Steroid regulation of growth hormone (GH) receptor and GH-binding protein messenger ribonucleic acids in the rat

In the rat, the GH receptor (GHR) and the GH-binding protein (GHBP), which arise from alternative splicing of the same gene, show a sexually dimorphic and GH-dependent expression pattern. Multiple alternative 5'-untranslated regions (UTRs) are present in GHR and GHBP transcripts in the rat, one of which, GHR1, has recently been shown to be liver specific and found at higher levels in females. We have measured the hepatic GHR1, GHR, and GHBP transcript levels, by RNase protection and solution hybridization assay, in animals with differing hormonal status, in which hepatic GHR binding and plasma GHBP have been previously assayed. Estradiol (E2) induced GHR1 in males, whereas ovariectomy or the antiestrogen tamoxifen reduced GHR1 expression in females. The induction of GHR1 by E2 was GH dependent, being lower in GH-deficient dwarf rats and absent in hypophysectomized rats, paralleling previous measurements of plasma GHBP and hepatic GHR binding in these animals. Significant changes in GHR1 could explain the trends seen in the same extracts when coding region probes were used. Short-term adrenalectomy had no effect on GHR and GHBP expression, but dexamethasone markedly reduced both protein and messenger RNA (mRNA) levels. Corticosterone treatment had no effect alone but reduced the E2-induced increase in GHR1 levels, whereas methylprednisolone administered orally reduced hepatic GH binding, plasma GHBP, and GHR1 mRNA levels. Thus, 5'-UTRs, encoded by different first exons, are involved in the regulation of hepatic GHR and GHBP expression and need to be considered when comparing effects of hormonal manipulation on the mRNA transcripts and protein products of the GHR gene. Previous studies have found discrepancies between levels of protein expression and mRNA transcripts measured only with coding region probes. Our results suggest that posttranscriptional differences related to 5'-UTR heterogeneity in the GHR gene explain some of these discrepancie

Steroid regulation of growth hormone (GH) receptor and GH-binding protein messenger ribonucleic acids in the rat

In the rat, the GH receptor (GHR) and the GH-binding protein (GHBP), which arise from alternative splicing of the same gene, show a sexually dimorphic and GH-dependent expression pattern. Multiple alternative 5\u27-untranslated regions (UTRs) are present in GHR and GHBP transcripts in the rat, one of which, GHR1, has recently been shown to be liver specific and found at higher levels in females. We have measured the hepatic GHR1, GHR, and GHBP transcript levels, by RNase protection and solution hybridization assay, in animals with differing hormonal status, in which hepatic GHR binding and plasma GHBP have been previously assayed. Estradiol (E2) induced GHR1 in males, whereas ovariectomy or the antiestrogen tamoxifen reduced GHR1 expression in females. The induction of GHR1 by E2 was GH dependent, being lower in GH-deficient dwarf rats and absent in hypophysectomized rats, paralleling previous measurements of plasma GHBP and hepatic GHR binding in these animals. Significant changes in GHR1 could explain the trends seen in the same extracts when coding region probes were used. Short-term adrenalectomy had no effect on GHR and GHBP expression, but dexamethasone markedly reduced both protein and messenger RNA (mRNA) levels. Corticosterone treatment had no effect alone but reduced the E2-induced increase in GHR1 levels, whereas methylprednisolone administered orally reduced hepatic GH binding, plasma GHBP, and GHR1 mRNA levels. Thus, 5\u27-UTRs, encoded by different first exons, are involved in the regulation of hepatic GHR and GHBP expression and need to be considered when comparing effects of hormonal manipulation on the mRNA transcripts and protein products of the GHR gene. Previous studies have found discrepancies between levels of protein expression and mRNA transcripts measured only with coding region probes. Our results suggest that posttranscriptional differences related to 5\u27-UTR heterogeneity in the GHR gene explain some of these discrepancie

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field