Saturated Fatty Acids Promote Endoplasmic Reticulum Stress and Liver Injury in Rats with Hepatic Steatosis
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《内分泌学杂志》
Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, Colorado 80523
Abstract
Nonalcoholic fatty liver disease is a relatively new hepatic sequela of obesity and type 2 diabetes. The pathogenesis of liver injury and disease progression in nonalcoholic fatty liver disease, however, is poorly understood. The present study examined the hypothesis that the composition of fatty acids in the steatotic liver promotes liver injury. Using dietary models of hepatic steatosis characterized by similar accumulation of total triglyceride but different composition of fatty acids, we show that hepatic steatosis characterized by increased saturated fatty acids is associated with increased liver injury and markers of endoplasmic reticulum stress (e.g. X-box binding protein-1 mRNA splicing and glucose-regulated protein 78 expression). These changes preceded and/or occurred independently of obesity and differences in leptin, TNF, insulin action, and mitochondrial function. In addition, hepatic steatosis characterized by increased saturated fatty acids reduced proliferative capacity in response to partial hepatectomy and increased liver injury in response to lipopolysaccharide. These data suggest that the composition of fatty acids in the steatotic liver is an important determinant of susceptibility to liver injury.
Introduction
THE STRIKING PREVALENCE of obesity worldwide is a significant health problem (1, 2). Nonalcoholic fatty liver disease (NAFLD) is a relatively new hepatic sequela of obesity and type 2 diabetes (3, 4, 5). NAFLD is initially characterized by a pure fatty liver (steatosis) with progression, in some, to nonalcoholic steatohepatitis (NASH) and liver failure (3, 5). NAFLD is now recognized as a common cause of chronic liver enzyme elevations and cryptogenic cirrhosis (6), but its pathogenesis remains uncertain. Steatosis is the earliest and most prevalent stage of NAFLD, often referred to as the "first hit." It has been proposed that steatosis increases the vulnerability of the liver to a "second hit," involving environmental and/or genetic factors, that ultimately can lead to end-stage liver disease (7).
Elevated serum free fatty acid levels contribute to the pathogenesis of obesity, the metabolic syndrome and cardiovascular disease (8, 9, 10). Whereas adipocytes have a unique capacity to store excess fatty acids in the form of triglyceride, nonadipose tissues, including liver, do not. Accumulation of lipids in nonadipose tissues can lead to cell dysfunction (e.g. insulin resistance) and cell death, a phenomenon known as lipotoxicity (11, 12, 13). Saturated and unsaturated fatty acids differ significantly in their contributions to lipotoxicity. Previous studies in Chinese hamster ovary cells, cardiac myocytes, pancreatic -cells, breast cancer cell lines, and hematopoietic precursor cell lines all suggest that lipotoxicity from accumulation of long-chain fatty acids is specific to or made more severe by saturated fatty acids (14, 15, 16, 17, 18). These data predict that the presence of increased circulating and/or hepatic saturated fatty acids may promote liver damage. This prediction was examined in the current study using dietary models of hepatic steatosis characterized by similar accumulation of total triglyceride but different composition of fatty acids. The data demonstrate that hepatic steatosis characterized by increased saturated fatty acids leads to increased caspase-3 activity, endoplasmic reticulum (ER) stress, and liver injury. In addition, hepatic steatosis characterized by increased saturated fatty acids reduced proliferative capacity in response to partial hepatectomy (PH) and increased liver injury in response to lipopolysaccharide.
Materials and Methods
Animals
Male Wistar Crl(WI)BR rats (Charles River, Wilmington, MA) weighing 180 g (7–8 wk of age) on arrival were provided free access to a purified high-starch diet (Research Diets, New Brunswick, NJ) (19) and water for 1 wk. Rats were housed individually in a temperature- and humidity-controlled environment with a 12-h light, 12-h dark cycle. All procedures were reviewed and approved by the Colorado State University institutional animal care committee.
Dietary models of hepatic steatosis
After the 1 wk acclimation period, rats remained on the high-starch diet (STD) (68% of energy from corn starch, 12% from corn oil, and 20% from casein) or were provided diets enriched in sucrose (HSD) (68% sucrose, 12% corn oil, and 20% casein), polyunsaturated fat (HPUFA) (35% corn starch, 45% corn oil, and 20% casein), or saturated fat (HSAT) (45% lard oil, 35% corn starch, and 20% casein) (Research Diets) (Refs.19 , 20). To minimize effects of obesity, energy intake in HSD, HPUFA, and HSAT was matched to that in the STD group (21).
Basal and euglycemic, hyperinsulinemic clamps
After 1, 4, or 24 wk of dietary treatment (n = 7–8 per diet per time), catheters were implanted in the carotid artery and jugular vein under general anesthesia as described previously (19, 22). Experiments were performed after 4–5 d of recovery, at which time body weight was more than 100% of presurgery body weight. During the recovery period, rats were fed their respective diets. Basal glucose kinetics were determined via infusion of HPLC-purified 3-3H-glucose over a 90 min period (19, 23). The final 30 min steady-state period was used for calculation of rates of glucose appearance and disappearance (19). After the 90 min basal period, euglycemic, hyperinsulinemic clamps (90 min) were initiated using a primed, continuous infusion of insulin (3 mU/kg·min). A variable glucose infusion (20% dextrose) was used to maintain plasma glucose at baseline values. To minimize changes in glucose-specific activity, the glucose infusate was spiked with 3-3H-glucose to a glucose-specific activity similar to the plasma glucose-specific activity in the basal period. Endogenous glucose appearance and glucose disappearance during the clamp period were calculated as described previously by Finegood et al. (24).
PH
Rats were provided STD, HSD, HPUFA, or HSAT for 1 wk. A 70% PH or sham (exposure of sternum and manipulation of the liver) surgery was then performed under metaphane anesthesia (25). Rats were killed at 1, 3, 7, or 14 d after PH or sham (n = 8 per diet per surgery per time).
Lipopolysaccharide (LPS) treatment
Rats were provided STD, HSD, HPUFA, or HSAT for 4 wk (n = 6 per diet group). Catheters were then implanted in the carotid artery and jugular vein under general anesthesia. Rats were allowed 4–5 d to recover, at which time they were more than 100% of presurgery body weight. During the recovery period, rats were fed their respective diets. On the day of study and before LPS injection, an arterial blood sample was drawn (pre-LPS). Escherichia coli LPS (Sigma, St. Louis, MO) was then delivered (5 mg/kg, iv), and blood and liver tissue were taken 12 h after delivery (post-LPS).
Isolation and evaluation of mitochondrial function
Mitochondria were isolated from rats undergoing dietary treatment for 1, 4, and 24 wk (n = 6 per diet per time) using a standard differential centrifugation protocol in a medium containing 0.25 M sucrose, 10 mM HEPES (potassium salt, pH = 7.4), 0.1 mM EGTA, and 5 mg/ml fatty acid-free BSA. The electrochemical proton gradient was estimated based on uptake of the fluorescent dye JC-1 (5,5'6,6'-tetrachloro-1,1',3,3'-tetraethylbenzamidazolocarbocyanine iodide) (Biomol, Plymouth Meeting, PA). A reduction of the electrochemical proton gradient indicates a deficiency of mitochondrial inner membrane integrity.
Hepatocyte isolation
Hepatocytes were isolated by collagenase perfusion with modifications (26, 27).
Analyses
Lipids.
Total lipids were extracted using the methods of Bligh and Dyer (28). Total triglyceride content was determined enzymatically (Sigma). Cholesterol content was determined by gas chromatography with automatic integration using coprostanol as an internal standard. Phospholipid species were measured in microsomal fractions after separation by two-dimensional thin-layer chromatography and quantified, as was total phospholipid content by the method of Ames and Dubin (29) and Podolin et al. (30). Fatty acid methyl esters were measured on a Hewlett-Packard (Palo Alto, CA) 5890 Series II gas chromatograph with flame ionization detection as described previously (21, 30). Total carcass lipid was determined as described previously (21).
Cell fractionation.
Liver or cell suspensions were homogenized in a chilled isolation buffer and centrifuged at 48,000 x g for 30 min (30). Microsomal membrane fractions were harvested from this supernatant via centrifugation at 100,000 x g for 45 min.
Membrane markers. Cytochrome c reductase was used as a microsomal membrane marker, and succinic dehydrogenase was used as an inner mitochondrial membrane marker (30). Enzyme activity and total protein were measured as described previously (30).
RNA isolation and PCR.
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). For PCR analysis of X-box binding protein-1 (XBP-1), a two-step protocol was used for RT-PCR using SuperScript II reverse transcriptase and Taq polymerase (31).
Western blotting.
Liver or freshly isolated hepatocytes were harvested using a lysis buffer containing 20 mM HEPES (pH 7.4), 1% Triton X-100, 10% glycerol, 2 mM EGTA, 1 mM sodium vanadate, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 mM -glycerophosphate, 3 mM benzamidine, 10 μM leupeptin, 5 μM pepstatin, and 10 μg/ml aprotinin, or isolated membrane fractions were used. Equivalent amounts of protein (50–100 μg) were subjected to SDS-PAGE and transferred to Hybond-P membranes (Amersham Biosciences, Piscataway, NJ), and membranes were incubated with antibodies against C/EBP homologous protein (CHOP) (Cell Signaling Technology, Beverly, MA), which is also called GADD153 (growth arrest and DNA damage-inducible gene 153) and glucose-regulated protein 78 (GRP78) (StressGen, San Diego, CA). Total protein was determined according to the methods Lowry et al. (32). Proteins were detected using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence reagent (Pierce, Rockford, IL). Density was quantified using a UVP Bioimaging system (UVP, Upland, CA).
Caspase-3 activity.
Caspase-3 activity was determined using a caspase-specific peptide conjugated to the color reporter p-nitroanaline (R & D Systems, Minneapolis, MN).
DNA synthesis.
Two hours before the rats were killed, they were injected with bromodeoxyuridine (BrdU). The incorporation of BrdU into nuclear DNA is a representative marker for S phase (33). Liver tissues were fixed in buffered formalin, embedded in paraffin, and sectioned. Hepatic nuclear BrdU incorporation was determined by light microscopy. The total number of hepatocyte nuclei and the number of BrdU-positive nuclei in 10 microscopic fields on each section were counted.
Plasma measures.
Glucose was measured with an automated analyzer (Beckman Instruments, Fullerton, CA). Insulin and leptin were analyzed by ELISA and TNF by immunoassay (Linco Research, St. Charles, MO). Alanine aminotransferase (AAT) and aspartate aminotransferase (AST) were measured using ThermoDMA (Arlington, TX) kits TR7111-125 and TR7011-125, respectively.
Data analysis
Data were analyzed using one- or two-way ANOVA or using the nonparametric ANOVA Kruskal-Wallis test. The two analyses, when compared, resulted in identical interpretations. Post hoc comparisons among means were made using the Scheffe’s or Tukey’s test. Rank sums were used from the Kruskal-Wallis test for post hoc comparisons. Differences were considered significant at P < 0.05. All data are reported as means ± SEM.
Results and Discussion
Dietary models of steatosis
Liver triglycerides were significantly increased in HSD, HPUFA, and HSAT compared with STD at 1, 4, and 24 wk (Fig. 1) (n = 8 per diet per time). The sum of saturated fatty acids in liver triglycerides and microsomal membrane phospholipids was significantly increased in HSD and HSAT compared with STD and HPUFA (Fig. 1). Microsomal membrane enrichment data are provided in Table 1. The cholesterol to phospholipid ratio was significantly increased in HSD, HPUFA, and HSAT compared with STD at 4 and 24 wk (data not shown). Energy intake was not significantly different among groups and averaged 91 ± 7 kcal/d in rats fed for 1 wk, 109 ± 9 kcal/d in rats fed for 4 wk, and 117 ± 10 kcal/d in rats fed for 24 wk. There was a significant time effect on body weight, fat mass, plasma leptin, and plasma TNF (Fig. 2) (n = 8 per diet per time). However, these variables were not significantly different among groups within each time point, with the exception of percentage fat mass, which was significantly increased in HSAT and HPUFA at 24 wk (Fig. 2). Plasma markers of liver injury AAT and AST were significantly increased in HSD and HSAT at 4 and 24 wk (Fig. 2).
Hepatic steatosis characterized by increased saturated fatty acids is associated with increased markers of ER stress and apoptosis
ER stress initiates a compensatory response, termed the unfolded protein response (UPR), which includes transcription of a set of genes whose protein products increase the capacity for protein folding (e.g. GRP78), ER-associated degradation (e.g. EDEM), and apoptosis (e.g. CHOP/GADD153) (34). ER stress-induced transcription is initiated, in part, via the splicing and activation of the transcription factor XBP-1 (35). Livers from HSD and HSAT rats were characterized by the presence of spliced XBP-1 (Fig. 3A), increased GRP78 (Fig. 3B) and CHOP protein (Fig. 3C), and increased caspase-3 activity (Fig. 3D) (n = 8 per diet per time).
Hepatic steatosis characterized by increased saturated fatty acids increases markers of ER stress and apoptosis in hepatocytes
To examine the contribution of hepatocytes to lipid-induced ER stress and apoptosis, RNA and cell lysates derived from freshly isolated hepatocytes were studied (n = 6 per diet per time). Spliced XBP-1 (Fig. 4A), increased GRP78 protein (Fig. 4B), and increased caspase-3 activity (Fig. 4B) were observed in hepatocytes isolated from HSD and HSAT compared with STD and HPUFA.
Insulin action and mitochondrial function are not selectively impaired by hepatic steatosis characterized by increased saturated fatty acids
Insulin action in the liver appears to be causally linked to hepatic fat accumulation (36) and ER stress (37, 38, 39). In addition, mitochondrial dysfunction, characterized by uncoupling of oxidative phosphorylation and extrusion of cytochrome c from the inner mitochondrial membrane, has been observed in models of fatty liver and is a component of the lipotoxic response to saturated fatty acids (12, 40, 41). Therefore, we next evaluated insulin action using the glucose clamp technique and mitochondrial function.
Basal glucose kinetics were not significantly different among groups (Table 2) (n = 7–8 per diet per time). During euglycemic, hyperinsulinemic clamps, the glucose infusion rate (Fig. 5A), insulin suppression of glucose appearance (Fig. 5B), and insulin-stimulated glucose disappearance (Fig. 5C) were significantly reduced in HSD, HPUFA, and HSAT when compared with STD, but there were no significant differences among HSD, HPUFA, or HSAT (Table 2 and Fig. 5) (n = 7–8 per diet per time).
JC-1 fluorescence was significantly reduced in mitochondria isolated from HSD, HPUFA, and HSAT at 4 and 24 wk when compared with STD, but there were no significant differences among HSD, HPUFA, and HSAT (Table 3) (n = 6 per diet per time).
Steatosis characterized by increased saturated fatty acids reduces hepatic proliferative capacity after PH
Healthy livers typically regenerate and recover completely from acute inflammation, but the normal regenerative response to injury is impaired in fatty liver (33, 42). Consistent with these previous observations, liver weights were not significantly different in sham- and PH-treated STD rats at 14 d but were significantly reduced at 14 d in PH-treated HSD, HPUFA, and HSAT rats compared with sham-treated counterparts (Fig. 6A) (n = 8 per diet per time after surgery). Recovery of liver weight was significantly reduced in PH-treated HSD and HSAT rats compared with STD and HPUFA (Fig. 6A). BrdU incorporation into nuclei, an estimate of cell proliferation, was significantly reduced in PH-treated HSD and HSAT rats compared with PH-treated STD and HPUFA rats (Fig. 6B). Body weight and energy intake during the 14 d regeneration period were not significantly different among diet groups or between sham and PH groups (data not shown).
Steatosis characterized by increased saturated fatty acids results in increased liver enzymes and ER stress markers in response to LPS treatment
LPS is a toxic component of cell walls of Gram-negative bacteria that has been postulated to be one factor that can promote progression from steatosis to NASH (4). Before injection of LPS, plasma AAT and AST were significantly greater in HSD and HSAT compared with STD and HPUFA (Fig. 7A) (n = 6 per diet). Acute injection of LPS increased plasma ALT and AST in all groups (Fig. 7A). However, the increase was significantly greater in HSD and HSAT compared with STD and HPUFA (Fig. 7, A and B). The LPS-induced increase in plasma AST was also significantly increased in HPUFA compared with STD (Fig. 7B). Hepatic caspase-3 activity was significantly increased in HSD and HSAT compared with STD and HPUFA after LPS injection (Fig. 7C). Caspase-3 activity was also significantly increased in HPUFA compared with STD (Fig. 7C).
A large majority of obese subjects have fatty liver, and it has been suggested that approximately 30% have NASH (5, 43). This link is noteworthy because of both the current obesity epidemic and the increased prevalence of liver damage in obese subjects (44). The mechanisms that promote the progression from steatosis to NASH and liver damage are poorly understood (3, 6). The prevailing hypothesis suggests that disease progression may be triggered when an acute inflammatory insult is superimposed (second hit) on hepatic steatosis (7). Using dietary models of hepatic steatosis in which fatty acid composition in the liver was varied, we have shown that hepatic steatosis characterized by increased saturated fatty acids is associated with increased markers of liver injury and ER stress, reduced proliferative capacity in response to PH, and increased susceptibility to LPS-mediated liver injury.
In the present study, hepatic steatosis was induced using diets enriched in sucrose (HSD), lard (HSAT), or corn oil (HPUFA). Although the magnitude of steatosis was similar among the diet groups (based on hepatic triglyceride concentration), HSD and HSAT were characterized by increased hepatic saturated fatty acids compared with HPUFA. Notably, increased liver injury, ER stress, and caspase-3 activity were observed in HSD and HSAT but not in HPUFA or STD. The presence of liver injury, ER stress, and increased caspase-3 activity in HSD and HSAT occurred before and independently of insulin action, body fat accumulation, and circulating leptin and TNF. These data suggest that the composition of fatty acids in the steatotic liver is an important determinant of liver injury and that the presence of increased saturated fatty acids in the steatotic liver may constitute an intrinsic second hit.
The regenerative response to liver injury is impaired in ob/ob mice with fatty livers (45), and it has been postulated that this contributes to NAFLD pathophysiology by inhibiting proliferation and increasing injury (42, 45, 46). Consistent with this postulate, recovery of liver weight and proliferative capacity in response to PH were reduced in all three dietary models of fatty liver (HSD, HPUFA, and HSAT). Remarkably, both the recovery of liver weight and proliferative capacity were also significantly reduced in HSD and HSAT compared with HPUFA and STD. Whether this is due to a persistent impairment caused by the fatty acid composition before PH or to the composition and magnitude of fatty acids in the regenerating liver is presently unknown. Increased liver injury and hepatic caspase-3 activity in response to LPS was observed in all three dietary models of fatty liver (i.e. HSD, HPUFA, and HSAT). However, more severe liver injury (based on plasma liver enzymes) and a greater increase in hepatic caspase-3 activity was observed in HSD and HSAT compared with HPUFA and STD. In total, these data support the notion that the composition of fatty acids in the steatotic liver is an important determinant of disease progression in NAFLD.
Recent studies have implicated the ER in obesity, insulin resistance, and diabetes (37, 38, 47, 48, 49). An essential function of the ER is the synthesis and processing of secretory and membrane proteins (50). Several pathologic stresses (e.g. calcium homeostasis, protein glycosylation, and oxidative and reductive stress) disrupt ER homeostasis and lead to the accumulation of unfolded proteins and protein aggregates in the ER lumen, which can be detrimental to cell survival (51, 52, 53). Disruption of ER homeostasis, collectively termed "ER stress," activates the UPR, a signaling pathway that links the ER lumen with the cytoplasm and nucleus (51, 53, 54). If the UPR is not sufficient to mitigate the imposed stress, caspase-dependent and -independent programmed cell death ensues (55). Cellular markers of ER stress include the splicing of the transcription factor XBP-1, up-regulation of the ER chaperone GRP78, and up-regulation of the proapoptotic gene CHOP (35, 51). In the present study, XBP-1 splicing and increased GRP78 protein, CHOP protein, and caspase-3 activity were observed in liver and hepatocytes from dietary models of hepatic steatosis characterized by increased saturated fatty acids (HSD and HSAT). We hypothesize that saturated fatty acids in the steatotic liver induce a stress to the ER that exceeds the capacity of the UPR, resulting in apoptosis and liver injury. Future studies will examine this hypothesis and the causal link(s) between saturated fatty acids, ER stress, and liver injury.
Hepatocyte apoptosis is significantly increased in patients with alcoholic hepatitis and NASH and correlates with disease severity and hepatic fibrosis (56, 57). It has been proposed that the uptake of apoptotic cells released from hepatocytes by Kupffer cells and hepatic stellate cells is mediated by lipid signals (57). Persistent activation of Kupffer and stellate cells can promote additional hepatic apoptosis and inflammation (57). Thus, it will be important to examine the sequential regulatory events that led to increased liver injury in these dietary models of hepatic steatosis.
A recent study has documented that both elevated peripheral fatty acids and de novo lipogenesis contribute to the accumulation of hepatic and lipoprotein fat in patients with NAFLD (58). The present results suggest that an increase in hepatic saturated fatty acids derived from either peripheral lipid (HSAT) or accelerated de novo lipogenesis (HSD) promotes ER stress and liver injury.
In summary, the data from the present study suggest that hepatic steatosis characterized by increased saturated fatty acids can promote liver injury, ER stress, and a proapoptotic environment. The fact that saturated fatty acids in the steatotic liver promote injury independently of obesity, insulin action, and TNF implies that they may be an important, early determinant of susceptibility for disease progression in NAFLD.
Acknowledgments
We thank the Hepatobiliary Center at the University of Colorado Health Sciences Center for assistance with partial hepatectomy, JC-1 fluorescence measurements, and lipid analyses, and the Colorado Clinical Nutrition Research Unit for assistance with insulin, leptin, and TNF measurements.
Footnotes
This work was supported by National Institutes of Health Grants DK47416 and DK072017.
D.W., Y.W., and M.J.P. have nothing to declare.
First Published Online November 3, 2005
Abbreviations: AAT, Alanine aminotransferase; AST, aspartate aminotransferase; BrdU, bromodeoxyuridine; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; HPUFA, high polyunsaturated fat diet; HSAT, high saturated fat diet; HSD, high sucrose diet; LPS, lipopolysaccharide; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PH, partial hepatectomy; STD, high-starch diet; UPR, unfolded protein response.
Accepted for publication October 24, 2005.
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Abstract
Nonalcoholic fatty liver disease is a relatively new hepatic sequela of obesity and type 2 diabetes. The pathogenesis of liver injury and disease progression in nonalcoholic fatty liver disease, however, is poorly understood. The present study examined the hypothesis that the composition of fatty acids in the steatotic liver promotes liver injury. Using dietary models of hepatic steatosis characterized by similar accumulation of total triglyceride but different composition of fatty acids, we show that hepatic steatosis characterized by increased saturated fatty acids is associated with increased liver injury and markers of endoplasmic reticulum stress (e.g. X-box binding protein-1 mRNA splicing and glucose-regulated protein 78 expression). These changes preceded and/or occurred independently of obesity and differences in leptin, TNF, insulin action, and mitochondrial function. In addition, hepatic steatosis characterized by increased saturated fatty acids reduced proliferative capacity in response to partial hepatectomy and increased liver injury in response to lipopolysaccharide. These data suggest that the composition of fatty acids in the steatotic liver is an important determinant of susceptibility to liver injury.
Introduction
THE STRIKING PREVALENCE of obesity worldwide is a significant health problem (1, 2). Nonalcoholic fatty liver disease (NAFLD) is a relatively new hepatic sequela of obesity and type 2 diabetes (3, 4, 5). NAFLD is initially characterized by a pure fatty liver (steatosis) with progression, in some, to nonalcoholic steatohepatitis (NASH) and liver failure (3, 5). NAFLD is now recognized as a common cause of chronic liver enzyme elevations and cryptogenic cirrhosis (6), but its pathogenesis remains uncertain. Steatosis is the earliest and most prevalent stage of NAFLD, often referred to as the "first hit." It has been proposed that steatosis increases the vulnerability of the liver to a "second hit," involving environmental and/or genetic factors, that ultimately can lead to end-stage liver disease (7).
Elevated serum free fatty acid levels contribute to the pathogenesis of obesity, the metabolic syndrome and cardiovascular disease (8, 9, 10). Whereas adipocytes have a unique capacity to store excess fatty acids in the form of triglyceride, nonadipose tissues, including liver, do not. Accumulation of lipids in nonadipose tissues can lead to cell dysfunction (e.g. insulin resistance) and cell death, a phenomenon known as lipotoxicity (11, 12, 13). Saturated and unsaturated fatty acids differ significantly in their contributions to lipotoxicity. Previous studies in Chinese hamster ovary cells, cardiac myocytes, pancreatic -cells, breast cancer cell lines, and hematopoietic precursor cell lines all suggest that lipotoxicity from accumulation of long-chain fatty acids is specific to or made more severe by saturated fatty acids (14, 15, 16, 17, 18). These data predict that the presence of increased circulating and/or hepatic saturated fatty acids may promote liver damage. This prediction was examined in the current study using dietary models of hepatic steatosis characterized by similar accumulation of total triglyceride but different composition of fatty acids. The data demonstrate that hepatic steatosis characterized by increased saturated fatty acids leads to increased caspase-3 activity, endoplasmic reticulum (ER) stress, and liver injury. In addition, hepatic steatosis characterized by increased saturated fatty acids reduced proliferative capacity in response to partial hepatectomy (PH) and increased liver injury in response to lipopolysaccharide.
Materials and Methods
Animals
Male Wistar Crl(WI)BR rats (Charles River, Wilmington, MA) weighing 180 g (7–8 wk of age) on arrival were provided free access to a purified high-starch diet (Research Diets, New Brunswick, NJ) (19) and water for 1 wk. Rats were housed individually in a temperature- and humidity-controlled environment with a 12-h light, 12-h dark cycle. All procedures were reviewed and approved by the Colorado State University institutional animal care committee.
Dietary models of hepatic steatosis
After the 1 wk acclimation period, rats remained on the high-starch diet (STD) (68% of energy from corn starch, 12% from corn oil, and 20% from casein) or were provided diets enriched in sucrose (HSD) (68% sucrose, 12% corn oil, and 20% casein), polyunsaturated fat (HPUFA) (35% corn starch, 45% corn oil, and 20% casein), or saturated fat (HSAT) (45% lard oil, 35% corn starch, and 20% casein) (Research Diets) (Refs.19 , 20). To minimize effects of obesity, energy intake in HSD, HPUFA, and HSAT was matched to that in the STD group (21).
Basal and euglycemic, hyperinsulinemic clamps
After 1, 4, or 24 wk of dietary treatment (n = 7–8 per diet per time), catheters were implanted in the carotid artery and jugular vein under general anesthesia as described previously (19, 22). Experiments were performed after 4–5 d of recovery, at which time body weight was more than 100% of presurgery body weight. During the recovery period, rats were fed their respective diets. Basal glucose kinetics were determined via infusion of HPLC-purified 3-3H-glucose over a 90 min period (19, 23). The final 30 min steady-state period was used for calculation of rates of glucose appearance and disappearance (19). After the 90 min basal period, euglycemic, hyperinsulinemic clamps (90 min) were initiated using a primed, continuous infusion of insulin (3 mU/kg·min). A variable glucose infusion (20% dextrose) was used to maintain plasma glucose at baseline values. To minimize changes in glucose-specific activity, the glucose infusate was spiked with 3-3H-glucose to a glucose-specific activity similar to the plasma glucose-specific activity in the basal period. Endogenous glucose appearance and glucose disappearance during the clamp period were calculated as described previously by Finegood et al. (24).
PH
Rats were provided STD, HSD, HPUFA, or HSAT for 1 wk. A 70% PH or sham (exposure of sternum and manipulation of the liver) surgery was then performed under metaphane anesthesia (25). Rats were killed at 1, 3, 7, or 14 d after PH or sham (n = 8 per diet per surgery per time).
Lipopolysaccharide (LPS) treatment
Rats were provided STD, HSD, HPUFA, or HSAT for 4 wk (n = 6 per diet group). Catheters were then implanted in the carotid artery and jugular vein under general anesthesia. Rats were allowed 4–5 d to recover, at which time they were more than 100% of presurgery body weight. During the recovery period, rats were fed their respective diets. On the day of study and before LPS injection, an arterial blood sample was drawn (pre-LPS). Escherichia coli LPS (Sigma, St. Louis, MO) was then delivered (5 mg/kg, iv), and blood and liver tissue were taken 12 h after delivery (post-LPS).
Isolation and evaluation of mitochondrial function
Mitochondria were isolated from rats undergoing dietary treatment for 1, 4, and 24 wk (n = 6 per diet per time) using a standard differential centrifugation protocol in a medium containing 0.25 M sucrose, 10 mM HEPES (potassium salt, pH = 7.4), 0.1 mM EGTA, and 5 mg/ml fatty acid-free BSA. The electrochemical proton gradient was estimated based on uptake of the fluorescent dye JC-1 (5,5'6,6'-tetrachloro-1,1',3,3'-tetraethylbenzamidazolocarbocyanine iodide) (Biomol, Plymouth Meeting, PA). A reduction of the electrochemical proton gradient indicates a deficiency of mitochondrial inner membrane integrity.
Hepatocyte isolation
Hepatocytes were isolated by collagenase perfusion with modifications (26, 27).
Analyses
Lipids.
Total lipids were extracted using the methods of Bligh and Dyer (28). Total triglyceride content was determined enzymatically (Sigma). Cholesterol content was determined by gas chromatography with automatic integration using coprostanol as an internal standard. Phospholipid species were measured in microsomal fractions after separation by two-dimensional thin-layer chromatography and quantified, as was total phospholipid content by the method of Ames and Dubin (29) and Podolin et al. (30). Fatty acid methyl esters were measured on a Hewlett-Packard (Palo Alto, CA) 5890 Series II gas chromatograph with flame ionization detection as described previously (21, 30). Total carcass lipid was determined as described previously (21).
Cell fractionation.
Liver or cell suspensions were homogenized in a chilled isolation buffer and centrifuged at 48,000 x g for 30 min (30). Microsomal membrane fractions were harvested from this supernatant via centrifugation at 100,000 x g for 45 min.
Membrane markers. Cytochrome c reductase was used as a microsomal membrane marker, and succinic dehydrogenase was used as an inner mitochondrial membrane marker (30). Enzyme activity and total protein were measured as described previously (30).
RNA isolation and PCR.
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). For PCR analysis of X-box binding protein-1 (XBP-1), a two-step protocol was used for RT-PCR using SuperScript II reverse transcriptase and Taq polymerase (31).
Western blotting.
Liver or freshly isolated hepatocytes were harvested using a lysis buffer containing 20 mM HEPES (pH 7.4), 1% Triton X-100, 10% glycerol, 2 mM EGTA, 1 mM sodium vanadate, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 mM -glycerophosphate, 3 mM benzamidine, 10 μM leupeptin, 5 μM pepstatin, and 10 μg/ml aprotinin, or isolated membrane fractions were used. Equivalent amounts of protein (50–100 μg) were subjected to SDS-PAGE and transferred to Hybond-P membranes (Amersham Biosciences, Piscataway, NJ), and membranes were incubated with antibodies against C/EBP homologous protein (CHOP) (Cell Signaling Technology, Beverly, MA), which is also called GADD153 (growth arrest and DNA damage-inducible gene 153) and glucose-regulated protein 78 (GRP78) (StressGen, San Diego, CA). Total protein was determined according to the methods Lowry et al. (32). Proteins were detected using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence reagent (Pierce, Rockford, IL). Density was quantified using a UVP Bioimaging system (UVP, Upland, CA).
Caspase-3 activity.
Caspase-3 activity was determined using a caspase-specific peptide conjugated to the color reporter p-nitroanaline (R & D Systems, Minneapolis, MN).
DNA synthesis.
Two hours before the rats were killed, they were injected with bromodeoxyuridine (BrdU). The incorporation of BrdU into nuclear DNA is a representative marker for S phase (33). Liver tissues were fixed in buffered formalin, embedded in paraffin, and sectioned. Hepatic nuclear BrdU incorporation was determined by light microscopy. The total number of hepatocyte nuclei and the number of BrdU-positive nuclei in 10 microscopic fields on each section were counted.
Plasma measures.
Glucose was measured with an automated analyzer (Beckman Instruments, Fullerton, CA). Insulin and leptin were analyzed by ELISA and TNF by immunoassay (Linco Research, St. Charles, MO). Alanine aminotransferase (AAT) and aspartate aminotransferase (AST) were measured using ThermoDMA (Arlington, TX) kits TR7111-125 and TR7011-125, respectively.
Data analysis
Data were analyzed using one- or two-way ANOVA or using the nonparametric ANOVA Kruskal-Wallis test. The two analyses, when compared, resulted in identical interpretations. Post hoc comparisons among means were made using the Scheffe’s or Tukey’s test. Rank sums were used from the Kruskal-Wallis test for post hoc comparisons. Differences were considered significant at P < 0.05. All data are reported as means ± SEM.
Results and Discussion
Dietary models of steatosis
Liver triglycerides were significantly increased in HSD, HPUFA, and HSAT compared with STD at 1, 4, and 24 wk (Fig. 1) (n = 8 per diet per time). The sum of saturated fatty acids in liver triglycerides and microsomal membrane phospholipids was significantly increased in HSD and HSAT compared with STD and HPUFA (Fig. 1). Microsomal membrane enrichment data are provided in Table 1. The cholesterol to phospholipid ratio was significantly increased in HSD, HPUFA, and HSAT compared with STD at 4 and 24 wk (data not shown). Energy intake was not significantly different among groups and averaged 91 ± 7 kcal/d in rats fed for 1 wk, 109 ± 9 kcal/d in rats fed for 4 wk, and 117 ± 10 kcal/d in rats fed for 24 wk. There was a significant time effect on body weight, fat mass, plasma leptin, and plasma TNF (Fig. 2) (n = 8 per diet per time). However, these variables were not significantly different among groups within each time point, with the exception of percentage fat mass, which was significantly increased in HSAT and HPUFA at 24 wk (Fig. 2). Plasma markers of liver injury AAT and AST were significantly increased in HSD and HSAT at 4 and 24 wk (Fig. 2).
Hepatic steatosis characterized by increased saturated fatty acids is associated with increased markers of ER stress and apoptosis
ER stress initiates a compensatory response, termed the unfolded protein response (UPR), which includes transcription of a set of genes whose protein products increase the capacity for protein folding (e.g. GRP78), ER-associated degradation (e.g. EDEM), and apoptosis (e.g. CHOP/GADD153) (34). ER stress-induced transcription is initiated, in part, via the splicing and activation of the transcription factor XBP-1 (35). Livers from HSD and HSAT rats were characterized by the presence of spliced XBP-1 (Fig. 3A), increased GRP78 (Fig. 3B) and CHOP protein (Fig. 3C), and increased caspase-3 activity (Fig. 3D) (n = 8 per diet per time).
Hepatic steatosis characterized by increased saturated fatty acids increases markers of ER stress and apoptosis in hepatocytes
To examine the contribution of hepatocytes to lipid-induced ER stress and apoptosis, RNA and cell lysates derived from freshly isolated hepatocytes were studied (n = 6 per diet per time). Spliced XBP-1 (Fig. 4A), increased GRP78 protein (Fig. 4B), and increased caspase-3 activity (Fig. 4B) were observed in hepatocytes isolated from HSD and HSAT compared with STD and HPUFA.
Insulin action and mitochondrial function are not selectively impaired by hepatic steatosis characterized by increased saturated fatty acids
Insulin action in the liver appears to be causally linked to hepatic fat accumulation (36) and ER stress (37, 38, 39). In addition, mitochondrial dysfunction, characterized by uncoupling of oxidative phosphorylation and extrusion of cytochrome c from the inner mitochondrial membrane, has been observed in models of fatty liver and is a component of the lipotoxic response to saturated fatty acids (12, 40, 41). Therefore, we next evaluated insulin action using the glucose clamp technique and mitochondrial function.
Basal glucose kinetics were not significantly different among groups (Table 2) (n = 7–8 per diet per time). During euglycemic, hyperinsulinemic clamps, the glucose infusion rate (Fig. 5A), insulin suppression of glucose appearance (Fig. 5B), and insulin-stimulated glucose disappearance (Fig. 5C) were significantly reduced in HSD, HPUFA, and HSAT when compared with STD, but there were no significant differences among HSD, HPUFA, or HSAT (Table 2 and Fig. 5) (n = 7–8 per diet per time).
JC-1 fluorescence was significantly reduced in mitochondria isolated from HSD, HPUFA, and HSAT at 4 and 24 wk when compared with STD, but there were no significant differences among HSD, HPUFA, and HSAT (Table 3) (n = 6 per diet per time).
Steatosis characterized by increased saturated fatty acids reduces hepatic proliferative capacity after PH
Healthy livers typically regenerate and recover completely from acute inflammation, but the normal regenerative response to injury is impaired in fatty liver (33, 42). Consistent with these previous observations, liver weights were not significantly different in sham- and PH-treated STD rats at 14 d but were significantly reduced at 14 d in PH-treated HSD, HPUFA, and HSAT rats compared with sham-treated counterparts (Fig. 6A) (n = 8 per diet per time after surgery). Recovery of liver weight was significantly reduced in PH-treated HSD and HSAT rats compared with STD and HPUFA (Fig. 6A). BrdU incorporation into nuclei, an estimate of cell proliferation, was significantly reduced in PH-treated HSD and HSAT rats compared with PH-treated STD and HPUFA rats (Fig. 6B). Body weight and energy intake during the 14 d regeneration period were not significantly different among diet groups or between sham and PH groups (data not shown).
Steatosis characterized by increased saturated fatty acids results in increased liver enzymes and ER stress markers in response to LPS treatment
LPS is a toxic component of cell walls of Gram-negative bacteria that has been postulated to be one factor that can promote progression from steatosis to NASH (4). Before injection of LPS, plasma AAT and AST were significantly greater in HSD and HSAT compared with STD and HPUFA (Fig. 7A) (n = 6 per diet). Acute injection of LPS increased plasma ALT and AST in all groups (Fig. 7A). However, the increase was significantly greater in HSD and HSAT compared with STD and HPUFA (Fig. 7, A and B). The LPS-induced increase in plasma AST was also significantly increased in HPUFA compared with STD (Fig. 7B). Hepatic caspase-3 activity was significantly increased in HSD and HSAT compared with STD and HPUFA after LPS injection (Fig. 7C). Caspase-3 activity was also significantly increased in HPUFA compared with STD (Fig. 7C).
A large majority of obese subjects have fatty liver, and it has been suggested that approximately 30% have NASH (5, 43). This link is noteworthy because of both the current obesity epidemic and the increased prevalence of liver damage in obese subjects (44). The mechanisms that promote the progression from steatosis to NASH and liver damage are poorly understood (3, 6). The prevailing hypothesis suggests that disease progression may be triggered when an acute inflammatory insult is superimposed (second hit) on hepatic steatosis (7). Using dietary models of hepatic steatosis in which fatty acid composition in the liver was varied, we have shown that hepatic steatosis characterized by increased saturated fatty acids is associated with increased markers of liver injury and ER stress, reduced proliferative capacity in response to PH, and increased susceptibility to LPS-mediated liver injury.
In the present study, hepatic steatosis was induced using diets enriched in sucrose (HSD), lard (HSAT), or corn oil (HPUFA). Although the magnitude of steatosis was similar among the diet groups (based on hepatic triglyceride concentration), HSD and HSAT were characterized by increased hepatic saturated fatty acids compared with HPUFA. Notably, increased liver injury, ER stress, and caspase-3 activity were observed in HSD and HSAT but not in HPUFA or STD. The presence of liver injury, ER stress, and increased caspase-3 activity in HSD and HSAT occurred before and independently of insulin action, body fat accumulation, and circulating leptin and TNF. These data suggest that the composition of fatty acids in the steatotic liver is an important determinant of liver injury and that the presence of increased saturated fatty acids in the steatotic liver may constitute an intrinsic second hit.
The regenerative response to liver injury is impaired in ob/ob mice with fatty livers (45), and it has been postulated that this contributes to NAFLD pathophysiology by inhibiting proliferation and increasing injury (42, 45, 46). Consistent with this postulate, recovery of liver weight and proliferative capacity in response to PH were reduced in all three dietary models of fatty liver (HSD, HPUFA, and HSAT). Remarkably, both the recovery of liver weight and proliferative capacity were also significantly reduced in HSD and HSAT compared with HPUFA and STD. Whether this is due to a persistent impairment caused by the fatty acid composition before PH or to the composition and magnitude of fatty acids in the regenerating liver is presently unknown. Increased liver injury and hepatic caspase-3 activity in response to LPS was observed in all three dietary models of fatty liver (i.e. HSD, HPUFA, and HSAT). However, more severe liver injury (based on plasma liver enzymes) and a greater increase in hepatic caspase-3 activity was observed in HSD and HSAT compared with HPUFA and STD. In total, these data support the notion that the composition of fatty acids in the steatotic liver is an important determinant of disease progression in NAFLD.
Recent studies have implicated the ER in obesity, insulin resistance, and diabetes (37, 38, 47, 48, 49). An essential function of the ER is the synthesis and processing of secretory and membrane proteins (50). Several pathologic stresses (e.g. calcium homeostasis, protein glycosylation, and oxidative and reductive stress) disrupt ER homeostasis and lead to the accumulation of unfolded proteins and protein aggregates in the ER lumen, which can be detrimental to cell survival (51, 52, 53). Disruption of ER homeostasis, collectively termed "ER stress," activates the UPR, a signaling pathway that links the ER lumen with the cytoplasm and nucleus (51, 53, 54). If the UPR is not sufficient to mitigate the imposed stress, caspase-dependent and -independent programmed cell death ensues (55). Cellular markers of ER stress include the splicing of the transcription factor XBP-1, up-regulation of the ER chaperone GRP78, and up-regulation of the proapoptotic gene CHOP (35, 51). In the present study, XBP-1 splicing and increased GRP78 protein, CHOP protein, and caspase-3 activity were observed in liver and hepatocytes from dietary models of hepatic steatosis characterized by increased saturated fatty acids (HSD and HSAT). We hypothesize that saturated fatty acids in the steatotic liver induce a stress to the ER that exceeds the capacity of the UPR, resulting in apoptosis and liver injury. Future studies will examine this hypothesis and the causal link(s) between saturated fatty acids, ER stress, and liver injury.
Hepatocyte apoptosis is significantly increased in patients with alcoholic hepatitis and NASH and correlates with disease severity and hepatic fibrosis (56, 57). It has been proposed that the uptake of apoptotic cells released from hepatocytes by Kupffer cells and hepatic stellate cells is mediated by lipid signals (57). Persistent activation of Kupffer and stellate cells can promote additional hepatic apoptosis and inflammation (57). Thus, it will be important to examine the sequential regulatory events that led to increased liver injury in these dietary models of hepatic steatosis.
A recent study has documented that both elevated peripheral fatty acids and de novo lipogenesis contribute to the accumulation of hepatic and lipoprotein fat in patients with NAFLD (58). The present results suggest that an increase in hepatic saturated fatty acids derived from either peripheral lipid (HSAT) or accelerated de novo lipogenesis (HSD) promotes ER stress and liver injury.
In summary, the data from the present study suggest that hepatic steatosis characterized by increased saturated fatty acids can promote liver injury, ER stress, and a proapoptotic environment. The fact that saturated fatty acids in the steatotic liver promote injury independently of obesity, insulin action, and TNF implies that they may be an important, early determinant of susceptibility for disease progression in NAFLD.
Acknowledgments
We thank the Hepatobiliary Center at the University of Colorado Health Sciences Center for assistance with partial hepatectomy, JC-1 fluorescence measurements, and lipid analyses, and the Colorado Clinical Nutrition Research Unit for assistance with insulin, leptin, and TNF measurements.
Footnotes
This work was supported by National Institutes of Health Grants DK47416 and DK072017.
D.W., Y.W., and M.J.P. have nothing to declare.
First Published Online November 3, 2005
Abbreviations: AAT, Alanine aminotransferase; AST, aspartate aminotransferase; BrdU, bromodeoxyuridine; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; HPUFA, high polyunsaturated fat diet; HSAT, high saturated fat diet; HSD, high sucrose diet; LPS, lipopolysaccharide; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PH, partial hepatectomy; STD, high-starch diet; UPR, unfolded protein response.
Accepted for publication October 24, 2005.
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