Regulation of hepatic glycogen in the insulin-like growth factor II-deficient mouse.

Insulin-like growth factor II (IGF-II), a polypeptide hormone with structural homologies to insulin-like growth factor I (IGF-I) and insulin, regulates the metabolism and growth of many tissues. In this study, we examined the role of IGF-II in hepatic glycogen metabolism in normal and growth-retarded IGF-II-deficient (knockout) mice. Liver glycogen content was significantly lower in the IGF-II knockout than in control livers during embryonic day 18 and postnatal day 0. Biochemical results were verified histologically using a glycogen-specific stain. The enzymatic activity of glycogen synthase, the rate-limiting enzyme for glycogen synthesis, was significantly lower in livers of knockout mice than in livers from wild-type controls on embryonic day 18 and postnatal day 0. The levels of glycogen synthase messenger RNA were not different between the two groups at any age studied, indicating that IGF-II acts posttranscriptionally. Hepatic glycogen content, measured in newborns after food withdrawal, was significantly lower in knockout mice compared with that in wild-type mice after 0, 3, and 6 h of fasting. Blood glucose was significantly lower in knockouts vs. wild-type newborn mice before fasting and was similar in both genotypes after 6 h of fasting. Consistent with this, only 23% of IGF-II knockout newborn mice survived fasting for 12 h, whereas 93% of wild-type mice survived this treatment. These results indicate that IGF-II is required for the regulation of glycogen metabolism of the mouse in the perinatal period, possibly via stimulation of glycogen synthase activity. IGF-II, via perinatal regulation of glycogen synthesis, may regulate fetal growth as well as play an important role in the transition from fetal to postnatal life by protecting the neonate against hypoglycemia during periods of fasting.

Expression and imprinting of the insulin-like growth factor II gene in neonatal mouse cerebellum.

Insulin-like growth factor II (IGF-II) plays significant roles in the growth and development of mammals through the regulation of mitogenesis and cell survival. Previously, IGF-II mRNA transcripts within the CNS were detected in the choroid plexus and leptomeninges (DeChiara et al., 1991). The objective of this study was to determine the expression pattern of IGF-II mRNA in different cell types of the cerebellum during development. We report here that the IGF-II gene is transcribed in granule and glial cells within the cerebellar parenchyma at various times during the early postnatal period in mice. IGF-II gene expression is further regulated by parent-specific imprinting such that only the paternal IGF-II allele is expressed in granule cells. In contrast, choroid plexus and leptomeninges express IGF-II mRNAs biallelically, indicating that cell type-specific regulation of genomic imprinting occurs within the mammalian CNS.

Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-beta-galactosidase fusion gene in transgenic mice.

To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene. Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

Regulation of vasoactive intestinal polypeptide and galanin mRNA stabilities.

The stabilities of vasoactive intestinal polypeptide (VIP) and galanin mRNAs were examined in a human neuroblastoma cell line (NBFL) treated with agents that alter second-messenger pathways. VIP and galanin mRNA stabilities were estimated by the decay of steady-state levels of transcripts following transcriptional arrest with actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In the presence of actinomycin D, phorbol ester treatment stabilized VIP mRNA while treatment with adenylate cyclase activators, calcium ionophore, or CNTF did not. In the presence of DRB, VIP mRNA was not stabilized in phorbol ester-treated cells but instead was stabilized in cells treated with adenylate cyclase activators. With either transcriptional inhibitor, stability of galanin mRNA was not significantly altered. The difference in the behavior of VIP mRNA in the presence of actinomycin D and DRB may result from their different mechanisms of action-actinomycin D intercalates into nucleic acids while DRB is a kinase inhibitor. Using an assay for RNA stability that did not require transcriptional inhibitors, an in vitro transcribed VIP RNA fragment was relatively stable in extracts from phorbol ester-treated cells. Although treatment with phorbol ester alone resulted in stabilization of VIP mRNA, treatment with a combination of phorbol ester and adenylate cyclase activator, calcium ionophore, or CNTF did not-implying a complex interaction of these second-messenger pathways in the regulation of RNA stability.

Insulin-like growth factor II affects the appearance and glycogen content of glycogen cells in the murine placenta.

The phenotype of mice with a targeted disruption of the insulin-like growth factor II gene (IGF-II null mice) is growth retardation of both fetus and placenta during the last two thirds of gestation (1). We have compared the placenta of IGF-II null and wild-type mice from days 9-18 of gestation. No morphological differences were detected until after day 12 of gestation, when a new population of placental cells, the glycogen cells, normally first appears. Fewer glycogen cells were present in the null placenta compared to the wild-type placenta on days 13, 15, and 18 of gestation. By day 15, glycogen cells constituted approximately 50% of the basal zone cells in the wild-type placenta, but only 20% of the basal zone cells in the null placenta (P < 0.01). By contrast, spongiotrophoblasts constituted 40% of the basal zone cells in the wild-type placenta and 70% of the basal zone cells in the null placenta. There were no differences in cell size at any time. These results suggest that glycogen cells do not efficiently differentiate in the absence of IGF-II. The differentiation of glycogen cells did not appear to be simply delayed in the null placenta, as the number of glycogen cells in the null placenta did not increase between days 15-18, and there was no change in the ratio of glycogen to spongiotrophoblasts in the basal zone (P > 0.50). The glycogen content of both spongiotrophoblasts and glycogen cells was significantly reduced in the null placenta, suggesting that IGF-II may be an important regulator of glycogen synthesis in the placenta. These results indicate that IGF-II regulates cell number in the placenta and may play an important role in the differentiation of glycogen cells and the production of glycogen by placental cells.

Quantitative analysis of the expression of a VIP transgene.

We have analyzed the expression of a transgene bearing 2 kilobases of the 5' flanking region of the human vasoactive intestinal polypeptide (VIP) gene coupled to beta-galactosidase. Expression was assayed by beta-galactosidase histochemistry and by mRNA quantitation using polymerase chain reaction (PCR)-mediated amplification; we compared beta-galactosidase activity against both transgene and endogenous VIP mRNA levels. We found that the human 5' flanking sequence in this construct is able to direct tissue-specific expression of beta-galactosidase similar to the pattern for endogenous VIP. However, the transgene is also expressed in smooth muscle and Schwann cells, where VIP mRNA is rare. In various tissues where the transgene and endogenous gene are both active, the ratio between their message levels differs dramatically--transgene mRNA is more abundant where VIP is relatively scarce, but is much less abundant than the endogenous message at sites where VIP mRNA is most concentrated. These results suggest that sequence elements that may restrict VIP transcription or cause tissue-specific VIP mRNA accumulation are missing from the transgene. In the testis there is a high level of transgene message but no significant beta-galactosidase activity; this discrepancy is caused by transcription from a cryptic promoter within the beta-galactosidase sequence.

Oligodendroglial development in human fetal cerebrum.

We have successfully established mixed glial cell primary cultures prepared from individual fetal human brains (15-18 weeks' gestation in age). Cultures were maintained for as long as 3 months in either 10% fetal calf serum (FCS) or serum-free chemically defined medium (CDM). By morphological and immunohistochemical criteria, the precursor cell for human oligodendrocytes (O-2A cell) was identified. This cell exhibited the bipolar morphology and A2B5-positive (A2B5+) immunoreactivity typical of the O-2A precursor cell. With time in culture, cells possessing a stellate morphology appeared, some of which stained with the O4 antibody, indicative of cell differentiation in the oligodendroglial lineage. At yet older culture age, arborized cells bearing the O1 (galactocerebroside, GC) immunohistochemical marker and displaying the morphological characteristics typical of more mature oligodendrocytes were found, confirming their oligodendroglial identity. Oligodendroglial differentiation was supported best by CDM rather than FCS. To complement these observations, double immunofluorescent studies were performed on parietal sections from human fetal brains at 20 to 22 weeks of gestation. Bipolar A2B5+, multipolar A2B5+/O4+, and arborized A2B5-/O1+ cells were found, thus confirming the presence of oligodendrocytes in human fetal brain at this stage of prenatal development and consistent with the observations made in cell culture.

Pancreatic Reg/pancreatic stone protein (PSP) gene expression does not correlate with beta-cell growth and regeneration in rats.

The Reg/pancreatic stone protein (PSP) gene is postulated to be an important regulator of pancreatic beta-cell growth. To investigate this hypothesis, we analysed the expression of the Reg/PSP gene following a 90% pancreatectomy and after chronic glucose infusion, two well-defined models of pancreatic beta-cell growth. There was a rapid induction of the Reg/PSP gene in the remnant pancreas after a 90% pancreatectomy in rats during the period of marked growth of the exocrine and islet tissue. However, a similar rapid, but smaller, induction of the Reg/PSP gene was observed in sham-operated rats and in non-surgical control rats in which there was no enhanced pancreatic growth. Furthermore, there was no pancreatic Reg/PSP gene induction in a model of selective beta-cell growth, the chronic glucose-infused rat. Thus, it is unlikely that Reg/PSP is a beta-cell specific growth factor, even though the function of this important pancreatic gene is still unknown.

Characterization of type I IGF receptor and IGF-I mRNA expression in cultured human and bovine glomerular cells.

Glomerular hypertrophy is reported in several endocrine disorders such as acromegaly and diabetes mellitus, where abnormalities of growth hormone and insulin-like growth factor (IGF-I) have been reported. In the present report, we have cultured bovine and human glomerular endothelial cells, and bovine glomerular epithelial and mesangial cells, and characterized the expression of IGF-I mRNA and its receptor in these cells. High affinity, specific receptors for IGF-I were identified in all three types of cells by radioreceptor assays. Receptor number (Ro) derived by Scatchard analysis revealed an unusually high number of Type I IGF receptors, approx. 1.2 x 10(5) receptors/cell in glomerular endothelial cells. Affinity crosslinking studies and immunoprecipitation with antibodies against the Type I IGF receptor identified the alpha-subunit of the IGF-I receptor as having a molecular mass of 140 kDa. Biologically, IGF-I was more potent than insulin or IGF-II in stimulating DNA synthesis in glomerular endothelial cells. Northern blot analysis showed that glomerular and aortic endothelial cells expressed IGF-1 mRNA of 1.7 kb. In contrast, renal glomeruli showed several IGF-1 mRNAs of 7.5, 1.7 and 1.2 kb. Thus, the demonstration of both a prepondence of Type I IGF receptors coupled with the growth promoting effects of IGF-I in glomerular endothelial and epithelial cells, as well as the local production of IGF-I mRNA suggests that IGF-I serves an important role as an autocrine or paracrine regulator of the growth of renal glomeruli.

Identification of an antisense transcript from the IGF-II locus in mouse.

We have identified a novel antisense RNA transcribed from the insulin-like growth factor-II (IGF-II) locus in mouse. This transcript was identified using probes derived from cloned genomic DNA lying between the insulin II locus and the IGF-II locus. S1 protection assays confirmed that this transcript is transcribed from the strand complementary to the stand encoding IGF-II. A 3.75 kb RNA was consistently detected on northern analysis of mouse tissue using different randomly primed DNA probes generated from this region. S1 nuclease protection analysis identified three exons contained in the transcript. Developmental northern analysis was performed using RNA from embryonic (E) and postnatal (P) tissues of E10, E13, E18, P1, P4, P10, and adult mice. The antisense RNA was most abundant in E13 and E18 mouse and was present in greatest amounts in skull, skeletal muscle, cardiac muscle, and placenta. No signal for this RNA was detected after the fourth day of life in any tissue studied.

Specific, temporally regulated expression of the insulin-like growth factor II gene during muscle cell differentiation.

We have compared the expression of insulin-like growth factor II (IGF-II) messenger RNA (mRNA) to the expression of other mRNAs encoding proteins known to play pivotal roles during the differentiation of continuously cultured, fusing muscle cell lines. These cell lines respond to changes in culture conditions by undergoing a well characterized alteration in gene expression which leads to a change in their phenotype from dividing, mononucleate myoblasts to fused, multinucleate myotubes. The hallmarks of this differentiation program include the induction of myogenic regulatory genes as well as the genes that encode the contractile proteins. We have found that the differentiation of these cells leads to the production of multiple IGF-II transcripts. In one of the cell lines studied, C2C12, IGF-II mRNA levels were rapidly induced during differentiation. Increases in IGF-II mRNA levels preceded the expression of the contractile protein genes but occurred only after the activation of the myogenic regulatory gene myogenin. The same regulated pattern of IGF-II mRNA expression was seen in both rapidly and slowly fusing subclones of this cell line, indicating a requirement for IGF-II at a specific point during muscle differentiation. These results suggest that IGF-II plays an important role during the terminal differentiation of skeletal muscle cells and are consistent with the existence of an autocrine loop through which IGF-II may act to regulate the differentiation process.

Expression of messenger ribonucleic acid for epidermal growth factor receptor and its ligands, epidermal growth factor and transforming growth factor-alpha, in human first- and second-trimester fetal ovary and uterus.

OBJECTIVES: The epidermal growth factor receptor, a 170 kd polypeptide with tyrosine kinase activity, is used as the common receptor by two homologous polypeptide growth factors, epidermal growth factor and transforming growth factor-alpha. The activation of the epidermal growth factor receptor results in effects including deoxyribonucleic acid synthesis and cellular differentiation. Epidermal growth factor, transforming growth factor-alpha, and the epidermal growth factor receptor are reported to be associated with adult reproduction. However, the pattern of gene expression for the epidermal growth factor receptor and its ligands in human fetal reproductive tissues has not been detailed previously. STUDY DESIGN: We studied the expression of messenger ribonucleic acid encoding three polypeptides, epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha, in 10-, 15-, 19-, and 22-week human fetal ovaries and uteri. Ribonucleic acid was prepared from the fetal tissues and made into complementary deoxyribonucleic acid by reverse transcription. The complementary deoxyribonucleic acid was amplified by polymerase chain reaction, using primers specific for the human epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha. RESULTS: We found that epidermal growth factor receptor messenger ribonucleic acid was present in all stages of ovarian and uterine tissues studied. In addition, both epidermal growth factor and transforming growth factor-alpha messenger ribonucleic acid was found in all four stages of ovarian development. Epidermal growth factor messenger ribonucleic acid was detected in all four stages of fetal uterine development. CONCLUSIONS: This is the first demonstration, to our knowledge, of epidermal growth factor receptor, epidermal growth factor, and transforming growth factor-alpha messenger ribonucleic acid expression in human first- and second-trimester uterus and ovary.

The ratio of mouse insulin I:insulin II does not reflect that of the corresponding preproinsulin mRNAs.

Rats and mice both express two, non-allelic, insulin genes. In the rat the ratio of the two preproinsulin mRNAs closely matches that of the mature insulin peptides. The experiments reported here demonstrate that this is not the case in the mouse. The relative amounts of the two murine proinsulin RNAs were measured by an S1 nuclease assay. The ratio of preproinsulin I mRNA to preproinsulin II mRNA was 4:1 in RNA extracted from the pancreas of mice fed ad libitum or fasted for 72 h. A similar value was found in mouse islets of Langerhans after maintenance in tissue culture for 48 h at either 2.8 or 16.7 mM glucose. The ratio of insulin I:insulin II peptides, assessed by separating the two insulins using reversed phase high-performance liquid chromatography, was approximately 1:3 in both pancreas and islets. Thus in the mouse, unlike the rat, the ratio of the two insulin peptides does not reflect that of the two preproinsulin mRNAs.

Messenger ribonucleic acid for transforming growth factor-alpha, but not for epidermal growth factor, is expressed in fetal and neonatal mouse brain.

OBJECTIVE: Little is known concerning the presence of transforming growth factor-alpha in fetal and neonatal tissues. Our objective was to analyze messenger ribonucleic acid expression for transforming growth factor-alpha and epidermal growth factor, the structural and biochemical analog of transforming growth factor-alpha in fetal and neonatal murine brain. STUDY DESIGN: Messenger ribonucleic acid was prepared from whole brains of mice from these developmental stages: embryonic days 14, 15, and 17, postnatal days 0, 4, 10, and adult. Polymerase chain reaction was performed on the complementary deoxyribonucleic acid obtained from reverse transcription of messenger ribonucleic acid with transforming growth factor-alpha and epidermal growth factor primers. In addition, ribonuclease protection assay was used to identify transforming growth factor-alpha transcripts. RESULTS: We found messenger ribonucleic acid encoding transforming growth factor-alpha in all stages of development used. Epidermal growth factor messenger ribonucleic acid was not found in any stage. Ribonuclease protection assay confirmed transforming growth factor-alpha transcripts in these tissues. CONCLUSION: Transforming growth factor-alpha may play an autocrine or paracrine role in the differentiation or maintenance of murine fetal and neonatal brains.

Processing of proinsulin by transfected hepatoma (FAO) cells.

Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV retroviral vector). Using the DOL vector, production of insulin immunoreactive material was stimulated up to 30-fold by dexamethasone (5 x 10(-7) M). For both proinsulins, fractional release of immunoreactive material relative to cellular content was high, in keeping with the absence of any storage compartment for secretory proteins in these cells. Pulse-chase experiments showed kinetics of release of newly synthesized products in keeping with release via the constitutive pathway. High performance liquid chromatography analysis showed immunoreactivity in the medium distributed between three peaks. For rat proinsulin II, the first coeluted with intact proinsulin; the second coeluted with des-64,65 split proinsulin (the product of endoproteolytic attack between the insulin A-chain and C-peptide followed by trimming of C-terminal basic residues by carboxypeptidase); the third (and minor peak) coeluted with native (fully processed) insulin. For human proinsulin, by contrast, the second peak coeluted with des-31,32 split proinsulin (split and trimmed at the B-chain/C-peptide junction). Analysis of cellular extracts showed intact proinsulin as the major product. The generation of the putative conversion intermediates and insulin was not due to proteolysis of proinsulin after its release but rather to an intracellular event. The data suggest that proinsulin, normally processed in secretory granules and released via the regulated pathway, may also be processed, albeit less efficiently, by the constitutive pathway conversion machinery. The comparison of the sites preferentially cleaved in rat II or human proinsulin suggests cleavage by endoprotease(s) with a preference for R/KXR/KR as substrate.

Insulin-like growth factor II expression in the developing human brain.

Insulin-like growth factor II (IGF-II) is a polypeptide hormone with insulin-like metabolic activity and neurotrophic activity in vitro that has been implicated in human brain development. In this study, we used northern blot analysis to examine the patterns of IGF-II mRNA expression in selected regions of 18 human brains from cases ranging in age from 20 gestational weeks to 2.5 years (median age 31 gestational weeks). The expression of IGF-II mRNA was widespread throughout the brain from midgestation through the perinatal period. Each region showed a distinct developmental pattern of expression and IGF-II mRNA levels varied considerably between regions. The highest levels of expression at all ages were in leptomeninges and choroid plexus. After two postnatal months, IGF-II mRNA virtually disappeared from parenchymal regions. Beyond the perinatal period, IGF-II expression persisted primarily in choroid plexus. Transcripts of both 6.0 and 4.8 kb were detected in most brain regions. A developmental change in the relative amounts of the two transcripts occurred in choroid plexus, leptomeninges and medulla. The expression of IGF-II mRNA in the brain parenchyma during the last half of gestation correlates with a period of major brain growth and supports the hypothesis that high levels of IGF-II stimulate the proliferation and differentiation of neural cells early in development.

Corruption of genomic databases with anomalous sequence.

We describe evidence that DNA sequences from vectors used for cloning and sequencing have been incorporated accidentally into eukaryotic entries in the GenBank database. These incorporations were not restricted to one type of vector or to a single mechanism. Many minor instances may have been the result of simple editing errors, but some entries contained large blocks of vector sequence that had been incorporated by contamination or other accidents during cloning. Some cases involved unusual rearrangements and areas of vector distant from the normal insertion sites. Matches to vector were found in 0.23% of 20,000 sequences analyzed in GenBank Release 63. Although the possibility of anomalous sequence incorporation has been recognized since the inception of GenBank and should be easy to avoid, recent evidence suggests that this problem is increasing more quickly than the database itself. The presence of anomalous sequence may have serious consequences for the interpretation and use of database entries, and will have an impact on issues of database management. The incorporated vector fragments described here may also be useful for a crude estimate of the fidelity of sequence information in the database. In alignments with well-defined ends, the matching sequences showed 96.8% identity to vector; when poorer matches with arbitrary limits were included, the aggregate identity to vector sequence was 94.8%.

Brief visual experience induces immediate early gene expression in the cat visual cortex.

Brief visual experience causes rapid physiological changes in the visual cortex during early postnatal development. A possible mediator of these effects is the immediate early genes whose protein products are involved in the rapid response of neurons to transsynaptic stimulation. Here we report evidence that the levels of immediate early gene mRNAs in the visual cortex can be altered by manipulating the visual environment. Specifically, we find that brief (1 h) visual experience in dark-reared cats causes dramatic transient inductions of egr1, c-fos, and junB mRNAs in the visual cortex but not in the frontal cortex. Levels of c-jun and c-myc mRNAs are unaffected. These results suggest that select combinatorial interactions of immediate early gene proteins are an important step in the cascade of events through which visually elicited activity controls visual cortical development.

Changes in immediate early gene expression during postnatal development of cat cortex and cerebellum.

Postnatal brain development involves interactions between extracellular signals and preprogrammed genetic events. Immediate early genes (IEGs) are a group of genes that are induced by extracellular signals and their protein products alter transcription by binding regulatory elements in other genes. Using Northern and slot blot analysis of total RNA isolated from visual cortex, frontal cortex, and cerebellum of cats, we have determined the postnatal development patterns of mRNA expression for 5 of these genes, c-fos, erg-1, c-jun, jun-B, and c-myc. Each gene had a distinct developmental pattern of mRNA expression, and for a given gene, these patterns were often different in different brain structures. These results suggest that temporal changes in the combinatorial interaction of different IEGs during early postnatal life are important for normal brain development.