Combined Role of Cell Cleanup and Fat-Breaking Enzyme Reduces Diet-Related Fat Build-Up in the Liver of Mice Missing a Specific Liver Protein

All experiments were conducted at the Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging in accordance with guidelines and regulations approved by the Institutional Animal Care and Use Committee of Tufts University. All mice starting at weaning were fed purified, chow diet (2016S, Teklad). Individually housed male PLIN2^fl/fl and PLIN2^LKO littermates were randomized into dietary treatment groups at 8 or 16 weeks of age where indicated. Animals received either an experimental, Western-type diet (WTD, Research Diets D15102305) containing 17% protein, 44% fat, and 39% carbohydrate by energy (kcal), or a micronutrient-matched low-fat control diet (LFD, Research Diets D15102307) containing 17% protein, 10% fat, and 73% carbohydrate by energy. The macronutrient composition of the WTD was modeled after a high-fat, high-sucrose diet with a demonstrated ability to induce hepatic steatosis after 12 weeks of feeding (diet composition available in Table S1 and S2) [26]. Following 12 weeks of dietary treatment animals were fasted for 6 h and euthanized via exsanguination under isoflurane anesthesia. Tissues were collected, snap frozen in liquid nitrogen, and stored at −80 °C. Blood was collected in EDTA coated tubes, spun at 2000× g for 20 min at 4 °C, and plasma fraction was collected and stored at −80 °C.

Body weight and body composition were analyzed by magnetic resonance imaging (EchoMRI-700) at baseline and 2, 4, 8, and 12 weeks following start of WTD feeding. Insulin tolerance tests (ITTs) were performed after 10 weeks of diet feeding in mice started on diet at 16 weeks of age. For ITTs animals were fasted for 6 h prior to intraperitoneal injection of 0.75 IU/kg insulin dissolved in sterile saline. Blood glucose was monitored from the tail vein via glucometer (OneTouch Ultra) at baseline (time 0) and 15, 30, 45, 60, 90, and 120 min following injection. Glucose tolerance tests (GTTs) were performed after 10 weeks of diet feeding in mice started on diet at 16 weeks of age. GTTs were performed similarly to ITTs with animals treated with 1.5 g/kg glucose dissolved in sterile saline delivered via intraperitoneal injection. As an estimate of insulin resistance, the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) was calculated as previously described using fasting insulin and blood glucose collected at time of sacrifice [16].

Following sacrifice, liver samples were fixed in either 4% zinc-buffered formaldehyde (Z-Fix) at room temperature for 48 h and then stored in 70% alcohol at 4 °C or frozen optimal cutting temperature solution and stored at −80 °C. Fixed samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and picrosirius red. Images were collected using an Olympus BX51-P polarizing light microscope with a ×40 objective. For histopathological analysis, sections were examined under light microscopy by one independent investigator who was blinded to the treatment groups. A ZEISS microscope with a PixeLINK USB 2.0 (PL-B623CU) digital Camera and PixeLINK μScope Microscopy Software was used for quantification and image capture for histopathological analyses. For each animal, 20 fields at a magnification of 10× were examined. Hepatic steatosis was graded based on the percentage of the liver section that was occupied by fat vacuoles (both macro- and micro-vesicular fat accumulation) at 100× magnification in 20 fields (grade 0 = < 5%; grade 1 = 5 to 25%; grade 2 = 26 to 50%; grade 3 = 51 to 75%; grade 4 = 76% to 100%). Inflammatory foci ((clusters of the recruited immune cells) and ballooning degeneration of hepatocytes were also examined.

All plasma measures were obtained from samples collected at time of sacrifice as described above. Plasma insulin was determined via ELISA (Ultra-Sensitive Mouse Insulin ELISA, Crystal Chem, Zaandam, Netherlands). Triglyceride (OSR6133, Beckman Coulter, Galway, Ireland), cholesterol (OSR6116, Beckman Coulter), HDL (OSR6195, Beckman Coulter), β-hydroxybutyrate (LiquiColor, StanBio, Boerne, TX, USA), and non-esterified fatty acids (NEFA-HR (2), Wako Diagnostics) were determined calorimetrically.

An amount of 50–75 mg of frozen, pulverized liver was mechanically homogenized (TissueLyser II, Qiagen, Milan, Italy) in ice-cold 2:1 chloroform-methanol and lipids were extracted following the methods of Folch et al. [27]. Lipid extract was dried under N₂ and solubilized in 1.5% Triton-X in phosphate-buffered saline. The protein fraction was isolated, dried under N₂, and digested in 1 N NaOH for 96 h at 4 °C. Triglyceride and hepatic non-esterified fatty acids (NEFA) were determined calorimetrically as described in Plasma Biochemistry above. Hepatic lipids were normalized per mg protein, as determined by BCA Assay (BCA Protein Assay Kit, Pierce).

Mouse primary hepatocytes were isolated from chow-fed, 3–9-month-old PLIN2^fl/fl and PLIN2^LKO animals following the collagenase perfusion method as previously described (18). Viability of isolated hepatocytes was routinely assessed via trypan blue exclusion and only isolations yielding 90% or greater viable cells were used for study. Following 4 h of plating, hepatocytes were incubated overnight in serum-free M199 media (23 mM HEPES, 26 mM sodium bicarbonate, 10 nM dexamethasone, 10 nM insulin, and 11 mM glucose) containing antibiotic/antimycotics. The following morning hepatocytes were pulsed for 2 h with serum-free M199 media containing 500 μM oleic acid and 0.1 µCi/mL ¹⁴C oleate. Following pulse cells were washed in PBS and chased in serum-free M199 media lacking insulin for 6 h. At the end of the experiment, media were collected and FA oxidation, as defined by the incorporation of ¹⁴C-oleate into CO₂ and acid-soluble metabolites, was determined as previously described [19]. Data were normalized per mg protein, as determined by harvesting cells at the termination of the experiment.

For ex vivo analysis, livers from mice fed a 60% high-fat diet (HFD, Research Diets D12492) for 12 weeks were harvested, washed briefly in PBS, minced on ice, and then placed in pre-warmed 12-well plates containing 37 °C M199 media supplemented with 20 mM NH₄Cl and 200 µM leupeptin. Following a 2 h incubation at 37 °C, well contents were collected, and tissue was pelleted via centrifugation. The supernatant was discarded, and pellet washed once in ice-cold PBS. Tissue was then homogenized as described below and Western blotting against the autophagic adaptor p62 was used to evaluate p62-autophagic flux. Immunostaining of endogenous LC3 and p62 was used to evaluate autophagic activity. PLIN2^fl/fl and PLIN2^LKO hepatocytes were incubated with complete medium, medium with 100 µM chloroquine (CQ), 30 µM ATGL-specific inhibitor ATGListatin (ATGLi) (Xcess Biosciences, San Diego, CA, USA), or medium containing both CQ and ATGLi treatments for 4 or 6 h (as specified in figure legends) and fixed in cold methanol. Hepatic tissues were embedded in OCT for immunohistochemistry and 10 μm cryosections were obtained, dried overnight, and stored at −80 °C until staining. Indirect immunofluorescence was performed following conventional procedures, as previously described [28,29]. ImageJ was used to calculate area occupied by autophagic vesicles per total cellular area.

Protein extract was prepared by mechanical homogenization of 50 mg of frozen, pulverized liver tissue in ice-cold TNET Buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.5% cholate) containing protease inhibitors (A32965, Thermo Scientific, Waltham, MA, USA). Protein concentration of extract was determined by BCA Assay as described above. Prior to gel loading, extracts were denatured by boiling at 95 °C for 5 min in an equal volume of 2× Laemelli Buffer (S3401-1VL, Sigma, St. Louis, MO, USA). An amount of 25–50 μg of protein was resolved in 10% polyacrylamide gels (Mini Protean TGX, BioRad, Hercules, CA, USA) and subsequently transferred to 0.45 μm nitrocellulose membranes (BioRad) at 300 mA for 2 h at 4 °C. Following transfer, membranes were blocked in 5% bovine serum albumin in phosphate-buffered saline containing 0.1% Tween-20 (PBST), washed three times in PBST, and incubated with primary antibody overnight. Blots were probed for PLIN2 (20R-AP002, Fitzgerald), PLIN3 (10694-1-AP, Proteintech, Rosemont, IL, USA), PLIN4 (rabbit anti-serum provided by Dr. Nathan Wollins), PLIN5 (GP44, Progen, Heidelberg, Germany) and GAPDH (14C10, Cell Signaling) using anti-rabbit secondary antibody (7074S, Cell Signaling, Frankfurt/Main, Germany). Proteins were detected by incubation with chemiluminescent reagent (34080, Thermo Scientific) and developed using x-ray film (34090, Thermo Scientific). Protein abundance was quantified densitometrically using ImageJ software and normalized to GAPDH.

An amount of 30–50 mg of pulverized liver tissue was mechanically homogenized in TRIzol (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted with RNeasy Mini columns (74106, Qiagen) as per the manufacturer’s instructions. RNA concentration and purity of extracts were determined using a Nanodrop 100 spectrophotometer. cDNA was generated from 2 µg RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR (qPCR) was performed using SYBR Green (Applied Biosystems) on an Applied Biosystems 7300 Real-Time PCR System. Relative expression was quantified using the 2-ΔΔCt method [20]. Primer sequences are listed in Table S3.

Values are expressed as the means and standard errors of the mean. Student’s t-test and two-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc testing was performed using R (Version 3.5.0). Values of p ≤ 0.05 were considered significant.

Mice with liver-specific deficiency of PLIN2 (PLIN2^LKO) were generated by mating mice harboring loxP segments flanking exon 5 of the Plin2 gene (PLIN2^fl/fl) [11] to mice carrying an albumin-Cre transgene. Western blot and qPCR analysis confirmed reduced protein and gene expression in livers PLIN2^LKO mice (Figure 1A,B). Consistent with tissue specificity of PLIN2 deficiency, PLIN2 protein levels were not changed in small-intestine and gonadal white adipose (gWAT) of PLIN2^LKO mice (Figure 1A,B). For our studies, 8-week-old PLIN2^fl/fl and PLIN2^LKO mice were randomized to either a low-fat (LFD) or a high-fat, Western-type diet (WTD), which has been demonstrated to induce hepatic steatosis by 12 weeks (Table S1) [26].

To determine whether hepatic PLIN2 deficiency altered the expression of other LD-associated proteins, we first performed RT-PCR analysis, which demonstrated no difference in Plin1, Plin3, Plin4, Plin5, Fsp27 and Cideb mRNA expression between the two lines of mice during both LFD and WTD feeding (Figure 1C). Since protein expression of the perilipin proteins is strongly regulated post-translationally by level of LD TAG [30], we performed Western blot analysis to determine changes in PLIN isoform abundance. We observed no difference in protein abundance of PLIN3 and PLIN4 between the two lines of mice following WTD feeding (Figure 1D,E). Surprisingly, the protein abundance of PLIN5 was increased by 50% in PLIN2^LKO livers. In summary, these results suggest that after 12 weeks of WTD feeding, liver-specific loss of PLIN2 does not affect expression of other LD proteins except for PLIN5, an isoform implicated in the regulation of lipid storage, oxidation and autophagy [31,32,33].

We next investigated whether hepatic PLIN2 deficiency could alter the onset or severity of comorbidities associated with NAFLD including obesity, insulin resistance and dyslipidemia. In contrast to whole-body and antisense-oligonucleotide treatment models of PLIN2 deficiency [11,15,16,20], we observed no alterations in body weight or body composition between PLIN2^fl/fl and PLIN2^LKO during fed LFD or WTD for 12 weeks (Figure 2A,B). Fasting serum lipid metabolites including TAG, non-esterified fatty acids (NEFA), cholesterol, and ketones were also not different between genotypes within each diet treatment (Figure 2C–F). In addition, after 12 weeks of diet treatment, we observed no difference in fasting serum glucose, insulin and HOMA-IR between PLIN2^fl/fl and PLIN2^LKO during both LFD and WTD feeding (Figure 2G–I). Similarly, no differences in insulin sensitivity or glucose tolerance were observed between PLIN2^fl/fl and PLIN2^LKO during WTD feeding (Figure 2J,K). In sum, these data demonstrate that reduced hepatic PLIN2 does not protect against the development of HFD-induced weight and fat mass gain or obesity-associated alterations in insulin–glucose homeostasis early in the progression of NAFLD.

We next sought to delineate the impact of hepatic-specific deficiency of PLIN2 on lipid accumulation after 12 weeks of WTD feeding. Liver weights of WTD-fed mice were significantly elevated compared to LFD-fed mice. However, there was no significant effect of genotype on liver weight (Figure 3A). PLIN2 deficiency reduced liver TAG content by more than 50% following WTD feeding as compared to PLIN2^fl/fl controls (Figure 3B). In addition, there was a strong trend (p = 0.09) for reduced hepatic lipids in LFD fed PLIN2^LKO mice compared to LFD fed controls. Hepatic non-esterified fatty acids (NEFA) were reduced more than 30% in livers of WTD PLIN2^LKO mice as compared to WTD fed controls (Figure 3C). There was also a trend (p = 0.08) towards a reduction in NEFA content in PLIN2^LKO livers following LFD treatment. Consistent with the observed reduction in liver TAG content in WTD fed PLIN2^LKO mice, hematoxylin and eosin-stained liver sections from WTD-fed mice showed a marked reduction in lipid droplets in PLIN2^LKO mice compared with controls (Figure 3D). Furthermore, histological scoring revealed a decreased prevalence of grade 3 steatosis in WTD fed PLIN2^LKO livers compared to controls (Table S4). In summary, these findings are consistent with other studies investigating hepatic PLIN2 deficiency during high-fat feeding which demonstrated a reduction in liver TAG accumulation [11,14,15,16,19,20].

The TAG content of the liver represents the sum of multiple metabolic pathways: lipolysis and oxidation, TAG secretion in VLDL, as well as TAG and FA uptake. To explore which of these mechanisms may underlie protection against hepatic TAG accumulation in PLIN2^LKO mice, we analyzed the expression of genes involved in lipid metabolism in livers of mice fed LFD and WTD for 12 weeks. Expression of lipogenic genes Dgat1, Dgat2, Mgat1, Scd1 as well as Srebp1c and its downstream genes, Fasn and Acc1, did not differ between genotypes within diet treatment groups (Figure 3E,F). Neither the mRNA expression of Mttp, an enzyme involved in export of TAG in VLDL, nor the expression of lipolytic enzymes Atgl and Hsl differed between genotypes within diet treatments. With regard to FA oxidative metabolism, mRNA expression of Acox1 and Cpt1a did not differ between genotypes (Figure 3F). FA transporters Fatp5, Fatp2 and Fabp1 were also unchanged. In total, these results suggest that transcriptional modulation of genes involved in lipogenesis, lipolysis, lipid secretion and fatty acid uptake are not responsible for the observed reduction in hepatic TAG content in the livers of PLIN2^LKO mice.

When expressed on the surface of the LD, PLIN2 prevents association of ATGL and proteins involved in autophagy with the LD, thereby blocking their catabolic actions [22,23]. Given the absence of changes in lipogenic gene expression, we sought to determine whether autophagic-dependent lipolysis, termed lipophagy, is increased in livers of HFD fed PLIN2^LKO mice.

We first performed immunohistochemical analysis of two different autophagic markers to visualize autophagic vesicles in liver sections. We observed significant increases in LC3- and p62-positive puncta in livers of PLIN2^LKO mice fed WTD (Figure 4A–D). Increased levels of these two markers suggest altered autophagic function in PLIN2^LKO liver, but do not indicate whether accumulation is due to accelerated biogenesis of autophagic vesicles (enhanced autophagy) or deficient clearance of those degradative vesicles (impaired autophagy). In order to elucidate between these two possibilities, we carried out functional analysis in primary hepatocytes and ex vivo analysis of autophagic function. PLIN2^LKO hepatocytes demonstrated a significant increase in the number of autophagosomes following treatment with chloroquine (CQ), which inhibits autophagic flux by decreasing autophagosome–lysosome fusion (Figure 4E,F). Furthermore, we observed a significant increase in LC3 flux, indicative of enhanced autophagic function, in PLIN2-deficient hepatocytes (Figure 4G). Consistent with the findings in primary cell culture, ex vivo analysis revealed a strong trend (p = 0.06) toward increased p62-dependent autophagic flux in the absence of hepatic PLIN2 (Figure 4H,I). In sum, using three different approaches we demonstrate that loss of hepatic PLIN2 enhances autophagic function in WTD-fed mice and suggest that accelerated lipophagy contributes to the reduced lipid accumulation observed in the liver of PLIN2^LKO mice.

We next investigated whether increased rates of autophagy contribute to increased levels of FA oxidation in PLIN2^LKO hepatocytes. We observed a significant 65% increase in the rate of FA oxidation in isolated hepatocytes from chow fed PLIN2^LKO mice compared to hepatocytes from PLIN2^fl/fl mice (Figure 5A). Remarkably, increases in FA oxidation in PLIN2^LKO hepatocytes were completely blunted in the presence of the autophagy inhibitor CQ (Figure 5A) indicating that increased FA oxidation observed in PLIN2-deficient hepatocytes was due, in part, to increased rates of lipophagy.

While lipophagy and ATGL-mediated lipolysis were classically thought to be distinct processes, recent work has suggested that ATGL activity is required to increased rates of autophagy [24,25]. To investigate the contribution of ATGL to increased rates of autophagy in PLIN2^LKO hepatocytes, we measured the abundance of autophagic markers following inhibition of ATGL with ATGListatin (ATGLi). We hypothesized that blocking ATGLi activity would reduce autophagic function in PLIN2^LKO primary hepatocytes. In support of this hypothesis, we observed a significant decrease in p62-dependent autophagic flux (CQ puncta–untreated puncta) and a trend towards reduced LC3 flux (p = 0.06) following inhibition of ATGL in PLIN2^LKO hepatocytes (Figure 5B–G) These results indicate that in PLIN2-deficient hepatocytes ATGL activity enhances autophagic function.

Having established that hepatic PLIN2 deficiency (1) enhances autophagy and (2) increases FA oxidation in an autophagic-dependent manner and (3) that ATGL activity contributes to enhanced autophagy, we next sought to determine the relative contribution of ATGL to increased FA oxidation observed in the absence of PLIN2. Our analysis demonstrated that inhibition of ATGL by ATGLi treatment reduced FA oxidation significantly by 54% compared to untreated hepatocytes, confirming that ATGL activity contributes to FA oxidation in the absence of PLIN2 (Figure 5H). We hypothesized that the contribution of ATGL activity to increased FA oxidation in PLIN2^LKO hepatocytes is likely due to both ATGL-mediated regulation of autophagy and the autophagy-independent (lipolytic) actions of ATGL. To elucidate a possible autophagic-independent role of ATGL activity, we treated PLIN2^LKO hepatocytes with both CQ and ATGLi to simultaneously inhibit both autophagy and ATGL. We found that inhibition of ATGL and autophagy did not further reduce FA oxidation compared to treatment with ATGLi alone, arguing against an autophagy-independent role of ATGL activity in mediating FA oxidation in PLIN2^LKO hepatocytes, and suggesting that ATGL and autophagy act through an integrated pathway to increase FA oxidation following hepatic-specific PLIN2 ablation.