Abstract Objectives Metabolic-associated fatty liver disease (MAFLD) is the most common chronic liver disease that can range from hepatic steatosis to non-alcoholic steatohepatitis (NASH), which can lead to fibrosis and cirrhosis. Recently, ketogenic diet (KD), a low carbohydrate diet, gained popularity as a weight-loss approach, although it has been reported to induce hepatic insulin resistance and steatosis in animal model systems via an undefined mechanism. Herein, we investigated the KD metabolic benefits and its contribution to the pathogenesis of NASH. Methods Using metabolic, biochemical and omics approaches, we identified the effects of a KD on NASH and investigated the mechanisms by which KD induces hepatic insulin resistance and steatosis. Results We demonstrate that KD can induce fibrosis and NASH regardless of body weight loss compared to high-fat diet (HFD) fed mice at thermoneutrality. At ambient temperature (23 °C), KD-fed mice develop a severe hepatic injury, inflammation, and steatosis. In addition, KD increases liver cholesterol, IL-6, and p-JNK and aggravates diet induced-glucose intolerance and hepatic insulin resistance compared to HFD. Pharmacological inhibition of IL-6 and JNK reverses KD-induced glucose intolerance, and hepatic steatosis and restores insulin sensitivity. Conclusions Our studies uncover a new mechanism for KD-induced hepatic insulin resistance and NASH potentially via IL-6-JNK signaling and provide a new NASH mouse model. Keywords: Ketogenic diet, Hepatic insulin resistance, NASH, MAFLD, IL6, JNK Graphical abstract Image 1 [41]Open in a new tab Highlights * • Ketogenic diet reduces body weight and induces NASH at thermoneutrality * • Ketogenic diet increased IL-6 and JNK signaling pathways * • IL-6 and JNK induced hepatic insulin resistance and steatosis in keteogenic diet fed mice * • IL-6 neutralization reverses hepatic steatosis in ketogenic diet-fed mice * • Ketogenic diet is a new useful NASH mouse model List of abbreviations MAFLD metabolic associated fatty liver disease NAFLD Non-alcohol fatty liver disease NASH non-alcoholic steatohepatitis KD ketogenic diet HFD high-fat diet FFA free fatty acid WAT white adipose tissue DIO diet-induced obesity IL-6 interleukin-6 CD chow diet JNK c-jun N-terminal kinase LPC lysophosphatidylcholine PE Phosphatidylethanolamine DG diglycerides PS phosphatidylserine PG phosphatidylglycerol PI phosphatidylinositol PEP PE-based plasmalogens Cer ceramides HexCer hexosylceramides TG Triglycerides CE cholesteryl ester PC Phosphatidylcholine SM sphingomyelin FC free cholesterol 1. Introduction Metabolic-associated fatty liver disease (MAFLD) is a hepatic manifestation of metabolic disorders that can range from steatosis to non-alcoholic steatohepatitis (NASH). NASH, the aggravated form of MAFLD [[42]1,[43]2], can progress to liver fibrosis, cirrhosis and hepatocellular carcinoma [[44]3]. MAFLD is affecting up to a third of adults in high-income countries [[45]3]. Many individuals with MAFLD have obesity [[46]3,[47]4], although, non-obese persons can also develop MAFLD [[48]5]. The imbalance between liver lipid storage and removal [[49]6], due to increased fat intake, elevated de novo lipogenesis, increased lipolysis in adipose tissue, decreased fat oxidation, and reduced hepatic very low-density lipoprotein secretion contributes to MAFLD [[50][7], [51][8], [52][9]]. Moreover, inflammatory signals from adipose tissue: such as TNFα, IL-6 and CCL2 can exacerbate liver inflammation, cell death, and fibrosis [[53]1,[54]2]. One-third of NASH patients develop fibrosis and MAFLD-related mortality is increased up to ten-fold in patients with NASH [[55]10]. Therefore, many pharmacological agents have been studied with the aim to improve liver inflammation and steatosis to prevent MAFLD [[56]11]. Nevertheless, weight loss remains the only standard intervention method for the management of NAFLD in patients [[57]12]. Studies investigated the mechanism behind MAFLD and reported that IL-6 induces free fatty acid (FFA) release from visceral adipocytes, thereby promoting diet-induced hepatic insulin resistance and steatosis [[58]13]. Visceral white adipose tissue (WAT) is a source for diet-induced circulating IL-6, while subcutaneous WAT contributes to basal IL-6 levels [[59]14]. Elevated IL-6 levels in mice with liver-specific overexpression of IKK-β were associated with glucose intolerance and hepatic insulin resistance [[60]15] and IL-6 neutralization in mice led to improved hepatic insulin resistance [[61]15], indicating that IL-6 involved in modulation of liver function [[62]14,[63]15]. The inflammatory c-jun N-terminal kinase (JNK), a member of the mitogen activated protein kinase (MAPK) family, is activated by TNFα, IL-6 and FFA, and can increase Ser/Thr phosphorylation of IRS1/2, that can lead to insulin resistance [[64][16], [65][17], [66][18]]. While, deletion of JNK1 in adipose tissue results in blunted IL-6 in DIO mice [[67]19] and increased hepatic insulin sensitivity [[68]20]. Furthermore, liver JNK1 knockout mice demonstrate decreased steatosis, suggesting that JNK activation in the liver can induces MAFLD [[69]21] and insulin resistance [[70]22]. Ketogenic diet (KD), a high-fat and low-carbohydrate diet, which is used in the treatment of epilepsy [[71]23] and has beneficial effect in autism and Alzheimer disease [[72]24], has become a popular weight loss strategy [[73]25,[74]26]. KD is inherently a low-protein diet [[75]27]. In lean healthy people without diabetes and pregnancy, KD induces ketosis after 2–4 days [[76]28]. In mice a KD containing 9.1% calories from protein significantly increased ketone bodies after 1 day [[77]29]. Clinical KD used in humans are restricted in protein content (5–10% of calories from protein compared to normal diets which contain 15–20% of protein) [[78]27]. However, most commonly used rodent KD contains less than 5% of energy from proteins [[79]27] which can lead to inconsistent results between human and animal studies [[80]30]. For instance, KD with low protein levels led to elevated FGF21 levels, independent of ketogenesis in mice [[81]31]. Based on these points it was proposed that for translational studies in mice, KD should contain 8–10% protein to ensure that the observations are due to low carbohydrate content, rather than due to protein restriction [[82]27]. Clinical studies reported that KD improved blood glucose, serum lipid and led to weight loss in obese subjects [[83]25,[84]26,[85]32]. However, a 3-day of KD consumption increased postprandial plasma glucose in healthy men [[86]33]. Four weeks of KD intervention also increased cholesterol and inflammatory markers in obese subjects [[87]34]. In mice, 12 or 22 weeks of KD increased steatosis, inflammation and glucose intolerance [[88]35,[89]36]. Moreover, hyperinsulinemic–euglycemic clamp studies demonstrated that 5 weeks of KD induced hepatic insulin resistance in mice [[90]37]. The underlying mechanisms behind KD-induced hepatic insulin resistance and steatosis, however, remain undefined. Here, we investigated the metabolic effects of a KD with 8.5% of protein at late timepoints to ensure a robust ketosis phenotype and its contribution to the pathogenesis of NASH, to identify the mechanisms underlying KD-induced hepatic insulin resistance, steatosis and fibrosis. 2. Materials and methods 2.1. Animal C57BL/6 mice were obtained from Charles River (Wilmington, MA, USA) and housed in a pathogen-free animal facility at 23 °C/30 °C under a 12-hour light/dark cycle with free access to water and standard chow diet (18% proteins, 4.5% fibers, 4.5% fat, 6.3% ashes of energy, #2222, Kliba-Nafag, Switzerland). After 12 weeks of chow, male mice were fed either a chow, a KD (8.5% protein, 4.3% fibers, 79.1% fat, 4.3% ashes, 3.8% carbohydrate of calories, [91]E15149 Snniff, Germany), or a high-fat diet (HFD) with 60% of energy derived from fat (23.9% protein, 3% fibers, 35% fat, 5.7% ashes, 23.2% carbohydrate, #3436, Kliba-Nafag, Switzerland). All animal studies conformed to the Swiss animal protection laws and were approved by the cantonal Veterinary Office in Zurich, Switzerland 2.2. Glucose tolerance test To investigate whether KD effect on glucose tolerance is acute or progressive 12 weeks old male mice were randomly assigned to either chow, KD or HFD for 3 and 7 days or 5 and 16 weeks. Mice were fasted for 6 h and baseline glucose levels were measured. Thereafter, glucose (2 g/kg body weight (d-glucose, Sigma in 0.9% saline)) was injected intraperitoneal (i.p.) and blood glucose concentration was measured from tail-tip blood after 15, 30, 45, 60, 90, and 120 min by using a glucometer (Accu-Check Aviva glucose strip system, #07400918016, #06453988016; Roche Diagnostics International, Basel, Switzerland). 2.3. IL-6 neutralization antibody For in vivo IL-6 neutralization 12 weeks old male mice were randomly assigned to either chow, KD or HFD for 3 days and received an i.p. injection of 100 μg rat anti-mouse IL-6 antibody (Cat# 16-7061, Clone MP5-20F3, Switzerland) in 0.1 ml Dulbecco's phosphate-buffered saline. Rat IgG1 (Cat# 16-4301, ThermoFisher, Switzerland) was administered as an isotype-matched control. Administration of the antibodies was conducted every day for consecutive 3 days. 2.4. JNK inhibitor treatment 12 weeks old male mice were randomly assigned to either chow, KD or HFD for 3 days for pharmacological inhibition of JNK, 10 mg/kg AS602801 (Cat# HY-14761, Med Chem Express, Switzerland) or vehicle alone (2:8:1:9 ratio of DMSO; PEG300; Tween-80; saline) was administered by i.p. injection once daily for consecutive 3 days. 2.5. Lipid extraction and mass spectrometric analysis Liver samples from mice fed a chow, KD or HFD for 3 days were fasted for 6 hrs and injected with glucose before sampling to mimic KD-Induced glucose intolerance. Liver samples were subjected to bead-based homogenization in H[2]O/MeOH = 1/1 supplemented with 1% SDS at a concentration of 0.05 mg wet weight/μL [[92]38]. The lipids were extracted from 2 mg of wet weight according to the method of Bligh and Dyer [[93]39] in the presence of not naturally occurring lipid species as internal standards. The following lipid species were added as internal standards: PC 14:0/14:0, PC 22:0/22:0, PE 14:0/14:0, PE 20:0/20:0 (di-phytanoyl), PS 14:0/14:0, PS 20:0/20:0 (di-phytanoyl), PI 17:0/17:0, LPC 13:0, LPC 19:0, LPE 13:0, Cer 18:1;O2/14:0, Cer 18:1;O2/17:0, D7-FC, CE 17:0, CE 22:0, TG 51:0, TG 57:0, DG 28:0 and DG 40:0. Chloroform phase was recovered by a pipetting robot (Tecan Genesis RSP 150) and vacuum dried. The residues were dissolved in either in 10 mM ammonium acetate in methanol/chloroform (3:1, v/v) (for low mass resolution tandem mass spectrometry) or chloroform/methanol/2-propanol (1:2:4 v/v/v) with 7.5 mM ammonium formate (for high resolution mass spectrometry). Data analysis was performed by wilcoxon pairwise comparisons with corrections for multiple testing, details in the supplementary. 2.6. Histological analysis For histological analysis, 12 weeks old male mice were randomly assigned to either chow, KD or HFD for 3 days or 14 and 16 weeks at ambient or thermoneutrality. Liver tissues from all mice were fixed with 4% paraformaldehyde in PBS (Gibco; pH 7.4) for 24 h at 4 °C, dehydrated and embedded in paraffin. Liver sections (3 μm thick) were stained with hematoxylin and eosin (H&E), Oil red O, van Giesson/granular osmiophilic material (GOM)/Masson's trichrome and cleaved caspase 3. Tissue processing was performed by Prof. Dr. Anja Kipar (Pathologist, Zurich University); liver sections were stained with auto strainers for histology, immunohistology and the images were acquired by digital slide scanners and quantified. Histological assessments were conducted in blinded conditions. For quantitative assessment, various parameters, including inflammation, steatosis, hepatocyte ballooning, fibrosis, NASH and NAFLD, the NASH Clinical Research Network Scoring System [[94]40] were assessed, taking into account murine NAFLD activity score according to [[95]41,[96]42]. 2.7. Plasma and tissue lysate parameters To study KD effects on inflammatory cytokines, 12 weeks old male mice were randomly assigned to either chow, KD or HFD for 3 days and 16 weeks. Mice were fasted for 6 h and injected glucose 30 min, or without glucose injections before tissue and blood sampling. Circulating IL-6 concentration, IL-6 in liver, mesenteric fat, and ingWAT lysate were measured by using Mouse IL-6 ELISA Kit (RayBiotech, #ELM-IL6, Luzern, Switzerland). Liver cholesterol was measured by Cobas Roche (Hitachi Kit #11877771, Roche Diagnostics International). Plasma cytokine Interferon gamma (IFN-γ), interleukin 1 beta (IL-1β), interleukin 2 (IL-2), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin-12 (IL-12p70), tumor necrosis factor alpha (TNF-α), and keratinocyte chemoattractant (KC)/growth-regulated oncogene (GRO) chemokines and pro-inflammatory chemokines were measured using the MSD technology (Meso Scale Discovery, Gaithersburg, MD, USA). All analyses were carried out according to manufacturer's protocols. 2.8. Quantification and statistical analysis A power calculation was performed based on the results of previous work by our group to calculate animal numbers [[97]43]. All data are expressed as mean ± standard error of the mean. The significance was determined using a two-tailed, unpaired Student's t-test, one-way ANOVA with Newman–Keuls correction for multiple group comparisons, or two-way ANOVA with Bonferroni multiple comparisons/Tukey's multiple comparison. Statistical tests were calculated using GraphPad Prism 8.0 (GraphPad Software, San Diego, USA). P-values <0.05 were considered significant. For further details regarding the materials and methods used, please refer to the [98]supplementary information. 3. Results 3.1. Three days of KD induces hepatic steatosis To investigate whether short-term KD can induce hepatic steatosis, we analyzed the physiological changes in response to 3 days of KD feeding in mice ([99]Figure 1A). Three days of KD reduced body weight compared to chow and HFD-fed mice ([100]Figure 1B). Importantly, 16 weeks ([101]Fig. S1A) of chow and KD led to a similar body weight gain compared to HFD ([102]Fig. S1B), indicating that long term KD feeding does not affect body weight in lean mice. As expected, 16 weeks of HFD significantly increased liver and fat tissue masses, whereas similar fat tissue weights were observed in chow and KD-fed mice ([103]Figs. S1C–D). KD significantly increased liver weight compared to chow-fed mice ([104]Fig. S1D). In addition, 3 days or 16 weeks of KD increased hepatic lipid droplet accumulation ([105]Figure 1C, [106]S1E) demonstrating that KD accelerates diet-induced hepatic steatosis. Figure 1. [107]Figure 1 [108]Open in a new tab Three days of ketogenic diet (KD) accelerates diet-induced hepatic steatosis. (A) Experimental scheme for KD or high fat diet (HFD) feeding in C57BL/6 WT mice. WT Chow (CD) fed mice (black): KD-fed mice (orange): and HFD groups (blue). Mice were fed a CD, KD or HFD for 3 days and fasted for 6-hrs and injected with glucose (2 g/kg body) 30 min before sampling. (B) Body weight measurement (CD n = 5; KD n = 8; HFD n = 8), values are presented as mean ± SEM. ∗∗∗(p < 0.001), ^### (p < 0.001) by 2-way ANOVA. (C) Representative images of liver sections stained with H&E from mice fed a CD, HFD and KD. Scale bar represents 100 μm. (D) Individual plots with 95% confidence ellipses of the lipid principal component analysis. (E) Lipid profile clustering plots with Ward's criterion and (F) Volcano plots for lipid analysis by pairwise comparisons. (G–-I) Volcano plot of lipid detected (G) yellow: 50 upregulated lipid species in KD, purple: 27 decreased lipid species in KD vs CD, black: NS. (H) yellow: 41 increased lipid species in KD, purple: 7 decreased lipid species in KD vs HFD, black: NS. (I) yellow: 14 increased lipid species in HFD, purple: 11 decreased lipid species in HFD vs CD, black: NS. For G-I p-values corresponding to a t-test of single lipid concentrations. (J) The distribution of the lipid species in the pool of lipids upregulated vs the total pool tested with Fisher's exact test for count data with Monte-Carlo simulated P-value 10,000 replicates; P < 0.01. TG = triglyceride. (K) Total concentrations of liver TG. (L) Free cholesterol. D-L number of mice (CD n = 5; KD n = 8; HFD n = 6). (For interpretation of the references