Prolonged Lipid Accumulation in Cultured Primary Human Hepatocytes Rather Leads to ER Stress than Oxidative Stress.

Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced.

Tick-tock hedgehog-mutual crosstalk with liver circadian clock promotes liver steatosis.

BACKGROUND & AIMS: The mammalian circadian clock controls various aspects of liver metabolism and integrates nutritional signals. Recently, we described Hedgehog (Hh) signaling as a novel regulator of liver lipid metabolism. Herein, we investigated crosstalk between hepatic Hh signaling and circadian rhythm. METHODS: Diurnal rhythms of Hh signaling were investigated in liver and hepatocytes from mice with ablation of Smoothened (SAC-KO) and crossbreeds with PER2::LUC reporter mice. By using genome-wide screening, qPCR, immunostaining, ELISA and RNAi experiments in vitro we identified relevant transcriptional regulatory steps. Shotgun lipidomics and metabolic cages were used for analysis of metabolic alterations and behavior. RESULTS: Hh signaling showed diurnal oscillations in liver and hepatocytes in vitro. Correspondingly, the level of Indian Hh, oscillated in serum. Depletion of the clock gene Bmal1 in hepatocytes resulted in significant alterations in the expression of Hh genes. Conversely, SAC-KO mice showed altered expression of clock genes, confirmed by RNAi against Gli1 and Gli3. Genome-wide screening revealed that SAC-KO hepatocytes showed time-dependent alterations in various genes, particularly those associated with lipid metabolism. The clock/hedgehog module further plays a role in rhythmicity of steatosis, and in the response of the liver to a high-fat diet or to differently timed starvation. CONCLUSIONS: For the first time, Hh signaling in hepatocytes was found to be time-of-day dependent and to feed back on the circadian clock. Our findings suggest an integrative role of Hh signaling, mediated mainly by GLI factors, in maintaining homeostasis of hepatic lipid metabolism by balancing the circadian clock. LAY SUMMARY: The results of our investigation show for the first time that the Hh signaling in hepatocytes is time-of-day dependent, leading to differences not only in transcript levels but also in the amount of Hh ligands in peripheral blood. Conversely, Hh signaling is able to feed back to the circadian clock.

Conditional loss of hepatocellular Hedgehog signaling in female mice leads to the persistence of hepatic steroidogenesis, androgenization and infertility.

The Hedgehog signaling pathway is known to be involved in embryogenesis, tissue remodeling, and carcinogenesis. Because of its involvement in carcinogenesis, it seems an interesting target for cancer therapy. Indeed, Sonidegib, an approved inhibitor of the Hedgehog receptor Smoothened (Smo), is highly active against diverse carcinomas, but its use is also reported to be associated with several systemic side effects. Our former work in adult mice demonstrated hepatic Hedgehog signaling to play a key role in the insulin-like growth factor axis and lipid metabolism. The current work using mice with an embryonic and hepatocyte-specific Smo deletion describes an adverse impact of the hepatic Hedgehog pathway on female fertility. In female SAC-KO mice, we detected androgenization characterized by a 3.3-fold increase in testosterone at 12 weeks of age based on an impressive induction of steroidogenic gene expression in hepatocytes, but not in the classic steroidogenic organs (ovary and adrenal gland). Along with the elevated level of testosterone, the female SAC-KO mice showed infertility characterized by juvenile reproductive organs and acyclicity. The endocrine and reproductive alterations resembled polycystic ovarian syndrome and could be confirmed in a second mouse model with conditional deletion of Smo at 8 weeks of age after an extended period of 8 months. We conclude that the down-regulation of hepatic Hedgehog signaling leads to an impaired hormonal balance by the induction of steroidogenesis in the liver. These effects of Hedgehog signaling inhibition should be considered when using Hedgehog inhibitors as anti-cancer drugs.

RNAi in murine hepatocytes: the agony of choice--a study of the influence of lipid-based transfection reagents on hepatocyte metabolism.

Primary hepatocyte cell cultures are widely used for studying hepatic diseases with alterations in hepatic glucose and lipid metabolism, such as diabetes and non-alcoholic fatty liver disease. Therefore, small interfering RNAs (siRNAs) provide a potent and specific tool to elucidate the signaling pathways and gene functions involved in these pathologies. Although RNA interference (RNAi) in vitro is frequently used in these investigations, the metabolic alterations elucidated by different siRNA delivery strategies have hardly been investigated in transfected hepatocytes. To elucidate the influence of the most commonly used lipid-based transfection reagents on cultured primary hepatocytes, we studied the cytotoxic effects and transfection efficiencies of INTERFERin(®), Lipofectamine(®)RNAiMAX, and HiPerFect(®). All of these transfection agents displayed low cytotoxicity (5.6-9.0 ± 1.3-3.4%), normal cell viability, and high transfection efficiency (fold change 0.08-0.13 ± 0.03-0.05), and they also favored the satisfactory down-regulation of target gene expression. However, when effects on the metabolome and lipidome were studied, considerable differences were observed among the transfection reagents. Cellular triacylglycerides levels were either up- or down-regulated [maximum fold change: INTERFERin(®) (48 h) 2.55 ± 0.34, HiPerFect(®) (24 h) 0.79 ± 0.08, Lipofectamine(®)RNAiMAX (48 h) 1.48 ± 0.21], and mRNA levels of genes associated with lipid metabolism were differentially affected. Likewise, metabolic functions such as amino acid utilization from were perturbed (alanine, arginine, glycine, ornithine, and pyruvate). In conclusion, these findings demonstrate that the choice of non-viral siRNA delivery agent is critical in hepatocytes. This should be remembered, especially if RNA silencing is used for studying hepatic lipid homeostasis and its regulation.

Cyclopamine and Rapamycin Synergistically Inhibit mTOR Signalling in Mouse Hepatocytes, Revealing an Interaction of Hedgehog and mTor Signalling in the Liver.

In the liver, energy homeostasis is mainly regulated by mechanistic target of rapamycin (mTOR) signalling, which influences relevant metabolic pathways, including lipid metabolism. However, the Hedgehog (Hh) pathway is one of the newly identified drivers of hepatic lipid metabolism. Although the link between mTOR and Hh signalling was previously demonstrated in cancer development and progression, knowledge of their molecular crosstalk in healthy liver is lacking. To close this information gap, we used a transgenic mouse model, which allows hepatocyte-specific deletion of the Hh pathway, and in vitro studies to reveal interactions between Hh and mTOR signalling. The study was conducted in male and female mice to investigate sexual differences in the crosstalk of these signalling pathways. Our results reveal that the conditional Hh knockout reduces mitochondrial adenosine triphosphate (ATP) production in primary hepatocytes from female mice and inhibits autophagy in hepatocytes from both sexes. Furthermore, in vitro studies show a synergistic effect of cyclopamine and rapamycin on the inhibition of mTor signalling and oxidative respiration in primary hepatocytes from male and female C57BL/6N mice. Overall, our results demonstrate that the impairment of Hh signalling influences mTOR signalling and therefore represses oxidative phosphorylation and autophagy.

Effect of glucose and insulin supplementation on the isolation of primary human hepatocytes.

Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue's energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation.

Mutual Zonated Interactions of Wnt and Hh Signaling Are Orchestrating the Metabolism of the Adult Liver in Mice and Human.

The Hedgehog (Hh) and Wnt/ß-Catenin (Wnt) cascades are morphogen pathways whose pronounced influence on adult liver metabolism has been identified in recent years. How both pathways communicate and control liver metabolic functions are largely unknown. Detecting core components of Wnt and Hh signaling and mathematical modeling showed that both pathways in healthy liver act largely complementary to each other in the pericentral (Wnt) and the periportal zone (Hh) and communicate mainly by mutual repression. The Wnt/Hh module inversely controls the spatiotemporal operation of various liver metabolic pathways, as revealed by transcriptome, proteome, and metabolome analyses. Shifting the balance to Wnt (activation) or Hh (inhibition) causes pericentralization and periportalization of liver functions, respectively. Thus, homeostasis of the Wnt/Hh module is essential for maintaining proper liver metabolism and to avoid the development of certain metabolic diseases. With caution due to minor species-specific differences, these conclusions may hold for human liver as well.

The Diurnal Timing of Starvation Differently Impacts Murine Hepatic Gene Expression and Lipid Metabolism - A Systems Biology Analysis Using Self-Organizing Maps.

Organisms adapt their metabolism and draw on reserves as a consequence of food deprivation. The central role of the liver in starvation response is to coordinate a sufficient energy supply for the entire organism, which has frequently been investigated. However, knowledge of how circadian rhythms impact on and alter this response is scarce. Therefore, we investigated the influence of different timings of starvation on global hepatic gene expression. Mice (n = 3 each) were challenged with 24-h food deprivation started in the morning or evening, coupled with refeeding for different lengths and compared with ad libitum fed control groups. Alterations in hepatocyte gene expression were quantified using microarrays and confirmed or complemented with qPCR, especially for lowly detectable transcription factors. Analysis was performed using self-organizing maps (SOMs), which bases on clustering genes with similar expression profiles. This provides an intuitive overview of expression trends and allows easier global comparisons between complex conditions. Transcriptome analysis revealed a strong circadian-driven response to fasting based on the diurnal expression of transcription factors (e.g., Ppara, Pparg). Starvation initiated in the morning produced known metabolic adaptations in the liver; e.g., switching from glucose storage to consumption and gluconeogenesis. However, starvation initiated in the evening produced a different expression signature that was controlled by yet unknown regulatory mechanisms. For example, the expression of genes involved in gluconeogenesis decreased and fatty acid and cholesterol synthesis genes were induced. The differential regulation after morning and evening starvation were also reflected at the lipidome level. The accumulation of hepatocellular storage lipids (triacylglycerides, cholesteryl esters) was significantly higher after the initiation of starvation in the morning compared to the evening. Concerning refeeding, the gene expression pattern after a 12 h refeeding period largely resembled that of the corresponding starvation state but approached the ad libitum control state after refeeding for 21 h. Some components of these regulatory circuits are discussed. Collectively, these data illustrate a highly time-dependent starvation response in the liver and suggest that a circadian influence cannot be neglected when starvation is the focus of research or medicine, e.g., in the case of treating victims of sudden starvation events.

Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a GLI-code associated with steatosis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and is increasing in prevalence. The pathomechanisms, however, are poorly understood. This study assessed the unexpected role of the Hedgehog pathway in adult liver lipid metabolism. Using transgenic mice with conditional hepatocyte-specific deletion of Smoothened in adult mice, we showed that hepatocellular inhibition of Hedgehog signaling leads to steatosis by altering the abundance of the transcription factors GLI1 and GLI3. This steatotic 'Gli-code' caused the modulation of a complex network of lipogenic transcription factors and enzymes, including SREBP1 and PNPLA3, as demonstrated by microarray analysis and siRNA experiments and could be confirmed in other steatotic mouse models as well as in steatotic human livers. Conversely, activation of the Hedgehog pathway reversed the "Gli-code" and mitigated hepatic steatosis. Collectively, our results reveal that dysfunctions in the Hedgehog pathway play an important role in hepatic steatosis and beyond.