L.N.J.L. Marlier

Effect of biotin on glucokinase activity, mRNA expression and insulin release in cultured beta-cells

Biotin is known to influence hepatic glucokinase (GK) expression both at a transcriptional and at a translational level. The aim of the present paper was to investigate the effect of biotin on pancreatic GK. For this purpose, RIN 1046-38 cells were cultured in the presence of different biotin concentrations for different times; there-after, GK mRNA expression, GK activity and insulin release were studied. Results demonstrated that biotin has a biphasic effect on GK mRNA expression, being stimulatory after short-term treatment and inhibitory after long-term treatment. GK activity was increased after long-term treatment. Insulin release was not affected by biotin treatment. These data suggest that biotin may influence glucose metabolism also by acting directly at the level of beta-cells.

Molecular and cellular characterization of the GABAA receptor in the rat pancreas

In the present study, we characterize the molecular structure of the GABAA receptor in pancreas, islets, alpha and beta cells, and in RIN 1046-38 cells. Using the polymerase chain reaction and specific primers for 11 out of the 15 subunits known so far, that may contribute to the composition of the GABAA receptors, we demonstrate that pancreas and its cellular components, as well RIN 1046-38 cells, might contain a GABAA receptor resulting from all the possible combinations in a pentameric configuration of the subtypes alpha 1, alpha 2, alpha 3 of the alpha subunit family, beta 1, beta 2, beta 3 subtypes of the beta subunit family, delta subunit and gamma 2 subtype of the gamma subunit family. The presence of the gamma 2 subunit renders the GABAA receptors potentially sensitive to allosteric modulators.

Distribution of glucocorticoid receptor mRNA in the rat spinal cord

Using in situ hybridization (ISH), we studied the distribution of rat glucocorticoid receptors (GR) mRNA in rat spinal cord. mRNA encoding for GR was abundant throughout the white matter and a clear pattern of distribution was detected within the grey matter. In the grey matter mRNA was primarily localized in the ventral horn, where motoneurones were strongly labelled. In the dorsal horn, the distribution appears more diffuse but the superficial layers (I and II) clearly exhibited a shigher signal. We conclude that, in rat spinal cord, GR are present in both glial and neuronal cells. In particular, both somatosensory and motor pathways contain GR.

Differential regulation of adrenocorticoid receptors in the hippocampus and spinal cord of adrenalectomized rats.

Using multiplex polymerase chain reaction assay and cytosolic receptor binding assay we studied type I, mineralocorticoid receptor (MR), and type II, glucocorticoid receptor (GR), adrenocorticoid receptors expression in rat hippocampus and spinal cord, at various times after adrenalectomy: 12 hr, 24 hr, 3 days, and 1 week. Analysis of the data demonstrates that in hippocampus the expression of MR and GR mRNA was not significantly affected by adrenalectomy. On the contrary, Bmax of MR was significantly increased at each time post‐surgery, with only slight modifications of Kd. Bmax and Kd for GR showed a significant increase after 3 days and 1 week. In the spinal cord, MR mRNA was increased 12 hr after adrenalectomy, reaching a maximum at 3 days. Bmax of MR was also significantly increased after 3 days, whereas its Kd remained unchanged for the entire duration of the study. Both GR mRNA and binding parameters were poorly affected by adrenalectomy. The results of the present experiments demonstrate that the absence of adrenocortical hormones influences differentially MR and GR expression in hippocampus and spinal cord, suggesting the existence of various and independent mechanisms of regulation of adrenocorticoid receptor.

Quantitative analysis of pancreatic glucokinase gene expression in cultured β cells by competitive polymerase chain reaction

Regulation of glucokinase (GK) gene expression in pancreatic beta cells has been poorly investigated, both due to low abundance of the gene and to difficulties in cells isolation. The present study describes the establishment of a competitive RT-PCR method for quantitative analysis of GK gene. The method has been applied to the analysis of GK mRNA expression RIN 1046-38 cells. We have monitored modifications of GK mRNA expression after different periods of time in culture and we have studied the effect induced by dexamethasone (DEX) treatment. We show that the method is very sensitive and requires very low amount of RNA. Data demonstrate that GK mRNA expression in RIN cells is reduced as a function of passages in culture and that the reduction is positively correlated with the decrease of insulin responsiveness observed in high passages cells. DEX treatment inhibits GK mRNA expression in RIN cells in a dose-dependent and time-dependent manner.

DBI mRNA is expressed in endocrine pancreas and its post-translational product DBI(33-50) inhibits insulin release.

It has been previously demonstrated that DBI is present in endocrine pancreas and it is able to inhibit insulin release in isolated rat islets. Its mechanism of action has been investigated, demonstrating the possible involvement of cAMP and ATP-dependent K+ channels. DB133–50, a post-translational product of DBI, is also able to inhibit insulin release, but its action has not been characterized. In the present study, we have investigated the presence of DBI mRNA in pancreas, islets and cultured ß cells. The possible mechanism of action of DBI33–50 and the involvement of BZ/GABAA receptors has been studied.

Corticosterone treatment differentially affects adrenocorticoid receptors expression and binding in the hippocampus and spinal cord of the rat.

We have studied the immediate and long-term effects of high doses of corticosterone (CORT) on mRNA expression and binding properties of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and spinal cord of rats. Animals were treated with corticosterone (10 mg/d subcutaneously) for 21 consecutive days, and mineralocorticoid and glucocorticoid receptors were studied either 24 h or 2 wk after the last injection. Major results show that corticosterone treatment reduces mineralocorticoid and glucocorticoid receptor maximum binding capacity (Bmax) in both the hippocampus and spinal cord and that this reduction is partially reversed after cessation of treatment. With respect to mRNA expression, in the hippocampus recovery after cessation of treatment is complete. By contrast, in the spinal cord, mineralocorticoid receptor mRNA expression is irreversibly increased after treatment, but the glucocorticoid receptor mRNA level remains unaffected during and after treatment. Thus, these data suggest the presence of distinct regulatory mechanisms for adrenocorticoid receptors in rat brain and spinal cord, in response to long-term exposure to high levels of circulating corticosterone and after recovery from treatment.

Distribution of Adrenocorticoid Receptors in the Rat CNS Measured by Competitive PCR and Cytosolic Binding

Combined quantitative polymerase chain reaction (PCR) and cytosolic binding assay techniques are used to measure mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA, Kd, and Bmax in various rat central nervous system (CNS) regions, namely amygdala, hypothalamus, hippocampus, cortex, pituitary, and cervical, thoracic, and lumbar spinal cord. Two internal standards (i.s.) cDNA were cloned for quantitative PCR purposes. The i.s. templates differed from the respective wild-type (wt) templates for a single base-pair mutation introduced by PCR that generated a unique restriction site, thus allowing amplification products arising from coamplification ofwt and i.s. to be distinguished. Results show that cerebellum, which displayed average Bmax values for both receptors, contained the highest level of MR and GR mRNA. Hippocampus also had a high level of MR mRNA. Low mRNA content was found in the hypothalamus for MR and GR as well as in the cortex for GR. High Bmax values for both MR and GR were found in the lumbar spinal cord, despite a modest mRNA content. The lowest Bmax values were found in the cortex for both receptors. It is, therefore, concluded that mRNA content and Bmax are not closely correlated in the rat CNS. These data suggest a differential regulation of various adrenocorticoid receptor isoforms. Moreover, this quantitative PCR method is very sensitive and can be used to assay small amounts of material in order to obtain absolute measurements of mRNA expression.