The origin story of frequently diagnosed diseases is a complicated one – there can be many distinct causes, some of which are rare and poorly understood. Once such rare underlying condition that can lead to common diseases is lipodystrophy, a disease state defined by the loss of functional adipose tissue (the medical term for fat) that leads to the gain of fat deposits within internal organs such as liver and skeletal muscle. Lipodystrophy can progress causing insulin resistance, diabetes, and in some instances, cancer. Dr. Nobunao Wakabayashi, a Senior Staff Scientist in Dr. Thomas Kensler’s lab, a part of Fred Hutchinson Cancer Center’s Public Health Sciences Division, is among a team of researchers working to better understand the molecular mechanisms driving this disease state, and importantly, working to develop experimental approaches for impeding its progression to cancer. To achieve these goals Dr. Kensler, Dr. Wakabayashi and colleagues have developed a lipodystrophy mouse model. “We previously established a lipodystrophy model in RosaNIC/NIC::AdiCre mice in which Notch signaling (through NIC [notch intracellular domain]) increases only in adipose tissue. The resultant lipoatrophy of the adipose tissue is followed in several months by a lipodystrophic phenotype of high serum triglycerides, diabetes, fatty liver, and by 6-7 months, the development of sarcomas,” explained Dr. Wakabayashi. By utilizing Keap1 knockdown mouse models, the researchers were able to enhance expression of NRF2, a transcription factor with target genes spanning many essential pathways including oxidative stress, homeostasis, and cell death, which altered the impact of lipodystrophy (KEAP1 is a dominant NRF2 chaperone). “Introduction of systemically enhanced NRF2 signaling into these mice clearly prevented the aforementioned effects except for the initial atrophy of adipose tissue (Keap1A/A::RosaNIC/NIC::AdiCre mice). Further, this protective effect was abrogated by the deletion of Nrf2 from this mouse (Nrf2-/-::Keap1A/A::RosaNIC/NIC::AdiCre mice),” said Dr. Wakabayashi, highlighting the potentially critical role of NRF2 in lipodystrophy.
However, findings from these previous research efforts noted that the liver continued to suffer damage from adipose deposits, pinpointing it as a key organ of interest. Describing their mission further, Dr. Wakabayashi said “since adipose tissue-specific enhancement of NRF2 signaling (Keap1B/B::RosaNIC/NIC::AdiCre mice) did not protect against hepatic damage, we hypothesized that the key target tissue for the prevention of lipodystrophic phenotype observed in the Keap1A/A::RosaNIC/NIC::AdiCre mouse might be liver. Therefore, we asked whether enhanced NRF2 signaling specifically in the liver of the RosaNIC/NIC::AdiCre mouse could prevent the hepatic phenotype. We used direct DNA delivery of pCAG expression vector systems to the liver by hydrodynamic tail vein injection. We therefore sought to probe the role of NRF2 in the liver to protect against the sequelae of lipodystrophy and to establish underlying mechanisms of protection.” This research was recently published in International Journal of Molecular Sciences.
This pCAG expression vector contained a dominant active recombinant NRF2, designed to prolong NRF2 activity and reduce the rate of degradation. Immunohistochemistry confirmed that expression of NRF2 and its target gene Nqo1 were significantly higher than in mice injected with a control vector. Likewise, immunoblotting analysis determined reduced levels of negatively regulated NRF2 target genes. These data suggest that tail vein injection of the vector could substantially reduce hepatic effects of lipodystrophy. Next, they injected their pCAG-Nqo1 and pCAG DA-Nrf2 vectors into 5-week-old mice that were then put on a high fat diet. The introduction of these vectors decreased levels of hepatomegaly (liver enlargement) and reduced hepatic triglyceride levels by 50% compared to control mice. Emphasizing the significance of these findings, Dr. Wakabayashi noted “this study established that elevated expression of NRF2 in the livers of RosaNIC/NIC::AdiCre mice could reduce the hepatomegaly and fatty liver evoked by the induction of lipodystrophy. Remarkably, singular expression of a canonical NRF2 target gene (Nqo1) in the liver provided dramatic protection against these early sequelae. Whether the diabetic and sarcoma outcomes would similarly be impacted remains to be determined. Liver-directed targeting of NRF2 or some of its target genes may offer therapeutic opportunities.”
So, what’s next for this group and their efforts to mitigate the effects of lipodystrophy? The authors plan to continue to enhance the effects of their current model system. “Our expression construct, DA-Nrf2, bears multiple point mutations in the primary domain normally mediating KEAP1-NRF2 binding. Based on in vitro cellular studies, the construct was expected to provide a highly dominant active function of endogenous NRF2 as KEAP1 facilitates the degradation of NRF2. However, it was only partially successful in enhancing NRF2 stability. Probably, other NRF2-degrasomes including b-TrCP need to be evaluated for improving our system,” said Dr. Wakabayashi. In parallel, the researchers are focused on continuing to provide insight into the key players in the NRF2 target gene family. “Forced hepatic expression of Nqo1, which is a direct NRF2 target gene, showed much stronger attenuation of hepatic lipid accumulation. We need to elucidate what unknown NQO1 function is at play here (perhaps it is not its enzymatic function but rather a molecular chaperon mechanism). We would like to expand our understanding of NRF2 in targeted therapies for management of steatotic liver disease. Given the known regulatory effect on de novo lipogenesis related gene expression through the NRF2-NQO1 signaling axis, it might be effective,” hypothesized Dr. Wakabayashi.
This work was funded by the Washington State Andy Hill CARE Fund, the National Institutes of Health, and the National Cancer Institute.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Dr. Thomas Kensler contributed to this work.
Wakabayashi N, Yagishita Y, Joshi T, Kensler TW. Forced Hepatic Expression of NRF2 or NQO1 Impedes Hepatocyte Lipid Accumulation in a Lipodystrophy Mouse Model. Int J Mol Sci. 2023 Aug 28;24(17):13345. doi: 10.3390/ijms241713345. PMID: 37686150; PMCID: PMC10487640