Hyperpolarized [1-13 C] pyruvate MRSI to detect metabolic changes in liver in a methionine and choline-deficient diet rat model of fatty liver disease.

PURPOSE: Nonalcoholic fatty liver disease is an important cause of chronic liver disease. There are limited methods for monitoring metabolic changes during progression to steatohepatitis. Hyperpolarized 13 C MRSI (HP 13 C MRSI) was used to measure metabolic changes in a rodent model of fatty liver disease. METHODS: Fifteen Wistar rats were placed on a methionine- and choline-deficient (MCD) diet for 1-18 weeks. HP 13 C MRSI, T2 -weighted imaging, and fat-fraction measurements were obtained at 3 T. Serum aspartate aminotransaminase, alanine aminotransaminase, and triglycerides were measured. Animals were sacrificed for histology and measurement of tissue lactate dehydrogenase (LDH) activity. RESULTS: Animals lost significant weight (13.6% ± 2.34%), an expected characteristic of the MCD diet. Steatosis, inflammation, and mild fibrosis were observed. Liver fat fraction was 31.7% ± 4.5% after 4 weeks and 22.2% ± 4.3% after 9 weeks. Lactate-to-pyruvate and alanine-to-pyruvate ratios decreased significantly over the study course; were negatively correlated with aspartate aminotransaminase and alanine aminotransaminase (r = -[0.39-0.61]); and were positively correlated with triglycerides (r = 0.59-0.60). Despite observed decreases in hyperpolarized lactate signal, LDH activity increased by a factor of 3 in MCD diet-fed animals. Observed decreases in lactate and alanine hyperpolarized signals on the MCD diet stand in contrast to other studies of liver injury, where lactate and alanine increased. Observed hyperpolarized metabolite changes were not explained by alterations in LDH activity, suggesting that changes may reflect co-factor depletion known to occur as a result of oxidative stress in the MCD diet. CONCLUSION: HP 13 C MRSI can noninvasively measure metabolic changes in the MCD model of chronic liver disease.

A genetic basis of mitochondrial DNAJA3 in nonalcoholic steatohepatitis-related hepatocellular carcinoma.

BACKGROUND AND AIMS: NAFLD is the most common form of liver disease worldwide, but only a subset of individuals with NAFLD may progress to NASH. While NASH is an important etiology of HCC, the underlying mechanisms responsible for the conversion of NAFLD to NASH and then to HCC are poorly understood. We aimed to identify genetic risk genes that drive NASH and NASH-related HCC. APPROACH AND RESULTS: We searched genetic alleles among the 24 most significant alleles associated with body fat distribution from a genome-wide association study of 344,369 individuals and validated the top allele in 3 independent cohorts of American and European patients (N=1380) with NAFLD/NASH/HCC. We identified an rs3747579-TT variant significantly associated with NASH-related HCC and demonstrated that rs3747579 is expression quantitative trait loci of a mitochondrial DnaJ Heat Shock Protein Family (Hsp40) Member A3 ( DNAJA3 ). We also found that rs3747579-TT and a previously identified PNPLA3 as a functional variant of NAFLD to have significant additional interactions with NASH/HCC risk. Patients with HCC with rs3747579-TT had a reduced expression of DNAJA3 and had an unfavorable prognosis. Furthermore, mice with hepatocyte-specific Dnaja3 depletion developed NASH-dependent HCC either spontaneously under a normal diet or enhanced by diethylnitrosamine. Dnaja3 -deficient mice developed NASH/HCC characterized by significant mitochondrial dysfunction, which was accompanied by excessive lipid accumulation and inflammatory responses. The molecular features of NASH/HCC in the Dnaja3 -deficient mice were closely associated with human NASH/HCC. CONCLUSIONS: We uncovered a genetic basis of DNAJA3 as a key player of NASH-related HCC.

Novel autoantibody targets identified in patients with autoimmune hepatitis (AIH) by PhIP-Seq reveals pathogenic insights.

Autoimmune hepatitis (AIH) is a severe autoimmune disease, characterized by the presence of autoantibodies. However, the role of autoantibodies in the pathophysiology of AIH remains uncertain. Here, we employed Phage Immunoprecipitation-Sequencing (PhIP-Seq) to identify novel autoantibodies in AIH. Using these results, a logistic regression classifier was able to predict which patients had AIH, indicating the presence of a distinct humoral immune signature. To further investigate the autoantibodies most specific to AIH, significant peptides were identified relative to a broad array of controls (298 patients with non-alcoholic fatty liver disease (NAFLD), primary biliary cholangitis (PBC), or healthy controls). Top ranked autoreactive targets included SLA, the target of a well-recognized autoantibody in AIH, and disco interacting protein 2 homolog A (DIP2A). The autoreactive fragment of DIP2A shares a 9-amino acid stretch nearly identical to the U27 protein of HHV-6B, a virus found in the liver. In addition, antibodies against peptides derived from the leucine rich repeat N-terminal (LRRNT) domain of the relaxin family peptide receptor 1 (RXFP1) were highly enriched and specific to AIH. The enriched peptides map to a motif adjacent to the receptor binding domain, which is required for RXFP1 signaling. RXFP1 is a G protein-coupled receptor that binds relaxin-2, an anti-fibrogenic molecule shown to reduce the myofibroblastic phenotype of hepatic stellate cells. Eight of nine patients with antibodies to RXFP1 had evidence of advanced fibrosis (F3 or greater). Furthermore, serum from AIH patients positive for anti-RFXP1 antibody was able to significantly inhibit relaxin-2 signaling in the human monocytic cell line, THP1. Depletion of IgG from anti-RXFP1 positive serum abrogated this effect. These data provide supporting evidence that HHV6 plays a role in the development of AIH and point to a potential pathogenic role for anti-RXFP1 IgG in some patients. Identification of anti-RXFP1 in patient serum may enable risk stratification of AIH patients for fibrosis progression and lead to the development of novel strategies for disease intervention.

Modeling and therapeutic targeting of inflammation-induced hepatic insulin resistance using human iPSC-derived hepatocytes and macrophages.

Hepatic insulin resistance is recognized as a driver of type 2 diabetes and fatty liver disease but specific therapies are lacking. Here we explore the potential of human induced pluripotent stem cells (iPSCs) for modeling hepatic insulin resistance in vitro, with a focus on resolving the controversy about the impact of inflammation in the absence of steatosis. For this, we establish the complex insulin signaling cascade and the multiple inter-dependent functions constituting hepatic glucose metabolism in iPSC-derived hepatocytes (iPSC-Heps). Co-culture of these insulin-sensitive iPSC-Heps with isogenic iPSC-derived pro-inflammatory macrophages induces glucose output by preventing insulin from inhibiting gluconeogenesis and glycogenolysis and activating glycolysis. Screening identifies TNFα and IL1ß as the mediators of insulin resistance in iPSC-Heps. Neutralizing these cytokines together restores insulin sensitivity in iPSC-Heps more effectively than individual inhibition, reflecting specific effects on insulin signaling and glucose metabolism mediated by NF-κB or JNK. These results show that inflammation is sufficient to induce hepatic insulin resistance and establish a human iPSC-based in vitro model to mechanistically dissect and therapeutically target this metabolic disease driver.

The Role of STING in Liver Injury Is Both Stimulus- and Time-Dependent.

STING, Tmem173, is involved in liver injury caused by both infectious and sterile inflammatory models. Its role in toxic liver injury and non-alcoholic fatty liver disease (NAFLD), however, is less clear. While a few groups have investigated its role in NAFLD pathogenesis, results have been conflicting. The objective of this study was to clarify the exact role of STING in toxic liver injury and NAFLD models. Goldenticket mice (Tmem173gt), which lack STING protein, were subjected to either a toxic liver injury with tunicamycin (TM) or one of two dietary models of non-alcoholic fatty liver disease: high fructose feeding or Fructose-Palmitate-Cholesterol (FPC) feeding. Three days after TM injection, Tmem173gt mice demonstrated less liver injury (average ALT of 54 ± 5 IU/L) than control mice (average ALT 108 ± 24 IU/L). In contrast, no significant differences in liver injury were seen between WT and Tmem173gt mice fed either high fructose or FPC. Tmem173gt mice only distinguished themselves from WT mice in their increased insulin resistance. In conclusion, while STING appears to play a role in toxic liver injury mediated by TM, it plays little to no role in two dietary models of NAFLD. The exact role of STING appears to be stimulus-dependent.

Mouse liver injury induces hepatic macrophage FGF23 production.

Fibroblast growth factor 23 (FGF23) is a bone marrow cell produced hormone that functions in the intestine and kidney to regulate phosphate homeostasis. Increased serum FGF23 is a well-established predictor of mortality in renal disease, but recent findings linking increased levels to hepatic and cardiac diseases have suggested that other organs are sources of FGF23 or targets of its effects. The potential ability of the liver to produce FGF23 in response to hepatocellular injury was therefore examined. Very low levels of Fgf23 mRNA and FGF23 protein were detected in normal mouse liver, but the amounts increased markedly during acute liver injury from the hepatotoxin carbon tetrachloride. Serum levels of intact FGF23 were elevated during liver injury from carbon tetrachloride. Chronic liver injury induced by a high fat diet or elevated bile acids also increased hepatic FGF23 levels. Stimulation of toll-like receptor (TLR) 4-driven inflammation by gut-derived lipopolysaccharide (LPS) underlies many forms of liver injury, and LPS induced Fgf23 in the liver as well as in other organs. The LPS-inducible cytokines IL-1ß and TNF increased hepatic Fgf23 expression as did a TLR2 agonist Pam2CSK3. Analysis of Fgf23 expression and FGF23 secretion in different hepatic cell types involved in liver injury identified the resident liver macrophage or Kupffer cell as a source of hepatic FGF23. LPS and cytokines selectively induced the hormone in these cells but not in hepatocytes or hepatic stellate cells. FGF23 failed to exert any autocrine effect on the inflammatory state of Kupffer cells but did trigger proinflammatory activation of hepatocytes. During liver injury inflammatory factors induce Kupffer cell production of FGF23 that may have a paracrine proinflammatory effect on hepatocytes. Liver-produced FGF23 may have systemic hormonal effects as well that influence diseases in in other organs.

Visceral Adipose Tissue Inflammation and Radiographic Visceral-to-Subcutaneous Adipose Tissue Ratio in Patients with Cirrhosis.

BACKGROUND AND AIMS: Accumulation of visceral adipose tissue is associated with hepatic inflammation and fibrosis, suggestive of its metabolic and inflammatory properties. We aimed to examine the histologic findings of visceral and subcutaneous adipose tissue and to associate these findings with clinical and radiologic characteristics in patients with cirrhosis. METHODS: Included were 55 adults with cirrhosis who underwent liver transplantation from 3/2017-12/2018 and had an abdominal computed tomography (CT) scan within 6 months prior to transplant. Visceral-to-subcutaneous adipose tissue ratio (VSR) was calculated using visceral (VATI) and subcutaneous adipose tissue index (SATI) quantified by CT at the L3-vertebral level and normalized for height (cm2/m2). VAT (greater omentum), SAT (abdominal wall), and skeletal muscle (rectus abdominis) biopsies were collected at transplant. RESULTS: Majority of patients had VAT inflammation (71%); only one patient (2%) had SAT inflammation. Patients with VAT inflammation had similar median VATI (42 vs 41 cm2/m2), lower median SATI (64 vs 97 cm2/m2), and higher median VSR (0.63 vs 0.37, p = 0.002) than patients without inflammation. In univariable logistic regression, VSR was associated with VAT inflammation (OR 1.47, 95%CI 1.11-1.96); this association remained significant even after adjusting for age, sex, BMI, HCC, or MELD-Na on bivariable analyses. CONCLUSION: In patients with cirrhosis undergoing liver transplantation, histologic VAT inflammation was common, but SAT inflammation was not. Increased VSR was independently associated with VAT inflammation. Given the emerging data demonstrating the prognostic value of VSR, our findings support the value of CT-quantified VSR as a prognostic marker for adverse outcomes in the liver transplant setting.

Doxycycline Significantly Enhances Induction of Induced Pluripotent Stem Cells to Endoderm by Enhancing Survival Through Protein Kinase B Phosphorylation.

BACKGROUND AND AIMS: Induced pluripotent stem cells (iPSCs) provide an important tool for the generation of patient-derived cells, including hepatocyte-like cells, by developmental cues through an endoderm intermediate. However, most iPSC lines fail to differentiate into endoderm, with induction resulting in apoptosis. APPROACH AND RESULTS: To address this issue, we built upon published methods to develop an improved protocol. We discovered that doxycycline dramatically enhances the efficiency of iPSCs to endoderm differentiation by inhibiting apoptosis and promoting proliferation through the protein kinase B pathway. We tested this protocol in >70 iPSC lines, 90% of which consistently formed complete sheets of endoderm. Endoderm generated by our method achieves similar transcriptomic profiles, expression of endoderm protein markers, and the ability to be further differentiated to downstream lineages. CONCLUSIONS: Furthermore, this method achieves a 4-fold increase in endoderm cell number and will accelerate studies of human diseases in vitro and facilitate the expansion of iPSC-derived cells for transplantation studies.

Hepatocyte-specific deletion of XBP1 sensitizes mice to liver injury through hyperactivation of IRE1α.

X-box binding protein-1 (XBP1) is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum (ER) stress. Under stress conditions, the transcriptionally active form of XBP1 is generated via splicing of Xbp1 mRNA by the ER-resident protein inositol-requiring enzyme-1 (IRE1α). Genetic deletion of XBP1 has multiple consequences: some resulting from the loss of the transcription factor per se, and others related to compensatory activation of IRE1α. The objective of the current study was to investigate the effects of XBP1 deletion in adult mouse liver and determine to what extent they are direct or indirect. XBP1 was deleted from hepatocytes in adult Xbp1fl/fl mice using AAV8-Transthyretin-Cre (Xbp1Δhep). Xbp1Δhep mice exhibited no liver disease at baseline, but developed acute biochemical and histologic liver injury in response to a dietary challenge with fructose for 4 weeks. Fructose-mediated liver injury in Xbp1Δhep mice coincided with heightened IRE1α activity, as demonstrated by Xbp1 mRNA splicing, JNK activation, and regulated IRE1α-dependent RNA decay (RIDD). Activation of eIF2α was also evident, with associated up-regulation of the pro-apoptotic molecules CHOP, BIM, and PUMA. To determine whether the adverse consequences of liver-specific XBP1 deletion were due to XBP1 loss or heightened IRE1α activity, we repeated a fructose challenge in mice with liver-specific deletion of both XBP1 and IRE1α (Xbp1Δhep;Ire1aΔhep). Xbp1Δhep;Ire1aΔhep mice were protected from fructose-mediated liver injury and failed to exhibit any of the signs of ER stress seen in mice lacking XBP1 alone. The protective effect of IRE1α deletion persisted even with long-term exposure to fructose. Xbp1Δhep mice developed liver fibrosis at 16 weeks, but Xbp1Δhep;Ire1aΔhep mice did not. Overall, the results indicate that the deleterious effects of hepatocyte-specific XBP1 deletion are due primarily to hyperactivation of IRE1α. They support further exploration of IRE1α as a contributor to acute and chronic liver diseases.

Ly6cLo non-classical monocytes promote resolution of rhesus rotavirus-mediated perinatal hepatic inflammation.

Perinatal hepatic inflammation can have devastating consequences. Monocytes play an important role in the initiation and resolution of inflammation, and their diverse functions can be attributed to specific cellular subsets: pro-inflammatory or classical monocytes (Ly6cHi) and pro-reparative or non-classical monocytes (Ly6cLo). We hypothesized that inherent differences in Ly6cHi classical monocytes and Ly6cLo non-classical monocytes determine susceptibility to perinatal hepatic inflammation in late gestation fetuses and neonates. We found an anti-inflammatory transcriptional profile expressed by Ly6cLo non-classical monocytes, and a physiologic abundance of these cells in the late gestation fetal liver. Unlike neonatal pups, late gestation fetuses proved to be resistant to rhesus rotavirus (RRV) mediated liver inflammation. Furthermore, neonatal pups were rendered resistant to RRV-mediated liver injury when Ly6cLo non-classical monocytes were expanded. Pharmacologic inhibition of Ly6cLo non-classical monocytes in this setting restored susceptibility to RRV-mediated disease. These data demonstrate that Ly6cLo monocytes promote resolution of perinatal liver inflammation in the late gestation fetus, where there is a physiologic expansion of non-classical monocytes, and in the neonatal liver upon experimental expansion of these cells. Therapeutic strategies directed towards enhancing Ly6cLo non-classical monocyte function may mitigate the detrimental effects of perinatal liver inflammation.

Macronutrients and the Adipose-Liver Axis in Obesity and Fatty Liver.

Macronutrient metabolism is a highly orchestrated process, with adipose tissue and liver each playing central roles in nutrient uptake, processing, transport, and storage. These 2 tissues form an important metabolic circuit, particularly as it relates to lipids as the primary storage form of excess energy. The function of the circuit is influenced by many factors, including the quantity and type of nutrients consumed and their impact on the overall health of the tissues. In this review we begin with a brief summary of the homeostatic disposition of lipids between adipose tissue and liver and how these processes can become dysregulated in obesity. We then explore how specific dietary nutrients and nutrient combinations can exert unique influences on the liver-adipose tissue axis.

Loss of Integrin αvß8 in Murine Hepatocytes Accelerates Liver Regeneration.

Recent fate-mapping studies in mice have provided substantial evidence that mature adult hepatocytes are a major source of new hepatocytes after liver injury. In other systems, integrin αvß8 has a major role in activating transforming growth factor (TGF)-ß, a potent inhibitor of hepatocyte proliferation. We hypothesized that depletion of hepatocyte integrin αvß8 would increase hepatocyte proliferation and accelerate liver regeneration after injury. Using Itgb8flox/flox;Alb-Cre mice to deplete hepatocyte αvß8, after partial hepatectomy, hepatocyte proliferation and liver-to-body weight ratio were significantly increased in Itgb8flox/flox;Alb-Cre mice compared with control mice. Antibody-mediated blockade of hepatocyte αvß8 in vitro, with assessment of TGF-ß signaling pathways by real-time quantitative PCR array, supported the hypothesis that integrin αvß8 inhibition alters hepatocyte TGF-ß signaling toward a pro-regenerative phenotype. A diethylnitrosamine-induced model of hepatocellular carcinoma, used to examine the possibility that this pro-proliferative phenotype might be oncogenic, revealed no difference in either tumor number or size between Itgb8flox/flox;Alb-Cre and control mice. Immunohistochemistry for integrin αvß8 in healthy and injured human liver demonstrated that human hepatocytes express integrin αvß8. Depletion of hepatocyte integrin αvß8 results in increased hepatocyte proliferation and accelerated liver regeneration after partial hepatectomy in mice. These data demonstrate that targeting integrin αvß8 may represent a promising therapeutic strategy to drive liver regeneration in patients with a broad range of liver diseases.

Race/ethnicity is an independent risk factor for autoimmune hepatitis among the San Francisco underserved.

Although autoimmune hepatitis (AIH) is more common in women and affects people of all races/ethnicities, there is currently limited information regarding the relationship between race/ethnicity and AIH, especially in the context of underserved populations. We aim to evaluate the relationship between race/ethnicity and AIH and better characterize its clinical features among different racial groups. We conducted a 15-year retrospective analysis, from January 2002 to June 2017, of patients seen at Zuckerberg San Francisco General Hospital (ZSFG). Sixty-three AIH patients and 2049 non-AIH controls were eligible for the study. The main predictor of interest was race/ethnicity, and the main outcome of interest was AIH diagnosis; other secondary measures recorded include clinical features such as ALT, bilirubin, and biopsy fibrosis at presentation. In a multivariable model adjusting for age and sex, we found that black (OR 9.6, 95% CI 1.8-178), Latino (OR 25.0, 95% CI 5.3-448), and Asian/Pacific Islander (API) (OR 10.8, 95% CI 2.2-196) race/ethnicity were associated with increased odds of an AIH diagnosis compared to the white reference group. Among people of colour with AIH, there were no significant differences in baseline ALT (p = .45), total bilirubin at presentation (p = .06), fibrosis at presentation (p = .74), and hospitalization (p = .27). Race/ethnicity is an independent risk factor for AIH. The clinical features of AIH did not differ significantly among black, Latino, and API patients.

CD18 deficiency improves liver injury in the MCD model of steatohepatitis.

Neutrophils and macrophages are important constituents of the hepatic inflammatory infiltrate in non-alcoholic steatohepatitis. These innate immune cells express CD18, an adhesion molecule that facilitates leukocyte activation. In the context of fatty liver, activation of infiltrated leukocytes is believed to enhance hepatocellular injury. The objective of this study was to determine the degree to which activated innate immune cells promote steatohepatitis by comparing hepatic outcomes in wild-type and CD18-mutant mice fed a methionine-choline-deficient (MCD) diet. After 3 weeks of MCD feeding, hepatocyte injury, based on serum ALT elevation, was 40% lower in CD18-mutant than wild-type mice. Leukocyte infiltration into the liver was not impaired in CD18-mutant mice, but leukocyte activation was markedly reduced, as shown by the lack of evidence of oxidant production. Despite having reduced hepatocellular injury, CD18-mutant mice developed significantly more hepatic steatosis than wild-type mice after MCD feeding. This coincided with greater hepatic induction of pro-inflammatory and lipogenic genes as well as a modest reduction in hepatic expression of adipose triglyceride lipase. Overall, the data indicate that CD18 deficiency curbs MCD-mediated liver injury by limiting the activation of innate immune cells in the liver without compromising intrahepatic cytokine activation. Reduced liver injury occurs at the expense of increased hepatic steatosis, which suggests that in addition to damaging hepatocytes, infiltrating leukocytes may influence lipid homeostasis in the liver.

Specific Macronutrients Exert Unique Influences on the Adipose-Liver Axis to Promote Hepatic Steatosis in Mice.

BACKGROUND & AIMS: The factors that distinguish metabolically healthy obesity from metabolically unhealthy obesity are not well understood. Diet has been implicated as a determinant of the unhealthy obesity phenotype, but which aspects of the diet induce dysmetabolism are unknown. The goal of this study was to investigate whether specific macronutrients or macronutrient combinations provoke dysmetabolism in the context of isocaloric, high-energy diets. METHODS: Mice were fed 4 high-energy diets identical in calorie and nutrient content but different in nutrient composition for 3 weeks to 6 months. The test diets contained 42% carbohydrate (sucrose or starch) and 42% fat (oleate or palmitate). Weight and glucose tolerance were monitored; blood and tissues were collected for histology, gene expression, and immunophenotyping. RESULTS: Mice gained weight on all 4 test diets but differed significantly in other metabolic outcomes. Animals fed the starch-oleate diet developed more severe hepatic steatosis than those on other formulas. Stable isotope incorporation showed that the excess hepatic steatosis in starch-oleate-fed mice derived from exaggerated adipose tissue lipolysis. In these mice, adipose tissue lipolysis coincided with adipocyte necrosis and inflammation. Notably, the liver and adipose tissue abnormalities provoked by starch-oleate feeding were reproduced when mice were fed a mixed-nutrient Western diet with 42% carbohydrate and 42% fat. CONCLUSIONS: The macronutrient composition of the diet exerts a significant influence on metabolic outcome, independent of calories and nutrient proportions. Starch-oleate appears to cause hepatic steatosis by inducing progressive adipose tissue injury. Starch-oleate phenocopies the effect of a Western diet; consequently, it may provide clues to the mechanism whereby specific nutrients cause metabolically unhealthy obesity.

Isocaloric manipulation of macronutrients within a high-carbohydrate/moderate-fat diet induces unique effects on hepatic lipogenesis, steatosis and liver injury.

Diets containing excess carbohydrate and fat promote hepatic steatosis and steatohepatitis in mice. Little is known, however, about the impact of specific carbohydrate/fat combinations on liver outcome. This study was designed to determine whether high-energy diets with identical caloric density but different carbohydrate and fat composition have unique effects on the liver. Four experimental diets were formulated with 60%kcal carbohydrate and 20%kcal fat, each in nearly pure form from a single source: starch-oleate, starch-palmitate, sucrose-oleate and sucrose-palmitate. The diets were fed to mice for 3 or 12 weeks for analysis of lipid metabolism and liver injury. All mice developed hepatic steatosis over 12 weeks, but mice fed the sucrose-palmitate diet accumulated more hepatic lipid than those in the other three experimental groups. The exaggerated lipid accumulation in sucrose-palmitate-fed mice was attributable to a disproportionate rise in hepatic de novo lipogenesis. These mice accrued more hepatic palmitate and exhibited more evidence of liver injury than any of the other experimental groups. Interestingly, lipogenic gene expression in mice fed the custom diets did not correlate with actual de novo lipogenesis. In addition, de novo lipogenesis rose in all mice between 3 and 12 weeks, without feedback inhibition from hepatic steatosis. The pairing of simple sugar (sucrose) and saturated fat (palmitate) in a high-carbohydrate/moderate-fat diet induces more de novo lipogenesis and liver injury than other carbohydrate/fat combinations. Diet-induced liver injury correlates positively with hepatic de novo lipogenesis and is not predictable by isolated analysis of lipogenic gene expression.