The NAD salvage pathway during the progression of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a major chronic liver disease and thus a main reason for liver-related morbidities and mortality. NAFLD covers a wide range of diseases starting with steatosis and frequently progressing to non-alcoholic steatohepatitis (NASH), which is an independent predictor for the development of the hepatocellular carcinoma (HCC). Nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme of the mammalian NAD salvage pathway, recycles nicotinamide to nicotinamide mononucleotide (NMN), which is further converted to nicotinamide adenine dinucleotide (NAD). NAD is not only an important redox partner but also a crucial co-substrate for NAD-dependent enzymes such as sirtuin 1 (SIRT1). Thus, NAD metabolism might be involved in the progression of NAFLD by regulating many cellular processes, such as apoptosis, de novo lipogenesis, glycolysis and gluconeogenesis, in the liver. Interestingly, tumor cells have a high NAD turnover due to their rapid proliferation and high activity of NAD-dependent enzymes. For these reasons, I hypothesized that the NAD salvage pathway is dysregulated during the progression of non-alcoholic fatty liver disease. Therefore, the first study of the present work deals with the role of the NAD salvage pathway in a diet-induced mouse model of hepatic steatosis. In mice fed a high-fat diet for 11 weeks hepatic NAMPT mRNA, protein abundance and activity as well as NAD levels were increased. Additionally, SIRT1 protein abundance was upregulated indicating a higher SIRT1 activity. This could be confirmed by detecting decreased acetylation or transcription of SIRT1 targets. For example, p53 and nuclear factor κB (NF-κB) were less acetylated demonstrating lower activity of key regulators of apoptosis and inflammation, respectively. In the second study of this thesis NAMPT activity was inhibited by applying its specific inhibitor FK866 in hepatocarcinoma cells to investigate whether or not NAMPT inhibition could be a potential novel therapeutic approach in HCC treatment. Hepatocarcinoma cells were more sensitive to NAMPT inhibition by FK866 than primary human hepatocytes, presenting a high number of apoptotic cells after FK866 treatment. FK866 induced NAD and ATP depletion which was associated with activation of the key regulator of energy metabolism 5’-AMP-activated protein kinase (AMPK) and decreased activity of its downstream target mammalian target of rapamycin (mTOR). This thesis shows that the NAD salvage pathway is involved in hepatic steatosis and HCC. During hepatic steatosis NAD metabolism is upregulated to potentially protect against adverse effects of the massive hepatic lipid accumulation. To repress the progression to NASH it might be useful to maintain the hepatic NAD levels during early disease stages by administration of NAD precursors, such as NMN. However, hepatocarcinoma cells have a higher activity of NAMPT and NAD-dependent enzymes. NAMPT inhibition by FK866 could be a potential therapeutic approach in HCC, especially due to the fact that NAD depletion is selectively induced in hepatocarcinoma cells, but not in primary human hepatocytes

The Novel Phosphatidylinositol-3-Kinase (PI3K) Inhibitor Alpelisib Effectively Inhibits Growth of PTEN-Haploinsufficient Lipoma Cells

Germline mutations in the tumor suppressor gene PTEN cause PTEN Hamartoma Tumor Syndrome (PHTS). Pediatric patients with PHTS frequently develop lipomas. Treatment attempts with the mTORC1 inhibitor rapamycin were unable to reverse lipoma growth. Recently, lipomas associated with PIK3CA-related overgrowth syndrome were successfully treated with the novel PI3K inhibitor alpelisib. Here, we tested whether alpelisib has growth-restrictive effects and induces cell death in lipoma cells. We used PTEN-haploinsufficient lipoma cells from three patients and treated them with alpelisib alone or in combination with rapamycin. We tested the effect of alpelisib on viability, proliferation, cell death, induction of senescence, adipocyte differentiation, and signaling at 1–100 M alpelisib. Alpelisib alone or in combination with rapamycin reduced proliferation in a concentrationand time-dependent manner. No cell death but an induction of senescence was detected after alpelisib incubation for 72 h. Alpelisib treatment led to a reduced phosphorylation of AKT, mTOR, and ribosomal protein S6. Rapamycin treatment alone led to increased AKT phosphorylation. This effect could be reversed by combining rapamycin with alpelisib. Alpelisib reduced the size of lipoma spheroids by attenuating adipocyte differentiation. Since alpelisib was well tolerated in first clinical trials, this drug alone or in combination with rapamycin is a potential new treatment option for PHTS-related adipose tissue overgrowth

Resveratrol differentially regulates NAMPT and SIRT1 in hepatocarcinoma cells and primary human hepatocytes

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells

The Novel Phosphatidylinositol-3-Kinase (PI3K) Inhibitor Alpelisib Effectively Inhibits Growth of PTEN-Haploinsufficient Lipoma Cells

Germline mutations in the tumor suppressor gene PTEN cause PTEN Hamartoma Tumor Syndrome (PHTS). Pediatric patients with PHTS frequently develop lipomas. Treatment attempts with the mTORC1 inhibitor rapamycin were unable to reverse lipoma growth. Recently, lipomas associated with PIK3CA-related overgrowth syndrome were successfully treated with the novel PI3K inhibitor alpelisib. Here, we tested whether alpelisib has growth-restrictive effects and induces cell death in lipoma cells. We used PTEN-haploinsufficient lipoma cells from three patients and treated them with alpelisib alone or in combination with rapamycin. We tested the effect of alpelisib on viability, proliferation, cell death, induction of senescence, adipocyte differentiation, and signaling at 1-100 \ub5M alpelisib. Alpelisib alone or in combination with rapamycin reduced proliferation in a concentration- and time-dependent manner. No cell death but an induction of senescence was detected after alpelisib incubation for 72 h. Alpelisib treatment led to a reduced phosphorylation of AKT, mTOR, and ribosomal protein S6. Rapamycin treatment alone led to increased AKT phosphorylation. This effect could be reversed by combining rapamycin with alpelisib. Alpelisib reduced the size of lipoma spheroids by attenuating adipocyte differentiation. Since alpelisib was well tolerated in first clinical trials, this drug alone or in combination with rapamycin is a potential new treatment option for PHTS-related adipose tissue overgrowth

Simvastatin induces apoptosis in PTEN‑haploinsufficient lipoma cells

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E‑binding protein (4E‑BP)‑1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator‑activated receptor (PPAR)γ expression. The small interfering RNA‑mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas

Visual and visuomotor interhemispheric transfer time in older adults

Older adults typically experience reductions in the structural integrity of the anterior channels of the corpus callosum. Despite preserved structural integrity in central and posterior channels, many studies have reported that interhemispheric transfer, a function attributed to these regions, is detrimentally affected by aging. In this study, we use a constrained event-related potential analysis in the theta and alpha frequency bands to determine whether interhemispheric transfer is affected in older adults. The crossed-uncrossed difference and lateralized visual evoked potentials were used to assess interhemispheric transfer in young (18–27) and older adults (63–80). We observed no differences in the crossed-uncrossed difference measure between young and older groups. Older adults appeared to have elongated transfer in the theta band potentials, but this effect was driven by shortened contralateral peak latencies, rather than delayed ipsilateral latencies. In the alpha band, there was a trend toward quicker transfer in older adults. We conclude that older adults do not experience elongated interhemispheric transfer in the visuomotor or visual domains and that these functions are likely attributed to posterior sections of the corpus callosum, which are unaffected by aging