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Relationship: 3366

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Decreased, INSIG1 protein activity leads to Increased, SREBP2 protein expression

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Decreased, INSIG1 protein activity

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increased, SREBP2 protein expression

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Activation, Pregnane-X receptor, NR1l2 leads to increased plasma low-density lipoprotein (LDL) cholesterol via increased cholesterol synthesis	adjacent	High		John Frisch (send email)	Under development: Not open for comment. Do not cite
Activation, Pregnane-X receptor, NR1l2 leads to increased plasma low-density lipoprotein (LDL) cholesterol via increased PCSK9 protein expression	adjacent	High		John Frisch (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
mammals	mammals	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
All life stages	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Insulin-induced gene 1 (INSIG1) is a transmembrane protein in the endoplasmic reticulum membrane (Ouyang et al. 2020). INSIG1 has an important role in lipid synthesis regulation. At low cholesterol levels, INSIG1 has lower binding affinity for SREBP cleavage-activating protein (SCAP), allowing free SCAP to bind to Coat Protein Complex II (COPII; Ouyang et al. 2020). The SCAP-COPII complex enables Sterol Regulatory Element Binding Protein (SREBPs) to move through the endoplasmic reticulum to the Golgi, where membrane-bound transcription factor site-1-protease (S1P) and site-2-protease (S2P) enable proteolytic processing that allows SREBPs to enter the nucleus (Yabe et al. 2002; Yang et al. 2002). In the nucleus, SREBP2 increase gene expression for enzymes involved in cholesterol synthesis (Ouyang et al. 2020; Itkonen et al., 2023). At high cholesterol levels, INSIG1 binds to SCAP, competitively inhibiting the ability of SCAP to bind to COPII (Ouyang et al. 2020). SREBPs are retained in the endoplasmic reticulum rather than being transferred to the Golgi, reducing levels of cholesterol synthesis (Ouyang et al. 2020; Itkonen et al., 2023).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

This Key Event Relationship was developed as part of an Environmental Protection Agency effort to represent putative AOPs from peer-reviewed literature which were heretofore unrepresented in the AOP-Wiki. Itkonen et al. (2023) focused on identifying Adverse Outcome Pathways that linked PXR activation to increased level of plasma low-density lipoprotein (LDL) cholesterol through review of existing literature, and provided initial network analysis.

Cited empirical studies are focused on decreased INSIG1 and resulting increased SREBP2 gene and protein expression in mammals, in support of development of AOP 545 for Itkonen et al. (2023) content.

Authors of KER 3366 did a further evaluation of published peer-reviewed literature to provide additional evidence in support of the key event relationship.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Insulin-induced gene 1 (INSIG1) and Sterol Regulatory Element Binding Protein-2 (SREBP2) have been studied in a variety of gene-knockout, gene transfection, and diet studies designed to disrupt maintenance of lipid homeostasis in laboratory mammals. Evidence from gene expression and protein expression studies show a consistent response in decrease of INSIG1 activity leading to an increase in Sterol Regulatory Element Binding Protein-2 (SREBP2) activity. In addition, study of cholesterol and lipid levels, as well as proteins SREBP cleavage-activating protein (SCAP) and Coat Protein Complex II (COPII), help to understand the mechanism for regulation of cholesterol synthesis in this key event relationship.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Species	Duration	Dose	Decreased INSIG1 activity?	Increased SREBP2 activity?	Summary	Citation
Human and laboratory mammal genes transfected into Chinese hamster (Cricetulus griseus)	3 days	Various gene transfection combinations.	yes	yes	Chinese hamster ovary cell experiments with various gene transfections to examine the relationship among INSIG1, SCAP, SREBP2 loci confirmed that decreased INSIG1 protein activity led to increased SREBP2 protein activity via decreased SCAP binding per immunoblot.	Yabe et al. (2002); Yang et al. (2002)
Mouse (Mus musculus)	2.5 days	Gene knockout study, high fat 2.0 % cholesterol diet	yes	yes	Mice with knockout INSIG1 gene had decreased INSIG1 protein expression leading to increased SREBP2 protein expression per immunoblot.	Engelking et al. (2005)
Human and laboratory mammal genes transfected into Chinese hamster (Cricetulus griseus)	24 hours	Various gene transfection combinations.	yes	yes	Chinese hamster ovary cell experiments with various gene transfection, as INSIG1 protein expression decreased, SREBP2 protein expression increased per immunoblot.	Gong et al. (2006)
Mouse (Mus musculus	20 weeks	Gene knockout study	yes	yes	Mice with knockout INSIG1 gene had 91-99 percent decreased gene expression to wild-type mice, leading to statistically significant increase in SREBP2 protein expression versus wild-type mice.	MacFarlaine et al. (2014)
Mouse (Mus musculus)	16 weeks	60% calories from fat (High fat diet) for 15 weeks, 50 mg/kg pregnenolone-16α-carbonitrile.	yes	yes	6 week old C57BL/6 N mice had statistically significant decrease in INSIG1 gene expression and lower but not significant INSIG1 protein expression, leading to statistically significant increase in SREBP2 gene and protein expression.	Karpale et al. (2021)

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Life Stage: All life stages.

Sex: Applies to both males and females.

Taxonomic: Primarily studied in humans and laboratory rodents.

References

List of the literature that was cited for this KER description. More help

Engelking, L.J., Liang, G., Hammer, R.E., Takaishi, K., Kuriyama, H., Evers, B.M., Li, W.-P., Horton, J.D., Goldstein, J.L., and Brown, M.S. 2005. Schoenheimer effect explained — feedback regulation of cholesterol synthesis in mice mediated by Insig proteins. The Journal of Clinical Investigation 115(9): 2489–2498.

Gong, Y., Lee, J.N., Lee, P.C.W., Goldstein, J.L., Brown, M.S., and Ye, J. 2006. Sterol-regulated ubiquitination and degradation of Insig-1 creates a convergent mechanism for feedback control of cholesterol synthesis and uptake. Cell Metabolism 3: 15–24.

Itkonen, A., Hakkola, J., and Rysa, J. 2023. Adverse outcome pathway for pregnane X receptor‑induced Hypercholesterolemia. Archives of Toxicology 97: 2861–2877.

Karpale, M. Karajamaki, A.J., Kummu, O., Gylling, H., Hyotylainen, T., Oresic, M., Tolonen, A., Hautajarvi, H., Savolainen, M.J., Ala-Korpela, M., Hukkanen, J., and Hakkola, J. 2021. Activation of pregnane X receptor induces atherogenic lipids and PCSK9 by a SREBP2-mediated mechanism. British Journal of Pharmacology 178: 2461–2481.

MacFarlaine, M.R., Liang, G., and Engelking, L.J. 2014. Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine. The Journal of Biological Chemistry 289(4): 2148-2156.

Ouyang, S., Mo, Z., Sun, S., Yin, K., and Lv, Y. 2020. Emerging role of Insig-1 in lipid metabolism and lipid disorders. Clinica Chimica Acta 508: 206–212. Yabe, D., Brown, M.S., and Goldstein, J.L. 2002. Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins. Proceedings of the National Academy of Sciences 99(20): 12753–12758.

Yang, T., Espenshade, P.J., Wright, M.E., Yabe, D., Gong, Y., Aebersold, R., Goldstein, J.L., and Brown, M.S. 2002. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110: 489–500.

NOTE: Italics indicate edits from John Frisch October 2024.