Johnson

Causal Roles of Hyperinsulinemia in Obesity and Altered Longevity

The epidemics of obesity, type 2 diabetes and related diseases threaten to overrun the global healthcare system. We know that obesity, insulin resistance, early type 2 diabetes are all highly correlated with each other and are all associated with a higher than normal release of insulin from the pancreas, but we still do not fully understand the causal relationship between these phenomena. The most commonly accepted view is that obesity first leads to insulin resistance, which then leads to a compensatory hypersecretion of insulin, which finally results in diabetes when insulin release from the pancreas fail to meet demands. However, these implied cause and effect relationships have been questioned and are impossible to formally test in humans using rigorous genetic loss-of-function approaches. Indeed, there has long been evidence that basal insulin hypersecretion can precede insulin resistance and even obesity and clinical studies have also pointed to anti-obesity effects of drugs that block insulin secretion. We are testing the hypothesis that pancreatic insulin causes obesity directly, by genetically eliminating half of the insulin gene from the pancreas. We expect our study to have an important impact on our fundamental understanding of obesity, which might change the way we diagnose and treat millions of people. This work is funded by the Canadian Institutes of Health Research.

Roles of Brain-Produced Insulin in Alzheimer’s Disease

Many previous reports have suggested that small amounts of insulin may be produced locally by adult central neurons of mammals, including humans. Johnson’s group has recently confirmed these reports using rigorous negative and positive controls, but the function of centrally produced insulin remains a mystery. In this project, we will selectively delete the insulin 2 gene from the mouse brain in order to determine the role of central insulin. Previous studies have suggested that brain insulin is reduced in Alzheimer’s disease, making these studies highly relevant to human disease. This work is funded by the Alzheimer’s Society of Canada.

Hyperinsulinemia and Insulin Signalling in Pancreatic Cancer

Pancreatic adenocarcinoma is the fourth most common cause of cancer death in Canada, but receives the lowest proportion of research funding of any major cancer. As a result, our understanding of the factors that initiate and drive the progression of this disease remains poor relative to our knowledge pertaining to other cancers. Diabetes mellitus and obesity are emerging as important risk factors for pancreatic cancer and the rapid rise in BMI foreshadows a rise in pancreatic cancer. Elevated insulin levels are a feature of both obesity and type 2 diabetes. Hyperinsulinemia has been investigated as a possible contributor to cancer initiation and progression. Lowering hyperinsulinemia with metformin was shown to reduce the risk of pancreatic cancer by 60%. Groups in Europe created headlines worldwide by showing an increased risk of cancer with use of long-acting insulin analogues. The question of whether elevated insulin can be a causal in the pathogenesis of pancreatic cancer has not been rigorously tested. To test this hypothesis, we have established models engineered to lack multiple alleles of their two Insulin genes. Mice with two of four Insulin alleles are hyperinsulinemic on a high-fat diet, whereas mice lacking all but one Insulin allele are hypoinsulinemic but not diabetic. We also have mouse models that will allow us to test whether genes involved in insulin-stimulated proliferation and anti-apoptosis in pancreatic islets, Raf-1, Akt or Pdx-1, participate in pancreatic cancer. Whether insulin signalling might synergize with Kras, the most frequently mutated gene in pancreatic cancer, is a key unanswered question. The overall goal is to test the hypothesis that hyperinsulinemia in diabetes can contribute to hyperproliferation in the exocrine pancreas, promote pre-cancerous lesions, and promote the survival of pancreatic cancer cells. We will also test the hypothesis that Raf-1, Akt and Pdx-1 are critical for insulin action in the pancreas. Together, these studies have the potential to increase our understanding of this devastating disease and increase avenues towards rational therapeutic intervention. This information will eventually be used to identify novel compounds capable of blocking the hyperproliferative and anti-apoptotic effects of insulin in primary human pancreatic tissue and pancreatic tumor cell lines. This work has been funded by the Cancer Research Society of Canada.

Insulin Receptor Trafficking and Insulin ‘Feedback’ Signalling in Type 1 and Type 2 Diabetes

Insulin is both a metabolic hormone and growth factor. The signal transduction cascades activated by insulin have been well studied in ‘insulin target tissues’ such as muscle and fat. However, many studies have revealed unexpected tissues where blocking insulin signalling has adverse consequences to glucose homeostasis. Surprisingly, along with the liver and brain, these studies show that the pancreatic beta-cell itself an important site of insulin action. In addition, islets from human type 2 diabetics appear to be ‘insulin resistant’. We have investigated the role and mechanism of insulin signalling in the beta-cell and we have uncovered exciting differences compared to other tissues. We are continuing to study the effects of insulin on primary human and mouse islets, focusing on the anti-apoptotic effects of insulin and the mechanism of these effects. We have focused on signalling pathways regulated by the Raf-1 kinase and related proteins. We are testing the hypothesis that altered insulin expression plays a role in type 1 diabetes. Recently, we have developed a new way of following the movements of functional insulin receptors in living cells, and used this technology to make insights into insulin signal transduction. This project has been supported by grants from the Juvenile Diabetes Research Foundation and the Canadian Institute for Health Research.

High-Content Screening for Molecules that Promote Beta-Cell Survival

Virtually any cure for type 1 diabetes will require strategies to protect beta-cells from death and dysfunction. With recent support from JDRF, they have optimized novel imaging technologies that allow for the first time the simultaneous, real-time analysis beta-cell function and programmed cell death, on a single-cell basis. At the same time, using bioinformatics and genomics, our laboratory has compiled and published a list of 234 locally produced secreted factors and 233 secreted factor receptors. High-throughput screening approaches now make it possible to examine simultaneously all of these potential survival and differentiation factors under multiple conditions related to the pathogenesis of type 1 diabetes. The overall goal of the proposed study is to identify the most powerful locally acting survival and/or differentiation factors in human islets. Such a factor could be harnessed to improve graft survival in clinical islet transplantation, improve the production surrogate beta-cells, and eventually treat patients with type 1 diabetes and/or their at-risk family members. This work has been funded by the JDRF.

High-Content Screening for Molecules that Regulate Beta-Cell Differentiation Status

A major goal of regenerative medicine is the generation of fully functional pancreatic beta-cells, either from residual beta-cells in the patient or from stem cells. Both of these therapeutic avenues require a thorough understanding of the process by which beta-cells go from being immature to fully functional beta-cells. With SCN support, we devised a method to examine the maturation of single beta-cells for the first time using powerful custom microscopes and state-of-the-art fluorescent markers. At the same time, we have built (using CFI funds) the infrastructure to perform these experiments in a massively parallel manner. In doing so, we have become one of Canada’s leading centres for image-based screening in the diabetes field. This work has been funded by the Stem Cell Network and the JDRF.

New Roles for RyR2-Mediated Calcium Flux in Cardiomyocyte Survival, Metabolism

Every heartbeat is composed of a complex cycle of highly orchestrated events. The cardiac ryanodine receptor calcium channel (RyR2) is central to this cycle, releasing calcium to cause heart muscle cell contraction with each heartbeat. Diseases such as arrhythmias and diabetic cardiomyopathy are associated with changes in RyR2 function. However, it has remained unclear whether the other cellular symptoms of these conditions are causes or consequences of the loss of RyR2 function. Working in other cell types, we have recently described unexpected roles for RyR2, namely the control of gene expression and cell survival. In this project we examine hearts with selective deletion of RyR2 calcium channels and determine which cellular functions are changed most directly as a result. These studies will provide new insight into the dysfunction and death of heart cells in disease.

Calcium-Dependent Signal Transduction in Pancreatic Beta-Cells

All cellular processes are controlled by signals. Defects in the transduction of these signals cause disease. Although we have learned a great deal about the events that control a variety of functions in pancreatic beta-cells, the signalling defects that cause diabetes remain to be elucidated. A major interest in the laboratory is the role of intracellular calcium stores, including those sensitive to IP3, ryanodine and NAADP, in beta-cell survival and function. Intracellular calcium homeostasis is vital to the survival of all cell types. We are particularly interested in the mechanisms by which dysfunctional intracellular calcium signalling leads to programmed cell death. Intracellular calcium stores have been linked to diabetes in previous studies. There is also strong evidence that ER-stress, resulting from lowered ER calcium levels in the beta-cell, plays a significant role in both rare and common forms of diabetes. We are currently using advanced biochemical and molecular techniques, including FRET-based imaging for calcium signals, to further this research.

Lipotoxicity and Gene-Environment Interactions in Type 2 Diabetes

One of the causes of type 2 diabetes is an increase in pancreatic beta-cell death, leading to insufficient insulin. Unfortunately, we still do not understand which genes are required for beta cell survival in the presence of high fat, so we are unable to design effective treatments to stop beta-cell death. For decades, medical research has primarily used a ‘candidate’ gene approach to study known proteins, one at a time, for their role in specific disease states. With the emergence of new technology and systems biology, it is now possible to simultaneously examine virtually all genes or proteins in a cell. These unbiased approaches reveal novel findings that could not have been predicted based on prior knowledge. We have used unbiased proteomic analysis to reveal part of the mechanism by which fatty acids kill beta-cells via ER-stress. The objective of this research program is to continue our proteomics-guided efforts to understand how fatty acids kill beta cells. In particular, we will focus how fatty acids alter the beta-cell’s quality control system for proteins such insulin. We use advanced molecular biology and microscopy to determine how fatty acids lead to the degradation of key proteins in beta-cells. This study will improve our understanding of the underlying causes of diabetes as we search for ways to prevent, manage and cure this disease. This project is supported by a grant-in-aid from the Canadian Diabetes Association.

Technical Expertise

• Optical single cell recording techniques, including multiple wavelength dye and fluorescent protein imaging (e.g. Fura calcium imaging, GFP-tagging and localization).
• Forster resonance energy transfer imaging (FRET), frequency domain fluorescence lifetime imaging microscopy (FLIM; FLIM-FRET).
• Total internal reflectance fluorescence imaging microscopy (TIRF).
• Advanced data analysis, techniques for image quantification and manipulation.
• 3D imaging and cell volume analysis in cardiomyocytes.
• High-content, high-throughput imaging.
• Proteomics (fluorescent 2D-DIGE), genomic analysis
• Primary culture of endocrine cells for physiological measurements of hormone secretion, production, gene expression, radioimmunoassay and ELISA.
• Immunohistochemistry, cell proliferation, apoptosis.
• Morphological identification of live cells.
• Single cell microinjection.
• Basic molecular biology, including shRNAi, gene manipulation.
• Basic cellular biochemistry, DNA ladders, Western blot.
• In vivo and in vitro phenotype analysis of transgenic and mutant mice.

1. Skovsø S, Panzhinskiy E, Kolic J, Cen HH, Dionne DA, Dai XQ, Sharma RB, Elghazi L, Ellis CE, Faulkner K, Marcil SAM, Overby P, Noursadeghi N, Hutchinson D, Hu X, Li H, Modi H, Wildi JS, Botezelli JD, Noh HL, Suk S, Gablaski B, Bautista A, Kim R, Cras-Méneur C, Flibotte S, Sinha S, Luciani DS, Nislow C, Rideout EJ, Cytrynbaum EN, Kim JK, Bernal-Mizrachi E, Alonso LC, MacDonald PE, Johnson JD (2022)  Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance Nat Commun 13, 735 (2022). https://doi.org/10.1038/s41467-022-28039-8
2. Zhang AMY, Magrill J, de Winter TJJ, Hu X, Skovsø S, Schaeffer DF, Kopp JL, Johnson JD (2019)  Endogenous hyperinsulinemia contributes to pancreatic cancer development Cell metabolism https://doi.org/10.1016/j.cmet.2019.07.003
3. Cen HH, Hussein B, Botezelli JD, Wang S, Zhang JA, Noursadeghi N, Jessen N, Rodrigues B, Timmons JA, Johnson JD (2022) Human and mouse muscle transcriptomic analyses identify insulin receptor mRNA downregulation in hyperinsulinemia‐associated insulin resistance FASEB https://doi.org/10.1096/fj.202100497RR
4. Templeman NM, Flibotte S, Chik JHL, Sinha S, Lim GE, Foster LJ, Nislow C, Johnson JD (2017) Reduced circulating insulin enhances insulin sensitivity in old mice and extends lifespan Cell reports https://doi.org/10.1016/j.celrep.2017.06.048.
5. Szabat M, Page MM, Panzhinskiy E, Skovsø S, Mojibian M, Fernandez-Tajes J, Bruin JE, Bround MJ, Lee JT, Xu EE, Taghizadeh F, O’Dwyer S, van de Bunt M, Moon KM, Sinha S, Han J, Fan Y, Lynn FC, Trucco M, Borchers CH, Foster LJ, Nislow C, Kieffer TJ, Johnson JD (2016) Reduced insulin production relieves endoplasmic reticulum stress and induces β cell proliferation Cell metabolism https://doi.org/10.1016/j.celrep.2017.06.048

Further publications can be found here.

Lab Members

• Dr. James D. Johnson, Ph.D. (Group Leader)
• Dr. Howard Cen, Ph.D.  (Postdoctoral Research Fellow)
• Dr. Jelena Kolic (Postdoctoral Research Fellow)
• Dr. Liam Hall (Postdoctoral Research Fellow)
• Dr. Taina Vuorio (Postdoctoral Research Fellow)
• Leanne Beet (Lab Manager)
• Connie Lap Ka Leung (Research Assistant/Technician)
• Xiaoke Betty Hu (Research Assistant/Technician)
• Anni Zhang Graduate (Research Assistant/Technician)
• Aurora Mattison (Graduate Research Assistant)
• Chieh Min Chu (Graduate Research Assistant)
• Michael Atser (Graduate Research Assistant)
• Natalie Nahirney (Graduate Research Assistant)
• Nilou Noursadeghi (Graduate Research Assistant)
• Peter Overby (Graduate Research Assistant)
• Sepehr Kamal (Graduate Research Assistant)
• Stella Baehring (Graduate Research Assistant)
• Jiashuo Zhang (Co-op Student)
• Grace Sun (Co-op Student)
• Yi Han Xia (Co-op Student)

Join the Lab

The Laboratory for Molecular Signalling in Diabetes is looking for exceptional students and post-doctoral fellows.

Potential applicants should be highly motivated. Potential students should have a strong interest in pursuing a career in research and be competitive for external scholarships. Post-doctoral applicants should have experience in a relevant research area, a strong publication record and be competitive for external fellowship awards.

Please contact Dr. Johnson directly by e-mail if you are interested. Include a curriculum vitae (with grades for students), a statement of career goals, and an outline of the specific research you see yourself doing in the lab.