James D Johnson

• Ph.D.
• University of British Columbia

Insulin is produced by pancreatic β cells, whose dysfunction and death are hallmarks of diabetes. Stem cell-derived β-like cells (SCβ cells) are a potential alternative to cadaveric islets for replacement therapy. However, SCβ cells face a multitude of stresses, including hypoxia, hyperglycemia, ER stress, inflammation, and autoimmunity that must b...

Bile acids (BAs) affect the growth of potentially pathogenic commensals, including those from the Enterobacteriaceae family, which are frequently overrepresented in inflammatory bowel disease (IBD). BAs are normally reabsorbed in the ileum for recycling and are often increased in the colonic lumina of patients with IBD, including those with Crohn’s...

Pyruvate dehydrogenase kinase (PDK) 1 is one of four isozymes that inhibit the oxidative decarboxylation of pyruvate to acetyl-CoA via pyruvate dehydrogenase. PDK activity is elevated in fasting or starvation conditions to conserve carbohydrate reserves. PDK has also been shown to increase mitochondrial fatty acid utilization. In cardiomyocytes, me...

Metabolic diseases, such as type 2 diabetes (T2D), insulin resistance, and obesity, often accompany pancreatic ductal adenocarcinoma (PDAC), and they are associated with reduced survival. Hyperinsulinemia is a common hallmark symptom shared by those disorders and is independently associated with reduced survival of PDAC patients. While it has been...

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches, we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiatio...

Dietary carbohydrates raise blood glucose levels, and limiting carbohydrate intake improves glycemia in patients with type 2 diabetes. Low carbohydrate intake (< 25 g) allows the body to utilize fat as its primary fuel. As a consequence of increased fatty acid oxidation, the liver produces ketones to serve as an alternative energy source. β-Hydroxy...

Population-level variation and mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized. We defined prototypical insulin secretion responses to three macronutrients in islets from 140 cadaveric donors, including those with type 2 diabetes. The majority of donors’ islets exhibited the highest insulin r...

Comprehensive molecular and cellular phenotyping of human islets can enable deep mechanistic insights for diabetes research. We established the Human Islet Data Analysis and Sharing (HI-DAS) consortium to advance goals in accessibility, usability, and integration of data from human islets isolated from donors with and without diabetes at the Albert...

Pancreatic β cells exist in low and high insulin gene activity states that are dynamic on a scale of hours to days. Cells with higher Ins2 gene activity have a mature β cell transcriptomic profile but are more fragile. Information remains unknown on the spatial relationship between these β cell states, their proteomic signatures, and the signaling...

Background Substantial weight loss in people living with type 2 diabetes (T2D) can reduce the need for glucose-lowering medications while concurrently lowering glycemia below the diagnostic threshold for the disease. Furthermore, weight-loss interventions have also been demonstrated to improve aspects of underlying T2D pathophysiology related to ec...

Dietary carbohydrates raise blood glucose and limiting carbohydrate intake improves glycemia in patients with T2D. Low carbohydrate intake (< 25 g) allows the body to utilize fat as its primary fuel. As a consequence of increased fatty-acid oxidation, the liver produces ketones to serve as an alternative energy source. Beta-hydroxybutyrate is the m...

The growing number of multi-omics studies demands clear conceptual workflows coupled with easy-to-use software tools to facilitate data analysis and interpretation. This protocol covers three key components involved in multi-omics analysis, including single-omics data analysis, knowledge-driven integration using biological networks and data-driven...

The rising incidence of pancreatic cancer is largely driven by increased prevalence of obesity and type 2 diabetes (T2D). Hyperinsulinemia is a cardinal feature of obesity and T2D, and is associated with increased cancer incidence and mortality. Whether insulin alone plays a causal role in increasing cancer risk by directly affecting the tumor cell...

Qualitative and quantitatively appropriate insulin secretion is essential for optimal control of blood glucose. Beta-cells of the pancreas produce and secrete insulin in response to glucose and non-glucose stimuli including amino acids. In this manuscript, we review the literature on amino acid-stimulated insulin secretion in oral and intravenous i...

The rising pancreatic cancer incidence due to obesity and type 2 diabetes is closely tied to hyperinsulinemia, an independent cancer risk factor. Previous studies demonstrated reducing insulin production suppressed pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in Kras-mutant mice. However, the pathophysiological and molecular m...

Background Obesity increases breast cancer risk and breast cancer-specific mortality, particularly for people with estrogen receptor (ER)-positive tumors. Body mass index (BMI) is used to define obesity, but it may not be the best predictor of breast cancer risk or prognosis on an individual level. Adult weight gain is an independent indicator of b...

Introduction Micronutrients perform a wide range of physiological functions essential for growth and development. However, most people still need to meet the estimated average requirement worldwide. Globally, 2 billion people suffer from micronutrient deficiency, most of which are co-occurring deficiencies in children under age five. Despite decade...

Pancreatic β-cells are critical for systemic glucose homeostasis, and most of them undergo cell death during the pathogenesis of type 1 diabetes. We previously showed that a Na+ channel inhibitor, carbamazepine, could protect β-cells in vitro and in vivo. Here, we confirmed the effects of carbamazepine and other Na+ channel inhibitors on human isle...

Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition. We now define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnos...

Genetic factors affect an individual's risk of developing obesity, but in most cases each genetic variant has a small effect. Discovery of genes that regulate obesity may provide clues about its underlying biological processes and point to new ways the disease can be treated. Pre-clinical animal models facilitate genetic discovery in obesity becaus...

Objective: Pancreatic β cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in β cells, but few studies have examined male-female differences in β cell stress responses and resilience across multiple contexts, including diabetes....

Sequencing the human genome empowers translational medicine, facilitating transcriptome-wide molecular diagnosis, pathway biology, and drug repositioning. Initially, microarrays are used to study the bulk transcriptome; but now short-read RNA sequencing (RNA-seq) predominates. Positioned as a superior technology, that makes the discovery of novel t...

Objectives Pancreatic cancer risk is elevated approximately two-fold in type 1 and type 2 diabetes. Islet amyloid polypeptide (IAPP) is an abundant beta-cell peptide hormone that declines with diabetes progression. IAPP has been reported to act as a tumour-suppressor in p53-deficient cancers capable of regressing tumour volumes. Given the decline o...

Background Lipoprotein lipase (LPL)‐derived fatty acid is a major source of energy for cardiac contraction. Synthesized in cardiomyocytes, LPL requires translocation to the vascular lumen for hydrolysis of lipoprotein triglyceride, an action mediated by endothelial cell (EC) release of heparanase. We determined whether flow‐mediated biophysical for...

Transcriptional and functional cellular specialization has been described for insulin-secreting β-cells of the endocrine pancreas. However, it is not clear whether β-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary...

A central goal of physiological research is the understanding of cell-specific roles of disease-associated genes. Cre-mediated recombineering is the tool of choice for cell type-specific analysis of gene function in pre-clinical models. In the type 1 diabetes research field, multiple lines of NOD mice have been engineered to express Cre recombinase...

Genetic factors affect an individual's risk of developing obesity, but in most cases each genetic variant has a small effect. Discovery of genes that regulate obesity may provide clues about its underlying biological processes and point to new ways the disease can be treated. Pre-clinical animal models facilitate genetic discovery in obesity becaus...

Objective Pancreatic β cells play a key role in glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in β cells, but few studies have examined male-female differences in β cell stress responses and resilience across multiple contexts, including diabetes. Here, we addre...

The rising incidence of pancreatic cancer is largely driven by increased prevalence of obesity and type 2 diabetes (T2D). Hyperinsulinemia is a cardinal feature of obesity and T2D, and is associated with increased cancer incidence and mortality. Genetic reduction of insulin production suppresses formation of pancreatic intraepithelial neoplasia (Pa...

Background Hyperinsulinemia is independently associated with increased risk and mortality of pancreatic cancer. We recently reported that genetically reduced insulin production resulted in ~ 50% suppression of pancreatic intraepithelial neoplasia (PanIN) precancerous lesions in mice. However, only female mice remained normoglycemic, and only the ge...

Insulin receptor (Insr) protein is present at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Here, we use an Ins1 cre knock-in allele to delete Insr specifically in β-cells of both female and male mice. We compare experimental mice to Ins1 cre -containing littermat...

Cre-mediated recombineering is the main tool of choice for cell type-specific analysis of gene function in pre-clinical models. In the type 1 diabetes research field, multiple lines of NOD mice have been generated that express Cre in pancreatic β-cells using insulin promoter fragments, but tissue promiscuity remains a concern. The constitutive Ins1...

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have demonstrated that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome‐wide level. Transcriptomic meta‐analysis i...

Hundreds of millions of people are affected by hyperinsulinaemia, insulin resistance, obesity and the dysglycaemia that mark a common progression from metabolic health to type 2 diabetes. Although the relative contribution of these features and the order in which they appear may differ between individuals, the common clustering and seemingly progre...

Breast cancer survivors treated with tamoxifen and aromatase inhibitors report weight gain and have an elevated risk of type 2 diabetes, especially if they have obesity. Pre-clinical reports are disconnected from patient experiences, but many used high doses of tamoxifen. We investigated the impact of breast cancer endocrine therapies in a pre-clin...

Diabetes therapeutic approaches continue to expand and to be refined. As the field moves toward more intensive insulin- and cell-based therapies, care must be taken to mimic healthy physiological insulin dynamics and avoid hyperinsulinemia, with its deleterious downstream complications.

Insulin resistance contributes to type 2 diabetes and can be driven by hyperinsulinemia. Insulin receptor (INSR) internalization and cell-surface dynamics at rest and during insulin exposure are incompletely understood in muscle cells. Using surfacing labeling and live-cell imaging, we observed robust basal internalization of INSR in C2C12 myoblast...

Breast cancer survivors treated with anti-estrogen therapies report weight gain and have an elevated risk of type 2 diabetes. Here, we show that current tamoxifen use did not influence body mass index but associated with larger breast adipocyte diameter only in women with obesity, suggesting adipose tissue may be targeted by breast cancer therapies...

Objectives Type 1 diabetes is characterized by the autoimmune destruction of insulin-secreting beta cells. Genetic variants upstream at the insulin (INS) locus contribute to ∼10% of type 1 diabetes heritable risk. Previous studies showed an association between rs3842753 C/C genotype and type 1 diabetes susceptibility, but the molecular mechanisms r...

The relative insufficiency of insulin secretion and/or insulin action causes diabetes. However, obesity and type 2 diabetes mellitus can be associated with an absolute increase in circulating insulin, a state known as hyperinsulinemia. Studies are beginning to elucidate the cause-effect relationships between hyperinsulinemia and numerous consequenc...

Hyperinsulinemia is independently associated with increased risk and mortality of pancreatic cancer. We recently reported that a ~50% reduction in pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in mice could be achieved with reduced insulin production. However, only female mice remained normoglycemic and only the gene dosage of...

The endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) helps decide cell survival in diabetes. The alternative eukaryotic initiation factor 2A (EIF2A) has been proposed to mediate EIF2S1-independent translation during cellular stress and viral infection, but its role in cells is unknown. EIF2A abundance is high in human and m...

Type 1 diabetes is characterized by the autoimmune destruction of insulin secreting beta-cells. Genetic variations upstream at the insulin ( INS ) locus contribute to ~10% of type 1 diabetes heritable risk. Multiple studies showed an association between rs3842753 C/C genotype and type 1 diabetes susceptibility. Three small studies have reported an...

Insulin receptor (Insr) protein can be found at higher levels in pancreatic β-cells than in most other cell types, but the consequences of β-cell insulin resistance remain enigmatic. Ins1 cre allele was used to delete Insr specifically in β-cells of both female and male mice which were compared to Ins1 cre -containing littermate controls at multipl...

Insulin receptor (Insr) protein can be found at higher levels in pancreatic β-cells than in most other cell types, but the consequences of β-cell insulin resistance remain enigmatic. Ins1cre allele was used to delete Insr specifically in β-cells of both female and male mice which were compared to Ins1cre-containing littermate controls at multiple a...

The incidence of new onset diabetes after transplant (NODAT) has increased over the past decade, likely due to calcineurin inhibitor-based immunosuppressants, including tacrolimus (TAC) and cyclosporin (CsA). Voclosporin (VCS), a next generation calcineurin inhibitor is reported to cause fewer incidences of NODAT but the reason is unclear. Whilst c...

Breast cancer survivors treated with anti-estrogen therapies report weight gain and have an elevated risk of type 2 diabetes. Here, we show that current tamoxifen use associated with larger breast adipocyte diameter only in women with a BMI >30 kg/m2. To understand the mechanisms behind these clinical findings, we investigated the impact of estroge...

Hyperinsulinemia plays a causal role in adipose expansion. Mice with reduced insulin have increased energy expenditure, but the mechanisms remained unclear. We investigated the effects of genetically reducing insulin production on uncoupling and oxidative mitochondrial proteins in liver, skeletal muscle, white adipose tissue (WAT), and brown adipos...

Evidence has mounted that insulin can be synthesized in various brain regions including the hypothalamus. However, the distribution and functions of insulin-expressing cells in the hypothalamus remain elusive. Herein, we show that in the mouse hypothalamus, the perikarya of insulin-positive neurons are located in the paraventricular nucleus (PVN) a...

In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on β-cell function and organism physiology. Cre-LoxP is a system wherein LoxP sites flanking a gene are recognized by Cre recombinase. Cre transgenic mice are the most prevalent technology used to deliver Cre but many models have caveats of off-target recombi...

Cryptococcus neoformans is the causative agent of cryptococcal meningitis, a disease responsible for ∼15% of all HIV-related deaths. Unfortunately, development of antifungal drugs is challenging because potential targets are conserved between humans and C. neoformans . In this context, we characterized a unique J-domain protein, Mrj1, which lacks o...

Insulin dysregulation independently underlies diabetes and Alzheimer’s Disease (AD) pathology. However, the former has also been shown to be a risk factor for the latter. The ancestral insulin gene (Ins2), but not the pancreas-specific Ins1gene, is transcribed locally within the brain in mice. We confirmed that neuronal expression of Ins2 is most p...

Context The incidence of new onset diabetes after transplant (NODAT) has increased over the past decade, likely due to calcineurin inhibitor-based immunosuppressants, including tacrolimus (TAC) and cyclosporin (CsA). Voclosporin (VCS), a next generation calcineurin inhibitor is reported to cause fewer incidences of NODAT but the reason is unclear....

Background and Aims Esophageal adenocarcinoma (EAC) develops from within Barrett’s esophagus (BE) concomitant with gastro-esophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial-mesenchymal transitions, may contribute to the development of the BE and the eventual migratory escape of metastatic cancer c...

Hyperinsulinemia plays a causal role in adipose tissue expansion. Mice with reduced insulin have increased energy expenditure, but the mechanisms remained unclear. Here we investigated the effects of genetically reducing insulin production on uncoupling and oxidative mitochondrial proteins in liver, skeletal muscle, white adipose tissue (WAT), and...

The incidence of cancers, including pancreatic ductal adenocarcinoma (PDAC), continues to rise alongside the global obesity and diabetes epidemics. This rise suggests that factors modulated by environment or lifestyle play a key role, but the mechanisms by which obesity and type 2 diabetes promote cancer remain unclear. Obesity and type 2 diabetes...

The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administr...

Background: A positive energy balance promotes white adipose tissue (WAT) expansion which is characterized by activation of a repertoire of events including hypoxia, inflammation and extracellular matrix remodelling. The transmembrane glycoprotein CD248 has been implicated in all these processes in different malignant and inflammatory diseases but...

Childhood obesity and early rapid growth increase the risk for type 2 diabetes. Such early overnutrition can be modeled in mice by reducing litter size. We investigated the effects of early overnutrition and increased dietary fat intake on β cell function in Swiss Webster mice. On a moderate-fat diet, early overnutrition accelerated weight gain and...

Hyperinsulinemia is often viewed as a compensatory mechanism for insulin resistance, but recent studies have shown that high levels of insulin may also contribute to insulin resistance. The precise mechanisms by which hyperinsulinemia contributes to insulin resistance remain poorly defined. To understand the direct effects of prolonged exposure to...

Obesity and early-stage type 2 diabetes (T2D) increase the risk for many cancers, including pancreatic ductal adenocarcinoma (PDAC). The mechanisms linking obesity and T2D to cancer have not been established, preventing targeted interventions. Arguments have been made that hyperinsulinemia, hyperglycemia, or inflammation could drive cancer initiati...

Exploring how proliferation and maturation of beta-cells can be impaired after birth will shed light on the origins of various forms of diabetes.

Insulin plays roles in lipid uptake, lipolysis, and lipogenesis, in addition to controlling blood glucose levels. Excessive circulating insulin is associated with adipose tissue expansion and obesity, yet a causal role for hyperinsulinemia in the development of mammalian obesity has proven controversial, with many researchers suggesting it as a con...

The PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) directly dephosphorylates and inactivates Akt2, the Akt isozyme that is a key transducer of insulin signaling. Elevated levels of PHLPP1 protein have been associated with obesity and insulin resistance in humans, suggesting PHLPP1 may be a novel therapeutic target to combat insulin re...

Pancreatic beta-cells are selectively destroyed by the host immune system in type 1 diabetes. Thus, drugs that preserve beta-cell mass and/or function have the potential to prevent or slow the progression of this disease. We recently reported that the use-dependent sodium channel blocker, carbamazepine, protects beta-cells from inflammatory cytokin...

Caloric restriction (CR) is the only environmental intervention with robust evidence that it extends lifespan and delays the symptoms of aging, but its mechanisms are incompletely understood. Based on the prolonged longevity of knockout models, it was hypothesized that the insulin-IGF pathway could be a target for developing a CR mimic. This study...

Excess circulating insulin is associated with obesity in humans and in animal models. However, the physiologic causality of hyperinsulinemia in adult obesity has rightfully been questioned because of the absence of clear evidence that weight loss can be induced by acutely reversing diet-induced hyperinsulinemia. Herein, we describe the consequences...

The causal relationships between insulin levels, insulin resistance, and longevity are not fully elucidated. Genetic downregulation of insulin/insulin-like growth factor 1 (Igf1) signaling components can extend invertebrate and mammalian lifespan, but insulin resistance, a natural form of decreased insulin signaling, is associated with greater risk...

Insulin modulates the biochemical pathways controlling lipid uptake, lipolysis and lipogenesis at multiple levels. Elevated insulin levels are associated with obesity, and conversely, dietary and pharmacological manipulations that reduce insulin have occasionally been reported to cause weight loss. However, the causal role of insulin hypersecretion...

Heparanase, a protein with enzymatic and nonenzymatic properties, contributes toward disease progression and prevention. In the current study, a fortuitous observation in transgenic mice globally overexpressing heparanase (hep-tg) was the discovery of improved glucose homeostasis. We examined the mechanisms that contribute toward this improved gluc...

Kaplan-Meir survival curve of male and female Lepob/ob or Lepwt/wt mice lacking 1, 2 or 3 insulin alleles monitored up to 14 weeks of age.

VO2 (A) and VCO2 (B) were measured by indirect calorimetry in male Ins1+/+;Ins2−/−;Lepob/ob and Ins1+/+;Ins2+/−;Lepob/ob mice at 5–6 weeks of age. Data are presented as unadjusted values. Heat (C) was also measured by indirect calorimetry in male Ins1+/+;Ins2−/−;Lepob/ob and Ins1+/+;Ins2+/−;Lepob/ob mice at 5–6 weeks of age and presented as adjuste...

Breeding scheme to generate Lepob/ob and Lepwt/wt mice lacking 1, 2 or 3 insulin alleles. A mini osmotic pump releasing 0.5 μg leptin/day was implanted in a male Lepob/ob mice to enable breeding with female Ins1+/−;Ins2−/−;Lepwt/wt mice for the first of three crosses used to generate experimental groups. Lepob/ob mice and Lepwt/wt littermates lacki...

Total fat mass (A-male, C-female) and lean mass (B-male, D-female) were assessed in Lepob/ob and Lepwt/wt mice at 5 weeks of age using dual X-ray absorptiometry. Values are presented as mean ± SEM. A One Way ANOVA was used to assess significance. *P < 0.05 Ins1+/+;Ins2+/−;Lepob/ob vs. all other groups; †P < 0.05 Ins1+/+;Ins2+/−;Lepob/ob vs. Ins1+/+...

Cardiac ryanodine receptor (Ryr2) Ca2+ release channels and cellular metabolism are both disrupted in heart disease. Recently, we demonstrated that total loss of Ryr2 leads to cardiomyocyte contractile dysfunction, arrhythmia, and reduced heart rate. Acute total Ryr2 ablation also impaired metabolism, but it was not clear whether this was a cause o...

Objective Hyperinsulinemia is commonly associated with obesity. Mice deficient in the adipose-derived hormone leptin (Lepob/ob) develop hyperinsulinemia prior to onset of obesity and glucose intolerance. Whether the excess of circulating insulin is a major contributor to obesity and impaired glucose homeostasis in Lepob/ob mice is unclear. It has b...

Multiple signaling pathways mediate the actions of metabolic hormones to control glucose homeostasis, but the proteins that coordinate such networks are poorly understood. We previously identified the molecular scaffold protein, 14-3-3ζ, as a critical regulator of in vitro β-cell survival and adipogenesis, but its metabolic roles in glucose homeost...

Anti-adiposity effects of caloric restriction (CR) are associated with reduced insulin/insulin-like growth factor 1 signalling, but it is unclear whether the effects of CR would be additive to genetically reducing circulating insulin. To address this question, we examined female Ins1(+/-):Ins2(-/-) mice and Ins1(+/+):Ins2(-/-) littermate controls o...

Insulin is an essential hormone with key roles in energy homeostasis and body composition. Mice and rats, unlike other mammals, have two insulin genes: the rodent-specific Ins1 gene and the ancestral Ins2 gene. The relationships between insulin gene dosage and obesity has previously been explored in male and female Ins2-/- mice with full or reduced...

Worldwide efforts are underway to replace or repair lost or dysfunctional pancreatic beta-cells to cure diabetes. However, it is unclear what the final product of these efforts should be, as beta-cells are thought to be heterogeneous. To enable the analysis of beta-cell heterogeneity in an unbiased and quantitative way, we developed model-free and...

Movie S3: Time lapse TIRF imaging of a Cav1 MIN6 cell expressing InsRA-TagRFP and Cav1-mTFP recorded at 25 mM glucose.

Movie S1: Time lapse TIRF imaging of HEK-293T cells expressing either InsRA-TagRFP (inter-domain tagged) or InsRA-C-eGFP (C-terminal label) constructs recorded at 25 mM glucose.

Movie S2: Live cell imaging of a MIN6 cell co-transfected with IGF1R-TagGFP2 (left) and InsRA-TagRFP (middle) (both interdomain tagged). TagGFP2 and TagRFP was inserted between two domains at bp 2809/aa 937, and bp 2812/aa 938, respectively.

Movie S4: Time lapse TIRF imaging of Cav1 MIN6 cell lines expressing various Cav1 mutants and inter-domain tagged InsRA-lum-eGFP constructs recorded at 25 mM glucose.