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Supaglutide alleviates hepatic steatosis in monkeys with spontaneous MASH

Diabetology & Metabolic Syndrome volume 16, Article number: 303 (2024) Cite this article

Abstract

Background

Glucagon-like peptide 1 (GLP-1) is an incretin hormone and plays an important role in regulating glucose homeostasis. GLP-1 has a short half-life due to degrading enzyme dipeptidyl peptidase-IV and rapid kidney clearance, which limits its clinical application as a therapeutic agent. We demonstrated previously that supaglutide, a novel long-acting GLP-1 analog, exerted hypoglycemic, hypolipidemic, and weight loss effects in type 2 diabetic db/db mice, DIO mice, and diabetic monkeys. In the present study, we investigated supaglutide’s therapeutic efficacy in rhesus monkeys with spontaneous metabolic dysfunction-associated steatohepatitis (MASH).

Methods

15 rhesus monkeys with biopsy-confirmed MASH were divided into three groups, receiving supaglutide 50 µg/kg, supaglutide 150 µg/kg, and placebo, respectively, by weekly subcutaneous injection for 3 months. Liver fat content quantified by magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF), liver pathology, and metabolic parameters were assessed.

Results

We found that once-weekly subcutaneous injections of supaglutide for 3 months significantly reduced hepatic fat accumulation, with a 40% percentage decrease in MRI-PDFF from baseline (P < 0.001 vs. Placebo). Treatment with supaglutide alleviated hepatic histological steatosis (nonalcoholic fatty liver disease activity score P < 0.001 vs. Placebo) without worsening of fibrosis, as assessed by ultrasound-guided liver biopsy. Supaglutide concomitantly ameliorated liver injury exemplified by a lowering tendency of hepatic alanine aminotransferase levels. Supaglutide also decreased body weight in a dose-dependent fashion accompanied by decreased food intake, improved lipid profile and glycemic control.

Conclusions

Supaglutide exerts beneficial effects on hepatic and metabolic outcomes in spontaneous MASH monkeys.

Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a spectrum of metabolic diseases exemplified by excess liver fat deposition, bland steatosis, and more severe lesions, including metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, and cirrhosis [1]. MASH, a more advanced form of MASLD, is characterized by marked hepatic steatosis, lobular inflammation, and hepatocyte ballooning, with or without fibrosis [2]. MASH is associated with an increased risk of cirrhosis, hepatocellular carcinoma, cardiovascular diseases (CVDs), and liver-related mortality [3]. Up until now, despite focused efforts and numerous studies, a definitive pharmacological treatment for MASH has not yet been established. Therefore, effective treatment for MASH remains a substantial unmet medical need.

Given the strong links between obesity, insulin resistance (IR), type 2 diabetes mellitus (T2DM), and the progression of MASLD to MASH or cirrhosis [4], a growing number of studies have focused on the efficacy of anti-hyperglycemia drugs for MASH. Glucagon-like peptide 1 receptor agonists (GLP-1RA), as a class of anti-diabetic agents mimicking the actions of GLP-1, have been approved for T2DM and weight management [5, 6]. There are contradictory data on the presence of GLP-1 receptor (GLP-1R) in the liver. Despite that the expression of the canonical GLP-1R in mouse hepatocytes was not detected [7, 8], preclinical studies revealed that GLP-1RAs treatment reduced fatty acid accumulation, decreased liver inflammation, diminished endoplasmic reticulum (ER) stress, and promoted autophagy [9,10,11,12,13,14,15,16], suggesting their beneficial effects on fatty liver. Gupta et al. further found that GLP-1R was present in human hepatocytes and that exposure of GLP-1RA to hepatocytes led to a reduction of fat load in these cells, as well as in HepG2 and Huh7 cell lines [17]. Clinical studies revealed that liraglutide and semaglutide treatment improved liver histology, increased MASH resolution, and decreased the rate of progression to fibrosis in subjects with biopsy-proven MASH [18, 19]. Overall, these findings suggest that the potentially direct effects of GLP-1RAs on the liver, together with their benefits on glucolipid homeostasis and weight management, might help prevent MASH progression.

Supaglutide is a novel GLP-1RA produced by gene engineering recombinant techniques by fusing human GLP-1 with human IgG2-Fc fragment, which retains GLP-1 activity and has prolonged half-life [20]. We have demonstrated that supaglutide treatment exerted significant hypoglycemic effects in T2DM db/db mice [21] and spontaneously diabetic monkeys [22]. Supaglutide treatment also reduced high-fat diet (HFD)-induced obesity and fatty liver associated with increased uncoupling protein 1 (Ucp1) in white adipose tissue in mice [23]. These findings suggested that supaglutide therapy might represent a novel therapeutic agent for treating MASH.

Rhesus monkeys spontaneously develop obesity and metabolic syndrome with aging [24, 25], and monkeys with metabolic syndrome are more susceptible to developing non-alcoholic fatty liver disease (NAFLD) spontaneously [26], which provide an ideal model for NAFLD. In this study, we aimed to investigate the effects of supaglutide, a novel long-acting human GLP-1RA, on the progression of MASH and relevant metabolic parameters in rhesus monkeys with spontaneous MASH.

Methods

Animals

Non-human primates are an ideal model for biomedical studies [27], due to their high similarity to human beings in genetics, physiology and biochemistry. A cohort of spontaneously dysmetabolic rhesus monkeys in PriMed Non-human Primate Research Center of Sichuan PriMed Shines Bio-tech Co., Ltd. shows similar clinical features with human patients [22, 28] and develops diabetic complications i.e. macular edema [29], blood-brain barrier dysfunction [30], cardiac dysfunction [31] and myocardial fibrosis [32].

In this study, we selected rhesus monkeys with spontaneous MASH from this cohort, which were housed in PriMed Non-human Primate Research Center (Ya’an, China). All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Primed Shines Bio-tech Co., Ltd (No. AW2110). No animals were sacrificed for the purposes of this work. Prior to this study, all monkeys had undergone liver ultrasound, magnetic resonance imaging (MRI) and liver biopsy for confirmation and stratification of liver lesions. Animals were quarantined and received physical examinations, two tuberculosis tests, and tests for parasite, Salmonella, Klebsiella and B virus before the experiment. All animal procedures were performed in strict accordance with the Institutional Animal Care and Utilization Committee (IACUC) of SPSB accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), comply with the national Animal Care and Use Committee Guidelines.

Monkeys were housed under controlled temperature (18–26℃), 12 h light:12 h darkness cycle, humidity of 40–70%, ventilation of fresh air at least eight times per hour, with free access to food and water. In addition to the chow diet (Primed Monkey Chow 4#, containing ~ 18% proteins, ~ 12% fat, ~ 62% carbohydrate), monkeys were provided with 50–300 g of green feed (fruits and vegetables) every day. As a reward after domestication, snacks such as peanuts, melon seeds and other nuts were also provided.

Inclusion criteria

The rhesus monkeys selected for this study were male, 11–23 years of age (equivalent to 30–70 years of human age), with abnormal lipid metabolism for more than 2 years. Every monkey had a moderate fatty liver as assessed by liver ultrasound, an intrahepatic fat content as quantified by MRI-estimated proton density fat fraction (MRI-PDFF) of ≥ 6%, and histologic evidence of NAFLD activity score (NAS) ≥ 3 and fibrosis score ≥ 1a within 6 months prior to the study.

Exclusion criteria

Animals meeting the following criteria were excluded: (1) uncontrolled hypertension (systolic blood pressure > 140 mmHg, diastolic blood pressure > 90 mmHg); (2) abnormal fluctuations of body weight; (3) severe hepatic or renal dysfunction, or electrolyte disorders; (4) hematological diseases including anemia, leukocytosis, and thrombocytopenia; (5) significant endocrine, immune, coagulation, urogenital tract abnormalities or diseases; (6) any other medical history that may influence the efficacy or safety evaluation.

Experimental design and drug administration

The experimental design was presented in Fig. 1. Rhesus monkeys with spontaneous MASH were domesticated for 4 weeks, during which glucose and lipid metabolism indicators were examined, liver biopsy was performed under ultrasound guidance, and liver fat content was quantitatively analyzed by MRI-PDFF. 15 rhesus monkeys were randomly divided into 3 groups (n = 5 each group) and received subcutaneous injections of placebo (normal saline, placebo control), 50 µg/kg or 150 µg/kg supaglutide (International Nonproprietary Name: Efsubaglutide Alfa; code: YN-011; Innogen Pharma, Shanghai, China) once a week (QW) for 3 months. MRI and liver biopsy were performed at the end of 3-month treatment as liver fat content and liver fibrosis were reported to be improved after 3-month treatment by GLP-1RAs or fibroblast growth factor 21 analogs [33].

Fig. 1
figure 1

Study design. The experiment consists of two periods: adaptation (4 weeks) and observation (13 weeks). The black dots indicate time points of physical examination and blood sampling for laboratory testing as indicated. The red dots indicate time points of MRI-PDFF as indicated. The triangles indicate the time points for the liver biopsy. The arrow indicates the time point for the titration of Supa from 100 µg/kg to 150 µg/kg. Abbreviations: MRI-PDFF, magnetic resonance imaging-proton density fat fraction; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score; MASH, metabolic dysfunction-associated steatohepatitis; QW, once weekly; SC, Subcutaneous; Supa, supaglutide

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An adaptive drug administration (the first dose was 100 µg/kg, and the subsequent 11 doses were 150 µg/kg) was applied in 150 µg/kg group to reduce potential gastrointestinal reactions.

MRI imaging acquisition and data processing

MRI biomarkers are emerging surrogates of liver biopsy in assessing diffuse liver lesions, and MRI-PDFF is considered as the most accurate and reproducible method to evaluate liver steatosis [34, 35]. In this study, MRI-PDFF was measured by a GE 3.0T MRI scanner (750W3T MRI, GE Healthcare) using IDEAL-IQ sequence scanning [36], which was performed on all monkeys once before and once at the end of administration. Monkeys were imaged in the supine position under anesthesia induced with 10 mg/kg intramuscular injection of ketamine hydrochloride and maintained by inhaled isoflurane with oxygen. Breath-hold was achieved by mechanical ventilation under deep anesthesia. Pulse rate, breath and oxygen saturation were monitored during and after the scan.

Experienced readers manually drew 3 regions of interests (ROIs) ranging between 90 and 110 mm2, all of which were selected in the right lobe of the liver on three consecutive slices showing the largest liver volumes. In the operational process, large vessels, bile ducts and gallbladder were avoided in case of volume effect. The mean of the three MRI-PDFF values were calculated for analyses.

Liver pathology examination

Liver biopsy was performed by an experienced surgeon to collect ≤ 2 percutaneous liver tissue sample from the monkeys, under direct ultrasound guidance (GE Vivid S5 ultrasound device) to ensure accurate localization and needle placement at the intended liver tissue area for biopsy. The biopsy specimens (approximately 2 cm in length) were collected before and after 89 days of dosing, and immediately fixed in 4% paraformaldehyde buffer for more than 24 h. After the liver puncture specimen was well fixed, it was directly dehydrated, soaked in wax, embedded, and sectioned. Liver sections were cut with a rotary microtome (Leica RM 2135, Leica Instruments, Nussloch, Germany), and mounted on glass slides followed by staining with hematoxylin-eosin (HE) and Masson’s trichrome. All sections were evaluated by experienced histopathologists based on the guidelines for the diagnosis and treatment of nonalcoholic fatty liver co-drafted by the American Society of Liver Diseases AASLD, American College of Gastroenterology ACG and American Gastroenterology Association AGA [1, 37]. Using HE-stained samples, the NAS were calculated ranging from 0 to 8 (unweighted sum of the scores for steatosis [assessed on a scale of 0 to 3], lobular inflammation [assessed on a scale of 0 to 3], and hepatocyte ballooning [assessed on a scale of 0 to 2]), with higher scores indicating an increased likelihood of MASH. Using Masson-stained samples, the assessments of fibrosis stages were defined as follows: F0, no fibrosis; F1, mild-to moderate zone 3 perisinusoidal fibrosis or portal or periportal fibrosis only; F2, zone 3 perisinusoidal fibrosis and portal or periportal fibrosis; F3, bridging fibrosis; and F4, cirrhosis. The fibrosis area fraction for each liver biopsy specimen was calculated using a QuantumPath (QPath) image analysis software. The fibrosis area fraction was the total area of fibrosis (Afib) divided by the total area of the section.

Metabolic measurements

Food intake was calculated (food intake = feed - discard - surplus) during the study period. Four feedings of total 200–700 g feeds were made every day. Fasting bodyweight was measured using METTLER TOLEDO electronic platform scale, once in the adaptation period (before grouping), seven times in the administration observation period (Week 1, Week 2, Week 4, Week 6, Week 8, Week 10 and Week 12), a total of eight times. Sitting height was measured when the animal kept lying on the left side after anesthetized. Body mass index (BMI) was calculated as fasting bodyweight divided by sitting height squared (kg/m2).

Serology studies

Blood samples were collected under fasting and non-anesthesia condition by 2.0 mL EDTA-2 K anticoagulant vacuum tubes and centrifuged at 3000 rpm for 10 min at 4 °C. Plasma glucose was detected by Roche Cobas 6000 analyzer series C501 (Roche Diagnostics GmbH). Roche Cobas 6000 analyzer series C501/E601 was used to detect fructosamine (FRA) and blood biochemical parameters including fasting plasma glucose (FPG), liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST] and γ-glutamyl transferase [GGT]) and lipid profile (low-density lipoprotein cholesterol [LDL-c], high-density lipoprotein cholesterol [HDL-c], total cholesterol [TC], triglyceride [TG]).

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics V26.0 and GraphPad Prism 8 (GraphPad Software). One-way analysis of variance (ANOVA) test was used to compare among three groups, and post-hoc least significant difference (LSD) test was used for multiple comparisons between two given groups. Repeated-measures ANOVA or paired t test was used as appropriate to perform statistical analysis on the indicators before and post-dosing. The change of MRI-PDFF was further reanalyzed among three treatment groups using analysis of covariance (ANCOVA), adjusting for change of bodyweight from baseline to the end of the treatment. Pearson correlation was conducted to analyze the association of change of different variables. Multiple linear regression analysis was performed with the change of MRI-PDFF as dependent variable and potential covariates as independent variables to identify independent determinants of MRI-PDFF changes. Data were presented as mean ± SEM. P values less than 0.05 were considered to be statistically significant.

Results

Baseline characteristics of MASH rhesus monkeys

The rhesus monkeys included for this study were male, 11–23 years of age (equivalent to 30–70 years of human age), weighted 13.63–22.85 kg, with abnormal lipid metabolism for 3–11 years. The intrahepatic fat content as quantified by MRI-PDFF was between of 7.8-11.9%, with histological NAS between 3 and 4 and fibrosis stage between 1a and 1c at baseline.

Supaglutide reduces liver fat content in MASH rhesus monkeys

To investigate the effects of supaglutide on liver steatohepatitis, MRI was performed to quantify the liver fat content before and after 80 days of dosing (Table 1; Fig. 2). On the baseline images, mean liver fat content assessed by MRI-PDFF was more than 9% and comparable among different groups. In the supaglutide groups, MRI-PDFF at Week 12 was significantly decreased from baseline (50 µg/kg group, 8.98% ± 0.4–4.98% ± 0.09%, P = 0.001; 150 µg/kg group, 9.38% ± 0.68–5.62% ± 0.65%, P = 0.004; Table 1), with the relative reduction of 44.0% and 39.8%, respectively (Fig. 2C). In contrast, MRI-PDFF increased from 8.86% ± 0.42–9.02% ± 0.51% (P = 0.617, Table 1) in the placebo group. Comparison among groups showed that the improvements in MRI-PDFF were significantly greater with supaglutide than placebo (both dosing groups P < 0.001; Table 1) but did not differ between 50 µg/kg and 150 µg/kg supaglutide treatment (P = 0.738; Fig. 2). This result revealed that supaglutide treatment reduced the liver fat deposition in MASH rhesus monkeys.

Table 1 Biochemical and hepatic outcomes of supaglutide versus placebo treatment
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Fig. 2
figure 2

Supaglutide decreases liver fat content as assessed by MRI-PDFF. A Representative liver MRI-PDFF images with supaglutide treatment. B Pre- and post-treatment liver fat content levels, and C Percentage change of liver fat content from baseline as assessed by MRI-PDFF. Data were expressed as Mean ± SEM. **, p < 0.01 compared to the baseline. ###, p < 0.001 compared to the placebo. Abbreviations: MRI-PDFF, magnetic resonance imaging-proton density fat fraction; Supa, supaglutide

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Supaglutide alleviated the MASH progression with no worsening of fibrosis in MASH rhesus monkeys

To investigate whether supaglutide treatment could lead to liver histological improvements, liver biopsy specimens were taken from all monkeys before and after 89 days of dosing. The degree of hepatic steatosis, lobular inflammation, hepatocyte ballooning, and fibrosis were scored. The histological evaluation of supaglutide versus placebo treatment is shown in Table 2; Fig. 3, and the representative images of liver lesions are shown in Fig. 4.

Table 2 Liver histological score of supaglutide versus placebo treatment
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Fig. 3
figure 3

Supaglutide alleviated MASH progression without worsening of fibrosis. A Change of NAFLD activity score from baseline. B Pre- and post-treatment fibrosis area fraction. Data were expressed as Mean ± SEM. ##, p < 0.01 compared to the placebo. Abbreviations: MAFLD, metabolic dysfunction-associated steatotic liver disease; Supa, supaglutide

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Fig. 4
figure 4

Supaglutide improves liver histopathology. Histological changes in hepatic steatosis, lobular inflammation, ballooning and fibrosis stage as assessed by A H&E, and B Masson staining in liver post- vs. pre-treatment biopsies. Blue arrows show inflammatory foci

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All monkeys had an aggregate NAS ≥ 3, with liver fibrosis stage ≥ 1a at baseline (Table 2). Administration of supaglutide for 12 weeks produced significant NAS reductions, when comparing the post- vs. pre-treatment biopsy. In the 150 µg/kg group, the mean NAS decreased from 3.6 ± 0.2 to 1.4 ± 0.2 at the end of administration (P < 0.001). The mean score of steatosis, inflammation, and ballooning was changed from 2.2 ± 0.2 to 0.6 ± 0.2 (P = 0.016), 1.0 ± 0.0 to 0.8 ± 0.2 (P = 0.374), and 0.4 ± 0.2 to 0.0 ± 0.0 (P = 0.178), respectively. In the 50 µg/kg group, the mean NAS decreased from 3.6 ± 0.2 to 1.6 ± 0.2 at the end of administration (P = 0.003). The mean steatosis, inflammation, and ballooning scores were changed from 1.2 ± 0.2 to 0.2 ± 0.2 (P < 0.05), 1.8 ± 0.4 to 1.0 ± 0.0 (P = 0.099), and 0.6 ± 0.2 to 0.4 ± 0.2 (P = 0.374), respectively. In contrast, no improvement in NAS or its component scores was identified in the placebo group. Comparison among groups showed that the improvements in NAS were significantly greater with supaglutide than placebo (both dosing groups P < 0.001) but did not differ between 50 µg/kg and 150 µg/kg supaglutide treatment (P > 0.05). All monkeys in the supaglutide groups showed improvements in liver steatosis, but not in the placebo group. 1 of the 5 monkeys in the 150 µg/kg supaglutide group, and 3 of the 5 monkeys in the 50 µg/kg supaglutide group showed improvements in inflammation. By contrast, 1 of the 5 monkeys in the placebo group showed improvements in inflammation. 1 of the 5 monkeys in the 150 µg/kg supaglutide group, 2 of the 5 monkeys in the 50 µg/kg supaglutide group showed improvements in ballooning, which was not observed in the placebo group (Figs. 3A and 4A). In addition, 1 of the 5 monkeys in the 150 µg/kg supaglutide group, and 3 of the 5 monkeys in the 50 µg/kg supaglutide group showed improvements in fibrosis. By contrast, 2 of the 5 monkeys in the placebo group showed worsening of fibrosis. Treatment with supaglutide displayed a dose-related lowering tendency of fibrosis area fraction at 12 weeks after administration, compared with an increased tendency of fibrosis area fraction with placebo (Figs. 3B and 4B, Fig. S1, and Table S1). The liver fibrosis of MASH monkey model used in this study was mild (1a-1c) at baseline, therefore, its effect on fibrosis might be difficult to determine within just 3-month treatment.

These findings demonstrate that supaglutide treatment mitigated the progression of MASH, primarily by improving hepatic steatosis, without exacerbating fibrosis in rhesus monkeys with MASH.

Supaglutide decreased liver injury biomarkers in MASH rhesus monkeys

We also evaluated the effects of supaglutide on liver injury biomarkers including ALT, AST and GGT throughout the study period (Table 1; Fig. 5A-C). Treatment with 50 µg/kg and 150 µg/kg supaglutide displayed a lowering tendency of ALT and AST up until 10 weeks after administration. During the study period, ALT and AST levels of MASH monkeys were nearly within the normal physiological range.

Fig. 5
figure 5

Supaglutide decreases liver injury biomarkers. Change of A ALT, B AST, and C GGT. All data were expressed as Mean ± SEM. #, ## p < 0.05 and 0.01 compared to the placebo. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyl transferase; Supa, supaglutide

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Supaglutide reduced food intake and bodyweight in MASH rhesus monkeys

The bodyweight of monkeys administered with supaglutide decreased progressively and continuously during the study period, with a mean 1.41 kg (8.75%) reduction in 50 µg/kg group and a mean 4.01 kg (22.25%) reduction in 150 µg/kg group after 3-month treatment. In comparison, monkeys in the placebo group displayed slight weight gain during the study period. Consistently, BMI reached a mean 4.00 kg/m2 (8.75%) reduction in 50 µg/kg group and 10.60 kg/m2 (22.25%) reduction in 150 µg/kg supaglutide group after 3-month treatment, compared with an increased tendency of BMI in the placebo group. On the whole, treatment with supaglutide significantly reduced bodyweight and BMI in a dose-dependent manner (Table 1; Fig. 6A-B).

Fig. 6
figure 6

Supaglutide decreases food intake and bodyweight. Percentage change of A bodyweight, B BMI, and C food intake. Data were expressed as Mean ± SEM. #, ## p < 0.05 and 0.01 compared to the placebo. Abbreviations: BMI, body mass index; Supa, supaglutide

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Significant reductions of food intake that is presented as total energy intake were also observed. During the intervention period, the mean food consumption maintained a 31-68% drop from baseline in 150 µg/kg supaglutide group and a 4-37% drop in 50 µg/kg supaglutide group. Different from the continuous reductions of bodyweight, the food intake of monkeys in supaglutide groups reached the maximal reduction at week 2 after the first injection, and then there was a slight rebound during the following treatment period (Table 1; Fig. 6C).

Supaglutide improved lipid profile in MASH rhesus monkeys

Supaglutide treatment produced significant improvements in lipid profiles, with the 150 µg/kg group typically presenting more significant improvements. In particular, 150 µg/kg supaglutide treatment demonstrated significant reductions of TC throughout the study, with the maximal reduction of 19.6% at week 4, and then plateaued for the following intervention period. Similar with TC, supaglutide also reduced TG and LDL-c levels in a dose-dependent manner. It should be mentioned that there was also a lowering tendency of HDL-c with supaglutide treatment in this study (Table 1; Fig. 7A-C). This might be attributed to the reduction of TC, which is a component of HDL-c, related to the reduced food and energy intake. These results suggested that supaglutide exerts beneficial effects in regulating lipid homeostasis in rhesus monkeys with MASH.

Fig. 7
figure 7

Supaglutide improves lipid and glucose profiles. A-C Percentage change of lipids from baseline. D-E Pre- and post-treatment blood glucose levels. Data were expressed as Mean ± SEM. #, ## p < 0.05 and 0.01 compared to the placebo. Abbreviations: FPG, fasting plasma glucose; FRA, fructosamine; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride; Supa, supaglutide

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During the intervention period, FPG levels of all groups fluctuated within the normal range, and no discernible between-group differences were detected after supaglutide and placebo administration (Fig. 7D). The plasma FRA, a validated biomarker that reflects the average levels of glucose control over the past 2–3 weeks [21, 38, 39], demonstrated a lowering tendency after supaglutide administration (Table 1; Fig. 7E). During the study period, FRA maintained a drop of 1–8 µmol/L from baseline in the 50 µg/kg group and a drop of 2–8 µmol/L in the 150 µg/kg supaglutide group.

Supaglutide decreases MRI-PDFF independent of bodyweight reduction

The correlations between the change in MRI-PDFF and other factors following 12 weeks of treatment are shown in Table 3. In the MASH rhesus monkeys, the changes of MRI-PDFF correlated significantly with the changes of bodyweight (r = 0.659, P = 0.008), while insignificantly with age (r = -0.394, P = 0.147), duration of metabolic disorder (r = -0.214, P = 0.443), baseline MRI-PDFF (r = -0.369, P = 0.176), and changes of TG (r = 0.413, P = 0.126), TC (r = 0.392, P = 0.148), LDL-c (r = 0.299, P = 0.278), and FRA levels (r = 0.505, P = 0.055) from baseline. Multiple regression analyses were further performed by introducing potential confounding factors into the model (Table 4). The change of MRI-PDFF was independently associated with the change of bodyweight (β = 0.647; P = 0.022) after adjusting for the effects of age, duration of metabolic disorder, baseline MRI-PDFF, and the change of FRA from baseline, whereas the other indices did not show significant associations with MRI-PDFF change after adjusting for other confounding factors.

Table 3 Correlations between change of MRI-PDFF with potential influencing factors
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Table 4 Multiple regression analysis of factors associated with the change of MRI-PDFF
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Interestingly, after adjusting for the bodyweight reduction, the differences of MRI-PDFF reduction among groups were still statistically significant (F = 9.293, P = 0.004). Multiple comparisons between groups indicated that the reductions of MRI-PDFF in 50 µg/kg and 150 µg/kg supaglutide groups were higher than that in the placebo group (50 µg/kg group, 4.0%, 95% CI 1.799–6.103%, P = 0.002; 150 µg/kg group, 3.5%, 95% CI -0.138-7.042%, P = 0.058). This result is consistent with previous findings that liraglutide treatment improved liver function and fibrosis in NAFLD and T2DM patients, regardless of BMI changes or obesity status [40].

Supaglutide was safe and well-tolerated in MASH rhesus monkeys

Throughout the study period, no noteworthy drug-related adverse events or abnormalities in animal’s behavior, mental state, and fur color were detected. No significant changes from baseline in other biochemical indicators were observed during the intervention period.

Discussion

GLP-1 is an incretin hormone with broad pharmacological potential. GLP-1RAs are successfully in clinical use for the treatment of T2D and obesity. Several GLP-1-based therapies are in the evaluation for the treatment of metabolic diseases. Supaglutide is a novel long-acting GLP-1 analog. In this preclinical study, we demonstrated that supaglutide significantly decreased the hepatic fat content and alleviated histological steatosis, without worsening of fibrosis in rhesus monkeys with MASH. Supaglutide also reduced bodyweight, improved lipid profiles and glucose control. This study provides preclinical validation for an innovative therapeutic approach to MASH and supports further studies of supaglutide for treating MASH and other metabolic diseases in humans.

In this study, treatment with supaglutide reduced liver fat content by about 40% from baseline, as quantified by MRI-PDFF which is the most accurate non-invasive method to evaluate liver steatosis [34]. This was further supported by significant reduction in NAS assessed by histological analysis, which was achieved mainly by an improvement in steatosis score. However, insignificant reduction of lobular inflammation or ballooning in the post- vs. pre-treatment biopsy was observed. Treatment with supaglutide also slightly alleviated liver fibrosis severity compared with placebo, but the between-group differences were not statistically significant. Of note, the liver fibrosis of MASH monkey model used in this study was mild at baseline, therefore, its effect on fibrosis might be difficult to determine within just 3-month treatment in such a small sample size. Further studies are required to confirm the effects of supaglutide on liver fibrosis with higher stages in lager sample sizes. In general, the efficacies of supaglutide on alleviating MASH are consistent with previous studies which supported that semaglutide and liraglutide displayed significant MASH resolution without worsening of fibrosis progression in MASH [18, 19].

Liver enzymes are the most commonly used serologic biomarkers in investigating the effectiveness of MASLD/MASH treatment [41]. In particular, ALT was shown to correlate well with liver fat deposition [42], while AST correlates well with liver inflammation [43]. Elevated GGT may also be present in MASH, but, similarly to AST, is less closely related to liver fat deposition than ALT [44]. In this study, supaglutide significantly improved ALT levels, but insignificantly improved AST and GGT levels compared with placebo, supporting its main benefits on reducing liver fat deposition. Consistent with our results, significant reductions in ALT, but not AST and GGT, were also observed [45] following dulaglutide treatment in T2DM patients with MASLD/MASH. Additionally, significant reductions in ALT and GGT following liraglutide treatment were also seen in T2DM patients [46]. In this study, the insignificant results may be partly attributed to the mild hepatocyte damage at baseline in the monkeys.

Management of dyslipidemia and dysglycemia is important for the resolution of MASH. In this study, supaglutide treatment exerted beneficial effects on improving lipid profiles, including TC, TG, and LDL-c levels. However, a lowering tendency of HDL-c was also detected, which might be attributed to the reduction of TC that is an important component of HDL-c. FRA, a validated biomarker that reflects the average levels of glucose control over the past 2–3 weeks [21], also demonstrated a lowering tendency after supaglutide administration. These results are consistent with our previous findings that supaglutide treatment exerted significant hypoglycemic effects in T2DM db/db mice [21] and diabetic monkeys [22] and improved lipid profiles in HFD induced obese mice [23]. We previously showed that supaglutide reduces HFD-induced obesity associated with increased Ucp1 in white adipose tissue in mice [23]. Ucp1 is a specific marker for fat browning in different fat depots, which has the capacity to increase thermogenesis and reverse obesity [47]. The knockout of Ucp1 also exacerbates glycemic dysregulation, hepatic steatosis, and inflammation, even in the absent of changes in bodyweight in western diet-induced mice [48, 49]. Therefore, the improvements in metabolic and hepatic dysfunction may be associated with the upregulated Ucp1 expression with supaglutide treatment.

Here, we observed a significant and dose-dependent decrease in bodyweight and BMI in monkeys treated with supaglutide, whereas a trend of weight gain was present with placebo. Different from the continuous reduction of bodyweight, the food intake of monkeys receiving supaglutide reached the maximal reduction at week 2 after administration, followed by a slight rebound later in the treatment. This suggests that reduced food intake is not solely responsible for weight loss.

We conducted correlation and multiple regression analyses with MRI-PDFF reduction and found that the reduction in liver fat content independently and significantly correlated with the weight reduction. Consistently, it was reported that weight loss of 4-14% resulted in relative reductions in intrahepatic fat of 35‐81% [50], and that weight loss of more than 5% led to significant histologic improvements of MASH [51,52,53]. The improvements in liver fat may result from both direct effects on liver fat deposition and indirect effects through weight reduction. To assess the direct impact of supaglutide on liver fat, we analyzed the change in MRI-PDFF normalized against body weight changes. Even after adjusting for weight, significant reductions in MRI-PDFF were observed in the supaglutide groups compared to placebo, suggesting potential weight-independent effects on hepatic steatosis. This finding aligns with previous studies showing that GLP-1RA reduced fat load in hepatocytes, as well as HepG2 and Huh7 cell lines [17]. Evidence also suggested that GLP-1RAs reduced fatty acid accumulation, diminished ER stress, attenuated hepatocyte apoptosis, promoted autophagy, and regulated the activity of enzymes involved in fatty acid metabolism [9,10,11,12,13,14,15,16, 54, 55]. Therefore, the direct effects of GLP-1RAs on liver, along with their benefits on glucolipid homeostasis and weight management, likely contribute to the amelioration of MASH. Given that the presence or absence of GLP-1R on hepatocytes is still controversial [7, 8, 17], a mechanistic understanding of how GLP-1 based therapies attenuate MASH remains to be further explored.

The effect of GLP-1RAs on liver fibrosis still remains unclear. Despite the significant improvements in noninvasive fibrosis markers [56, 57], only two clinical studies evaluated the effect of GLP-1RAs on histologically detected fibrosis. It showed that treatment with liraglutide for 48 weeks and semaglutide for 72 weeks was associated with lower progression of liver fibrosis compared to placebo, however, no significant effect on regression of pre-existing liver fibrosis was observed [18, 19]. Therefore, early treatment of liver fibrosis with GLP-1RAs may be more effective.

To our knowledge, this is the first study to evaluate the effects of supaglutide on treating MASH in a nonhuman primate model. A strength of this study is the use of liver biopsy, which remains the gold standard for evaluating MASH features, providing reliable data on hepatocyte steatosis, inflammation, ballooning, and fibrosis. Another strength is the use of MRI-PDFF, a precise and validated imaging biomarker for liver fat quantification [34]. However, there are several limitations to this study. While the effects of supaglutide on MASH were observed, the underlying molecular mechanisms remain unclear and warrant further investigation. Additionally, the liver fibrosis in MASH monkey model was mild at baseline, making it challenging to fully assess the impact on fibrosis within the short treatment period. Furthermore, due to the scarcity of monkeys with spontaneous MASH and limited evidence to inform sample size calculations, the sample size was small. Future research should include larger sample size and alternative MASH models to build on these findings.

Conclusion

In conclusion, we demonstrated that supaglutide, a novel long-acting GLP-1RA, significantly reduced hepatic fat fraction as assessed by MRI-PDFF and improved histological steatosis without exacerbating fibrosis in rhesus monkeys with MASH. Additionally, Supaglutide showed beneficial effects on liver metabolism and metabolic parameters. These findings suggest that supaglutide could be a promising therapeutic option for treating MASH and associated metabolic disorders. However, further studies are warranted to clarify the underlying mechanisms and to determine whether supaglutide could be effective in treating MASH in humans.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

GLP1:

Glucagon-like peptide 1

MASH:

Metabolic dysfunction associated steatohepatitis

MRI-PDFF:

Magnetic resonance imaging-estimated proton density fat fraction

MASLD:

Metabolic dysfunction associated steatotic liver disease

NAFLD:

Nonalcoholic fatty liver disease

CVDs:

Cardiovascular diseases

IR:

Insulin resistance

T2DM:

Type 2 diabetes mellitus

GLP-1RA:

GLP-1 receptor agonists

GLP-1R:

GLP-1 receptor

ER:

Endoplasmic reticulum

HFD:

High-fat diet

UCP1:

Uncoupling protein 1

MRI:

Magnetic resonance imaging

IACUC:

Institutional Animal Care and Utilization Committee

AAALAC:

Association for Assessment and Accreditation of Laboratory Animal Care

NAS:

NAFLD activity score

QW:

Once a week

ROIs:

Regions of interests

HE:

Hematoxylin-eosin

BMI:

Body mass index

FRA:

Fructosamine

FPG:

Fasting plasma glucose

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

GGT γ:

Glutamyl transferase

LDL-c:

Low-density lipoprotein cholesterol

TC:

Total cholesterol

TG:

Triglyceride

ANOVA:

Analysis of variance

LSD:

Least significant difference

ANCOVA:

Analysis of covariance

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Acknowledgements

We thank the authors responsible for published data that were included in this article. We are grateful to Prof. Li Zhang and Na Zhang for their expert opinion and technical assistance.

Funding

This study was sponsored by Innogen Pharmaceutical Co. Ltd., Shanghai, China.

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Authors and Affiliations

  1. Shanghai Innogen Pharmaceutical Co., Ltd, Shanghai, China

    Qinghua Wang, Yan-Ru Lou & Gerald J. Prud’homme

  2. Department of Endocrinology and Metabolism, Shanghai Medical School, Huashan Hospital, Fudan University, Shanghai, China

    Qinghua Wang, Yue Zhou, Yunzhi Ni & Zhihong Wang

  3. PriMed Non-human Primate Research Center of Sichuan PriMed Shines Bio-tech Co., Ltd, Ya’an, Sichuan Province, China

    Zunyuan Yang, Li Gong, Yinan Liang & Wen Zeng

  4. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

    Gerald J. Prud’homme

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Contributions

Q W contributed to the conception and design of the study. Z Y and W Z contributed to performing the major body of the experiments. Y Z, Y N, Z W and Z Y analyzed the data. Y Z, Y N, Y-R L, G P and Q W contributed to writing the manuscript. All authors reviewed and approved the manuscript.

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Correspondence to Qinghua Wang or Wen Zeng.

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All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Primed Shines Bio-tech Co., Ltd (No. AW2110).

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Supplementary Material 1: Additional file 1 Figure S1 Treatment with supaglutide showed a lowering tendency of fibrosis area fraction as assessed by Masson staining in liver biopsies. Table S1 Fibrosis quantification.

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Wang, Q., Zhou, Y., Ni, Y. et al. Supaglutide alleviates hepatic steatosis in monkeys with spontaneous MASH. Diabetol Metab Syndr 16, 303 (2024). https://doi.org/10.1186/s13098-024-01513-7

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Keywords

Diabetology & Metabolic Syndrome

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