A major obstacle to successful islet transplantation for both type 1 and 2 diabetes is an inadequate supply of insulin-producing tissue. This need for transplantable human islets has stimulated efforts to expand existing pancreatic islets and͞or grow new ones. To test the hypothesis that human adult duct tissue could be expanded and differentiated in vitro to form islet cells, digested pancreatic tissue that is normally discarded from eight human islet isolations

Differentiated pancreatic ␤ cells are unique in their ability to secrete insulin in response to a rise in plasma glucose. We have proposed that the unique constellation of genes they express may be lost in diabetes due to the deleterious effect of chronic hyperglycemia. To test this hypothesis, Sprague-Dawley rats were submitted to a 85-95% pancreatectomy or sham pancreatectomy. One week later, the animals developed mild to severe chronic hyperglycemia that was stable for the next 3 weeks, without significant alteration of plasma nonesterified fatty acid levels. Expression of many genes important for glucose-induced insulin release decreased progressively with increasing hyperglycemia, in parallel with a reduction of several islet transcription factors involved in ␤ cell development and differentiation. In contrast, genes barely expressed in sham islets (lactate dehydrogenase A and hexokinase I) were markedly increased, in parallel with an increase in the transcription factor c-Myc, a potent stimulator of cell growth. These abnormalities were accompanied by ␤ cell hypertrophy. Changes in gene expression were fully developed 2 weeks after pancreatectomy. Correction of blood glucose by phlorizin for the next 2 weeks normalized islet gene expression and ␤ cell volume without affecting plasma nonesterified fatty acid levels, strongly suggesting that hyperglycemia triggers these abnormalities. In conclusion, chronic hyperglycemia leads to ␤ cell hypertrophy and loss of ␤ cell differentiation that is correlated with changes in c-Myc and other key transcription factors. A similar change in ␤ cell differentiation could contribute to the profound derangement of insulin secretion in human diabetes.Pancreatic ␤ cells are highly specialized cells that secrete insulin in response to a variety of stimuli, the most important being glucose (1, 2). Their correct function is dependent on the expression of a unique set of genes that allow ␤ cells to respond to even small increases in plasma glucose levels by releasing appropriate amounts of insulin into the circulation (3). Type 2 diabetes is characterized by the combination of insulin resistance and profound alteration in glucose-stimulated insulin secretion (4). The latter can be ascribed, at least in part, to the deleterious effect of even mild chronic hyperglycemia and elevated plasma nonesterified fatty acids (NEFA) 1 on pancreatic ␤ cell function, a process often referred to as "gluco-lipotoxicity" (5, 6). In islets isolated from animal models of diabetes, several defects have been identified at the level of gene expression and/or enzymatic activity, but none by itself can entirely account for the ␤ cell defect characteristic of type 2 diabetes (5,7,8).Recently, the study of the transcriptional regulation of the insulin gene has led to the identification of several ␤ cell/islet transcription factors that are important for the development of the endocrine pancreas, the tissue-specific expression of key ␤ cell genes and maintenance of ␤ cell differentiation (9, 10). In islets ...

Doxorubicin (Adriamycin) is an anthracycline chemotherapy agent effective in treating a wide range of malignancies with a well–established dose–response cardiotoxic side effect that can lead to heart failure. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient–specific human induced pluripotent stem cell–derived cardiomyocytes (hiPSC–CMs) can recapitulate individual patients’ predilection to DIC at the single cell level. hiPSC–CMs derived from breast cancer patients who suffered clinical DIC are consistently more sensitive to doxorubicin toxicity, demonstrating decreased cell viability, mitochondrial and metabolic function, calcium handling, and antioxidant pathway activity, along with increased reactive oxygen species (ROS) production compared to hiPSC–CMs from patients who did not experience DIC. Together, our data indicate that hiPSC–CMs are a suitable platform for identifying and verifying the genetic basis and molecular mechanisms of DIC.

The regenerative process in the pancreas is of particular interest because diabetes results from an inadequate number of insulinproducing beta cells and pancreatic cancer may arise from the uncontrolled growth of progenitor/stem cells. Continued and substantial growth of islet tissue occurs after birth in rodents and humans, with additional compensatory growth in response to increased demand. In rodents there is clear evidence of pancreatic regeneration after some types of injury, with proliferation of preexisting differentiated cell types accounting for some replacement. Additionally, neogenesis or the budding of new islet cells from pancreatic ducts has been reported, but the existence and identity of a progenitor cell have been debated. We hypothesized that the progenitor cells are duct epithelial cells that after replication undergo a regression to a less differentiated state and then can form new endocrine and exocrine pancreas. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, we generated transgenic mice expressing human carbonic anhydrase II (CAII) promoter: Cre recombinase (Cre) or inducible CreER TM to cross with ROSA26 loxP-Stop-loxP LacZ reporter mice. We show that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor of all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes.diabetes ͉ islets of Langerhans ͉ lineage tracing

Inadequate ␤-cell function is an essential component of all forms of diabetes. The most obvious problem is a failure to maintain sufficient ␤-cell mass and function to cope with whatever insulin resistance is present. The most striking functional defect is a loss of acute glucose-induced insulin secretion (GIIS). This review discusses the ways in which ␤-cells successfully adapt to increased demand and then decompensate as diabetes develops. Successful adaptation is achieved through increased ␤-cell mass and increased insulin secretion. The hypothesis is explored that ␤-cells exposed to the diabetic milieu lose their differentiation, which leads to loss of specialized functions such as GIIS. This concept has been strengthened by the finding of dedifferentiation of ␤-cells in a rat model of partial pancreatectomy that includes a reduction of insulin gene expression, which may further contribute to decreased insulin production. Another finding was increased expression of c-Myc, which probably contributes to an increase in the expression of lactate dehydrogenase and the development of ␤-cell hypertrophy. Arguments are developed that the ␤-cell changes found in diabetes are better correlated with increased glucose levels than with nonesterified fatty acid levels, thus supporting the importance of glucose toxicity. Diabetes 50 (Suppl.

Major insulin gene transcription factors, such as PDX-1 or NeuroD1, have equally important roles in pancreatic development and the differentiation of pancreatic endocrine cells. Previously, we identified and cloned another critical insulin gene transcription factor MafA (RIPE3b1) and reported that other Maf factors were expressed in pancreatic endocrine cells. Maf factors are important regulators of cellular differentiation; to understand their role in differentiation of pancreatic endocrine cells, we analyzed the expression pattern of large-Maf factors in the pancreas of embryonic and adult mice. Ectopically expressed large-Maf factors, MafA, MafB, or cMaf, induced expression from insulin and glucagon reporter constructs, demonstrating a redundancy in their function. Yet in adult pancreas, cMaf was expressed in both alpha- and beta-cells, and MafA and MafB showed selective expression in the beta- and alpha-cells, respectively. Interestingly, during embryonic development, a significant proportion of MafB-expressing cells also expressed insulin. In embryos, MafB is expressed before MafA, and our results suggest that the differentiation of beta-cells proceeds through a MafB+ MafA- Ins+ intermediate cell to MafB- MafA+ Ins+ cells. Furthermore, the MafB to MafA transition follows induction of PDX-1 expression (Pdx-1(high)) in MafB+ Ins+ cells. We suggest that MafB may have a dual role in regulating embryonic differentiation of both beta- and alpha-cells while MafA may regulate replication/survival and function of beta-cells after birth. Thus, this redundancy in the function and expression of the large-Maf factors may explain the normal islet morphology observed in the MafA knockout mice at birth.

Background: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. Methods: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. Results: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. Conclusions: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

Tyrosine kinase inhibitors (TKIs), despite efficacy as anti-cancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen FDA-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a “cardiac safety index” to assess cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that VEGFR2/PDGFR-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. Using phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Upregulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during co-treatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anti-cancer TKIs, correlating with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.