Alleviation of Hepatic Steatosis by Alpha-Defensin Is Associated with Enhanced Lipolysis

Background and Objectives: The neutrophilic peptide, alpha-defensin, is considered an evolving risk factor intimately linked with lipid mobilization. It was previously linked to augmented liver fibrosis. Here, we assess a potential association between alpha-defensin and fatty liver. Materials and Methods: A cohort of transgenic C57BL/6JDef+/+ male mice that overexpress the human neutrophil-derived alpha-defensin in their polymorphonuclear neutrophils (PMNs) were assessed for liver steatosis and fibrosis development. Wild type (C57BL/6JDef.Wt) and transgenic (C57BL/6JDef+/+) mice were maintained on a standard rodent chow diet for 8.5 months. At the termination of the experiment, systemic metabolic indices and hepatic immunological cell profiling were assessed. Results: The Def+/+ transgenic mice exhibited lower body and liver weights, lower serum fasting glucose and cholesterol, and significantly lower liver fat content. These results were associated with impaired liver lymphocytes count and function (lower CD8, NK cells, and killing marker CD107a). The metabolic cage demonstrated dominant fat utilization with a comparable food intake in the Def+/+ mice. Conclusions: Chronic physiological expression of alpha-defensin induces favorable blood metabolic profile, increased systemic lipolysis, and decreased hepatic fat accumulation. Further studies are needed to characterize the defensin net liver effect.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease worldwide. NAFLD is a complex disease associated with hepatic steatosis, which is affected by a variety of environmental and genetic factors [1][2][3][4][5]. It is largely considered a hepatic manifestation of the metabolic syndrome. This disorder encompasses a wide range of diseases, from simple steatosis, which is relatively benign, to hepatic inflammation, hepatocyte injury, and fibrosis, a syndrome referred to as non-alcoholic steatohepatitis (NASH), that can progress to cirrhosis [6][7][8][9]. adheres to Israeli guidelines and follows the NIH/USA animal care and use protocols. Mice were housed on hardwood chip bedding in individual ventilation cages (IVC) under a 12 h light/dark cycle at 21-23 • C, given tap water and standard rodent chow diet that contained 4.5% fat (PMI5010, Harlan, Rehovot, Israel) ad libitum since weaning day (3 weeks old) until the end of the experiment at the age of 8.5 months. In each experimental group, 6-8 mice of the C57BL/6 transgenic (Def +/+ ) and its control (WT) were assessed. This study was conducted in the years 2017-2018. Notably, the expression of alpha-defensin in the transgenic mice was confirmed by measuring the concentrations of alpha-defensin in plasmas and sera via enzyme-linked immunosorbent assay (ELISA) as described previously (22).

Plasma Analysis
Blood samples were collected at the age of 8.5 months, after 12 h fasting, via direct cardiac puncture. Plasma glucose, triglycerides, total cholesterol, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured using the Cobas ® 6000 analyzer series (ROCHE diagnostics). Plasma samples were diluted 1:10 with PBS for measurement of ALT and AST.

Quantification of Total Hepatic Lipid
Total hepatic lipid was determined using a protocol adapted from Mopuri et al. [24] with minor modifications. Livers were obtained at the ages of 4 months and 8.5 months and were kept at −80 • C. Frozen liver tissue (100 mg) was homogenized in 4 mL of 2:1 chloroform-methanol solution, at 10,000-15,000 rpm, under 4 • C cooling for 10-30 s, until a homogenous texture was obtained. Samples were kept for 2 h at 4 • C, and then centrifuged at 5000 to 7000 rpm for 10 min to facilitate the separation of the upper phase (aqueous methanol dragging) and the lower phase (chloroform phase) containing the lipids. A volume of 1 mL of the chloroform phase was transferred into a tube previously weighed, and the solution was evaporated by drying using a nitrogen stream. The tube was weighed again, and the amount of fat was extracted. Total lipid per 100 mg liver tissue was calculated. Finally, lipids were dissolved in isopropanol and triglycerides measured using spectrophotometric method.

Liver Fat Fraction Quantification Using EchoMRI
The total ex vivo liver fat contents were evaluated using the EchoMRI™-100H (Echo. Medical Systems, Houston, TX, USA) at 4 and 8.5 months. The mice were sacrificed via cervical dislocation and the livers were completely removed for EchoMRI evaluation. The tube was thoroughly sanitized using 10% bleach solution and then rinsed and dried after each use.

Tissue Processing and Histological Analyses
All animals were housed in a temperature-controlled room (23-24 • C) on a 12 h light/dark cycle and had free access to food and water. Mice were sacrificed via cervical dislocation. After fixation in 10% neutral buffered formalin, a slice of the median lobe of the liver was trimmed, processed, sectioned into slices approximately 5 µm thick, and mounted on a glass slide. Hematoxylin and eosin stains, liver immunofluorescence staining with Hsp60 and β-Catenin, and electron microscope images were used for morphological analyses, and Masson's trichrome and Sirius red stains (0.1% Sirius red F3B in saturated picric acid (both from Sigma, Inc. (St. Louis, MO, USA))) were used for assessment of hepatic fibrosis. Histopathological analysis was performed by a pathologist who was not exposed to the study groups. Data were derived from blinded analysis of 5 sections from each of the 8 animals in each group. We used the NAFLD activity score (NAS) system to estimate steatosis grade and fibrosis stage.

Cell Isolation, Staining, and Flow Cytometric Analysis
The spleen was homogenized, and lymphocytes were washed and counted before staining for flow-cytometric analysis. Intrahepatic lymphocytes were isolated via perfusion of the liver with digestion buffer. After perfusion, the liver was homogenized and incubated at 37 • C for 30 min. The digested liver cell suspension was centrifuged to remove hepatocytes and cell clumps. The supernatant was then centrifuged to obtain a pellet of cells depleted of hepatocytes to a final volume of 1 mL. Lymphocytes were then isolated from this cell suspension using 24% metrizamide gradient separation. Liver natural killer (NK) cells, CD4+, and CD8+ subsets were isolated using DYNAL Biotech kits according to manufacturer's instructions.

Metabolic Caging Analysis
Metabolic rate of the control and Def +/+ mice on standard diet (STD) was measured using the Promethion Metabolic Phenotyping System by Sable Systems International, by incorporating sub-systems for open-circuit indirect calorimetry, feeding, water intake, activity, running wheel, body mass, and core temperature measurements in conventional live-in home cages, mice experienced minimized stress (Sable Instruments, Inc., Las Vegas, NV, USA); this fully automated system is the "one-test" solution for simultaneous multiparameter assessment for any metabolic, behavioral, and physiological research. While in the metabolic chambers, the mice had free access to food and water. We analyzed 8 individually caged mice at the same time. Mice were housed in these cages for 7 days. These were conventional live-in home cages with regular bedding. The parameters provided by the metabolic cages included food intake, respiratory quotient, total energy expenditure, fat oxidation, and carbohydrate oxidation.

Statistical Analysis
Continuous variables are presented as a mean and standard deviation, and categorical data are presented as percentages. Categorical variables were compared using Pearson's chi-square test, while continuous variables were compared using Student's t-test. All statistical analyses were performed on IBM SPSS version 26. Statistical significance was set at the 2-tailed 0.05 level, without multiplicity adjustment.

Quantification of Total Hepatic Lipid
The hepatic profiles of the fat accumulation of the mice livers are given in Figure 2I. At 4 months of age, the hepatic lipid composition was similar in both groups. At 8.5 months of age, the lipid content in Def +/+ mice was marginally affected with even slight, non-significant decrease in the total TG content. However, the total lipid content and total triglyceride/liver were significantly higher in the WT group (p < 0.05) at the age of 8.5 months.

Quantification of Total Hepatic Lipid
The hepatic profiles of the fat accumulation of the mice livers are given in Figure 2A. At 4 months of age, the hepatic lipid composition was similar in both groups. At 8.5 months of age, the lipid content in Def +/+ mice was marginally affected with even slight, nonsignificant decrease in the total TG content. However, the total lipid content and total triglyceride/liver were significantly higher in the WT group (p < 0.05) at the age of 8.5 months.

Liver Fat Fraction Quantification using EchoMRI
The EchoMRI results of the fat mass composition in the livers were in line with the biochemical findings. The MRI detected slightly higher fat mass percentage in the 4-monthold WT mice, which significantly increased at the age of 8.5 months, reaching 23.3% ± 3 compared to the 10.7% ± 1 (p < 0.05) fat mass in the Def +/+ mice ( Figure 2B).

Lymphocyte Isolation
The immune system was shown to play a key role in the development of liver fibrosis. Different subtypes of lymphocytes are involved in liver fibrosis as well as in the clearance of necrotic cells during inflammation.

Liver Fat Fraction Quantification using EchoMRI
The EchoMRI results of the fat mass composition in the livers were in line wi biochemical findings. The MRI detected slightly higher fat mass percentage in month-old WT mice, which significantly increased at the age of 8.5 months, rea 23.3% ± 3 compared to the 10.7% ± 1 (p < 0.05) fat mass in the Def +/+ mice ( Figure 2II 3.4. Lymphocyte Isolation The immune system was shown to play a key role in the development of liver sis. Different subtypes of lymphocytes are involved in liver fibrosis as well as in the ance of necrotic cells during inflammation.

Tissue Processing and Histological Profile
The histological examination demonstrated a low steatosis grade in the Def +/+ transgenic mice relative to the age-matched WT mice (1 ± 0.0 vs. 1.9 ± 0.4, p < 0.05, respectively), with the latter exhibiting a diffuse mixed macro and microvesicular steatosis ( Figure 3). Notably, both mice groups started with minimal steatosis at the early age of 4 months ( Figure 2D). The Sirius red and Masson blue staining for fibrosis disclosed mildly increased fibrosis stain in the Def +/+ group, with a near absence of fibrosis in the WT group (Figure 4). In addition, the electron microscopy demonstrated a lower fat accumulation, and a higher density of glycogen deposition in the Def +/+ mice.
with the latter exhibiting a diffuse mixed macro and microvesicular steatosis ( Figure  Notably, both mice groups started with minimal steatosis at the early age of 4 mon ( Figure 2IV). The Sirius red and Masson blue staining for fibrosis disclosed mildly creased fibrosis stain in the Def +/+ group, with a near absence of fibrosis in the WT gro ( Figure 4). In addition, the electron microscopy demonstrated a lower fat accumulati and a higher density of glycogen deposition in the Def +/+ mice.

Metabolic Caging Analysis
The Def +/+ mice show a different respiratory profile compared to their WT littermates. The alpha-defensin transgenic mice demonstrated a lower respiratory quotient ( Figure  5A). Moreover, the overexpression of alpha-defensin does not change the total energy expenditure (TEE; Figure 5B). On the other hand, the overexpression of alpha-defensin is

Metabolic Caging Analysis
The Def +/+ mice show a different respiratory profile compared to their WT littermates. The alpha-defensin transgenic mice demonstrated a lower respiratory quotient ( Figure 5A). Moreover, the overexpression of alpha-defensin does not change the total energy expenditure (TEE; Figure 5B). On the other hand, the overexpression of alpha-defensin is reflected by a higher fat oxidation ( Figure 5C) and a lower carbohydrate oxidation ( Figure 5D).

Discussion
Non-alcoholic fatty liver disease (NAFLD) is a worldwide looming epidemic. It is associated with a wide range of pathological manifestations at the liver level [25], as well as extrahepatic complications, including cardiovascular disease [26].
Effective therapeutic approaches for NAFLD patients are limited, and lifestyle modification remains the mainstay of treatment. Hence, the identification of innovative targets

Discussion
Non-alcoholic fatty liver disease (NAFLD) is a worldwide looming epidemic. It is associated with a wide range of pathological manifestations at the liver level [25], as well as extrahepatic complications, including cardiovascular disease [26].
Effective therapeutic approaches for NAFLD patients are limited, and lifestyle modification remains the mainstay of treatment. Hence, the identification of innovative targets and therapies is imperative.
Over the last years, the appreciation of the role of inflammation in NAFLD has burgeoned. Compelling evidence indicates that insights gained from the link between inflammation and NAFLD can yield predictive and prognostic information of considerable clinical utility. Previously, we described the role of alpha-defensin dimerization with LDL in accelerating cholesterol clearance from the circulation, and promoting its internalization, deposition, and retention in the vessel wall, as well as in the liver [22]. Alpha-defensin overexpression eventually induced atherosclerosis despite normal levels of plasma cholesterol, and lower glucose levels. In vitro, alpha-defensins were implicated in the activation of macrophages [27,28], and in the production of TNFα. The latter was proven to play a critical role in the induction and progression of NAFLD [29][30][31].
The above findings prompted our initial speculation for the Def +/+ mice as a potential trackable link between inflammation and the development of steatosis. Animal models used to study NAFLD mostly involve diets and different insults that do not simulate the metabolic or inflammatory context of human NAFLD. In this paper, we test the unprovoked, long-term effect of alpha-defensin expression on liver fat deposition. The old WT mice exhibited moderate liver steatosis, as opposed to negligible fat accumulation in the Def +/+ mice; this finding was concordant with significantly lower Def +/+ body weight.
In an attempt to reconcile the reduced liver fat content, we used the metabolic cages for metabolic and behavioral phenotyping. We found a similar food intake of the WT and Def +/+ mice. Surprisingly, although alpha-defensin overexpression did not affect the total energy expenditure, it prompted a significantly higher fat oxidation and lower carbohydrate consumption. The reduced carbohydrate utilization might explain the abundance of glycogen in the Def +/+ mice livers seen in EM.
The Ibusuki group [19] used a pan-tissue alpha-defensin expression model plus a CDAA diet and proved a significant fibrotic effect of alpha-defensin with a null effect on steatosis at the age of 17 weeks. The current study mainly demonstrates a substantial anti-steatotic effect of physiological alpha-defensin expression, which is obviously not a result of advanced liver fibrosis. We believe both "hits" of non-physiological alpha-defensin overexpression and a CDAA pro-fibrotic diet amplified the fibrotic effect of alpha-defensin in the Ibusuki study. In the same line, Ibusuki and colleagues measured lower levels of serum cholesterol and triglycerides in the alpha-transgenic mice that were ascribed to advanced hepatic fibrosis. In our model, we found using the blood coagulation tests that more sensitive clues of hepatic dysfunction/fibrosis were preserved in the Def +/+ mice. The described low cholesterol is potentially the result of active cholesterol shuttling and augmented utilization.
Golabi and colleagues [32] recently introduced the entity of "lean NAFLD"; this group comprises 10-20% of NAFLD patients, also termed metabolically unhealthy non-obese NAFLD. Our model of the physiological expression of alpha-defensin, free from dietary insults, ends with lean, normoglycemic and normolipidemic non-fatty liver mice. These lower indexes are neither related to energy expenditure nor to decreased food intake. Innovatively, the Def +/+ mice are dominant fat metabolizers with extremely low glucose consumption. This finding may explain both the lower fat contents with higher "lipolysis derived" plasma TG levels and residual glycogen deposits in the Def +/+ mice livers via a mechanism that is yet to be clarified.

Conclusions
To our knowledge, this is the first study to show that alpha-defensin might evolve as a hepatic anti-steatotic factor. As fat accumulation in the liver is considered a prerequisite for the development of NAFLD/NASH, future research investigating the metabolic impacts of alpha-defensin seems imperative.