Dulaglutide Alone and in Combination with Empagliflozin Attenuate Inflammatory Pathways and Microbiome Dysbiosis in a Non-Diabetic Mouse Model of NASH

Dysregulation of glucose homeostasis plays a major role in the pathogenesis of non-alcoholic steatohepatitis (NASH) as it activates proinflammatory and profibrotic processes. Beneficial effects of antiglycemic treatments such as GLP-1 agonist or SGLT-2 inhibitor on NASH in patients with diabetes have already been investigated. However, their effect on NASH in a non-diabetic setting remains unclear. With this aim, we investigated the effect of long-acting GLP1-agonist dulaglutide and SGLT-2 inhibitor empagliflozin and their combination in a non-diabetic mouse model of NASH. C57BL/6 mice received a high-fat-high-fructose (HFHC) diet with a surplus of cholesterol for 16 weeks. After 12 weeks of diet, mice were treated with either dulaglutide, empagliflozin or their combination. Dulaglutide alone and in combination with empagliflozin led to significant weight loss, improved glucose homeostasis and diminished anti-inflammatory and anti-fibrotic pathways. Combination of dulaglutide and empagliflozin further decreased MoMFLy6CHigh and CD4+Foxp3+ T cells. No beneficial effects for treatment with empagliflozin alone could be shown. While no effect of dulaglutide or its combination with empaglifozin on hepatic steatosis was evident, these data demonstrate distinct anti-inflammatory effects of dulaglutide and their combination with empagliflozin in a non-diabetic background, which could have important implications for further treatment of NASH.


Table of content
Germany in the drinking water ad libidum for 16 weeks. After 12 weeks of the diet, mice were randomly divided into 5 treatment groups for the last 4 weeks. All animals were included into the study. Sample size was calculated by biostatistical analyses based on previous experiments. Mice were either treated with dulaglutide (10nmol/kg/3xper week; Eli Lilly, Bad Homburg, Germany) (n=10) in saline intraperitoneally (i.p.) or treated with saline i.p. as control group in the same volume (n=10). Another treatment group (n=10) was treated daily with empagliflozin (10mg/kg; Boehringer Ingelheim, Ingelheim am Rhein, Germany) in 0,5% Hydroxyethylcellulose (Natrosol, Caelo, Hilden, Germany) administered by oral gavage. The control group (n=10) was treated with 0,5% Hydroxyethylcellulose by oral gavage with the same volume. Intragastric gavage administration was carried out with conscious animals, using straight gavage needles appropriate for the animal size. The biological effect of empagliflozin was proven by measuring the urine glucose content (Combur-Test, Roche, Basel, Schweiz). Another group (n=9) was treated both with daily empagliflozin p.o.(10mg/kg; Boehringer Ingelheim, Ingelheim am Rhein, Germany) and with dulaglutide (10nmol/kg/3xper week; Eli Lilly, Bad Homburg, Germany). The doses in dulaglutide and empagliflozine were selected according to previous studies [1,2]. All mice were sacrificed after 16 weeks of diet by CO2 inhalation followed by cervical dislocation to minimize animal discomfort. Blood was collected via puncture of the retrobulbar plexus just after death. Liver, spleen and ectopic adipose tissue (brown adipose tissue-BAT, white adipose tissue-WAT, visceral adipose tissue-VAT) were collected for further investigations. Mice were weighed non-fasted.

Intraperitoneal glucose-tolerance test
After 16 weeks of HFHC diet, an intraperitoneal glucose-tolerance test (IPGTT) was performed. After 12 hours of fasting, fasting blood glucose levels were measured using blood from the tail vein (One Touch Ultra, LifeScan, Milpitas, CA, USA). Following this, 1mg glucose/g bodyweight was administered intraperitoneally (i.p.). Blood glucose levels were measured at 30, 60, 90 and 120 minutes after injection, using a blood glucose test strip (One Touch Ultra/LifeScan, Milpitas, CA; USA)). For the bioinformatics analysis, sequencing was performed using the Illumina MiSeq platform and bioinformatic processing of paired end reads were done. Raw reads were merged and subsequently aligned using MOTHUR (gotoh algorithm using the SILVA reference database) prior to pre-clustering (diffs=2) [5]. Phylotypes exhibiting an average abundance of ≥ 0.001% and a sequence length ≥ 250bp were considered for follow up analysis. Phylotypes were classified using RDP's naive Bayesian classifier applying a confidence threshold of 80% [6].

Flow cytometry analyses
Sequences from phylotypes that were not annotated down to the genus level were subjected to RDP's Seqmatch allowing for some additional manual annotations at this level as previously described [4]. Sequence counts were merged at taxonomic levels within each sample. Count tables were subsampled to lowest number of sequences using the function rarefy_even_depth (rngseed=TRUE) from R's package phyloseq.

Supplementary figures and tables
Supplementary Figure S1    To prove the biological activity of empagliflozin, we tested the glucosuria using Combur 9 test.
Here, we could show increased glucosuria after empagliflozin gavage compared to natrosol gavage, which proves the biological activity of empagliflozin.