UC Berkeley

In mammalian cells and tissues, endoplasmic reticulum (ER) stress due to a disruption in the organelle's protein folding homeostasis is implicated in various metabolic diseases, notably obesity, type 2 diabetes (T2D), non-alcoholic fatty liver disease (NAFLD), and atherosclerosis. Inositol-requiring enzyme 1α (IRE1α), an ER-resident transmembrane protein, senses unfolded proteins, initiating the unfolded protein response (UPR) for adaptive transcriptional programs to restore ER balance. Additionally, IRE1α mediates inflammatory pathways, potentially driving the progression of metabolic impairments. Recent findings highlight IRE1α's ability to sense both unfolded-protein stress and lipid stress through distinct structural elements. Alleviating ER stress is considered protective against metabolic diseases, but the specific role of IRE1α, particularly in individual tissues or cell types, in driving metabolic disorders remains unexplored. This dissertation aims to 1) investigate IRE1α's role in metabolic dysfunctions, focusing on T2D, NAFLD, and atherosclerosis, and 2) generate IRE1α cell lines with specifically perturbed stress-sensing abilities.

Chapter 1 explores the impact of systemic IRE1α inhibition through both a small-molecule drug and genetic deletion of hepatic IRE1α in the context of T2D. The study demonstrates that both IRE1α inhibition and hepatocyte-specific deletion effectively prevent hyperglycemia and enhance hepatic insulin responsiveness. Transcriptional analysis of livers from both models identifies a set of downregulated genes associated with IRE1α inhibition/deletion, notably including those encoding for ER chaperones. Intriguingly, these genes, which are highly upregulated in obesity, are downregulated once more when IRE1α activity is blocked. This phenomenon, which we have termed "overchaperoning," is proposed as a potential cell-intrinsic mechanism driving insulin resistance.

Chapter 2 delves into the role of IRE1α in myeloid cells in mediating NAFLD pathogenesis. The studies demonstrate that the genetic deletion of IRE1α in myeloid cells effectively protects mice from NAFLD-associated steatosis and fibrosis. This observation suggests a mechanistic crosstalk between macrophages, hepatocytes, and hepatic stellate cells in the context of NAFLD.

Chapter 3 investigates the consequences of genetic IRE1α deletion in myeloid cells within the context of atherosclerosis. The findings indicate that myeloid-specific IRE1α deletion led to reduced plaque expansion, plaque vascular smooth muscle cell (VSMC) content, and cellular proliferation in the aorta. These results suggest that IRE1α deletion in macrophages induces alterations in the vascular niche that ameliorate the progression of atherosclerosis in mice.

Chapter 4 focuses on genomic editing of IRE1α in a mouse macrophage cell line. Using a CRISPR-based approach, we successfully generated and validated an IRE1α functional knockout (KO) in an immortalized mouse macrophage cell line. In addition, we designed and validated the feasibility of the construct design for both a luminal domain deletion and a transmembrane domain mutation of IRE1 α, which were sufficient to disable induction of the UPR in response to unfolded protein accumulation vs. lipid excess, respectively.