Jihui Zhang

In vivo effects of insulin‐like growth factor‐I (IGF‐I) on prenatal and early postnatal development of the central nervous system

The in vivo actions of insulin‐like growth factor‐I (IGF‐I) on prenatal and early postnatal brain development were investigated in transgenic (Tg) mice that overexpress IGF‐I prenatally under the control of regulatory sequences from the nestin gene. Tg mice demonstrated increases in brain weight of 6% by embryonic day (E) 18 and 27% by postnatal day (P) 12. In Tg embryos at E16, the volume of the cortical plate was significantly increased by 52% and total cell number was increased by 54%. S‐phase labeling with 5‐bromo‐2′‐deoxyuridine revealed a 13–15% increase in the proportion of labeled neuroepithelial cells in Tg embryos at E14. In Tg mice at P12, significant increases in regional tissue volumes were detected in the cerebral cortex (29%), subcortical white matter (52%), caudate‐putamen (37%), hippocampus (49%), dentate gyrus (71%) and habenular complex (48%). Tg mice exhibited significant increases in the total number of neurons in the cerebral cortex (27%), caudate‐putamen (27%), dentate gyrus (69%), medial habenular nucleus (61%) and lateral habenular nucleus (36%). In the cerebral cortex and subcortical white matter of Tg mice, the total numbers of glial cells were significantly increased by 37% and 42%, respectively. The numerical density of apoptotic cells in the cerebral cortex, labeled by antibodies against active caspase‐3, was reduced by 26% in Tg mice at P7. Our results demonstrate that IGF‐I can both promote proliferation of neural cells in the embryonic central nervous system in vivo and inhibit their apoptosis during postnatal life.

Insulin-like growth factor type 1 receptor signaling in the cells of oligodendrocyte lineage is required for normal in vivo oligodendrocyte development and myelination

Insulin‐like growth factor‐I (IGF‐I) has been shown to be a potent agent in promoting the growth and differentiation of oligodendrocyte precursors, and in stimulating myelination during development and following injury. To definitively determine whether IGF‐I acts directly on the cells of oligodendrocyte lineage, we generated lines of mice in which the type 1 IGF receptor gene (igf1r) was conditionally ablated either in Olig1 or proteolipid protein expressing cells (termed IGF1Rpre‐oligo‐ko and IGF1Roligo‐ko mice, respectively). Compared with wild type mice, IGF1Rpre‐oligo‐ko mice had a decreased volume (by 35–55%) and cell number (by 54–70%) in the corpus callosum (CC) and anterior commissure at 2 and 6 weeks of age, respectively. IGF1Roligo‐ko mice by 25 weeks of age also showed reductions, albeit less marked, in CC volume and cell number. Unlike astrocytes, the percentage of NG2+ oligodendrocyte precursors was decreased by ∼13% in 2‐week‐old IGF1Rpre‐oligo‐ko mice, while the percentage of CC1+ mature oligodendrocytes was decreased by ∼24% in 6‐week‐old IGF1Rpre‐oligo‐ko mice and ∼25% in 25‐week‐old IGF1Roligo‐ko mice. The reduction in these cells is apparently a result of decreased proliferation and increased apoptosis. These results indicate that IGF‐I directly affects oligodendrocytes and myelination in vivo via IGF1R, and that IGF1R signaling in the cells of oligodendrocyte lineage is required for normal oligodendrocyte development and myelination. These data also provide a fundamental basis for developing strategies with the potential to target IGF‐IGF1R signaling pathways in oligodendrocyte lineage cells for the treatment of demyelinating disorders.

Expression of insulin-like growth factor system genes during the early postnatal neurogenesis in the mouse hippocampus

Insulin‐like growth factor‐1 (IGF‐1) is essential to hippocampal neurogenesis and the neuronal response to hypoxia/ischemia injury. IGF (IGF‐1 and ‐2) signaling is mediated primarily by the type 1 IGF receptor (IGF‐1R) and modulated by six high‐affinity binding proteins (IGFBP) and the type 2 IGF receptor (IGF‐2R), collectively termed IGF system proteins. Defining the precise cells that express each is essential to understanding their roles. With the exception of IGFBP‐1, we found that mouse hippocampus expresses mRNA for each of these proteins during the first 2 weeks of postnatal life. Compared to postnatal day 14 (P14), mRNA abundance at P5 was higher for IGF‐1, IGFBP‐2, ‐3, and ‐5 (by 71%, 108%, 100%, and 98%, respectively), lower for IGF‐2, IGF‐2R, and IGFBP‐6 (by 65%, 78%, and 44%, respectively), and unchanged for IGF‐1R and IGFBP‐4. Using laser capture microdissection (LCM), we found that granule neurons and pyramidal neurons exhibited identical patterns of expression of IGF‐1, IGF‐1R, IGF‐2R, IGFBP‐2, and ‐4, but did not express other IGF system genes. We then compared IGF system expression in mature granule neurons and their progenitors. Progenitors exhibited higher mRNA levels of IGF‐1 and IGF‐1R (by 130% and 86%, respectively), lower levels of IGF‐2R (by 72%), and similar levels of IGFBP‐4. Our data support a role for IGF in hippocampal neurogenesis and provide evidence that IGF actions are regulated within a defined in vivo milieu.

Hepatic mRNAs up-regulated by starvation: an expression profile determined by suppression subtractive hybridization

Delineating the molecular basis for the metabolic switch from the well‐fed state to starvation is crucial to understanding nutritionally regulated metabolic abnormalities. We have examined the molecular events associated with nutrient deprivation, using suppression subtractive hybridization to define the transcriptional programs up‐regulated in rat liver by starvation. Of the genes that displayed significant increases in their hepatic mRNA levels following 48‐h starvation, most could be assigned to one of five major functional classes. We found up‐regulation of genes involved in energy and protein metabolism, genes that respond to stress, and genes encoding nutrient transporters or signaling transducers. The genes with functions in energy and protein metabolism have roles in initiating gluconeogenesis, switching fuel sources from carbohydrates to fatty acids, and protein turnover. A variety of stress response genes, including acute‐phase reactants, exhibited a marked increased in expression, indicating an attempt to restore homeostasis. The expression of several integrated membrane nutrient transporters that supply essential metabolic substrates was increased dramatically. Some known cytosolic signal transducers, likely involved in the metabolic shift from an anabolic to a catabolic state and in the stress response, were significantly enhanced as well. We also observed increased expression of a variety of other known and novel genes. Collectively, our findings indicate that starvation stimulates multiple signaling pathways, which likely lead to extensive metabolic alterations in the liver. These data should serve to enhance our understanding of the molecular mechanisms underlying energy and nitrogen expenditure in the starved state.

A novel mutation (E767K) in the second extracellular loop of the calcium sensing receptor in a family with autosomal dominant hypocalcemia

Autosomal dominant hypocalcemia resulting from gain‐of‐function mutations of the calcium sensing receptor (CASR) is a rare familial disorder that can become evident at any age. We report a novel mutation (E767K) of the CASR in a family with autosomal dominant hypocalcemia. Ten members of the family had a history of hypocalcemia. The index case exhibited marked hypocalcemia and seizures in the newborn period, while her father who also has hypocalcemia, was largely asymptomatic except for a myocardial infarction‐like event at 21 years of age, a new presentation of the disorder. The E767K mutation, which resides in the second extracellular loop adjacent to the fifth transmembrane domain, co‐segregated with hypocalcemia in these two individuals. Both subjects are heterozygous for the mutation. The proband is also heterozygous for the previously reported CASR polymorphism of G990R in the intracellular domain, while her father is homozygous. The co‐segregation of this naturally occurring mutation with autosomal dominant hypocalcemia supports the previously reported experimental model in which it was proposed that the three acidic residues (767, 758, and 759) in exo‐loop 2 in CASR help maintain an inactive conformation of the receptor.

Divergent regulation of proteasomes by insulin-like growth factor I and growth hormone in skeletal muscle of rats made catabolic with dexamethasone

Insulin-like growth factor I (IGF-I) and growth hormone (GH) exert their anabolic actions by increasing protein synthesis, but only IGF-I has been reported to impede protein breakdown. Using a model of myofibrillar catabolism produced by dexamethasone (Dex) we have reported that IGF-I down-regulates Dex-induced mRNAs for Ubiquitin (Ub) and Ub-conjugating enzymes (E2) in skeletal muscle, whereas GH had no significant effect. In the present study, we used the same model to determine whether IGF-I (0.35 mg/100 g BW) and/or GH (0.3 mg/100 g BW) have effects on proteasome subunit mRNAs in skeletal muscles of rats treated with Dex (0.5 mg/100 g BW) for 3 days. Dex caused significant increases in C-2, -3, and -8 proteasome subunit mRNAs (6.0-, 4.0-, and 6.6-fold increases, respectively). Injections of IGF-I in Dex-treated animals caused significant suppression of transcripts for C-2, -3, and -8 (32%, 42%, and 40%, respectively). GH restored the serum IGF-I levels in Dex treated animals, but caused further increases in proteasome subunit mRNAs (C-2, 35%; C-3, 34.5%; C-8, 33%; C-6, 42%; C-5, 32%; C-9, 37%). Administration of IGF-I in the Dex/GH-treated animals decreased the mRNAs of proteasome subunits in a manner and degree similar to those observed in the Dex/IGF-I group. Surprisingly, injection of GH alone in normal animals increased proteasome subunit mRNAs in skeletal muscle (C-2, 85%; C-3, 109%; C-8, 91%). This effect of GH on proteasome subunit mRNAs was also observed in liver. These findings suggest, therefore, that suppression of Dex-induced expression of proteasome subunit mRNAs in skeletal muscle is one of the mechanisms by which IGF-I exerts its antiproteolytic activity in catabolic states. On the other hand, the biological function of GH in regulating proteasome subunits needs further investigation.

Down-regulation of 14-3-3 η gene expression by IGF-I in mouse cerebellum during postnatal development

Insulin-like growth factor I (IGF-I) overexpression in the postnatal cerebellum of transgenic (Tg) mice results in remarkable cerebellar overgrowth characterized by a near doubling of granule cell number that is predominantly due to inhibition of apoptosis. Using this Tg model we set out to investigate IGF-I anti-apoptotic mechanisms by defining the influence of IGF-I on gene expression. Using a cDNA array technique, we screened a total of 243 mouse apoptosis-related genes, and found that 14-3-3 eta gene expression was significantly reduced in the cerebella of Tg mice compared with their wild-type (Wt) littermates. Using Northern blot analysis to corroborate our microarray finding, we showed that 14-3-3 eta mRNA abundance was decreased from postnatal day P5 through P17. Nonetheless, the expression pattern of 14-3-3 eta in Tg mice followed the same pattern observed in Wt mice, and was indistinguishable from that in Wt mice at P20 and P23. 14-3-3 eta protein abundance, as determined by Western immunoblot analyses, showed similar decreases in the cerebella of Tg mice. In situ hybridization demonstrated that 14-3-3 eta was predominantly, if not exclusively, expressed and regulated in Purkinje cells. 14-3-3 proteins have multiple functions, including participation in pathways that favor cell survival. Our finding of IGF-I-induced down-regulation of 14-3-3 eta expression in Purkinje cell at a time when IGF-I promotes granule cell survival leads us to speculate that down-regulation of 14-3-3 eta may: (a) serve a negative feedback role to modulate Purkinje cell survival, i.e. limit Purkinje cell number, and/or (b) function as part of a distinct signaling mechanism, perhaps one that augments the capacity of Purkinje cells to promote granule cell survival.

Pitfalls of PCR-based strategy for genotyping cre-loxP mice

Since the description of the application of the bacteriophage cre-loxP system to mammalian cells (1) and the subsequent generation of the first cre-loxP mutant mouse (2), the cre-loxP system has been widely used to generate conditional knockout mice. Accurate genotyping of cre-loxP mice is critical to characterizing these mice. Because Southern blot analysis is tedious and time-consuming, genotyping often uses PCR strategies (3,4). While detection of a cre transgene in mouse genome by PCR is straightforward, errors in differentiating among wild-type (wt), lox, and the targeted deletion (del) alleles can be confounding. Here we report pitfalls that we have encountered in the course of generating mice with a conditional knockout of the type 1 insulin-like growth factor receptor (igf1r) in the central nervous system (CNS). n nTo direct cre expression to the CNS, we utilized the intron 2 of human nestin gene and a minimal viral promoter to drive transgene cre expression. Nestin intron 2 contains a CNS-specific enhancer that is widely used to target transgene expression to the CNS (5). A construct containing human nestin intron 2 sequences was kindly provided by Dr. Claudia Kappen of the Mayo Clinic. Details of the generation of nestin-cre transgenic line will be described elsewhere. The igf1r-loxP line, in which the exon 3 of igf1r is flanked by two loxP sites (6), was the gift of Dr. Argiris Efstratiadis, Columbia University. Protocols involving these animals were approved by the Institutional Animal Care and Use Committee. Routinely we bred the igf1r-loxP homozygote (lox/lox) with cre transgenic, igf1r-loxP heterozygotes (wt/lox/cre) to generate progeny of desired genotypes. To genotype the igf1r alleles, primer 1 (P1), 5′-CTCCAGAGCACATACTGACTCC-3′, and P2, 5′-AGCCAAATAAGCCCCAGTAACC-3′, were used to detect the wt allele (371 bp) and the lox allele (421 bp); while P3, 5′-AGGAACCCACAGTACTAGGAAC-3′, and P4, 5-GACTAACAGAGACTGCCAACAC-3′, were used to detect the del allele (2.5 kb), the wt allele (5.0 kb), and the lox allele (5.1 kb) (Figure 1A). To genotype the cre transgene, two primers (5′-GCCAGCTAAACATGCTTCATC-3′ and 5′-ATTGCCCCTGTTTCACTATCC-3) were utilized to amplify a cre-specific product of 727 bp. PCR parameters were 35 (for igf1r) or 30 (for cre) cycles of 94°C for 45 s, 65°C (for igf1r P1 and P2, P3 and P4) or 62°C (for cre) for 45 s, 72°C for 45s (for igf1r P1 and P2, and cre) or 2 min and 45 s (for igf1r P3 and P4). Genotyping was performed using genomic DNA extracted from tails. n n n nFigure 1 n nGenotyping of igf1r alleles and cre transgene by PCR n n n nAs expected, PCR amplification of the 727-bp cre-specific product reliably identified transgenic mice. Using P1 and P2 primers to genotype igf1r alleles, a non-cre-transgenic wt/lox heterozygote was identified by the PCR products of expected size (wt, 371 bp; lox, 421 bp). Both wt/wt and lox/lox homozygotes were also reliably identified. When wt/lox heterozygotes were cre-positive, however, considerable variability in the intensity of the 421-bp lox allele product was evident. While in some mice this lox allele product was only moderately reduced, in others it was so diminished as to be difficult to identify (Figure 1B). The latter could lead to erroneous genotyping of wt/lox mice as wt/wt mice. The apparent preferential amplification of the wt allele suggested a loss of the lox allele in tails of cre transgenic wt/lox mice. n nThese findings prompted us to hypothesize that cre-mediated and loxP-dependent genomic recombination occurred in the tail, resulting in the loss of the lox alleles. To test this hypothesis, we performed PCR with primers spanning the loxP sites (P3 and P4) using DNA from tail and cerebellum (a targeted site of the igf1r deletion). PCRs from cerebellar DNA of a cre-positive wt/lox heterozygous mouse yielded the expected 2.5-kb del product. As predicted, this product was digested by BamHI into three fragments of 1.8, 0.53, and 0.17 kb. Consistent with our hypothesis, the 2.5-kb del product with the same pattern of BamHI digestion also was detected in tail DNA. Furthermore, our data indicate that amplification of the del product is dependent on the presence of the cre transgene and at least one lox allele. First, in non-cre-transgenic loxP mice (wt/lox or lox/lox) the del product was not detected in either cerebellar or tail DNA; instead, we found PCR products consistent in size with the 5.0 kb wt allele or the 5.1 kb lox allele. Secondly, in cre-transgenic wt/wt mice, only the 5.0 kb wt allele, but not the del allele, was detected (Figure 1C). These findings confirm that cre-mediated, specific deletion of igf1r indeed occurred in tails of cre transgenic wt/lox mice. n nA number of reports suggest that the cre transgene in our nestin-cre-igf1r-loxP mice is expressed in tail tissues. Nestin has been shown to be expressed in neural progenitor cells, developing myogenic and mensenchymal cells, hair follicle cells, proliferating endothelial cells, and nascent blood vessels (7). When the CNS-specific intron 2 enhancer was used to drive the expression of green fluorescent protein (GFP) in a transgenic line, GFP was highly expressed in hair follicle progenitor cells (8). Such GFP-labeled hair follicle progenitor cells were shown to be capable of forming neurons and blood vessels (9,10). These findings indicate that hair follicle progenitors and newly formed vascular cells in tails of our nestin-cre-igf1r-loxP mice express Cre recombinase and account for our results. n nWe believe that our findings are not limited to the nestin-cre-igf1r-loxP mice used in this study. Cre transgenic lines may exhibit varying degrees of cre expression in nontarget tissues. Such nonspecific expression can be due to the influence of the chromosomal integration site, known as positional effects or transgene leakage, which is often independent of the promoter/enhancer used in the transgene. More commonly, however, nonspecific sites of expression result from use of a promoter/enhancer that does not function as specifically as expected. As more cre-loxP conditional knockout mice are generated, especially using tissue/cell-specific promoters/enhancers that have not been thoroughly studied, more undesirable cre-mediated gene deletions will likely occur. In such cases, the genotyping of cre-loxP mice may be affected, and these undesired knockout effects can confound the interpretation of the function of the gene of interest. n nOur experience indicates that caution should be exercised when a two-primer PCR strategy is used to detect wt and lox alleles for genotyping cre-loxP mice. The detection of a del allele indicates the presence of the lox allele and the cre transgene. Leneuve et al. (4) have designed an elegant PCR strategy using three primers to simultaneously detect wt, lox, and del alleles in target tissues. This strategy also enables assessment of the efficiency of cre-mediated recombination. Because cre transgene expression in nontarget tissues is not anticipated, two-primer strategies usually are used to detect wt and lox alleles with DNA from nontarget tissues. Our findings point to the importance of the three-primer PCR approach to genotype cre-loxP mice even when DNA from nontarget tissues is studied.

Hepatic reduction of insulin-like growth factor (IGF)-I and IGF binding protein-3 that results from fasting is not attenuated in genetically obese rats

Fasting or caloric restriction causes substantial reductions in serum IGF-I in normal weight humans and animals, and reductions of liver IGF-I and IGFBP-3 mRNAs in animals. Obese humans, however, have attenuated and delayed decrements in IGF-I in serum when subjected to caloric restriction. Obese Zucker rats show a clear tendency to preserve body protein during fasting. To determine whether obesity opposes the effects of fasting on IGF-I and IGFBP-3, and thereby contributes to preservation of lean tissue, we have examined the effect of 72 h of fasting on IGF-I and IGFBP-3 in lean and obese Zucker rats. We observe that between lean and obese animals, fasting for 72 h produces similar decrements in body weight, serum IGF-I, liver IGF-I mRNA, serum IGFBP-3 and liver IGFBP-3 mRNA. Our finding that the reduction of IGF-I and IGFBP-3 in liver that results from 72 h of fasting is not attenuated in obese Zucker rats raises the possibility that conservation of lean tissue in these animals during fasting is not related to the hepatic production of IGF-I and IGFBP-3.