Arun Sharma

Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.

Differentiated pancreatic β 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 β 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 β 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 β cell volume without affecting plasma nonesterified fatty acid levels, strongly suggesting that hyperglycemia triggers these abnormalities. In conclusion, chronic hyperglycemia leads to β cell hypertrophy and loss of β cell differentiation that is correlated with changes in c-Myc and other key transcription factors. A similar change in β cell differentiation could contribute to the profound derangement of insulin secretion in human diabetes.

Induction of c-Myc Expression Suppresses Insulin Gene Transcription by Inhibiting NeuroD/BETA2-mediated Transcriptional Activation

Insulin biosynthesis and secretion are critical for pancreatic β-cell function, but both are impaired under diabetic conditions. We have found that hyperglycemia induces the expression of the basic helix-loop-helix transcription factor c-Myc in islets in several different diabetic models. To examine the possible implication of c-Myc in β-cell dysfunction, c-Myc was overexpressed in isolated rat islets using adenovirus. Adenovirus-mediated c-Myc overexpression suppressed both insulin gene transcription and glucose-stimulated insulin secretion. Insulin protein content, determined by immunostaining, was markedly decreased in c-Myc-overexpressing cells. In gel-shift assays c-Myc bound to the E-box in the insulin gene promoter region. Furthermore, in βTC1, MIN6, and HIT-T15 cells and primary rat islets, wild type insulin gene promoter activity was dramatically decreased by c-Myc overexpression, whereas the activity of an E-box mutated insulin promoter was not affected. In HeLa and HepG2 cells c-Myc exerted a suppressive effect on the insulin promoter activity only in the presence of NeuroD/BETA2 but not PDX-1. Both c-Myc and NeuroD can bind the E-box element in the insulin promoter, but unlike NeuroD, the c-Myc transactivation domain lacked the ability to activate insulin gene expression. Additionally p300, a co-activator of NeuroD, did not function as a co-activator of c-Myc. In conclusion, increased expression of c-Myc in β-cells suppresses the insulin gene transcription by inhibiting NeuroD-mediated transcriptional activation. This mechanism may explain some of the β-cell dysfunction found in diabetes.

Identification of Markers for Newly Formed β-Cells in the Perinatal Period: A Time of Recognized β-Cell Immaturity

Markers of β-cell maturity would be useful in staging the differentiation of stem/progenitor cells to β-cells whether in vivo or in vitro. We previously identified markers for newly formed β-cells in regenerating rat pancreases after 90% partial pancreatectomy. To test the generality of these markers of newly formed β-cells, we examined their expression during the perinatal period, a time of recognized β-cell immaturity. We show by semiquantitative RT-PCR and immunostaining over the time course from embryonic day 18/20 to birth, 1 day, 2 days, 3 days, 7 days, and adult that MMP-2, CK-19, and SPD are truly markers of new and immature β-cells and that their expression transiently peaks in the perinatal period and is not entirely synchronous. The shared expression of these markers among fetal, newborn, and newly regenerated β-cells, but not adult, strongly supports their use as potential markers for new β-cells in the assessment of both the maturity of stem cell–derived insulin-producing cells and the presence of newly formed islets (neogenesis) in the adult pancreas.

Induction of Mad expression leads to augmentation of insulin gene transcription

Insulin gene transcription is critical for the maintenance of pancreatic beta-cell differentiation and insulin production. In this study, we found that the basic helix-loop-helix transcription factor Mad, which usually acts as a repressor to c-Myc, enhances insulin gene transcription. In isolated rat islets adenoviral overexpression of Mad augmented insulin mRNA expression and insulin protein content, as well as glucokinase and GLUT2 mRNA expression. Also, Mad overexpression upregulated insulin promoter activity in beta-cell-derived cell lines, MIN6 and betaTC1, as well as in non-insulin producing liver cell line, HepG2. Mad overexpression in rat islets enhanced PDX-1 expression and its DNA binding activity. We found that Mad mediated increased PDX-1 expression by an E-box dependent transcriptional regulation of the PDX-1 gene. That the effects of Mad on insulin expression were mediated through PDX-1 was further substantiated by studies showing inhibition of insulin promoter activation by Mad in the presence of mutated PDX-1 binding site. Although Mad functions as a negative regulatory factor for multiple target genes, these studies establish the fact that Mad can also function as a positive regulatory factor for insulin gene transcription. Such regulation of insulin expression by Mad with modulation of PDX-1 expression and DNA binding activity could offer useful therapeutic and/or experimental tools to promote insulin production in appropriate cell types.

Generation of Beta Cells from Pancreatic Duct Cells and/or Stem Cells

Since diabetes is caused by the loss of the insulin-producing β cells, its reversal by replacement of these cells by transplantation or by replenishment from endogenous sources seems straightforward. In the pancreas, two mechanisms for β-cell growth are replication of preexisting β cells and neogenesis or the differentiation of new β cells from progenitor/stem cells that were not β cells. Replication and neogenesis are not mutually exclusive, and there is no biological reason for there to be only one mechanism for replenishment of the islet cells. This chapter focuses on the renewed interest in the identification, expansion, and differentiation of adult pancreatic progenitor/stem cells that can lead to more β cells, either in vivo or in vitro.

AMINO ACID CONSTITUTION OF CHROMOSOMES.

The present experiment was designed to demonstrate the presence of two protein-bound amino acids, viz. tryptophan and tyrosine, in the different, cellular constituents with particular reference to chromosomes. The materials chosen were the root-tips of Allium cepa and Vicia faba. Studies were made from the smeared tissue preparation. For tryptophan, indole reactions following two methods, viz., that of Gurr (1958) and of Glenner and Lillie (1957) were followed and the latter yielded satisfactory results; while for tyrosine, Bensley and Gershs modified Millons reaction (1953) was adopted. The results of the reactions show that tryptophan is present in much less amount than tyrosine in the protein component of the chromosomes. The same is true with cytoplasm as well. The strongly positive tyrosine reaction of nuclear membrane, with distinct strands towards the cell membrane, has been attributed to its role in nucleocytoplasmic transfer and synthesis of cytoplasmic protein. The less positive reaction of nuclear membrane with tryptophan suggests its comparatively little importance in transfer. Only in case of tyrosine, the differential reaction of the euchromatic and heterochromatic parts could he made out, the latter giving negative response. It has been suggested that it might appear only during the time of transfer through the nucleolus. Tryptophan on the other hand shows uniform distribution throughout the chromosome length.