis (4.08; 95%CI, 2.54 to 6.56,
p < 0.00001) was statistically significant [
59]. In our study, 3-year SGLT2is treatment significantly improved HSI and APRI in patients who were not treated by GLP-1RAs, suggesting that long-term SGLT2is treatments improve hepatic steatosis and fibrosis as well as GLP-1RAs.
Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are insulin-sensitizing agents and improve lipid metabolism, and are widely investigated in MASDL. In the PIVENS trial [
60], pioglitazone significantly reduced hepatic steatosis, lobular inflammation, and hepatic enzymes as compared with vitamin E and placebo for 96 weeks in patients with MASH. A meta-analysis showed that pioglitazone significantly improves liver fibrosis as compared with placebo in MASLD patients [
61]. Pioglitazone is recommended in persons with type 2 diabetes and biopsy-proven MASH by the American Association of Clinical Endocrinologists [
62]. Long-term pioglitazone treatment significantly improved liver-to-spleen attenuation ratio and serum aminotransferase [
50]. However, the use of PPARγ agonists is associated with poor bone quality, edema, and weight gain [
63]. In this study, there was no difference in the effects of 3-year SGLT2is treatment on HSI and APRI with or without the concomitant use of pioglitazone.
Metformin can activate AMPK by inhibiting mitochondrial complex 1, and activated AMPK can reduce FA synthesis [
64]. In addition, metformin can reduce reactive oxygen species production by inhibiting mitochondrial complex 1, which can reduce liver damage. Therefore, metformin has been found to alleviate MASLD [
64]. The meta-analysis including 10 RCTs with 459 patients showed that, compared to the controls, metformin could effectively reduce the serum fasting glucose and insulin levels and the HOMA-IR in MASLD patients at the 6-month follow-up and ALT and the HOMA-IR index at the 12-month follow-up [
65]. Another meta-analysis showed that metformin decreased ALT by −10.84 (95% CI, −21.85 to 0.16;
p = 0.05), AST by −4.82 (95% CI, −9.33 to −0.30;
p = 0.04), and HOMA-IR by −0.42 (95% CI, −0.82 to −0.02;
p = 0.04) in MASLD patients [
66]. In our study, significant reductions in both HSI and APRI were observed in patients treated with a combination of SGLT2is and metformin, and APRI was not reduced in patients without metformin, indicating that the combination of SGLT2is with metformin has a synergistic effect on improving MASLD. The reports on a synergistic effect of the combination of SGLT2is and metformin on improving MASLD are very limited. There are only two reports, and one using mice, showing the combination therapy additively improved MASH as compared with SGLT2i or metformin alone [
67]. In patients with type 2 diabetes, the combination of empagliflozin and metformin ameliorated liver steatosis, ALT levels, body weight, and glycated hemoglobin after a 6-month follow-up, as compared with the metformin monotherapy [
68]. Both SGLT2is and metformin activate AMPK and inactivate ACC, reducing hepatic FA synthesis and increasing FA oxidation which results in a decrease in hepatic FA accumulation. The combination treatment may potentiate such a cascade which is beneficial for the development of MASLD. However, to prove this hypothesis, further studies should be performed in the future.
We have to mention the limitations of our study. All subjects in this study were Japanese, and the results obtained in our study may not necessarily be obtained in other ethnicities. There was no liver imaging and histological examination to prove the existence of hepatic steatosis and hepatic fibrosis. The patients’ lifestyle changes such as diet and exercise might have influenced our result, which was not investigated. We investigated differences in changes in HSI and APRI after the start of SGLT2is with and without metformin, pioglitazone, GLP-1RAs, insulin, RAS inhibitors, and statins. However, the study did not take into account the effects of the possibility that the patients studied had taken multiple classes of drugs at the same time. Our patients had poor glycemic control; therefore, SGLT2is may have shown a significant improvement in MASLD.