Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Insulin‐like signalling in neurons controls lifespan in C. elegans

Insulin‐like signalling controls C. elegans lifespan, development and metabolism. Mutations that weaken this insulin‐like signalling pathway extend lifespan. Severe mutations abolishing insulin‐like signalling cause animals to arrest development as dauer larvae, a larval form specialized for stress resistance and long‐term survival. A number of the genes acting in this pathway have been cloned, including daf‐2, which encodes a homolog of vertebrate insulin/IGF‐I receptors, and age‐1, encoding the C. elegans homolog of the PI(3)K p110 catalytic subunit. In order to identify cells from which insulin‐like signalling controls lifespan and development, transgenic animals were constructed which possessed insulin‐like signalling only in specific cell types. To achieve this, cell‐type specific promoters were used to drive expression of daf‐2 or age‐1 cDNAs in daf‐2(–/–) or age‐1(–/–) backgrounds, respectively. By utilizing this strategy, we could restore wild‐type daf‐2 or age‐1 activity only in cells that are capable of expressing each transgene. Restoring insulin‐like signalling to the nervous system of daf‐2 or age‐1 mutants could rescue long lifespan. This result was specific for transgenes restoring insulin‐like signalling to the nervous system. Expressing daf‐2 or age‐1 cDNAs from muscle‐ or intestinally‐restricted promoters was insufficient to rescue lifespan. In contrast, age‐1 and daf‐2 expression in either neuronal or non‐neuronal cell types rescued dauer larval arrest in the mutants. These findings demonstrate that insulin‐like signalling pathways in the nervous system control C. elegans lifespan.     

Phosphatidylinositol 3‐kinase‐mediated regulation of neuronal apoptosis and necrosis by insulin and IGF‐I

We examined effects of two insulin‐like growth factors, insulin and insulin‐like growth factor‐I (IGF‐I), against apoptosis, excitotoxicity, and free radical neurotoxicity in cortical cell cultures. Like IGF‐I, insulin attenuated serum deprivation‐induced neuronal apoptosis in a dose‐dependent manner at 10–100 ng/mL. The anti‐apoptosis effect of insulin against serum deprivation disappeared by addition of a broad protein kinase inhibitor, staurosporine, but not by calphostin C, a selective protein kinase C inhibitor. Addition of PD98059, a mitogen‐activated protein kinase kinase (MAPKK) inhibitor, blocked insulin‐induced activation of extracellular signal‐regulated protein kinases (ERK1/2) without altering the neuroprotective effect of insulin. Cortical neurons underwent activation of phosphatidylinositol (PI) 3‐kinase as early as 1 min after exposure to insulin. Inclusion of wortmannin or LY294002, selective inhibitors of PI 3‐K, reversed the insulin effect against apoptosis. In contrast to the anti‐apoptosis effect, neither insulin nor IGF‐I protected excitotoxic neuronal necrosis following continuous exposure to 15 μM N‐methyl‐d ‐aspartate or 40 μM kainate for 24 h. Surprisingly, concurrent inclusion of 50 ng/mL insulin or IGF‐I aggravated free radical‐induced neuronal necrosis over 24 h following continuous exposure to 10 μM Fe²⁺ or 100 μM buthionine sulfoximine. Wortmannin or LY294002 also reversed this potentiation effect of insulin. These results suggest that insulin‐ like growth factors act as anti‐apoptosis factor and pro‐oxidant depending uon the activation of PI 3‐kinase. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 536–546, 1999     

pkc-1 regulates daf-2 insulin/IGF signalling-dependent control of dauer formation in Caenorhabditis elegans

In Caenorhabditis elegans, the insulin/IGF pathway participates in the decision to initiate dauer development. Dauer is a diapause stage that is triggered by environmental stresses, such as a lack of nutrients. Insulin/IGF receptor mutants arrest constitutively in dauer, an effect that can be suppressed by mutations in other elements of the insulin/IGF pathway or by a reduction in the activity of the nuclear hormone receptor daf‐12. We have isolated a pkc‐1 mutant that acts as a novel suppressor of the dauer phenotypes caused by insulin/IGF receptor mutations. Interactions between insulin/IGF mutants and the pkc‐1 suppressor mutant are similar to those described for daf‐12 or the DAF‐12 coregulator din‐1. Moreover, we show that the expression of the DAF‐12 target daf‐9, which is normally elevated upon a reduction in insulin/IGF receptor activity, is suppressed in a pkc‐1 mutant background, suggesting that pkc‐1 could link the daf‐12 and insulin/IGF pathways. pkc‐1 has been implicated in the regulation of peptide neurosecretion in C. elegans. Although we demonstrate that pkc‐1 expression in the nervous system regulates dauer formation, our results suggest that the requirement for pkc‐1 in neurosecretion is independent of its role in modulating insulin/IGF signalling. pkc‐1 belongs to the novel protein kinase C (nPKC) family, members of which have been implicated in insulin resistance and diabetes in mammals, suggesting a conserved role for pkc‐1 in the regulation of the insulin/IGF pathway.     

Temporal pattern of neuronal insulin release during Caenorhabditis elegans aging: Role of redox homeostasis

The insulin‐IGF‐1/DAF‐2 pathway has a central role in the determination of aging and longevity in Caenorhabditis elegans and other organisms. In this paper, we measured neuronal insulin secretion (using INS‐22::Venus) during C. elegans lifespan and monitored how this secretion is modified by redox homeostasis. We showed that INS‐22::Venus secretion fluctuates during the organism lifetime reaching maximum levels in the active reproductive stage. We also demonstrate that long‐lived daf‐2 insulin receptor mutants show remarkable low levels of INS‐22::Venus secretion. In contrast, we found that short‐lived mutant worms that lack the oxidation repair enzyme MSRA‐1 show increased levels of INS‐22::Venus secretion, specifically during the reproductive stage. MSRA‐1 is a target of the insulin‐IGF‐1/DAF‐2 pathway, and the expression of this antioxidant enzyme exclusively in the nervous system rescues the mutant insulin release phenotype and longevity. The msra‐1 mutant phenotype can also be reverted by antioxidant treatment during the active reproductive stage. We showed for the first time that there is a pattern of neuronal insulin release with a noticeable increment during the peak of reproduction. Our results suggest that redox homeostasis can modulate longevity through the regulation of insulin secretion, and that the insulin‐IGF‐1/DAF‐2 pathway could be regulated, at least in part, by a feedback loop. These findings highlight the importance of timing for therapeutic interventions aimed at improving health span.     

Central insulin‐like growth factor‐1 (IGF‐1) restores whole‐body insulin action in a model of age‐related insulin resistance and IGF‐1 decline

Low insulin‐like growth factor‐1 (IGF‐1) signaling is associated with improved longevity, but is paradoxically linked with several age‐related diseases in humans. Insulin‐like growth factor‐1 has proven to be particularly beneficial to the brain, where it confers protection against features of neuronal and cognitive decline. While aging is characterized by central insulin resistance in the face of hyperinsulinemia, the somatotropic axis markedly declines in older humans. Thus, we hypothesized that increasing IGF‐1 in the brain may prove to be a novel therapeutic alternative to overcome central insulin resistance and restore whole‐body insulin action in aging. Utilizing hyperinsulinemic‐euglycemic clamps, we show that old insulin‐resistant rats with age‐related declines in IGF‐1 level demonstrate markedly improved whole‐body insulin action, when treated with central IGF‐1, as compared to central vehicle or insulin (P < 0.05). Furthermore, central IGF‐1, but not insulin, suppressed hepatic glucose production and increased glucose disposal rates in aging rats (P < 0.05). Taken together, IGF‐1 action in the brain and periphery provides a ‘balance’ between its beneficial and detrimental actions. Therefore, we propose that strategies aimed at ‘tipping the balance’ of IGF‐1 action centrally are the optimal approach to achieve healthy aging and longevity in humans.     

Hormone replacement therapy enhances IGF‐1 signaling in skeletal muscle by diminishing miR‐182 and miR‐223 expressions: a study on postmenopausal monozygotic twin pairs

MiRNAs are fine‐tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co‐twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen‐based hormone replacement therapy (HRT) to explore estrogen‐dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62‐years‐old monozygotic female twin pairs discordant for HRT (median 7 years). MCF‐7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR‐182, miR‐223 and miR‐142‐3p expressions in HRT using than in their nonusing co‐twins. Insulin/IGF‐1 signaling emerged one common pathway targeted by these miRNAs. IGF‐1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR‐182 and miR‐223 on IGF‐1R and FOXO3A mRNA as well as a dose‐dependent miR‐182 and miR‐223 down‐regulations concomitantly with up‐regulation of FOXO3A and IGF‐1R expression. Novel finding is the postmenopausal HRT‐reduced miRs‐182, miR‐223 and miR‐142‐3p expression in female skeletal muscle. The observed miRNA‐mediated enhancement of the target genes' IGF‐1R and FOXO3A expression as well as the activation of insulin/IGF‐1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.     

PI3K‐p110‐alpha‐subtype signalling mediates survival,proliferation and neurogenesis of cortical progenitor cells via activation of mTORC2

Development of the cerebral cortex is controlled by growth factors among which transforming growth factor beta (TGFβ) and insulin‐like growth factor 1 (IGF1) have a central role. The TGFβ‐ and IGF1‐pathways cross‐talk and share signalling molecules, but in the central nervous system putative points of intersection remain unknown. We studied the biological effects and down‐stream molecules of TGFβ and IGF1 in cells derived from the mouse cerebral cortex at two developmental time points, E13.5 and E16.5. IGF1 induces PI3K, AKT and the mammalian target of rapamycin complexes (mTORC1/mTORC2) primarily in E13.5‐derived cells, resulting in proliferation, survival and neuronal differentiation, but has small impact on E16.5‐derived cells. TGFβ has little effect at E13.5. It does not activate the PI3K‐ and mTOR‐signalling network directly, but requires its activity to mediate neuronal differentiation specifically at E16.5. Our data indicate a central role of mTORC2 in survival, proliferation as well as neuronal differentiation of E16.5‐derived cortical cells. mTORC2 promotes these cellular processes and is under control of PI3K‐p110‐alpha signalling. PI3K‐p110‐beta signalling activates mTORC2 in E16.5‐derived cells but it does not influence cell survival, proliferation and differentiation. This finding indicates that different mTORC2 subtypes may be implicated in cortical development and that these subtypes are under control of different PI3K isoforms.

     

Phosphatidylinositol‐3‐OH kinase regulatory subunits are differentially expressed during development of the rat cerebellum

Recent evidence implicates a central role for PI3K signalling in mediating cell survival during the process of neuronal differentiation. Although PI3K activity is stimulated by a wide range of growth factors and cytokines in different cell lines and tissues, activation of this pathway by insulin‐like growth factor I (IGF‐I) most likely represents the main survival signal during neuronal differentiation. IGF‐I is highly expressed during development of the central nervous system, and thus is a critical factor for the development and maturation of the cerebellum. Upon ligand binding, the IGF‐I receptor phosphorylates tyrosine residues in SHC and insulin receptor substrates (IRSs) initiating two main signalling cascades, the MAP kinase and the phosphatidylinositol 3‐kinase (PI3K) pathways. Activated PI3K is composed of a catalytic subunit (p110α or β) associated with one of a large family of regulatory subunits (p85α, p85β, p55γ, p55α, and p50α). To evaluate the contributions of these various regulatory subunits to neuronal differentiation, we have used antibodies specific for each of the PI3K subunits. Using these antisera, we now demonstrate that PI3K subunits are differentially regulated in cerebellar development, and that the expression level of the p55γ regulatory subunit reaches a maximum during postnatal development, decreasing thereafter to low levels in the adult cerebellum. Furthermore, our studies reveal that the distribution of the various PI3K regulatory subunits varies during development of the cerebellum. Interestingly, p55γ is expressed in both glial and neuronal cells; moreover, in Purkinje neurones, this subunit colocalises with the IGF‐IR. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 39–50, 2001     

Lipidomics Analysis of Timosaponin BII in INS‐1 Cells Induced by Glycolipid Toxicity and Its Relationship with Inflammation

Anemarrhena asphodeloides Bunge is a traditional Chinese medicine. The timosaponin BII is one of the most abundant and widely studied active ingredients in Anemarrhena asphodeloides Bunge . Related studies have shown that timosaponin BII has potential value for development and further utilization. The protective effect of timosaponin BII on islet β cells under type 2 diabetes was investigated in the glycolipid toxic INS‐1 cell model and possible biomarkers were explored by lipidomics analysis. Timosaponin BII was isolated from Anemarrhena asphodeloides Bunge by polyamide resin and Sephadex LH‐20. Then, the glycolipid toxicity INS‐1 cell model was established to investigate the protective effect of timosaponin BII. The results showed that timosaponin BII could significantly influence the levels of malondialdehyde (MDA) and glutathione (GSH), thereby restoring the insulin secretion ability and cell viability of model cells. Lipidomics analysis was combined with multivariate statistical analysis for marker selection. The four most common pathological and pharmacological lipid markers were phosphatidylserine (PS), suggesting that timosaponin BII had protective effects on model cells related to the reduction oxidative stress and macrophage inflammation. RAW264.7 macrophages were stimulated by LPS to establish a model of inflammation and study the effect of timosaponin BII on the nodes of NOD‐like receptor P3 (NLRP3) inflammasome pathway in the model cells. In conclusion, timosaponin BII may have the effect of protecting INS‐1 pancreatic β cells through reducing IL‐1β (interleukin‐1β) production by inhibiting the NLRP3 inflammasome in macrophage and restoring the insulin secretion ability and cell viability by reducing oxidative stress.     

Tumor Necrosis Factor‐α Stimulates Cell Proliferation in Adipose Tissue‐Derived Stromal‐Vascular Cell Culture: Promotion of Adipose Tissue Expansion by Paracrine Growth Factors

Objective: Elevated levels of tumor necrosis factor‐α (TNF‐α) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF‐α has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF‐α, alone or together with insulin‐like growth factor‐I (IGF‐I), support preadipocyte development. Research Methods and Procedures: Cultured stromal‐vascular cells from rat inguinal fat depots were exposed to serum‐free media containing insulin and 0.2 nM TNF‐α, 2.0 nM TNF‐α, or 0.2 nM TNF‐α + 1.0 nM IGF‐I at different times during 7 days of culture. Results: TNF‐α inhibited adipocyte differentiation as indicated by a reduction in both immunocytochemical reactivity for the preadipocyte‐specific antigen (AD3; early differentiation marker) and glycerol‐3‐phosphate dehydrogenase activity (late differentiation marker). Early exposure (Days 1 through 3 of culture) to 0.2 nM TNF‐α did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF‐α from Days 1 through 7 of culture resulted in a 75% increase in cell number from control. There was a synergistic effect of 0.2 nM TNF‐α + 1 nM IGF‐I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. Discussion: These data suggest that paracrine levels (0.2 nM) of TNF‐α alone or in combination with IGF‐I may support adipose tissue development by increasing the total number of stromal‐vascular and/or uncommitted cells within the tissue. These cells may then be recruited to become preadipocytes or may alternatively serve as infrastructure to support adipose tissue growth.     

Longevity in response to lowered insulin signaling requires glycine N‐methyltransferase‐dependent spermidine production

Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. We here show that, in Drosophila, reduced IIS activity modulates methionine metabolism, through tissue‐specific regulation of glycine N‐methyltransferase (Gnmt), and that this regulation is required for full IIS‐mediated longevity. Furthermore, fat body‐specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt‐dependent increase in spermidine levels. We also show that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, we found that increased expression of Gnmt occurs in the liver of liver‐specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue‐specific modulators of IIS‐mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.     

Lentinan protects pancreatic β cells from STZ‐induced damage

Pancreatic β‐cell death or dysfunction mediated by oxidative stress underlies the development and progression of diabetes mellitus (DM). In this study, we evaluated the effect of lentinan (LNT), an active ingredient purified from the bodies of Lentinus edodes, on pancreatic β‐cell apoptosis and dysfunction caused by streptozotocin (STZ) and the possible mechanisms implicated. The rat insulinoma cell line INS‐1 were pre‐treated with the indicated concentration of LNT for 30 min. and then incubated for 24 hrs with or without 0.5 mM STZ. We found that STZ treatment causes apoptosis of INS‐1 cells by enhancement of intracellular reactive oxygen species (ROS) accumulation, inducible nitric oxide synthase (iNOS) expression and nitric oxide release and activation of the c‐jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) signalling pathways. However, LNT significantly increased cell viability and effectively attenuated STZ‐induced ROS production, iNOS expression and nitric oxide release and the activation of JNK and p38 MAPK in a dose‐dependent manner in vitro. Moreover, LNT dose‐dependently prevented STZ‐induced inhibition of insulin synthesis by blocking the activation of nuclear factor kappa beta and increasing the level of Pdx‐1 in INS‐1 cells. Together these findings suggest that LNT could protect against pancreatic β‐cell apoptosis and dysfunction caused by STZ and therefore may be a potential pharmacological agent for preventing pancreatic β‐cell damage caused by oxidative stress associated with diabetes.     

Altered longevity‐assurance activity of p53:p44 in the mouse causes memory loss,neurodegeneration and premature death

The longevity‐assurance activity of the tumor suppressor p53 depends on the levels of Δ40p53 (p44), a short and naturally occurring isoform of the p53 gene. As such, increased dosage of p44 in the mouse leads to accelerated aging and short lifespan. Here we show that mice homozygous for a transgene encoding p44 (p44^+/+) display cognitive decline and synaptic impairment early in life. The synaptic deficits are attributed to hyperactivation of insulin‐like growth factor 1 receptor (IGF‐1R) signaling and altered metabolism of the microtubule‐binding protein tau. In fact, they were rescued by either Igf1r or Mapt haploinsufficiency. When expressing a human or a ‘humanized’ form of the amyloid precursor protein (APP), p44^+/+ animals developed a selective degeneration of memory‐forming and ‐retrieving areas of the brain, and died prematurely. Mechanistically, the neurodegeneration was caused by both paraptosis‐ and autophagy‐like cell deaths. These results indicate that altered longevity‐assurance activity of p53:p44 causes memory loss and neurodegeneration by affecting IGF‐1R signaling. Importantly, Igf1r haploinsufficiency was also able to correct the synaptic deficits of APP_695/swe mice, a model of Alzheimer’s disease.