Obesity causes metabolic dysfunctions that lead to debilitating chronic diseases including type 2 diabetes, fatty liver disease, and cirrhosis. Diet-induced obese (DIO) rodents are popular models for studying obesity and pre-diabetes. In 1953, Fenton and Dowling manipulated nutrient composition across a range of refined rodent diets, demonstrating that inbred weanling mice could be induced to obesity on an accelerated timeline1. Since then, DIO mice have become staples of preclinical metabolic disease research. Modern DIO formulations are highly purified and typically provide 40 to 60% fat by caloric input. After as little as six weeks on diet, the inbred male C57BL/6 (B6) DIO mouse presents conditions within the metabolic syndrome cluster, including visceral obesity, hyperglycemia, and triglyceridemia. It also develops insulin resistance, an indicator of chronic inflammation that occurs with excessive adiposity. This tried and true model continues to help researchers characterize novel mechanisms implicated in metabolic disease and uncover translational drug targets.
A Promising New Approach to Treating Metabolic Dysfunction
A recent report published in Nature Communications documents an inspired translational breakthrough from scientists at the Scripps Research Institute (TSRI) in La Jolla2. The study stems from a collaboration between the laboratories of Enrique Saez and Luke Wiseman in the Department of Molecular Medicine. Their teams harnessed the classic DIO B6 mouse to activate a complicated cellular signaling pathway involved in adaptive remodeling to cellular stresses. Through careful pharmacologic manipulation, they induced adaptations that are beneficial to resolving metabolic syndrome, while avoiding triggering maladaptive effects that promote inflammation and cell death if the pathway is overactivated.The IRE1/XBP1s Pathway
The endoplasmic reticulum (ER) is vulnerable to cellular disturbances arising from lipid overload and inflammation due to obesity. The liver is particularly sensitive to ER stress because hepatocytes are highly secretory cells, producing cholesterol and plasma proteins. The ER contains three embedded sensors that respond to misfolded protein accumulation. Through them, the unfolded protein response (UPR) acts to restore homeostasis, but it can also induce apoptosis if a cell is irremediably damaged.The TSRI teams homed in on one particular sensor and a downstream effector. Inositol-requiring enzyme 1 (IRE1) is an autoinducing RNAse that mediates unconventional splicing of mRNA for X-box binding protein 1 (XBP1). XBP1s is a transcription factor that enhances ER protein folding, secretion, and autophagy3. This cellular remodeling can restore functionality to a cell under duress. Sustained activation of IRE1, however, signals the cell is past the point of no return. IRE1 then engages in promiscuous degradation of RNA, a phenomenon known as IRE1-dependent decay (RIDD), which depletes microRNA repressors of proapoptotic Caspase-24,5. Persistently activated IRE1 will also directly interact with tumor necrosis factor receptor-associated factor 2 to initiate a signaling cascade that culminates in apoptosis6. In addition to killing metabolically critical hepatocytes and insulin-producing pancreatic β islet cells, this generates further inflammation that can irreversibly damage liver and pancreatic tissue.
Fine-tuning the UPR Toward Clinical Benefit in the DIO B6 Mouse
The detrimental role of IRE1 hyperactivity in metabolic disease has made it a target for pharmacological treatments geared toward its inhibition7-9. The TSRI scientists approached from a different angle. Appreciating that the relative activities of the three IRE1-regulated pathways are critical in dictating tissue-specific remodeling in the context of obesity-linked diseases, they aimed to show that mild IRE1 activation could actually have therapeutic benefits.The researchers deployed a novel small molecule compound, IXA4, which was uncovered through the Institute's high-throughput screening program for IRE1 activators10. IXA4 is able to induce modest, transient activation of IRE1 without stimulating RIDD or TRAF2 cascades. They treated DIO B6 mice with 50 mg/kg IXA4 daily for 8 weeks in a dosing scheme enabling IRE1 to turn off for periods of time. The compound is also aided by favorable pharmacokinetics, as it reaches a desirable set of tissues including the liver and pancreas.
In conversation with Taconic Biosciences about the publication, Drs. Saez and Wiseman emphasized the value of the DIO mouse as a proof of concept model to showcase that there are beneficial outcomes for selectively activating IRE1. The model recapitulates many of the features of human obesity better than some of the extreme genetic models like db/db (the leptin receptor knockout). There were already good published data that showed XPB1s overexpression in the liver of DIO mice ameliorates metabolic phenotypes and steatosis11,12.
Using their pharmacologic approach, they were able to improve blood glucose levels through reductions in hepatic gluconeogenesis and improved insulin sensitivity. Accordingly, this also reduced hepatic steatosis, reflected through decreased liver triglycerides and expression of lipogenic genes. Dosing failed to invoke any of the pathologic warning signs of pharmacology gone awry, including release of serum markers ALT and AST, macrophage activation, or fibrosis; furthermore, pancreatic insulin production was unperturbed. IXA4 treatment also showed negligible to mild effects on inducing genes regulated through the alternate arms of the UPR, confirming IXA4 shows exquisite bias toward the beneficial IRE1-regulated pathway. Interestingly, the DIO B6 mice did not achieve significant weight loss or reductions to serum triglycerides. In spite of this, the mice presented other metabolic endpoints that were demonstrably druggable, showcasing the versatility of this classic model across more granular indications relevant to the pharmacodynamics of an unconventional therapeutic approach. Importantly, the DIO B6 model is affordable, widely accessible through commercial supply chains, and technically simple, offering a low entry barrier to initiate drug candidates through their preclinical development.
What's Next?
Additional DIO studies are likely, as the group's pipeline contains compounds that specifically target other systems, including adipose tissue. Drs. Saez and Wiseman acknowledge a key limitation of the DIO model is sex differentiation: females are conspicuously protected from metabolic and inflammatory consequences from a high-fat diet feeding using conventional study designs13. They would be interested in interrogating the response at thermoneutrality, which exacerbates disease progression and is conducive to females developing full disease characteristics14. Nonalcoholic steatohepatitis is another obvious metabolic disease target to address since the fatty livers of DIO mice do not present fibrosis.
They are also keen to point out that early compound characterization, done in collaboration with Dr. Jeff Kelly from the Department of Chemistry, showed IXA4 could decrease secretion of the pathologic amyloid precursor protein cleavage produce amyloid-β10. This, and other published work, suggests potential benefits for IXA4-dependent IRE1/XBP1s activation for neurodegenerative diseases including Alzheimer's disease.
They are also keen to point out that early compound characterization, done in collaboration with Dr. Jeff Kelly from the Department of Chemistry, showed IXA4 could decrease secretion of the pathologic amyloid precursor protein cleavage produce amyloid-β10. This, and other published work, suggests potential benefits for IXA4-dependent IRE1/XBP1s activation for neurodegenerative diseases including Alzheimer's disease.
