Genetic analysis of type-1 insulin-like growth factor receptor signaling through insulin receptor substrate-1 and -2 in pancreatic beta cells

Signaling by receptor tyrosine kinases regulates pancreatic β cell function. Inactivation of insulin receptor (InsR), IGF1 receptor (Igf1r), or Irs1 in β cells impairs insulin secretion. Conversely, Irs2 ablation impairs β cell replication. In this study, we examined aspects of the Igf1r regulatory signaling cascade in β cells. To examine genetically the involvement of Irs1 and Irs2 in Igf1r signaling, we generated double mutant mice lacking Igf1r specifically in pancreatic β cells in an Irs1- or Irs2-null background. We show that Igf1r/Irs1 double mutants do not differ phenotypically from Irs1 single mutants and exhibit hyperinsulinemia, while maintaining normal β cell mass and glucose tolerance. In contrast, lack of Igf1r function in β cells aggravates the consequences of Irs2 ablation in double mutants and results in lethal diabetes by 6 weeks of age. This additivity of phenotypic manifestations indicates that Irs2 serves a pathway that is largely independent of Igf1r signaling. Consistent with the view that the latter is the InsR pathway, we show that combined β cell-specific knock-out of both Insr and Igf1r results in a phenocopy of double mutants lacking Igf1r and Irs2. We conclude that Igf1r signals primarily through Irs1 and affects insulin secretion, whereas β cell proliferation is mainly regulated by InsR using Irs2 as a downstream signaling effector. The insulin and IGF pathways appear to control β cell functions independently and selectively.     

GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.     

Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth

To examine the relationship between growth hormone (GH) and insulin-like growth factor 1 (IGF1) in controlling postnatal growth, we performed a comparative analysis of dwarfing phenotypes manifested in mouse mutants lacking GH receptor, IGF1, or both. This genetic study has provided conclusive evidence demonstrating that GH and IGF1 promote postnatal growth by both independent and common functions, as the growth retardation of double Ghr/Igf1 nullizygotes is more severe than that observed with either class of single mutant. In fact, the body weight of these double-mutant mice is only approximately 17% of normal and, in absolute magnitude ( approximately 5 g), only twice that of the smallest known mammal. Thus, the growth control pathway in which the components of the GH/IGF1 signaling systems participate constitutes the major determinant of body size. To complement this conclusion mainly based on extensive growth curve analyses, we also present details concerning the involvement of the GH/IGF1 axis in linear growth derived by a developmental study of long bone ossification in the mutants.     

Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress

Appropriate regulation of the Integrated stress response (ISR) and mTORC1 signaling are central for cell adaptation to starvation for amino acids. Halofuginone (HF) is a potent inhibitor of aminoacylation of tRNA^Pro with broad biomedical applications. Here, we show that in addition to translational control directed by activation of the ISR by general control nonderepressible 2 (GCN2), HF increased free amino acids and directed translation of genes involved in protein biogenesis via sustained mTORC1 signaling. Deletion of GCN2 reduced cell survival to HF whereas pharmacological inhibition of mTORC1 afforded protection. HF treatment of mice synchronously activated the GCN2-mediated ISR and mTORC1 in liver whereas Gcn2-null mice allowed greater mTORC1 activation to HF, resulting in liver steatosis and cell death. We conclude that HF causes an amino acid imbalance that uniquely activates both GCN2 and mTORC1. Loss of GCN2 during HF creates a disconnect between metabolic state and need, triggering proteostasis collapse.     

Reductions in serum IGF‐1 during aging impair health span

In lower or simple species, such as worms and flies, disruption of the insulin‐like growth factor (IGF)‐1 and the insulin signaling pathways has been shown to increase lifespan. In rodents, however, growth hormone (GH) regulates IGF‐1 levels in serum and tissues and can modulate lifespan via/or independent of IGF‐1. Rodent models, where the GH/IGF‐1 axis was ablated congenitally, show increased lifespan. However, in contrast to rodents where serum IGF‐1 levels are high throughout life, in humans, serum IGF‐1 peaks during puberty and declines thereafter during aging. Thus, animal models with congenital disruption of the GH/IGF‐1 axis are unable to clearly distinguish between developmental and age‐related effects of GH/IGF‐1 on health. To overcome this caveat, we developed an inducible liver IGF‐1‐deficient (iLID) mouse that allows temporal control of serum IGF‐1. Deletion of liver Igf ‐1 gene at one year of age reduced serum IGF‐1 by 70% and dramatically impaired health span of the iLID mice. Reductions in serum IGF‐1 were coupled with increased GH levels and increased basal STAT5B phosphorylation in livers of iLID mice. These changes were associated with increased liver weight, increased liver inflammation, increased oxidative stress in liver and muscle, and increased incidence of hepatic tumors. Lastly, despite elevations in serum GH, low levels of serum IGF‐1 from 1 year of age compromised skeletal integrity and accelerated bone loss. We conclude that an intact GH/IGF‐1 axis is essential to maintain health span and that elevated GH, even late in life, associates with increased pathology.     

Suppressor of cytokine signaling-2 deficiency induces molecular and metabolic changes that partially overlap with growth hormone-dependent effects

Suppressor of cytokine signaling-2 (SOCS2)-deficient (SOCS2-/-) mice grow significantly larger than their littermates, suggesting that SOCS2 is important in the negative regulation of the actions of GH and/or IGF-I. The aim of this study was to identify genes and metabolic parameters that might contribute to the SOCS2-/- phenotype. We demonstrate that although SOCS2 deficiency induces significant changes in hepatic gene expression, only a fraction of these overlap with known GH-induced effects in the liver, suggesting that SOCS2 might be an important regulator of other growth factors and cytokines acting on the liver. However, an important role of GH and IGF-I in the phenotype of these animals was demonstrated by an overexpression of IGF-binding protein-3 mRNA in the liver and increased levels of circulating IGF-binding protein-3. Other GH-like effects included diminished serum triglycerides and down-regulation of lipoprotein lipase in adipose tissue. Interestingly, SOCS2-/- mice did not differ from their wild-type littermates in glucose or insulin tolerance tests, which is in contrast with the known diabetogenic effects of GH. Furthermore, there was no evidence of impaired insulin signaling in primary hepatocytes isolated from SOCS2-/- mice. Moreover, increased expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA was detected in skeletal muscle, which might contribute to normal glycemic control despite the apparent overactivity of the GH/IGF-I axis. Our data indicate that SOCS2 deficiency partially mimics a state of increased GH activity, but also results in changes that cannot be related to known GH effects.     

Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis

Amino acid availability is sensed by various signaling molecules, including general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). However, it is unclear how these sensors are associated with cancer cell survival under low amino acid availability. In the present study, we investigated AKT activation in non-small cell lung cancer (NSCLC) cells deprived of each one of 20 amino acids. Among the 20 amino acids, deprivation of glutamine, arginine, methionine, and lysine induced AKT activation. AKT activation was induced by GCN2/ATF4/REDD1 axis-mediated mTORC2 activation under amino acid deprivation. In CRISPR-Cas9-mediated REDD1-knockout cells, AKT activation was not induced by amino acid deprivation, indicating that REDD1 plays a major role in AKT activation under amino acid deprivation. Knockout of REDD1 sensitized cells cultured under glutamine deprivation conditions to radiotherapy. Taken together, GCN2/ATF4/REDD1 axis induced by amino acid deprivation promotes cell survival signal, which might be a potential target for cancer therapy.Subject terms: Cancer metabolism, Cell death     

IGF‐I regulates the age‐dependent signaling peptide humanin

Aging is influenced by endocrine pathways including the growth hormone/insulin‐like growth factor‐1 (GH/IGF) axis. Mitochondrial function has also been linked to the aging process, but the relevant mitochondrial signals mediating the effects of mitochondria are poorly understood. Humanin is a novel signaling peptide that acts as a potent regulator of cellular stress responses and protects from a variety of in vitro and in vivo toxic and metabolic insults. The circulating levels of humanin decline with age in mice and humans. Here, we demonstrate a negative correlation between the activity of the GH‐IGF axis and the levels of humanin, as well as a positive correlation between humanin and lifespan in mouse models with altered GH/IGF‐I axis. Long‐lived, GH‐deficient Ames mice displayed elevated humanin levels, while short‐lived GH‐transgenic mice have reduced humanin levels. Furthermore, treatment with GH or IGF‐I reduced circulating humanin levels in both mice and human subjects. Our results indicate that GH and IGF are potent regulators of humanin levels and that humanin levels correlate with lifespan in mice. This suggests that humanin represents a circulating mitochondrial signal that participates in modulating the aging process, adding a coordinated mitochondrial element to the endocrine regulation of aging.     

Systems-based Discovery of Tomatidine as a Natural Small Molecule Inhibitor of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. To address this problem, we used a systems-based discovery strategy to search for a small molecule whose mRNA expression signature negatively correlates to mRNA expression signatures of human skeletal muscle atrophy. This strategy identified a natural small molecule from tomato plants, tomatidine. Using cultured skeletal myotubes from both humans and mice, we found that tomatidine stimulated mTORC1 signaling and anabolism, leading to accumulation of protein and mitochondria, and ultimately, cell growth. Furthermore, in mice, tomatidine increased skeletal muscle mTORC1 signaling, reduced skeletal muscle atrophy, enhanced recovery from skeletal muscle atrophy, stimulated skeletal muscle hypertrophy, and increased strength and exercise capacity. Collectively, these results identify tomatidine as a novel small molecule inhibitor of muscle atrophy. Tomatidine may have utility as a therapeutic agent or lead compound for skeletal muscle atrophy.     

The rapid activation of protein synthesis by growth hormone requires signaling through mTOR

An important function of growth hormone (GH) is to promote cell and tissue growth, and a key component of these effects is the stimulation of protein synthesis. In this study, we demonstrate that, in H4IIE hepatoma cells, GH acutely activated protein synthesis through signaling via the mammalian target of rapamycin (mTOR) and specifically through the rapamycin-sensitive mTOR complex 1 (mTORC1). GH treatment enhanced the phosphorylation of two targets of mTOR signaling, 4E-BP1 and ribosomal protein S6. Phosphorylation of S6 and 4E-BP1 was maximal at 30-45 min and 10-20 min after GH stimulation, respectively. Both proteins modulate components of the translational machinery. The GH-induced phosphorylation of 4E-BP1 led to its dissociation from eIF4E and increased binding of eIF4E to eIF4G to form (active) eIF4F complexes. The ability of GH to stimulate the phosphorylation of S6 and 4E-BP1 was blocked by rapamycin. GH also led to the dephosphorylation of a third translational component linked to mTORC1, the elongation factor eEF2. Its regulation followed complex biphasic kinetics, both phases of which required mTOR signaling. GH rapidly activated both the MAP kinase (ERK) and PI 3-kinase pathways. Signaling through PI 3-kinase alone was, however, sufficient to activate the downstream mTORC1 pathway. Consistent with this, GH increased the phosphorylation of TSC2, an upstream regulator of mTORC1, at sites that are targets for Akt/PKB. Finally, the activation of overall protein synthesis by GH in H4IIE cells was essentially completely inhibited by wortmannin or rapamycin. These results demonstrate for the first time that mTORC1 plays a major role in the rapid activation of protein synthesis by GH.     

Effects of mutations in the insulin-like growth factor signaling system on embryonic pancreas development and beta-cell compensation to insulin resistance

Insulin and insulin-like growth factors (IGF) play overlapping and complementary roles in pancreatic beta-cell function and peripheral metabolism. In this study, we have analyzed mice bearing loss-of-function mutations of the insulin/IGF signaling systems. Combined inactivation of insulin receptor (Insr) and Igf1 receptor (Igf1r), but not of either receptor alone, resulted in a 90% decrease in the size of the exocrine pancreas, because of decreased cellular proliferation. In contrast to the findings in the exocrine compartment, endocrine alpha- and beta-cell development was unperturbed. Combined ablation of Igf1 and Igf2, the ligands for these two receptors, resulted in an identical phenotype. We also examined the effect of heterozygous null Igf1r mutations on glucose homeostasis in adult mice. Igf1r haploinsufficiency did not affect insulin action and compensatory beta-cell growth in insulin-resistant mice with combined Insr and Igf1r heterozygous null mutations, resulting in a considerably milder phenotype than combined haploinsufficiency for Insr and its main signaling substrates, Irs1 and Irs2. We conclude that Igf1r and Insr are required for embryonic development of the exocrine but not of the endocrine pancreas and that defects of Igf1r do not alter glucose homeostasis as long as the insulin receptor system remains intact.     

Insulin‐like growth factor 1 deficiency exacerbates hypertension‐induced cerebral microhemorrhages in mice,mimicking the aging phenotype

Clinical and experimental studies show that aging exacerbates hypertension‐induced cerebral microhemorrhages (CMHs), which progressively impair neuronal function. There is growing evidence that aging promotes insulin‐like growth factor 1 (IGF‐1) deficiency, which compromises multiple aspects of cerebromicrovascular and brain health. To determine the role of IGF‐1 deficiency in the pathogenesis of CMHs, we induced hypertension in mice with liver‐specific knockdown of IGF‐1 (Igf1^f/f + TBG‐Cre‐AAV8) and control mice by angiotensin II plus l ‐NAME treatment. In IGF‐1‐deficient mice, the same level of hypertension led to significantly earlier onset and increased incidence and neurological consequences of CMHs, as compared to control mice, as shown by neurological examination, gait analysis, and histological assessment of CMHs in serial brain sections. Previous studies showed that in aging, increased oxidative stress‐mediated matrix metalloprotease (MMP) activation importantly contributes to the pathogenesis of CMHs. Thus, it is significant that hypertension‐induced cerebrovascular oxidative stress and MMP activation were increased in IGF‐1‐deficient mice. We found that IGF‐1 deficiency impaired hypertension‐induced adaptive media hypertrophy and extracellular matrix remodeling, which together with the increased MMP activation likely also contributes to increased fragility of intracerebral arterioles. Collectively, IGF‐1 deficiency promotes the pathogenesis of CMHs, mimicking the aging phenotype, which likely contribute to its deleterious effect on cognitive function. Therapeutic strategies that upregulate IGF‐1 signaling in the cerebral vessels and/or reduce microvascular oxidative stress, and MMP activation may be useful for the prevention of CMHs, protecting cognitive function in high‐risk elderly patients.     

Growth hormone modulates hypothalamic inflammation in long‐lived pituitary dwarf mice

Mice in which the genes for growth hormone (GH) or GH receptor (GHR^?/?) are disrupted from conception are dwarfs, possess low levels of IGF‐1 and insulin, have low rates of cancer and diabetes, and are extremely long‐lived. Median longevity is also increased in mice with deletion of hypothalamic GH‐releasing hormone (GHRH), which leads to isolated GH deficiency. The remarkable extension of longevity in hypopituitary Ames dwarf mice can be reversed by a 6‐week course of GH injections started at the age of 2 weeks. Here, we demonstrate that mutations that interfere with GH production or response, in the Snell dwarf, Ames dwarf, or GHR^?/? mice lead to reduced formation of both orexigenic agouti‐related peptide (AgRP) and anorexigenic proopiomelanocortin (POMC) projections to the main hypothalamic projection areas: the arcuate nucleus (ARH), paraventricular nucleus (PVH), and dorsomedial nucleus (DMH). These mutations also reduce hypothalamic inflammation in 18‐month‐old mice. GH injections, between 2 and 8 weeks of age, reversed both effects in Ames dwarf mice. Disruption of GHR specifically in liver (LiGHRKO), a mutation that reduces circulating IGF‐1 but does not lead to lifespan extension, had no effect on hypothalamic projections or inflammation, suggesting an effect of GH, rather than peripheral IGF‐1, on hypothalamic development. Hypothalamic leptin signaling, as monitored by induction of pStat3, is not impaired by GHR deficiency. Together, these results suggest that early‐life disruption of GH signaling produces long‐term hypothalamic changes that may contribute to the longevity of GH‐deficient and GH‐resistant mice.     

IGF‐1 deficiency impairs neurovascular coupling in mice: implications for cerebromicrovascular aging

Aging is associated with marked deficiency in circulating IGF‐1, which has been shown to contribute to age‐related cognitive decline. Impairment of moment‐to‐moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of age‐related cognitive impairment. To establish the link between IGF‐1 deficiency and cerebromicrovascular impairment, neurovascular coupling mechanisms were studied in a novel mouse model of IGF‐1 deficiency (Igf1^f/f‐TBG‐Cre‐AAV8) and accelerated vascular aging. We found that IGF‐1‐deficient mice exhibit neurovascular uncoupling and show a deficit in hippocampal‐dependent spatial memory test, mimicking the aging phenotype. IGF‐1 deficiency significantly impaired cerebromicrovascular endothelial function decreasing NO mediation of neurovascular coupling. IGF‐1 deficiency also impaired glutamate‐mediated CBF responses, likely due to dysregulation of astrocytic expression of metabotropic glutamate receptors and impairing mediation of CBF responses by eicosanoid gliotransmitters. Collectively, we demonstrate that IGF‐1 deficiency promotes cerebromicrovascular dysfunction and neurovascular uncoupling mimicking the aging phenotype, which are likely to contribute to cognitive impairment.     

Pregnancy-associated Plasma Protein A (PAPP-A) Modulates the Early Developmental Rate in Zebrafish Independently of Its Proteolytic Activity

Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.