Ling Qi, Ph.D. - US grants

DESCRIPTION (provided by applicant): Chronic inflammation observed in obesity has been implicated in the development of medically important complications, particularly atherosclerosis, cancer, insulin resistance and non-alcoholic fatty liver disease. Macrophages, key mediators of inflammation, have been shown to contribute significantly to the development of these disorders. Despite considerable attention, however, there is little information thus far on the mechanism through which macrophages are differentially regulated by various inflammation- related adipokines (e.g., adiponectin and resistin) secreted by adipocytes. Is there a common molecular mediator in macrophages for various adipokines? Do various adipokines signal through distinctive pathways in macrophages? Remarkably, our recent data support the notion that macrophage TORC2 (for Transducers Of Regulated CREB activity 2) controls macrophage activation via a unique mechanism, involving nuclear-cytosolic shuttling of TORC2, upon stimulation by specific adipokines. Our two hypotheses are: (i) the adipokine signaling pathways mediated by adiponectin and resistin converge on macrophage TORC2;(ii) nuclear-cytosolic shuttling of TORC2 determines whether macrophages are activated, an event which controls inflammation and influences the pathophysiology of insulin resistance. These hypotheses place TORC2 as the key player that links systemic inflammatory effects of obesity to insulin resistance. Using the state-of-art biochemical and immunological approaches together with metabolic phenotyping, we will test these hypotheses with the following three Specific Aims: (1) to delineate the signaling pathway by which adipokines regulate TORC2 activity in macrophages, focusing on identification of the responsive kinase and phosphatase;(2) to validate the physiological importance of TORC2 in inflammation and insulin resistance in a macrophage-specific transgenic mouse model expressing a constitutively active TORC2 mutant;(3) to further elucidate the role of TORC2 in inflammation and insulin resistance in a loss-of-function TORC2 mouse model generated using bone marrow transplantation. Confirmation of the hypotheses will identify key molecules required for adipokine signaling of the macrophages and intracellular events leading to macrophage activation. Relevance to human health: Delineating the signaling pathway(s) will establish a pivotal mechanism for obesity- induced insulin resistance and related chronic diseases. It will provide one or more candidate targets for drug intervention in conditions exacerbated by chronic inflammation, and possibly establish TORC2 or its interacting partners as early markers of the onset of chronic inflammation. PUBLIC HEALTH RELEVANCE: This proposal investigates the molecular basis of adipokine signaling in macrophages in the context of obesity and its-related inflammatory disorders including atherosclerosis, cancer and diabetes. Delineating the signaling pathway(s) will establish a pivotal mechanism for obesity-induced insulin resistance and related chronic diseases. It will provide one or more candidate targets for drug intervention in conditions exacerbated by chronic inflammation, and possibly establish TORC2 or its interacting partners as early markers of the onset of chronic inflammation.

DESCRIPTION (provided by applicant): Alcoholism threatens the health of millions of Americans in the United States. Alcoholic fatty liver disease, or steatosis, is one of the earliest and most common consequences of excess alcohol consumption and can lead to more severe forms of liver injury, including cirrhosis, diabetes, hepatitis, fibrosis and hepatocellular carcinoma. Therefore, understanding the pathogenesis of alcohol-induced fatty liver disease is of great clinical and basic importance. Recent studies have suggested that response to stress in the endoplasmic reticulum (ER), or unfolded protein response (UPR) may be involved in the pathogenesis of alcoholic liver disease. This is a very attractive model because the ER is the predominant site for lipid biosynthesis, lipid droplet biogenesis, and alcohol detoxification, and because hepatic ER has been reported to expand in alcoholics. However, the causal relationship between ER stress and alcoholic fatty liver has yet to be established. We and others have recently shown that the IRE11-XBP1 branch of the UPR pathway plays a critical role in lipid metabolism. Excitingly, our preliminary data reveal that acute alcohol challenge doubled the activity of IRE11 in the liver within minutes, suggesting that ER stress occurs at an early stage following alcohol consumption. Moreover, ER stress was observed in two chronic-binge drinking mouse models with more severe forms of liver injury. Hence, the goal of this R21 proposal is to delineate the role of the IRE11-XBP1 branch in the pathogenesis of alcoholic fatty liver disease. We hypothesize that the IRE11-XBP1 branch of UPR directly regulates the lipogenic program in the liver in response to chronic-binge alcohol drinking, thereby playing a key role in the pathogenesis of alcoholic fatty liver disease. This hypothesis identifies the IRE11-XBP1 branch and ER homoeostasis as key components linking chronic-binge alcohol drinking and alcoholic fatty liver disease. Using state of the art methodology (e.g. adenoviral shRNA to achieve gene- and liver-specific temporal knockdown, Phos-tag-based method to quantitate ER stress under physiological conditions and tissue ChIP-qPCR to quantitate the binding of a transcription factor to specific gene promoters in the liver), we will test this hypothesis with the following Aims: (1) To determine how modulation of the IRE11-XBP1 pathway affects the pathogenesis of alcoholic fatty liver; (2) To explore the fundamental mechanism by which the IRE11-XBP1 pathway regulates lipogenic genes in response to alcohol. If successful, this study will establish, for the first time to our knowledge, a causal relationship between the UPR and alcoholic fatty liver disease. Relevance to human health: Excessive alcohol intake is a leading cause of chronic liver disease worldwide. Of those who drink (over 50% adults in US), about 29% report binge drinking on multiple occasions each month (i.e. chronic-binge drinkers), which results in about 1.5 billion episodes of binge drinking in the US each year. Fatty liver disease, one of the earliest and most common consequences of excessive heavy or binge drinking, can lead to more severe forms of liver injuries, including hepatitis, cirrhosis, and hepatocellular carcinoma. Our study will provide critical insights into the pathogenesis of alcohol-induced fatty liver disease. This study, if successful, may delineate key signaling pathways in alcoholic liver disease and identify new targets for preventing and treating alcoholic liver diseases as well as other forms of liver diseases. PUBLIC HEALTH RELEVANCE: Endoplasmic reticulum (ER) is a dynamic cellular structure that expands upon chronic alcohol drinking. This proposal tests a novel hypothesis that the activation of the IRE11-XBP1 pathway of the ER stress response upon alcohol consumption contributes significantly to the pathogenesis of alcoholic hepatic steatosis (faty liver) through direct regulation of the lipogenic program. This study, if successful, may establish a causal relationship between ER stress and alcoholic fatty liver, and provide one or more candidate targets for drug intervention in conditions exacerbated by excessive heavy or binge drinking.

DESCRIPTION (provided by applicant): Our long-term goal is to delineate the molecular mechanisms underlying the maintenance of endoplasmic reticulum (ER) homeostasis by two key quality-control systems in the cell, ER-associated degradation (ERAD) and unfolded protein response (UPR). Recently, we have identified a novel regulator of IRE1?, the most conserved sensor of the UPR (He et al. Dev Cell 2012), and reported the generation and characterization of inducible Sel1L knockout (Sel1LIKO) mouse and cell models (Sun et al. PNAS 2014), a cofactor of the ubiquitin ligase Hrd1 in mammalian ERAD. In the preliminary data of this application, we discovered a unique crosstalk between UPR and ERAD, namely the regulation of IRE1? stability by the Sel1L-Hrd1 ERAD complex. Here we showed that loss of Sel1L-Hrd1 ERAD function leads to a dramatic accumulation of IRE1??protein in various tissues and cell types including pancreas, colon, spleen, adipose tissue, MEFs, macrophages and etc. IRE1? accumulation in the absence of Sel1L is independent of transcriptional regulation, pointing to a post-transcriptional control. Indeed, IRE1? interacts with Sel1L and is significantly stabilized in the absence of Sel1L or Hrd1. Thus, these data point to IRE1? as a misfolding- prone Sel1L-Hrd1 ERAD substrate. Here we propose to test the hypotheses that IRE1? is an ERAD substrate and that the Sel1L-Hrd1 ERAD complex negatively regulates the amplitude of IRE1? signaling by mediating its degradation. Taking advantage of systems and tools that we have generated for both Sel1L ERAD and IRE1?, we will accomplish the following Aims: (1) Determine the biological significance of IRE1? ERAD on IRE1? signaling and cell survival; (2) Determine how misfolded IRE1? protein is recognized and delivered to the Sel1L-Hrd1 ERAD complex; and (3) Elucidate how misfolded IRE1? protein is degraded by the Sel1L-Hrd1 ERAD complex. Successful completion of this study may not only provide key insights into IRE1? and ERAD biology, but also uncover a novel regulatory mechanism for IRE1? signaling. This study will provide an unprecedented opportunity to investigate the complicated mechanism of ERAD using an endogenous substrate with great physiological significance, thus exerting a powerful influence on our views of physiological ERAD and UPR biology.

? DESCRIPTION (provided by applicant): Our long-term goal is to delineate the underlying molecular mechanisms and physiological significance of the maintenance of endoplasmic reticulum (ER) homeostasis by three key quality-control systems, ERassociated degradation (ERAD), autophagy and unfolded protein response (UPR). Although recent studies have implied a possible role of UPR in the pathogenesis of obesity and type-2 diabetes, the maintenance and physiological significance of ER homeostasis in adipocytes remains enigma. Our recent data demonstrate that Sel1L, a key adaptor protein for the E3 ligase Hrd1 in mammalian ERAD, plays a critical role in adipocytes and metabolic regulation. Adipocyte-specific Sel1L-deficient mice (Sel1L?adipo) are protected against diet-induced obesity with elevated UPR and autophagy, but uncoupled from inflammation and cell death in WAT. Moreover, our data demonstrate a critical requirement of Sel1L for the secretion of lipoprotein lipase (LPL), which may account for postprandial hypertriglyceridemia of Sel1L?adipo mice. Thus, our data point to an indispensable role of Sel1L in adipocyte function in the pathogenesis of obesity. However, underlying molecular mechanism(s) by which Sel1L affects adipocyte function and obesity remain largely unclear. We hypothesize that Sel1L regulates adipocyte function and metabolism via both Hrd1/ERAD-dependent and -independent mechanisms, and via the crosstalk among three ER qualitycontrol systems (Sel1L-Hrd1 ERAD, UPR and autophagy) in adipocytes. Using an array of adipocytespecific knockout mouse models coupled with in vitro mechanistic studies, we will determine mechanistically how Se1L regulates adipocyte function and metabolism with a particular emphasis on Hrd1/ERAD dependency in Aim 1 and on the crosstalk among three ER quality-control systems in adipocytes in Aim 2. The generation and characterization of several adipocyte-specific double knockout mouse models with various levels of ER stress will elucidate not only the functional crosstalk among key ER quality-control machineries, but also the cellular and pathological consequences of perturbed ER homeostasis in the pathogenesis of obesity and type-2 diabetes. Finally, this study may identify a novel endogenous substrate of the Sel1L-Hrd1 ERAD complex and establish a novel mechanism underlying a feedback regulatory loop between Sel1L-Hrd1 ERAD and IRE1??signaling. RELEVANCE TO HUMAN HEALTH: Protein misfolding is detrimental to the cell and has been linked to the pathogenesis of several human diseases. Despite nearly a decade of effort, the role of ER homeostasis in adipocytes in the pathogenesis of obesity remains vague. Our preliminary data point to adipocyte Sel1L as a key regulator in ER homeostasis and metabolism. A successful completion of this study will have a powerful impact on our understanding of the physiological significance of ER quality-control systems and the maintenance of ER homeostasis in adipocytes in the context of diet-induced obesity.

ABSTRACT Inadequate insulin secretion triggers diabetes. Normally, pancreatic ß-cells synthesize more than 300,000 molecules per minute of the insulin precursor, proinsulin, and these molecules must rapidly fold before their export from the endoplasmic reticulum (ER) ? for eventual processing to insulin. Unfortunately, proinsulin folding in the ER always leaves a small subset of misfolded proinsulin molecules (in particular, those forming mispaired disulfide bonds) that can attack ?bystander proinsulin?, triggering ?-cell ER stress, as occurs for the ~30 different INS gene coding sequence mutations in patients suffering from the autosomal dominant Mutant INS-gene induced Diabetes of Youth (MIDY). Significantly, islet ?-cells of individuals with wild-type INS genes also have proinsulin misfolding. In this multi-P.I. R-01 proposal, the applicants' central hypothesis is that normally, the amount of misfolded proinsulin is kept at sub-threshold levels by active ER-associated degradation (ERAD) of proinsulin that prevents excessive accumulation of misfolded proinsulin forms. Failure of efficient ERAD allows the low-level misfolded proinsulin to accumulate and trigger many of the same phenotypes seen in MIDY. Thus ? ironically ? efficient ERAD of proinsulin (in one subset of molecules) is actually coupled to proper folding of proinsulin (in another subset of molecules). Thus, ?--cell secretory capacity depends on the efficiency of ERAD. If correct, then if proinsulin ERAD should become impaired, misfolded proinsulin may accumulate, triggering pancreatic ?-cell dysfunction. Remarkably, our preliminary data establish that ?-cell-specific knockout of the ERAD gene product, SEL1, triggers a dramatic reduction of islet insulin, accompanied by a striking intracellular accumulation of proinsulin in ?-cells. As a consequence of diminished insulin production, mice with ?-cell SEL1 deficiency are predisposed to diabetes. To our knowledge, no other groups have pursued ?-cell ERAD as the critical homeostatic regulator of proinsulin quality control. We wish to define and quantify the molecular pathways regulating this process, and study the process in physiologically relevant diabetes models, including human islets. The group is uniquely qualified to address this central premise: Dr. Tsai is an expert in molecular mechanisms of protein retrotranslocation for ERAD; Dr. Qi is a leader in the development of whole animal models that can directly test the pathophysiologic role of ERAD in tissues; Dr. Liu is a world's leader in preproinsulin translocation across the ER membrane and along with Dr. Arvan, they have elucidated our current molecular understanding of the pathogenic mechanism underlying MIDY. The focus on disposal of misfolded proinsulin is the critical nexus shared by all of us. A more complete understanding of the molecular steps leading to ß-cell failure is critical to the development of new therapies for diabetes. With this in mind, we believe that work proposed by this group will be paradigm- shifting for the field.

Defining the Central Role of ER-Associated Degradation (ERAD) in Neuroendocrine Cells SUMMARY My laboratory has a long-standing interest in protein folding and degradation within the endoplasmic reticulum (ER) by defining the physiological and pathological importance of mammalian ER quality-control machineries in vivo. ER-associated degradation (ERAD) is the principal protein quality-control mechanism responsible for targeting misfolded proteins in the ER for cytosolic proteasomal degradation. Failure to clear misfolded proteins in the ER presumably activates the unfolded protein response, or UPR. We showed that Sel1L-Hrd1 ERAD modulates the activation of UPR sensor IRE1a by mediating its turnover (Sun et al. 2015 Nat Cell Biol). In two recent studies, we reported that impaired ERAD function may be directly linked to the pathogenesis of metabolic diseases, thereby holding significant therapeutic potential (Shi et al. 2017 and Kim et al. 2018 J CIin Invest). Specifically, we reported that mice with Sel1L deficiency in either AVP or POMC neurons exhibit diabetes insipidus and early-onset obesity, respectively. We showed that Sel1L-Hrd1 ERAD controls the maturation of two prohormones, proAVP and POMC, within the ER by targeting the misfolded forms for proteasomal degradation, thereby preventing the aggregation of a large proportion of native prohormones. We now propose to test the overarching hypothesis that the Sel1L-Hrd1 ERAD protein complex plays a critical role in neuroendocrine cells by directly recruiting misfolded prohormones for proteasomal degradation and by coordinating the activation of other ER quality-control machineries, such as UPR and autophagy, to ensure proper prohormone maturation and neuronal homeostasis. This model challenges the current paradigm in ER biology by placing ERAD at the center of cellular function in normal physiology and disease pathogenesis. Using POMC neurons as a model system, we will accomplish the following Aims: (1) determine the underlying molecular mechanisms and therapeutic potential of ERAD-POMC interactions; (2) demonstrate the pathophysiological importance and mechanisms underlying the crosstalk between ERAD and autophagy in POMC neurons; and (3) demonstrate the pathophysiological importance and mechanisms underlying the crosstalk between ERAD and IRE1? in POMC neurons. This study will provide unprecedented insights into ERAD function and prohormone biology in neuroendocrine cells, which has direct clinical implications for human diseases that are associated with defects of prohormone folding and export. RELEVANCE TO HUMAN HEALTH: All neuropeptides are synthesized as precursor proteins known as ?prohormones? in the ER; however, molecular mechanisms underlying their maturation within the ER remain poorly understood. This study will establish the pathophysiological significance of ERAD in coordinating ER homeostasis during prohormone maturation and explore the therapeutic potential of targeting ERAD in the treatment of diseases attributed to defects in prohormone maturation, such as diabetes insipidus, early-onset obesity, and other rare diseases such as Prader-Willi Syndrome.

SUMMARY Pancreatic ? cell regeneration is a promising approach for the treatment of insulin dependent type-1 (T1D) and -2 diabetes (T2D). However, the nature of the signaling pathway(s) responsible for the age-dependent decline of ? cell proliferation remains an enigma, a significant roadblock in diabetes therapy. The Kulkarni laboratory at Joslin Diabetes Center recently reported that SerpinB1 secreted from the liver promotes ? cell proliferation (El Ouaamari et al. Cell Metabolism 2016) while the Qi laboratory at the University of Michigan showed that Toll- Like Receptors 2 and 4 (TLR2/TLR4) signaling pathways, two redundant innate immune signaling pathways, block diet-induced ? cell replication (Nat Immunol, under revision). In this application, the Kulkarni and Qi laboratories will team up to test an overarching hypothesis that the antagonistic interplay between SerpinB1 and TLR2/TLR4 maintains a balance in favor of ? cell regeneration in both mice and humans under diabetogenic stimuli. We propose that TLR2/TLR4 activation in diet-induced obesity blocks SerpinB1-mediated ? cell replication while simultaneous disruption of TLR2/TLR4 signaling pathways on ? cells may promote ? cell proliferation via SerpinB1-dependent manner. To this end, we will (i) perform lineage tracing to test the hypothesis that TLR2/TLR4 regulates ? cell replication in a ? cell autonomous manner; (ii) delineate the interplay between TLR2/TLR4 and SerpinB1 and the underlying mechanism in the regulation of ? cell regeneration; and (iii) determine the therapeutic potential of targeting TLR2/TLR4 and SerpinB1 in ? cell proliferation and regeneration using human ? cells and islets. Hence, this study will be instrumental in demonstrating that metabolic and innate immune systems, two primitive systems critical for the long-term homeostasis of multi-cellular organisms, have evolved to promote cooperative, adaptive responses against diverse environmental challenges such as overnutrition. If successful, this study will help realize therapeutic potential of our discoveries and benefit millions of diabetic patients worldwide by delivering novel, invaluable therapeutics for the treatment of widespread diabetes. RELEVANCE TO HUMAN HEALTH: This application, with parallel mouse models and human islets studies, will reveal novel molecular and cellular mechanisms, shaped by metabolic and innate immunity signaling pathways, which govern ? cell replication and metabolic homeostasis. Hence, this study may fundamentally change our views of metabolic-innate immune interactions, and hold tremendous promise to uncover both novel disease mechanisms and pharmacological targets aimed at treating and reversing the underlying metabolic imbalances in diabetes.

Novel Role of Hepatic SEL1L-HRD1 ERAD in FGF21 Gene Transcription SUMMARY The liver regulates growth and systemic energy homeostasis through inter-organ communication via the secretion of growth factors, hormones and peptides. Fibroblast growth factor 21 (FGF21) is a liver-derived, fasting-induced hormone with broad effects on growth, nutrient metabolism and insulin sensitivity. The Qi and Fang laboratories have been interested in the physiological roles of SEL1L and HRD1, respectively, focusing on different cell types. They are best known for their SEL1L-HRD1 protein complex in the endoplasmic reticulum (ER)-associated degradation (ERAD), a process responsible for the recruitment and retrotranslocation of ER proteins for cytosolic proteasomal degradation. In addition, studies have suggested that SEL1L may have HRD1- independent functions, and vice versa. In this study, the two laboratories are teaming up to define the role of SEL1L and HRD1 in hepatocytes. Remarkably, recent studies from our laboratories independently linked both SEL1L and HRD1 to FGF21 expression. Indeed, mice with hepatocyte-specific deletion of SEL1L or HRD1 exhibit strikingly similar phenotypes including growth retardation and female infertility with markedly elevated FGF21 levels in the liver and circulation. Mechanistically, we independently identified the ER-resident transcription factor CREBH as a possible molecular link between SEL1L/HRD1 on the ER membrane and FGF21 in the nucleus. Based on these strong preliminary data, we will test the hypothesis that hepatic SEL1L or HRD1 functions as an ERAD complex and together, controls systemic energy metabolism at least in part through the CREBH-FGF21 axis. To this end, we plan to independently and collaboratively test the following Aims: Aim 1 to determine whether hepatic SEL1L-HRD1 ERAD regulates systemic metabolism via modulation of FGF21 levels; and Aim 2 to demonstrate whether hepatic SEL1L-HRD1 ERAD regulates FGF21 gene transcription by targeting CREBH for proteasomal degradation. This study will not only establish the importance of SEL1L-HRD1 ERAD in the liver in the regulation of systemic energy metabolism, but also reveals a novel hepatic ?ERAD-CREBH-FGF21? axis directly linking ER protein turnover to FGF21 gene transcription and systemic metabolic regulation. RELEVANCE TO HUMAN HEALTH: FGF21 is an important metabolic hepatokine that controls systemic energy metabolism and insulin sensitivity. This study will define the pathophysiological significance of SEL1L- HRD1 ERAD in hepatocytes and identify a novel hepatic ?ERAD-CREBH-FGF21? axis directly linking the function of ER protein degradation machinery to FGF21 gene transcription and systemic metabolic regulation.

Regulation of Mitochondrial Dynamics by ERAD ABSTRACT Cells face a complex challenge of balancing protein folding and degradation in the endoplasmic reticulum (ER), a multifunctional organelle that is central to human health. Further, dysregulation of this balance accounts for the pathogenesis of many human diseases. ER-associated degradation (ERAD) is a principal quality-control mechanism used by the cells to target misfolded proteins in the ER for proteasomal degradation in the cytosol. However, the physiological function of distinct mammalian ERAD components remain largely unclear. In the last several years, we have explored the physiological importance of cell type-specific ERAD in normal physiology and disease, and have identified molecular substrates and pathways underpinning ERAD- associated pathophysiology. While ERAD expression is known to be controlled by IRE1a signaling of the UPR, we recently discovered a negative feedback loop in which the Sel1L-Hrd1 protein complex of mammalian ERAD restrains IRE1a signaling and activation under the steady state by targeting IRE1a for proteasomal degradation. This study demonstrates an intimate crosstalk between the two most conserved ER quality- control systems. Surprisingly, our recent data in brown adipocytes reveals that Sel1L-Hrd1 ERAD may regulate mitochondrial dynamics, in part via IRE1a. Sel1L-deficient brown adipocytes exhibit a profound morphological alteration of mitochondria in response to cold exposure, which can be partially rescued upon the deletion of IRE1a. One of the major goals for the next five years is to delineate the molecular mechanism underlying the regulation of mitochondrial dynamics by ERAD by testing the overarching hypothesis that Sel1L-Hrd1 ERAD regulates mitochondrial dynamics and function via IRE1a. We will explore whether and how the ?Sel1L- Hrd1 ERAD-IRE1a? axis of ER quality control machineries exerts control over mitochondrial fission-fusion balance. This study may not only reveal the significance of an ?ERAD-UPR? crosstalk at the core of normal cellular function and physiology, but may also provide exciting insights into the organelle crosstalk, a largely mysterious process. With funding support from NIGMS, we have made great progress towards the understanding of the ERAD- UPR biology in mammals in the past several years. Hence, we are uniquely positioned to lead this project with innovation, passion and dedication to scientific discovery. The R35 grant mechanism will give us the intellectual freedom, time and resources to direct our energy for exploration into discovery and will open up new directions to provide unprecedented insights into the role of ER quality-control machineries in mitochondrial biology.

Pancreatic beta-cells synthesize large quantities of insulin. Growing evidence indicates that any of a number of deficiencies in insulin biosynthesis (genetic, or acquired) can lead to diabetes. We know that insulin biosynthesis begins with translation of preproinsulin. This short-lived precursor must be translocated into the endoplasmic reticulum (ER), signal peptide excised, and proinsulin properly folded in order to undergo successful export from the ER for delivery to the distal secretory pathway in which proinsulin-to-insulin processing and insulin storage in secretory granules finally occurs. In contrast, unsuccessful molecules may be degraded before they are even translocated into the ER, or may be restrained from anterograde export from the ER ? indeed, strong evidence indicates that misfolded proinsulin molecules are targeted for degradation. Secretory pathway protein degradation also involves other endogenous substrates that contribute to the differentiated pancreatic beta cell phenotype. The competing continuation of this multi-P.I. R01 will help clarify how three major mechanisms of secretory pathway protein disposal ? pre-translocation degradation; ER-Associated Degradation (ERAD); and Autophagy ? are all critical for proper beta-cell function. This proposal continues the longstanding association of three tightly collaborative investigators (Qi, Tsai, Arvan) that are experts in exactly these processes: preproinsulin translocation into the ER lumen with the subsequent folding/misfolding of proinsulin, secretory pathway protein degradation via ERAD, and an ER-to-lysosome degradative pathway that we believe is primarily ER-autophagy (ER-phagy). We have strong reason to believe that defects in these quality control mechanisms are linked to type 2 diabetes (T2D) as a result of insulin insufficiency, and this belief is supported by preliminary data. In this proposal, we seek to examine three interlinked areas related to the early secretory pathway of pancreatic beta-cells. For one, we will pursue studies in which infidelity of preproinsulin translocation across the ER membrane is directly linked to deficient proinsulin and insulin biosynthesis, leading directly to diabetes. Second, we will follow-up on some remarkable preliminary data demonstrating that de-differentiation of pancreatic beta-cells is triggered by a loss of efficient ERAD function, also leading directly to insulin-deficient diabetes. Finally, we not only delve deeply into the ER factors that trigger ER-phagic degradation of misfolded proinsulin, but we also propose a deeper understanding of how ineffective or improper ER-phagy can trigger beta cell failure, which also leads directly to insulin-deficient diabetes. These new research directions lead us to pursue a novel therapeutic approach to beta-cell secretory pathway dysfunction focused on stimulating intracellular protein clearance mechanisms, in order to prevent diabetes onset and/or limit its progression.