Draft:Ernesto Bernal-Mizrachi

One Sentence Summary:

Ernesto Bernal-Mizrachi is a physician-scientist at the University of Miami Leonard M. Miller School of Medicine, whose research is focused on the factors that promote pancreatic β-cell proliferation [1-2].

Early Life and Education:

Bernal-Mizrachi is originally from Colombia, and completed his doctorate in medicine (MD) from the University of Valle in the city of Cali, Valle del Cuenca, Colombia in 1989 [2]. He continued his medical education with a three-year residency in internal medicine in Miami at the Leonard M. Miller School of Medicine from 1993-1996, and a subsequent three-year fellowship in endocrinology and metabolism at the Washington University School of Medicine in St. Louis from 1996-1999 [2]. During this time, Bernal-Mizrachi was mentored by one of the leading figures in the genetics behind diabetes, M. Alan Permutt, who became a professor at the Washington University School of Medicine in St. Louis in 1985 and directed the Diabetes Research and Training Center during Bernal-Mizrachi's fellowship years [3-4]. That focus on diabetes stemmed from Bernal-Mizrachi's passion to further elucidate the molecular biology behind the chronic ailment and assist in treatment and identification efforts for those in less-developed countries [2].

Research and Career:

Breakthrough Linkage between Genetics and Diabetes Pathogenesis

One of Bernal-Mizrachi's landmark successes came at the beginnings of his research career. In 1998, Bernal-Mizrachi, alongside a team of scientists under the mentorship of Permutt, published their findings in Nature (journal) regarding a mutation that led to Wolfram syndrome (WFS), or childhood-onset insulin-dependent diabetes [4]. The study used linkage analysis in order to characterize WFS amongst 3 Japanese and 2 European families. D4S500 and D4S431 were located on the same yeast artificial chromosome (YAC) as previously identified in the Stanford Chromosome 4 YAC mapping project. Using human P1 and bacterial artificial chromosome (BAC) clones, a contiguous fragment was constructed encompassing the WFS critical region to appropriately map the region. A method called exon trapping, which is now replaced by computational-based cDNA sequencing, was used to determine potential exons in unknown DNA fragments through insertion into a splicing vector [4]. Large-scale genomic sequencing enabled for characterization of 180 kb, which was eventually narrowed to an ORF of 2672 nt (890 aa) via PCR amplification and corresponding northern blotting with a poly A+ RNA probe. This gene was named WFS1, and was compared in patients with and without Wolfram syndrome [4]. The primary conclusion was that a homozygous frameshift mutation led to the creation of an elongated 937 amino acid product, among a host of other polymorphisms [4]. WFS1, as Bernal-Mizrachi helped determine, was crucial for appropriate pancreatic β cell function, and an accumulation of mutated WFS1 isoforms induced early-onset insulin deficient diabetes mellitus [4].

Akt Pathway Signaling and Effect on β cell Mass

In 2001, Bernal-Mizrachi turned his attention towards specific signaling pathways and their effects on β cell function, publishing his work in the Journal of Clinical Investigation [5]. His goal was to study the in-vivo effects of the phosphoinositide 3-kinase (PI3K) Akt/PKB pathway [5]. Akt is a serine-threonine kinase that phosphorylates other targets that lead to cell growth and proliferation, and is activated by PI3K. Bernal-Mizrachi used a transgenic mouse model, in which a constitutively active Ak1 gene, which lacked a PH domain that otherwise mediates interaction with PI3K, was fused with a hemagglutinin tag (HA) for checking expression levels [5]. An pre-prepared RIP/β-globin expression vector was created, and the AK1 construct was inserted at an EcoRI restriction site. This chimeric gene was then injected into embryonic stem cells in fertilized mice eggs [5].

To approximate β-cell mass in control and transgenic mice, the pancreas was excised and immunohistochemistry detected insulin-positive cells in tissue sections [5]. A guinea pig anti-human insulin antibody was hybridized to insulin, and 3-amino-9-ethyl carbazole (AEC) was the conjugated chromophore that could be detected via fluorescence microscopy. Image analysis software from BioQuant allowed for a determination of the ratio of β cell area to total area, which was multiplied by pancreatic gross weight to quantify β cell mass. Glucose levels were also measured after injection following a 6-hour fast using a rat insulin ELISA kit [5]. Results indicated that β cell mass in transgenic mice increased eight to nine-fold, suggesting Akt overexpression drives β cell neogenesis pathways [5]. Additionally, the fasting state provoked a 1.7 fold increase in plasma insulin, leading to enhanced glucose tolerance. The findings obtained helped solidify Bernal-Mizrachi's focus on the Akt pathway, which he continued to study throughout his career.

Subsequent Significant Discoveries (2002 - Present)

Building upon his prior work with the Akt pathway, Bernal-Mizrachi took a closer look at the dysregulation in insulin secretion that resulted from reduced pathway activation. The negative control, or kinase-dead Akt1, was inserted into a RIP-I/β-globin expression vector, and then injected into fertilized mice eggs [6]. This transgene was confirmed to be expressed in β cells using immunofluorescence staining and Western blot analysis with anti-HA (hemagglutinin) and anti-insulin Ab. Using an in-vitro kinase assay, the phosphorylation of two proteins (S6K and Foxo1) could be observed, and kinase dead Akt lysates, as expected, had significantly lower rates of phosphorylation. His key finding was that abnormal patterns of insulin secretion resulted from Akt dysregulation [6]. A series of islet perfusion experiments were performed, where groups of islets were bathed in various glucose concentrations (2 mM basal, 20 mM). Upon this higher glucose concentration, reductions in insulin secretion were observed, even when treated with a Ca2+ ionophore ionomycin that increases Ca2+ intracellular concentration. The influx of Ca2+ forms part of a cascade that triggers insulin secretion. Therefore, the role of the Akt pathway was concluded to be related to the exocytosis mechanism of insulin [6].

Given the role of Ca2+ in insulin release, a primary target merited further analysis: calcineurin [7]. Calcineurin serves as a key biological regulator of intracellular responses, and is another serine-threonine phosphatase that is dependent on Ca2+ influx [7]. As a result, Bernal-Mizrachi designed a study to explore the role of calcineurin in insulin response. A transgenic mice model was again used, with a constitutively active calcineurin protein lacking its regulatory domain. Site-directed mutagenesis was used to introduce a stop codon, and a similar RIP-I/β-globin expression vector was used to generate copies of the chimeric gene [7]. In 8-12 week old mice, glucose levels remained higher and insulin levels were lower in the mutant mice compared to the WT, from intraperitoneal testing of the tail vein. Insulin levels were also analyzed using cell culture methods in 2 mM glucose for 1 hour, and were lower in the calcineurin mutants [7]. Staining against Ki-67 (protein) helped identify the decreases in mutant β-cell mass, revealing increased rates of apoptosis. The primary conclusions of this study, from analyzing glucose and insulin levels, was that constant calcineurin activation was detrimental to insulin production and β-islet morphology and function [7]. Overactivation, leading to persistent depolarization and Ca2+ influx, was linked to higher apoptosis rates and reductions in β-cell mass, a potentially relevant underlying mechanism for the development of diabetes [7].

Bernal-Mizrachi expanded upon prior work of the Akt signaling pathway to look at one of its key targets for phosphorylation: glycogen synthase kinase-3 [8]. GSK3 is a serine-threonine kinase inhibited by Akt and is important for large-scale cellular responses such as proliferation and apoptosis [8]. The goal was to examine if GSK3β affected β-islet cell mass and insulin response. A transgene was created using a rat insulin-1 promoter and human GSK3β(S9A) cDNA, fused in a pBS-KS vector, and injected into fertilized mice eggs to create recombinant chimeric lines [8]. The line had a constitutively active GSK3β using a serine to alanine mutant, and an HA tag was used to verify the mutant gene's presence via Western blotting. Levels were 2-3 fold higher of GSK3β, which is phosphorylated to inactivation in the traditional insulin receptor mediated response pathway [8]. Intraperitoneal glucose testing was performed by sampling blood plasma using the tail vein, and insulin levels were lower in the mutant mice at the age of 5 months compared to WT individuals. This diminished glucose tolerance and insulin level was a result of lower β-cell mass and proliferation rather than GSK3β's direct effect on insulin synthesis [8]. Additionally, GSK3β was observed to inhibit the production of PDX1, essential for β-cell maturation, as treatment with lithium, a GSK3 inhibitor, conferred higher PDX1 levels. Inhibiting PDX1, therefore, was highlighted by Bernal-Mizrachi and his team to be the mechanism of GSK3β to reduce β-cell growth proliferation and promote apoptosis [8]. Therefore, this molecular target led to lower insulin levels by affecting the entire cells rather than the insulin production pathway itself [8].

More recently, Bernal-Mizrachi worked specifically with MTORC1, and how its deregulation can also lead to the development of diabetes. MTORC1 modulates the EIF4E binding proteins, which are eukaryotic translational initiation factors [9]. Thus, impairing its appropriate functioning could lead to autophagy and problems with cell proliferation. A transgenic mice line with a deletion of the raptor element led to the creation of non-functional MTORC1 units [9]. A similar mechanism of a homozygous deletion of the locus of interest, in this case that of the raptor element, was carried out. The study used in-vivo methods of determining blood plasma glucose and insulin levels from the tail vein as metrics for the severity of diabetes [9]. Moving to the key results, β-cell mass was reduced by over 40% compared to the WT after just 30 days, yet staining for the pancreatic β-cell differentiation factor PDX1 did not indicate substantial differences [9]. ELISA was used to measure insulin content, whereas the TUNEL assay was used to determine apoptosis. The aforementioned findings suggested that MTORC1 played a crucial role in β-cell mass maintenance, but not in cell maturation as did GSK3 [8-9]. A new mouse line with tamoxifen inducible raptor deletion led to difficulties in glucose-stimulated insulin secretion following TMX administration. Apoptotic rates increased whereas β-cell size decreased [9]. One interesting result was that when performing immunostaining for proinsulin compared to insulin, the raptor-knockout mouse line maintained high levels of proinsulin. For that reason, MTORC1 and its associated downstream targets were concluded by Bernal-Mizrachi to likely play a significant role in this processing step [9]. Flow cytometry enabled a determination of insulin content per cell using existent fluorescence signals, which could then be related to proinsulin levels. The key conclusion was that through eIF4E, MTORC1 regulates the translation of specific proteins responsible for the conversion of proinsulin to insulin [9].

Ultimately, Bernal-Mizrachi's work as a whole has shed light on the unique molecular pathways involved in glucose-intolerance. Type II Diabetes is a devastating condition, and better understanding the role of genes and proteins in pancreatic β-islet cells will offer novel therapeutic approaches.

Primary Honors and Awards

Bernal-Mizrachi is a member of the American Society for Clinical Investigation (ASCI) since 2010, and won a Junior Faculty and Career Development Award from the American Diabetes Association [2, 10]. He also is a recipient of multiple NIH grants as well as the Veterans Affairs Biomedical Laboratory R&D Senior Clinician Scientist Investigator Award in 2022 [10].

Furthermore, Bernal-Mizrachi is also board-certified in internal medicine and endocrinology [2]. In addition to research, he works as a physician at the Miami Veterans Administration Hospital.

Current Lab Focus

After spending decades elucidating the PI3K/AKT/mTOR pathway-dependent induction of β cell proliferation and mass, Bernal-Mizrachi has expanded his focus towards the broader impacts of type 2 diabetes treatment [2]. He is also interested in how to mitigate cell death with β-cell transplantation and has worked to develop transgenic mice models for transplantation [11]. The ultimate goal of his lab, which complements his clinical practice, is to further understanding of pancreatic cell specialization and probe novel remedies (pharmacologic and transplant-based) to help eliminate the need for standard medicine/insulin-based therapies [1-2, 11].

References:

[1] Our Research. Welcome. Retrieved May 11, 2024, from https://betacellsignaling.com/new-page-4

[2] Ernesto BernalMizrachi MD. Retrieved May 11, 2024, from https://med.miami.edu/faculty/ernesto-bernalmizrachi-md

[3] M. Alan Permutt, MD. (2020, October 26). Division of Endocrinology, Metabolism & Lipid Research. https://endocrinology.wustl.edu/about/our-history/m-alan-permutt-md/

[4] Inoue, H., Tanizawa, Y., Wasson, J., Behn, P., Kalidas, K., Bernal-Mizrachi, E., et al. (1998) A gene encoding a transmembrane protein is mutated in patients with diabetes mellitus and optic atrophy (Wolfram syndrome). Nat Genet, Nature Publishing Group 20, 143–148 https://doi.org/10.1038/2441

[5] Bernal-Mizrachi, E., Wen, W., Stahlhut, S., Welling, C. M. and Permutt, M. A. (2001) Islet β cell expression of constitutively active Akt1/PKBα induces striking hypertrophy, hyperplasia, and hyperinsulinemia. J Clin Invest, American Society for Clinical Investigation 108, 1631–1638 https://doi.org/10.1172/JCI13785

[6] Bernal-Mizrachi, E., Fatrai, S., Johnson, J. D., Ohsugi, M., Otani, K., Han, Z., et al. (2004) Defective insulin secretion and increased susceptibility to experimental diabetes are induced by reduced Akt activity in pancreatic islet β cells. J Clin Invest, American Society for Clinical Investigation 114, 928–936 https://doi.org/10.1172/JCI20016

[7] Bernal-Mizrachi E, Cras-Méneur C, Ye BR, Johnson JD, Permutt MA (2010) Transgenic Overexpression of Active Calcineurin in β-Cells Results in Decreased β-Cell Mass and Hyperglycemia. PLoS ONE 5(8): e11969. https://doi.org/10.1371/journal.pone.0011969

[8] Liu, Z., Tanabe, K., Bernal-Mizrachi, E. and Permutt, M. A. (2008) Mice with beta cell overexpression of glycogen synthase kinase-3β have reduced beta cell mass and proliferation. Diabetologia 51, 623–631 https://doi.org/10.1007/s00125-007-0914-7

[9] Blandino-Rosano, M., Barbaresso, R., Jimenez-Palomares, M., Bozadjieva, N., Werneck-de-Castro, J. P., Hatanaka, M., et al. (2017) Loss of mTORC1 signalling impairs β-cell homeostasis and insulin processing. Nat Commun, Nature Publishing Group 8, 16014 https://doi.org/10.1038/ncomms16014

[10] Ernesto Bernal Mizrachi. DRIF. Retrieved May 11, 2024, from https://diabetesresearch.org/ernesto-bernal-mizrachi/

[11] Ernesto Bernal-Mizrachi—Laboratory | Sylvester Comprehensive Cancer Center. Retrieved May 11, 2024, from https://umiamihealth.org/sylvester-comprehensive-cancer-center/research/labs/bernal-mizrachi-lab