Alex M. DePaoli

Evolution of β-Cell Dysfunction in the Male Zucker Diabetic Fatty Rat

The molecular basis for the β-cell dysfunction that characterizes non-insulin-dependent diabetes mellitus (NIDDM) is unknown. The Zucker diabetic fatty (ZDF) male rat is a rodent model of NIDDM with a predictable progression from the prediabetic to the diabetic state. We are using this model to study β-cell function during the development of diabetes with the goal of identifying genes that play a key role in regulating insulin secretion and, thus, may be potential targets for therapeutic intervention aimed at preserving or improving β-cell function. As a first step, we have characterized morphology, insulin secretion, and pattern of gene expression in islets from prediabetic and diabetic ZDF rats. The development of diabetes was associated with changes in islet morphology, and the islets of diabetic animals were markedly hypertrophic with multiple irregular projections into the surrounding exocrine pancreas. In addition, there were multiple defects in the normal pattern of insulin secretion. The islets of prediabetic ZDF rats secreted significantly more insulin at each glucose concentration tested and showed a leftward shift in the dose-response curve relating glucose concentration and insulin secretion. Islets of prediabetic animals also demonstrated defects in the normal oscillatory pattern of insulin secretion, indicating the presence of impairment of the normal feedback control between glucose and insulin secretion. The islets from diabetic animals showed further impairment in the ability to respond to a glucose stimulus. Changes in gene expression were also evident in islets from prediabetic and diabetic ZDF rats compared with age-matched control animals. In prediabetic animals, there was no change in insulin mRNA levels. However, there was a significant 30–70% reduction in the levels of a large number of other islet mRNAs including glucokinase, mitochondrial glycerol-3-phosphate dehydrogenase, voltage-dependent Ca2+ and K+ channels, Ca2+-ATPase, and transcription factor Islet-1 mRNAs. In addition, there was a 40–50% increase in the levels of glucose-6-phosphatase and 12-lipoxygenase mRNAs. There were further changes in gene expression in the islets from diabetic ZDF rats, including a decrease in insulin mRNA levels that was associated with reduced islet insulin levels. Our results indicate that multiple defects in β-cell function can be detected in islets of prediabetic animals well before the development of hyperglycemia and suggest that changes in the normal pattern of gene expression contribute to the development of β-cell dysfunction.

Differential expression of messenger RNAs for somatostatin receptor subtypes SSTR1, SSTR2 and SSTR3 in adult rat brain: Analysis by RNA blotting and in situ hybridization histochemistry

The messenger RNAs encoding three somatostatin receptor subtypes, SSTR1, SSTR2 and SSTR3, were detected in rat by RNA blotting and in situ hybridization histochemistry to identify the sites of synthesis and expression of these somatostatin receptor subtypes. RNA blotting revealed that SSTR1 messenger RNA of 3.8 kilobases was highly expressed in cerebral cortex, hippocampus, midbrain and hypothalamus. In situ hybridization histochemistry revealed that SSTR1 messenger RNA was localized to discrete layers of the cerebral cortex, the piriform cortex and the dentate gyrus of the hippocampus. SSTR1 messenger RNA was expressed at low levels in the cerebellum and pituitary and was not detectable in striatum or other peripheral organs. At least two SSTR2 messenger RNAs were detected by RNA blotting of 2.4 and 2.8 kilobases which correspond to the size of the spliced and unspliced forms of this receptor messenger RNA. SSTR2 messenger RNA detected by in situ hybridization is diffusely expressed in cerebral cortex and amygdala but is discretely localized to dentate gyrus in the hippocampus, medial habenula and ventromedial and dorsomedial nuclei and arcuate nucleus of the hypothalamus. The levels of SSTR2 messenger RNA are very low in the cerebellum and were not observed in the striatum or peripheral tissues other than the pituitary or adrenal gland. A single SSTR3 messenger RNA of 4.0 kilobases was seen in hippocampus, cerebral cortex, midbrain, hypothalamus and pituitary. However, the tissue with the highest levels of SSTR3 messenger RNA is the cerebellum with messenger RNA localized to the granule cell layer. The expression of the three different somatostatin receptor messenger RNAs are distinct but overlapping. Such distinct expression may contribute to the selective biological roles of the receptor subtypes.

Expression of Calcium Channel mRNAs in Rat Pancreatic Islets and Downregulation After Glucose Infusion

Recent studies have shown that two different voltage-dependent Ca2+ channels are expressed in pancreatic islets, the β-cell/neuroendocrine-brain and the cardiac subtypes. The effects of chronic hyperglycemia on the levels in pancreatic islets of the mRNAs encoding the α1-subunits of the β-cell and cardiac subtype Ca2+ channels were studied in rats made hyperglycemic by infusion of glucose for 48 h. A competitive reverse transcriptase-polymerase chain reaction procedure was used to obtain quantitative data on the levels of these two transcripts in islets obtained from individual rats. The quantitative polymerase chain reaction data indicate that the levels of mRNA encoding the α 1-subunit of the β-cell Ca2+ channel are 2.5-fold > those for the cardiac subtype. The levels of β-cell Ca2+ channel mRNA were 72.9% lower in the glucose-infused animals when compared with the saline-infused animals (P < 0.005) and those of the cardiac channel were 72.1% lower in the animals infused with glucose (P < 0.02). In contrast, glucose infusion resulted in a twofold increase in insulin mRNA levels and did not significantly alter levels of β-actin mRNA. In situ hybridization studies revealed that the mRNAs for these two Ca2+ channels are expressed at higher levels in normal rat islets than in the surrounding acinar tissue, which suggests that the observed changes in mRNA levels occur within cells of the pancreatic islet. To assess the possible functional consequences of this reduction in expression of mRNA for the Ca2+ channels, the insulin secretory responses of perfused pancreases to the Ca2+ channel agonist Bay K8644 were studied. In pancreases from glucose-infused animals, the relative incremental insulin secretory response to Bay K8644 was reduced, and a similar blunting of the acute response to glucose also was seen. In summary, the predominant voltage-dependent Ca2+ channels subtype expressed in islets under basal conditions and after glucose infusion is the β-cell form with lower levels of the cardiac subtype. Glucose infusion for 48 h results in a significant reduction in the mRNA levels for both the β-cell and cardiac subtypes, and this is associated with enhanced basal secretion but reduced responses to glucose and the Ca2+ channel agonist Bay K8644.

Distribution of κ Opioid Receptor mRNA in Adult Mouse Brain: An in Situ Hybridization Histochemistry Study

The distribution of the kappa opioid receptor mRNA in adult mouse brain has been determined using the technique of in situ hybridization histochemistry. The mRNA for the kappa opioid receptor was expressed in distinct areas throughout the brain. The telencephalon showed high levels of expression in the deeper layers of the parietal and temporal cortex, olfactory tubercle, nucleus accumbens, claustrum, endopiriform nucleus, nucleus of the vertical and horizontal limb of the diagonal band, and medial and central nuclei of the amygdala. In the diencephalon, kappa opioid receptor mRNA was present in multiple medial thalamic nuclei including the centromedial, paraventricular, parafasicular, central, and peritenial nuclei, as well as in most hypothalamic nuclei including the ventromedial, periventricular, supraoptic, arcuate, and dorsomedial nuclei. The mesencephalon showed highest levels of kappa receptor mRNA in the substantia nigra pars compacta, ventral tegmental area, zona incerta, interpeduncular nucleus, superior colliculus, inferior colliculus, central grey, and the raphe nucleus. In the metencephalon, kappa opioid receptor mRNA was expressed in the parabrachial nuclei, locus coeruleus, dorsal and ventral tegmental nuclei, and the raphe pontine nuclei. The distribution of the kappa receptor mRNA closely coincides with the localization of binding sites in rat brain for [3H]U-69,593, a specific kappa 1 opioid receptor ligand. The mRNA distribution also correlates with neuroanatomical sites of actions of kappa agonists and distribution of the endogenous kappa receptor ligand dynorphin.

G Protein-Activated Inwardly Rectifying Potassium Channel (GIRK1/KGA) mRNA in Adult Rat Heart and Brain by in Situ Hybridization Histochemistry

GIRK1/KGA (referred to as GIRK1) is a member of the inwardly rectifying K+ channel family and is activated by G protein-linked receptors. The activation of this channel leads to hyperpolarization of the plasma membrane and is fundamental to the control of atrial and neuronal excitability. RNA blotting studies have shown that GIRK1 is expressed in the atrium of the heart and in the brain. We have used in situ hybridization histochemistry to characterize the pattern of expression of GIRK1 mRNA in adult rat heart and brain. In heart, expression of GIRK1 mRNA is homogeneous throughout the atria. There is no significant ventricular expression, although the conduction bundles were not specifically identified. GIRK1 mRNA expression in the brain is widespread with highest levels in the cortex, septum, hippocampus, thalamus, amygdala, cerebellum, and many nuclei of the midbrain and hindbrain, including red nucleus, inferior colliculus, pontine nucleus, nucleus of the solitary tract, and multiple cranial nerve nuclei (motor and sensory components). This detailed map of GIRK1 mRNA expression provides a basis for further study of this important new family of K+ channels.

Graft-versus-Host Disease and Liver Transplantation

Excerpt To the Editors: We offer another case in support of the supposition by Collins and colleagues (1) that graft-versus-host disease (GVHD) in liver transplantation is more common than previous...

Lymphoma in acquired generalized lipodystrophy.

Acquired generalized lipodystrophy (AGL) is a rare disease thought to result from autoimmune destruction of adipose tissue. Peripheral T-cell lymphoma (PTCL) has been reported in two AGL patients. We report five additional cases of lymphoma in AGL, and analyze the role of underlying autoimmunity and recombinant human leptin (metreleptin) replacement in lymphoma development. Three patients developed lymphoma during metreleptin treatment (two PTCL and one ALK-positive anaplastic large cell lymphoma), and two developed lymphomas (mycosis fungoides and Burkitt lymphoma) without metreleptin. AGL is associated with high risk for lymphoma, especially PTCL. Autoimmunity likely contributes to this risk. Lymphoma developed with or without metreleptin, suggesting metreleptin does not directly cause lymphoma development; a theoretical role of metreleptin in lymphoma progression remains possible. For most patients with AGL and severe metabolic complications, the proven benefits of metreleptin on metabolic disease will likely outweigh theoretical risks of metreleptin in lymphoma development or progression.