Michelle Kirby

High-fructose, medium chain trans fat diet induces liver fibrosis and elevates plasma coenzyme Q9 in a novel murine model of obesity and nonalcoholic steatohepatitis†

Diets high in saturated fat and fructose have been implicated in the development of obesity and nonalcoholic steatohepatitis (NASH) in humans. We hypothesized that mice exposed to a similar diet would develop NASH with fibrosis associated with increased hepatic oxidative stress that would be further reflected by increased plasma levels of the respiratory chain component, oxidized coenzyme Q9 (oxCoQ9). Adult male C57Bl/6 mice were randomly assigned to chow, high‐fat (HF), or high‐fat high‐carbohydrate (HFHC) diets for 16 weeks. The chow and HF mice had free access to pure water, whereas the HFHC group received water with 55% fructose and 45% sucrose (wt/vol). The HFHC and HF groups had increased body weight, body fat mass, fasting glucose, and were insulin‐resistant compared with chow mice. HF and HFHC consumed similar calories. Hepatic triglyceride content, plasma alanine aminotransferase, and liver weight were significantly increased in HF and HFHC mice compared with chow mice. Plasma cholesterol (P < 0.001), histological hepatic fibrosis, liver hydroxyproline content (P = 0.006), collagen 1 messenger RNA (P = 0.003), CD11b‐F4/80+Gr1+ monocytes (P < 0.0001), transforming growth factor β1 mRNA (P = 0.04), and α‐smooth muscle actin messenger RNA (P = 0.001) levels were significantly increased in HFHC mice. Hepatic oxidative stress, as indicated by liver superoxide expression (P = 0.002), 4‐hydroxynonenal, and plasma oxCoQ9 (P < 0.001) levels, was highest in HFHC mice. Conclusion: These findings demonstrate that nongenetically modified mice maintained on an HFHC diet in addition to developing obesity have increased hepatic ROS and a NASH‐like phenotype with significant fibrosis. Plasma oxCoQ9 correlated with fibrosis progression. The mechanism of fibrosis may involve fructose inducing increased ROS associated with CD11b+F4/80+Gr1+ hepatic macrophage aggregation, resulting in transforming growth factor β1–signaled collagen deposition and histologically visible hepatic fibrosis. (HEPATOLOGY 2010)

Vertical sleeve gastrectomy reduces hepatic steatosis while increasing serum bile acids in a weight-loss-independent manner

Our objective was to investigate the role of bile acids in hepatic steatosis reduction after vertical sleeve gastrectomy (VSG).

A Surgical Model in Male Obese Rats Uncovers Protective Effects of Bile Acids Post-Bariatric Surgery

Bariatric surgery elevates serum bile acids. Conjugated bile acid administration, such as tauroursodeoxycholic acid (TUDCA), improves insulin sensitivity, whereas short-circuiting bile acid circulation through ileal interposition surgery in rats raises TUDCA levels. We hypothesized that bariatric surgery outcomes could be recapitulated by short circuiting the normal enterohepatic bile circulation. We established a model wherein male obese rats underwent either bile diversion (BD) or Sham (SH) surgery. The BD group had a catheter inserted into the common bile duct and its distal end anchored into the middistal jejunum for 4-5 weeks. Glucose tolerance, insulin and glucagon-like peptide-1 (GLP-1) response, hepatic steatosis, and endoplasmic reticulum (ER) stress were measured. Rats post-BD lost significantly more weight than the SH rats. BD rats gained less fat mass after surgery. BD rats had improved glucose tolerance, increased higher postprandial glucagon-like peptide-1 response and serum bile acids but less liver steatosis. Serum bile acid levels including TUDCA concentrations were higher in BD compared to SH pair-fed rats. Fecal bile acid levels were not different. Liver ER stress (C/EBP homologous protein mRNA and pJNK protein) was decreased in BD rats. Bile acid gavage (TUDCA/ursodeoxycholic acid [UDCA]) in diet-induced obese rats, elevated serum TUDCA and concomitantly reduced hepatic steatosis and ER stress (C/EBP homologous protein mRNA). These data demonstrate the ability of alterations in bile acids to recapitulate important metabolic improvements seen after bariatric surgery. Further, our work establishes a model for focused study of bile acids in the context of bariatric surgery that may lead to the identification of therapeutics for metabolic disease.

Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A

Previous studies showed that Fanconi anemia (FA) murine stem cells have defective reconstitution after bone marrow (BM) transplantation. The mechanism underlying this defect is not known. Here, we report defective homing of FA patient BM progenitors transplanted into mouse models. Using cells from patients carrying mutations in FA complementation group A (FA-A), we show that when transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) recipient mice, FA-A BM cells exhibited impaired homing activity. FA-A cells also showed defects in both cell-cell and cell-matrix adhesion. Complementation of FA-A deficiency by reexpression of FANCA readily restored adhesion of FA-A cells. A significant decrease in the activity of the Rho GTPase Cdc42 was found associated with these defective functions in patient-derived cells, and expression of a constitutively active Cdc42 mutant was able to rescue the adhesion defect of FA-A cells. These results provide the first evidence that FA proteins influence human BM progenitor homing and adhesion via the small GTPase Cdc42-regulated signaling pathway.

Mixed lineage kinase 3 deficient mice are protected against the high fat high carbohydrate diet-induced steatohepatitis

C‐Jun N‐terminal kinase (JNK) activation is pivotal in the development of nonalcoholic steatohepatitis (NASH). Mixed lineage kinase 3 (MLK) 3 is one of the mitogen activated protein kinase kinase kinase (MAP3K) that mediates JNK activation in the liver. Despite this concept, the role of MLK3 in modulating liver injury during nutrient excess has not been explored. Our aim was to determine if MLK3 deficient mice were protected against high fat high carbohydrate (HFHC) diet‐induced NASH.

Ectopic HOXB4 overcomes the inhibitory effect of tumor necrosis factor-α on Fanconi anemia hematopoietic stem and progenitor cells

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha, we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution.

Identification and Characterization of Mutations in FANCL Gene: a Second Case of Fanconi Anemia Belonging to FA-L Complementation Group

Fanconi anemia (FA) is a rare autosomal recessive or X‐linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA crosslinking agents. Eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and FANCM) and three non‐FA proteins (FAAP100, FAAP24 and HES1) form an FA nuclear core complex, which is required for monoubiquitination of the FANCD2‐FANCI dimer upon DNA damage. FANCL possesses a PHD/RING‐finger domain and is a putative E3 ubiquitin ligase subunit of the core complex. In this study, we report an FA patient with an unusual presentation belonging to the FA‐L complementation group. The patient lacks an obvious FA phenotype except for the presence of a café‐au‐lait spot, mild hypocellularity and a family history of leukemia. The molecular diagnosis and identification of the FA subgroup was achieved by FA complementation assay. We identified bi‐allelic novel mutations in the FANCL gene and functionally characterized them. To the best of our knowledge, this is the second reported case belonging to the FA‐L complementation group.

Insulin Concentration Modulates Hepatic Lipid Accumulation in Mice in Part via Transcriptional Regulation of Fatty Acid Transport Proteins

Background Fatty liver disease (FLD) is commonly associated with insulin resistance and obesity, but interestingly it is also observed at low insulin states, such as prolonged fasting. Thus, we asked whether insulin is an independent modulator of hepatic lipid accumulation. Methods/Principal Findings In mice we induced, hypo- and hyperinsulinemia associated FLD by diet induced obesity and streptozotocin treatment, respectively. The mechanism of free fatty acid induced steatosis was studied in cell culture with mouse liver cells under different insulin concentrations, pharmacological phosphoinositol-3-kinase (PI3K) inhibition and siRNA targeted gene knock-down. We found with in vivo and in vitro models that lipid storage is increased, as expected, in both hypo- and hyperinsulinemic states, and that it is mediated by signaling through either insulin receptor substrate (IRS) 1 or 2. As previously reported, IRS-1 was up-regulated at high insulin concentrations, while IRS-2 was increased at low levels of insulin concentration. Relative increase in either of these insulin substrates, was associated with an increase in liver-specific fatty acid transport proteins (FATP) 2&5, and increased lipid storage. Furthermore, utilizing pharmacological PI3K inhibition we found that the IRS-PI3K pathway was necessary for lipogenesis, while FATP responses were mediated via IRS signaling. Data from additional siRNA experiments showed that knock-down of IRSs impacted FATP levels. Conclusions/Significance States of perturbed insulin signaling (low-insulin or high-insulin) both lead to increased hepatic lipid storage via FATP and IRS signaling. These novel findings offer a common mechanism of FLD pathogenesis in states of both inadequate (prolonged fasting) and ineffective (obesity) insulin signaling.

Hepatic natural killer T‐cell and CD8+ T‐cell signatures in mice with nonalcoholic steatohepatitis

Hepatic inflammation is a key pathologic feature of nonalcoholic steatohepatitis (NASH). Natural killer T (NKT) cells and clusters of differentiation (CD)8+ T‐cells are known to play an important role in obesity‐related adipose tissue inflammation. We hypothesized that these same inflammatory phenotypes would be present in progressive NASH. We used a previously established high‐fat high‐carbohydrate (HFHC) murine obesogenic diet model of progressive NASH to investigate the role of NKT cells and CD8+ T‐cells in C57Bl6/J mice. To better understand the impact of these cell populations, CD1d‐deficient and CD8+ T‐cell‐depleted mice were subjected to an HFHC diet for 16 weeks. C57Bl6/J mice fed an HFHC diet had increased body weight, liver triglyceride content, serum alanine aminotransferase levels, and increased NKT‐cell and CD8+ T‐cell infiltration in the liver. In addition, human liver sections from patients with NASH showed increased CD8+ T‐cells. In comparison, CD1d‐deficient and CD8 T‐cell‐depleted mice fed an HFHC diet had a lower hepatic triglyceride content, lower alanine aminotransferase levels, lower activated resident macrophages and infiltrating macrophages, improved nonalcoholic fatty liver disease activity scores, and reduced α‐smooth muscle actin, collagen type 1 alpha 1, and collagen type 1 alpha 2 messenger RNA expression. Further, while CD1d‐deficient mice were protected against weight gain on the HFHC diet, CD8 T‐cell‐depleted mice gained weight on the HFHC diet. Conclusion: We found that NASH has an immunological signature that includes hepatic infiltrating NKT and CD8+ T‐cells. Depletion of these cells resulted in reduced NASH progression and thus presents novel therapeutic avenues for the treatment of NASH. (Hepatology Communications 2017;1:299–310)

Mo1043 A Chronic High Fructose Diet Induces Progressive Murine NASH With Early Mitochondrial Aging

Background Non-alcoholic fatty liver disease (NAFLD) and the more serious sub-category non-alcoholic steatohepatitis (NASH), have quickly become recognized as one of the most common causes of liver disease in both adults and children in the world. Diets high in saturated fat and fructose are factors leading to obesity and NASH in humans. Case reports demonstrate mitochondria ultra-structural changes obtained from humans with histological NASH with progressing development to uncover mechanisms in development of NASH, diagnostic tools to differentiate the severity and progression of the disease, and ultimately treatment options.