Jeffery S. Tessem

The development of diabetes in E2f1/E2f2 mutant mice reveals important roles for bone marrow-derived cells in preventing islet cell loss

Our studies of mice deficient for the E2F1 and E2F2 transcription factors have revealed essential roles for these proteins in the cell cycle control of pancreatic exocrine cells and the regulation of pancreatic beta cell maintenance. Pancreatic exocrine cells in E2f1/E2f2 mutant mice become increasingly polyploid with age, coinciding with severe exocrine atrophy. Furthermore, mice deficient for both E2F1 and E2F2 develop nonautoimmune, insulin-dependent diabetes with high penetrance. Surprisingly, transplantation of wild-type bone marrow can prevent or rescue diabetes in E2f1-/-E2f2-/- mice. We hypothesize that exocrine degeneration results in a destructive environment for beta cells, which can be alleviated by restoration of the hematopoietic system that is also defective in E2f1-/-E2f2-/- mice. The demonstration that beta cell maintenance under conditions of stress is influenced by bone marrow-derived cells may provide important insight into the design of therapies to boost islet mass and function in diabetic patients.

Critical roles for macrophages in islet angiogenesis and maintenance during pancreatic degeneration

OBJECTIVE— Chronic pancreatitis, characterized by pancreatic exocrine tissue destruction with initial maintenance of islets, eventually leads to insulin-dependent diabetes in most patients. Mice deficient for the transcription factors E2F1 and E2F2 suffer from a chronic pancreatitis-like syndrome and become diabetic. Surprisingly, onset of diabetes can be prevented through bone marrow transplantation. The goal of the described studies was to determine the hematopoietic cell type responsible for maintaining islets and the associated mechanism of this protection. RESEARCH DESIGN AND METHODS— Mouse models of acute and chronic pancreatitis, together with mice genetically deficient for macrophage production, were used to determine roles for macrophages in islet angiogenesis and maintenance. RESULTS— We demonstrate that macrophages are essential for preventing endocrine cell loss and diabetes. Macrophages expressing matrix metalloproteinase-9 migrate to the deteriorating pancreas. E2f1/E2f2 mutant mice transplanted with wild-type, but not macrophage-deficient colony stimulating factor 1 receptor mutant (Csf1r−/−), bone marrow exhibit increased angiogenesis and proliferation within islets, coinciding with increased islet mass. A similar macrophage dependency for islet and islet vasculature maintenance is observed during caerulein-induced pancreatitis. CONCLUSIONS— These findings demonstrate that macrophages promote islet angiogenesis and protect against islet loss during exocrine degeneration, could explain why most patients with chronic pancreatitis develop diabetes, and suggest an avenue for preventing pancreatitis-associated diabetes.

Nkx6.1 regulates islet β-cell proliferation via Nr4a1 and Nr4a3 nuclear receptors

Significance Loss of pancreatic islet β cells occurs in both major forms of diabetes, and strategies for restoring β cells are needed. The homeobox transcription factor NK6 homeobox 1 (Nkx6.1) activates β-cell proliferation and insulin secretion when overexpressed in pancreatic islets, but the molecular pathway involved in the proliferative response is unknown. We show that Nkx6.1 induces expression of orphan nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3), which stimulate proliferation via two mechanisms: (i) increased expression of the cell cycle inducers E2F transcription factor 1 and cyclin E1; and (ii) induction of anaphase-promoting complex elements, and degradation of the cell cycle inhibitor p21. These studies reveal a new bipartite pathway for activation of β-cell proliferation that could guide development of therapeutic strategies for diabetes. Loss of functional β-cell mass is a hallmark of type 1 and type 2 diabetes, and methods for restoring these cells are needed. We have previously reported that overexpression of the homeodomain transcription factor NK6 homeobox 1 (Nkx6.1) in rat pancreatic islets induces β-cell proliferation and enhances glucose-stimulated insulin secretion, but the pathway by which Nkx6.1 activates β-cell expansion has not been defined. Here, we demonstrate that Nkx6.1 induces expression of the nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3) orphan nuclear receptors, and that these factors are both necessary and sufficient for Nkx6.1-mediated β-cell proliferation. Consistent with this finding, global knockout of Nr4a1 results in a decrease in β-cell area in neonatal and young mice. Overexpression of Nkx6.1 and the Nr4a receptors results in increased expression of key cell cycle inducers E2F transcription factor 1 and cyclin E1. Furthermore, Nkx6.1 and Nr4a receptors induce components of the anaphase-promoting complex, including ubiquitin-conjugating enzyme E2C, resulting in degradation of the cell cycle inhibitor p21. These studies identify a unique bipartite pathway for activation of β-cell proliferation, suggesting several unique targets for expansion of functional β-cell mass.

Aurora Kinase A is critical for the Nkx6.1 mediated β-cell proliferation pathway

Type 1 and type 2 diabetes are ultimately characterized by depleted β-cell mass. Characterization of the molecular pathways that control β-cell proliferation could be harnessed to restore these cells. The homeobox β-cell transcription factor Nkx6.1 induces β-cell proliferation by activating the orphan nuclear receptors Nr4a1 and Nr4a3. Here, we demonstrate that Nkx6.1 localizes to the promoter of the mitotic kinase AURKA (Aurora Kinase A) and induces its expression. Adenovirus mediated overexpression of AURKA is sufficient to induce proliferation in primary rat islets while maintaining glucose stimulated insulin secretion. Furthermore, AURKA is necessary for Nkx6.1 mediated β-cell proliferation as demonstrated by shRNA mediated knock down and pharmacological inhibition of AURKA kinase activity. AURKA preferentially induces DNA replication in β-cells as measured by BrdU incorporation, and enhances the rate of histone H3 phosphorylation in primary β-cells, demonstrating that AURKA induces the replicative and mitotic cell cycle phases in rat β-cells. Finally, overexpression of AURKA results in phosphorylation of the cell cycle regulator p53, which targets p53 for degradation and permits cell cycle progression. These studies define a pathway by which AURKA upregulation by Nkx6.1 results in phosphorylation and degradation of p53, thus removing a key inhibitory factor and permitting engagement of the β-cell proliferation pathway.

Cdk5r1 Overexpression Induces Primary β-Cell Proliferation

Decreased β-cell mass is a hallmark of type 1 and type 2 diabetes. Islet transplantation as a method of diabetes therapy is hampered by the paucity of transplant ready islets. Understanding the pathways controlling islet proliferation may be used to increase functional β-cell mass through transplantation or by enhanced growth of endogenous β-cells. We have shown that the transcription factor Nkx6.1 induces β-cell proliferation by upregulating the orphan nuclear hormone receptors Nr4a1 and Nr4a3. Using expression analysis to define Nkx6.1-independent mechanisms by which Nr4a1 and Nr4a3 induce β-cell proliferation, we demonstrated that cyclin-dependent kinase 5 regulatory subunit 1 (Cdk5r1) is upregulated by Nr4a1 and Nr4a3 but not by Nkx6.1. Overexpression of Cdk5r1 is sufficient to induce primary rat β-cell proliferation while maintaining glucose stimulated insulin secretion. Overexpression of Cdk5r1 in β-cells confers protection against apoptosis induced by etoposide and thapsigargin, but not camptothecin. The Cdk5 kinase complex inhibitor roscovitine blocks islet proliferation, suggesting that Cdk5r1 mediated β-cell proliferation is a kinase dependent event. Overexpression of Cdk5r1 results in pRb phosphorylation, which is inhibited by roscovitine treatment. These data demonstrate that activation of the Cdk5 kinase complex is sufficient to induce β-cell proliferation while maintaining glucose stimulated insulin secretion.

β-Cell deletion of Nr4a1 and Nr4a3 nuclear receptors impedes mitochondrial respiration and insulin secretion

β-Cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle, and adipose. Previously, we have demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic β-cells. In this study, we examined whether Nr4a expression impacts pancreatic β-cell mitochondrial function. Here, we show that β-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in β-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16 mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose-stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP and ultimately inhibits glucose-stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for β-cell mitochondrial function and insulin secretion.

Monomeric cocoa catechins enhance β-cell function by increasing mitochondrial respiration

A hallmark of type 2 diabetes (T2D) is β-cell dysfunction and the eventual loss of functional β-cell mass. Therefore, mechanisms that improve or preserve β-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on β-cell function. INS-1 832/13-derived β-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract and with oligomeric or polymeric procyanidin-rich fraction demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in β-cell fuel utilization. Mitochondrial complex III, IV and V components are up-regulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with nuclear factor, erythroid 2 like 2 (Nrf2) nuclear localization and expression of Nrf2 target genes including nuclear respiratory factor 1 (Nrf1) and GA binding protein transcription factor alpha subunit (GABPA), essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and up-regulation of genes critical for mitochondrial respiration, glucose-stimulated insulin secretion and ultimately improved β-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients.

Nkx6.1-mediated insulin secretion and β-cell proliferation is dependent on upregulation of c-Fos.

Understanding the molecular pathways that enhance β‐cell proliferation, survival, and insulin secretion may be useful to improve treatments for diabetes. Nkx6.1 induces proliferation through the Nr4a nuclear receptors, and improves insulin secretion and survival through the peptide hormone VGF. Here we demonstrate that Nkx6.1‐mediated upregulation of Nr4a1, Nr4a3, and VGF is dependent on c‐Fos expression. c‐Fos overexpression results in activation of Nkx6.1 responsive genes and increases β‐cell proliferation, insulin secretion, and cellular survival. c‐Fos knockdown impedes Nkx6.1‐mediated β‐cell proliferation and insulin secretion. These data demonstrate that c‐Fos is critical for Nkx6.1‐mediated expansion of functional β‐cell mass.

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

Common gut microbial metabolites of dietary flavonoids exert potent protective activities in β-cells and skeletal muscle cells

Flavonoids are dietary compounds with potential anti-diabetes activities. Many flavonoids have poor bioavailability and thus low circulating concentrations. Unabsorbed flavonoids are metabolized by the gut microbiota to smaller metabolites, which are more bioavailable than their precursors. The activities of these metabolites may be partly responsible for associations between flavonoids and health. However, these activities remain poorly understood. We investigated bioactivities of flavonoid microbial metabolites [hippuric acid (HA), homovanillic acid (HVA), and 5-phenylvaleric acid (5PVA)] in primary skeletal muscle and β-cells compared to a native flavonoid [(-)-epicatechin, EC]. In muscle, EC was the most potent stimulator of glucose oxidation, while 5PVA and HA simulated glucose metabolism at 25 μM, and all compounds preserved mitochondrial function after insult. However, EC and the metabolites did not uncouple mitochonndrial respiration, with the exception of 5PVA at10 μM. In β-cells, all metabolites more potently enhanced glucose-stimulated insulin secretion (GSIS) compared to EC. Unlike EC, the metabolites appear to enhance GSIS without enhancing β-cell mitochondrial respiration or increasing expression of mitochondrial electron transport chain components, and with varying effects on β-cell insulin content. The present results demonstrate the activities of flavonoid microbial metabolites for preservation of β-cell function and glucose utilization. Additionally, our data suggest that metabolites and native compounds may act by distinct mechanisms, suggesting complementary and synergistic activities in vivo which warrant further investigation. This raises the intriguing prospect that bioavailability of native dietary flavonoids may not be as critical of a limiting factor to bioactivity as previously thought.