Prolonged Insulin Treatment Research Articles

EPDR1 is a noncanonical effector of insulin-mediated angiogenesis regulated by an endothelial-specific TGF-β receptor complex

Insulin signaling in blood vessels primarily functions to stimulate angiogenesis and maintain vascular homeostasis through the canonical PI3K and MAPK signaling pathways. However, angiogenesis is a complex process coordinated by multiple other signaling events. Here, we report a distinct crosstalk between the insulin receptor and endoglin/activin receptor-like kinase 1 (ALK1), an endothelial cell–specific TGF-β receptor complex essential for angiogenesis. While the endoglin–ALK1 complex normally binds to TGF-β or bone morphogenetic protein 9 (BMP9) to promote gene regulation via transcription factors Smad1/5, we show that insulin drives insulin receptor oligomerization with endoglin–ALK1 at the cell surface to trigger rapid Smad1/5 activation. Through quantitative proteomic analysis, we identify ependymin-related protein 1 (EPDR1) as a major Smad1/5 gene target induced by insulin but not by TGF-β or BMP9. We found endothelial EPDR1 expression is minimal at the basal state but is markedly enhanced upon prolonged insulin treatment to promote cell migration and formation of capillary tubules. Conversely, we demonstrate EPDR1 depletion strongly abrogates these angiogenic effects, indicating that EPDR1 is a crucial mediator of insulin-induced angiogenesis. Taken together, these results suggest important therapeutic implications for EPDR1 and the TGF-β pathways in pathologic angiogenesis during hyperinsulinemia and insulin resistance.

High Glucose Impairs Insulin Signaling in the Glomerulus: An In Vitro and Ex Vivo Approach.

ObjectiveChronic hyperglycaemia, as seen in type II diabetes, results in both morphological and functional impairments of podocytes in the kidney. We investigated the effects of high glucose (HG) on the insulin signaling pathway, focusing on cell survival and apoptotic markers, in immortalized human glomerular cells (HGEC; podocytes) and isolated glomeruli from healthy rats.Methods and FindingsHGEC and isolated glomeruli were cultured for various time intervals under HG concentrations in the presence or absence of insulin. Our findings indicated that exposure of HGEC to HG led to downregulation of all insulin signaling markers tested (IR, p-IR, IRS-1, p-Akt, p-Fox01,03), as well as to increased sensitivity to apoptosis (as seen by increased PARP cleavage, Casp3 activation and DNA fragmentation). Short insulin pulse caused upregulation of insulin signaling markers (IR, p-IR, p-Akt, p-Fox01,03) in a greater extent in normoglycaemic cells compared to hyperglycaemic cells and for the case of p-Akt, in a PI3K-dependent manner. IRS-1 phosphorylation of HG-treated podocytes was negatively regulated, favoring serine versus tyrosine residues. Prolonged insulin treatment caused a significant decrease of IR levels, while alterations in glucose concentrations for various time intervals demonstrated changes of IR, p-IR and p-Akt levels, suggesting that the IR signaling pathway is regulated by glucose levels. Finally, HG exerted similar effects in isolated glomeruli.ConclusionsThese results suggest that HG compromises the insulin signaling pathway in the glomerulus, promoting a proapoptotic environment, with a possible critical step for this malfunction lying at the level of IRS-1 phosphorylation; thus we herein demonstrate glomerular insulin signaling as another target for investigation for the prevention and/ or treatment of diabetic nephropathy.

Prolonged insulin treatment sensitizes apoptosis pathways in pancreatic β cells.

Insulin resistance results from impaired insulin signaling in target tissues that leads to increased levels of insulin required to control plasma glucose levels. The cycle of hyperglycemia and hyperinsulinemia eventually leads to pancreatic cell deterioration and death by a mechanism that is yet unclear. Insulin induces ROS formation in several cell types. Furthermore, death of pancreatic cells induced by oxidative stress could be potentiated by insulin. Here, we investigated the mechanism underlying this phenomenon. Experiments were done on pancreatic cell lines (Min-6, RINm, INS-1), isolated mouse and human islets, and on cell lines derived from nonpancreatic sources. Insulin (100nM) for 24h selectively increased the production of ROS in pancreatic cells and isolated pancreatic islets, but only slightly affected the expression of antioxidant enzymes. This was accompanied by a time- and dose-dependent decrease in cellular reducing power of pancreatic cells induced by insulin and altered expression of several ER stress response elements including a significant increase in Trb3 and a slight increase in iNos The effect on iNos did not increase NO levels. Insulin also potentiated the decrease in cellular reducing power induced by H2O2 but not cytokines. Insulin decreased the expression of MCL-1, an antiapoptotic protein of the BCL family, and induced a modest yet significant increase in caspase 3/7 activity. In accord with these findings, inhibition of caspase activity eliminated the ability of insulin to increase cell death. We conclude that prolonged elevated levels of insulin may prime apoptosis and cell death-inducing mechanisms as a result of oxidative stress in pancreatic cells.

Impaired insulin signaling affects renal organic anion transporter 3 (Oat3) function in streptozotocin-induced diabetic rats.

Organic anion transporter 3 (Oat3) is a major renal Oats expressed in the basolateral membrane of renal proximal tubule cells. We have recently reported decreases in renal Oat3 function and expression in diabetic rats and these changes were recovered after insulin treatment for four weeks. However, the mechanisms by which insulin restored these changes have not been elucidated. In this study, we hypothesized that insulin signaling mediators might play a crucial role in the regulation of renal Oat3 function. Experimental diabetic rats were induced by a single intraperitoneal injection of streptozotocin (65 mg/kg). One week after injection, animals showing blood glucose above 250 mg/dL were considered to be diabetic and used for the experiment in which insulin-treated diabetic rats were subcutaneously injected daily with insulin for four weeks. Estrone sulfate (ES) uptake into renal cortical slices was examined to reflect the renal Oat3 function. The results showed that pre-incubation with insulin for 30 min (short term) stimulated [3H]ES uptake into the renal cortical slices of normal control rats. In the untreated diabetic rats, pre-incubation with insulin for 30 min failed to stimulate renal Oat3 activity. The unresponsiveness of renal Oat3 activity to insulin in the untreated diabetic rats suggests the impairment of insulin signaling. Indeed, pre-incubation with phosphoinositide 3-kinase (PI3K) and protein kinase C zeta (PKCζ) inhibitors inhibited insulin-stimulated renal Oat3 activity. In addition, the expressions of PI3K, Akt and PKCζ in the renal cortex of diabetic rats were markedly decreased. Prolonged insulin treatment in diabetic rats restored these alterations toward normal levels. Our data suggest that the decreases in both function and expression of renal Oat3 in diabetes are associated with an impairment of renal insulin-induced Akt/PKB activation through PI3K/PKCζ/Akt/PKB signaling pathway.

Myricetin attenuates hyperinsulinemia-induced insulin resistance in skeletal muscle cells

Previous studies have shown that hyperinsulinemia is not only a marker of insulin resistance, but also the causative factor of peripheral tissue insulin resistance. It also has been suggested that prolonged high-dose insulin treatment can mimic the effects of hyperinsulinemia and exacerbate insulin resistance in skeletal muscle cells. However, how to prevent or reverse insulin resistance induced by hyperinsulinemia remains largely unclear. In the past few decades, the use of myricetin as an anti-diabetic agent has gained much attention, but little information is available regarding the effects of myricetin on glucose uptake and hyperinsulinemia-induced insulin resistance in skeletal muscle cells. The present study focuses on the effect of myricetin on insulin signaling in skeletal muscle cell line C2C12 myotubes. Initially, the effect of myricetin under normal condition was determined. We found that myricetin’s enhancement in glucose uptake coincided with both protein kinase B (Akt) and AMP-activated protein kinase (AMPK) activities. After that, the role of hyperinsulinemia was investigated. It was showed that prolonged high-dose insulin treatment inhibited both Akt and AMPK activities. As the results, the low-dose insulin stimulation of glucose uptake was inhibited by hyperinsulinemia. However, the treatment of myricetin improved low-dose insulin-stimulated glucose uptake in the hyperinsulinemic state, and this effect essentially depended on the AMPK signal pathway. Together, our data suggest a putative link between hyperinsulinemia and insulin resistance in C2C12 myotubes, and the myricetin treatment stimulates glucose uptake and attenuates insulin resistance.

Phosphoinositide 3-kinase as a novel functional target for the regulation of the insulin signaling pathway by SIRT1

The protein deacetylase SIRT1, and its activator resveratrol, exert beneficial effects on glucose metabolism. Different SIRT1 targets have been identified, including PTP1B, AMPK, FOXO, PGC-1α and IRS2. The latter may underscore a tight link between SIRT1 and insulin signaling components. However, whether SIRT1 has a direct effect on insulin resistance and whether resveratrol acts directly or indirectly in this context is still a matter of controversy and this question has not been addressed in muscle cells.Here, we show that SIRT1 protein expression is decreased in muscle biopsies and primary myotubes derived from type 2 diabetic patients, suggesting a contribution of diminished SIRT1 in the determination of muscle insulin resistance. To investigate the functional impact of SIRT1 on the insulin pathway, the activation of insulin downstream effector PKB was evaluated after SIRT1 inactivation by RNAi, SIRT1 overexpression, or resveratrol treatments. In muscle cells and HEK293 cells, downregulation of SIRT1 reduced, while overexpression increased, insulin-induced PKB activatory phosphorylation. Further molecular characterisation revealed that SIRT1 interacts in an insulin-independent manner with the PI3K adapter subunit p85. We then investigated whether resveratrol may improve insulin signaling in muscle cells via SIRT1, or alternative targets. Incubation of muscle cells with resveratrol reverted the insulin-resistant state induced by prolonged TNFα or insulin treatment. Resveratrol-dependent improvement of insulin-resistance occurred through inhibition of serine phosphorylation of IRS1/2, implicating resveratrol as a serine kinase inhibitor. Finally, a functional interaction between PI3K and SIRT1 was demonstrated in C. elegans, where constitutively active PI3K – mimicking increased IIS signaling – lead to shortened lifespan, while removal of sir-2.1 abolished PI3K-induced lifespan shortening.Our data identify SIRT1 as a positive modulator of insulin signaling in muscle cells through PI3K, and this mechanism appears to be conserved from C. elegans through humans.

Blocking O-Linked GlcNAc Cycling in Drosophila Insulin-producing Cells Perturbs Glucose-Insulin Homeostasis

A dynamic cycle of O-linked GlcNAc (O-GlcNAc) addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively, in a process that serves as the final step in a nutrient-driven "hexosamine-signaling pathway." Evidence points to a role for O-GlcNAc cycling in diabetes and insulin resistance. We have used Drosophila melanogaster to determine whether O-GlcNAc metabolism plays a role in modulating Drosophila insulin-like peptide (dilp) production and insulin signaling. We employed transgenesis to either overexpress or knock down Drosophila Ogt(sxc) and Oga in insulin-producing cells (IPCs) or fat bodies using the GAL4-UAS system. Knockdown of Ogt decreased Dilp2, Dilp3, and Dilp5 production, with reduced body size and decreased phosphorylation of Akt in vivo. In contrast, knockdown of Oga increased Dilp2, Dilp3, and Dilp5 production, increased body size, and enhanced phosphorylation of Akt in vivo. However, knockdown of either Ogt(sxc) or Oga in the IPCs increased the hemolymph carbohydrate concentration. Furthermore, phosphorylation of Akt stimulated by extraneous insulin in an ex vivo cultured fat body of third instar larvae was diminished in strains subjected to IPC knockdown of Ogt or Oga. Knockdown of O-GlcNAc cycling enzymes in the fat body dramatically reduced neutral lipid stores. These results demonstrate that altered O-GlcNAc cycling in Drosophila IPCs modulates insulin production and influences the insulin responsiveness of peripheral tissues. The observed phenotypes in O-GlcNAc cycling mimic pancreatic β-cell dysfunction and glucose toxicity related to sustained hyperglycemia in mammals.

Physiological and clinical role of insulin in the neonate

In the newborn infant, insulin secretion has to adjust in response to the switch from a regulated and continuous placental supply of glucose in utero to the delivery of intermittent oral feeds postnatally. Changes in insulin secretion must reflect its primary role for maintaining glucose homeostasis, but also its roles in promoting growth and anabolism and in the newborn disorders of insulin secretion or sensitivity, which present with hyperglycemia and impaired growth. Recent elucidation of the genetic basis of neonatal diabetes has helped to provide valuable insights into the molecular mechanisms of β-cell function and the potential for treatment of some patients with oral hypoglycemic agents, although the majority require prolonged subcutaneous insulin treatment, which may prove challenging. The recent development of insulin pump therapy has significantly improved the clinical management of these infants. Although they do not have neonatal diabetes, the preterm or very-low-birthweight infant, subjected to the combined effects of insulin resistance owing to the impact of intensive care, and relative insulin deficiency related to prematurity, may have long periods of hyperglycemia and impaired growth, which have been associated with adverse clinical outcomes. Although these infants often require insulin treatment, the optimal management of glucose control and use of insulin has not been determined and remains controversial.

Insulin Induces REDD1 Expression through Hypoxia-inducible Factor 1 Activation in Adipocytes

REDD1 (regulated in development and DNA damage responses) is essential for the inhibition of mTORC1 (mammalian target of rapamycin complex) signaling pathway in response to hypoxia. REDD1 expression is regulated by many stresses such as hypoxia, oxidative stress, and energy depletion. However, the regulation of REDD1 expression in response to insulin remains unknown. In the present study, we demonstrate that in murine and in human adipocytes, insulin stimulates REDD1 expression. Insulin-induced REDD1 expression occurs through phosphoinositide 3-kinase/mTOR-dependent pathways. Moreover, using echinomycin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, and HIF-1alpha small interfering RNA, we demonstrate that insulin stimulates REDD1 expression only through the transcription factor HIF-1. In conclusion, our study shows that insulin stimulates REDD1 expression in adipocytes.

Central Insulin Signaling Is Attenuated by Long-Term Insulin Exposure via Insulin Receptor Substrate-1 Serine Phosphorylation, Proteasomal Degradation, and Lysosomal Insulin Receptor Degradation

Central insulin signaling is critical for the prevention of insulin resistance. Hyperinsulinemia contributes to insulin resistance, but it is not yet clear whether neurons are subject to cellular insulin resistance. We used an immortalized, hypothalamic, clonal cell line, mHypoE-46, which exemplifies neuronal function and expresses the components of the insulin signaling pathway, to determine how hyperinsulinemia modifies neuronal function. Western blot analysis indicated that prolonged insulin treatment of mHypoE-46 cells attenuated insulin signaling through phospho-Akt. To understand the mechanisms involved, time-course analysis was performed. Insulin exposure for 4 and 8 h phosphorylated Akt and p70-S6 kinase (S6K1), whereas 8 and 24 h treatment decreased insulin receptor (IR) and IR substrate 1 (IRS-1) protein levels. Insulin phosphorylation of S6K1 correlated with IRS-1 ser1101 phosphorylation and the mTOR-S6K1 pathway inhibitor rapamycin prevented IRS-1 serine phosphorylation. The proteasomal inhibitor epoxomicin and the lysosomal pathway inhibitor 3-methyladenine prevented the degradation of IRS-1 and IR by insulin, respectively, and pretreatment with rapamycin, epoxomicin, or 3-methyladenine prevented attenuation of insulin signaling by long-term insulin exposure. Thus, a sustained elevation of insulin levels diminishes neuronal insulin signaling through mTOR-S6K1-mediated IRS-1 serine phosphorylation, proteasomal degradation of IRS-1 and lysosomal degradation of the IR.

Insulin-Induced Laminin Expression Promotes a Hypercontractile Airway Smooth Muscle Phenotype

Airway smooth muscle (ASM) plays a key role in the development of airway hyperresponsiveness and remodeling in asthma, which may involve maturation of ASM cells to a hypercontractile phenotype. In vitro studies have indicated that long-term exposure of bovine tracheal smooth muscle (BTSM) to insulin induces a functional hypercontractile, hypoproliferative phenotype. Similarly, the extracellular matrix protein laminin has been found to be involved in both the induction and maintenance of a contractile ASM phenotype. Using BTSM, we now investigated the role of laminins in the insulin-induced hypercontractile, hypoproliferative ASM phenotype. The results demonstrate that insulin-induced hypercontractility after 8 days of tissue culture was fully prevented by combined treatment of BTSM-strips with the laminin competing peptides Tyr-Ile-Gly-Ser-Arg (YIGSR) and Arg-Gly-Asp-Ser (RGDS). YIGSR also prevented insulin-induced increases in sm-myosin expression and abrogated the suppressive effects of prolonged insulin treatment on platelet-derived growth factor-induced DNA synthesis in cultured cells. In addition, insulin time-dependently increased laminin alpha2, beta1, and gamma1 chain protein, but not mRNA abundance in BTSM strips. Moreover, as previously found for contractile protein accumulation, signaling through PI3-kinase- and Rho kinase-dependent pathways was required for the insulin-induced increase in laminin abundance and contractility. Collectively, our results indicate a critical role for beta1-containing laminins, likely laminin-211, in the induction of a hypercontractile, hypoproliferative ASM phenotype by prolonged insulin exposure. Increased laminin production by ASM could be involved in the increased ASM contractility and contractile protein expression in asthma. Moreover, the results may be of interest for the use of inhaled insulin administrations by diabetics.

Prolonged exposure to high insulin impairs the endothelial PI3-kinase/Akt/nitric oxide signalling

Hyperinsulinemia predicts future cardiovascular events, but may also contribute to atherosclerosis. We therefore studied the consequences of prolonged insulin treatment of human umbilical vein endothelial cells (HUVEC) on the phosphatidylinositol-3'-kinase(PI3K)/Akt/nitric oxide(NO)-dependent insulin signaling, together with the expression of the pro-atherogenic molecule vascular cell adhesion molecule (VCAM)-1. HUVEC were incubated with insulin (10(-11) to 10(-7) M) in short- (30 min) and long-term (24 h to 3 days) incubations. In short-term incubations, insulin did not affect constitutive Akt and eNOS at any concentration, but significantly increased their active phosphorylated forms, and NO production. In long-term incubations, however, such insulin effects on the phosphorylated forms, as well as NO production, were attenuated, promoting an effect of insulin also at concentrations otherwise ineffective. Such effects were accompanied by a boosting of insulin effect on VCAM-1 surface expression. In contrast, under similar conditions, insulin did not exert any significant effect on the surface expression of ICAM-1 and E-selectin. Therefore, prolonged exposure of HUVEC to high insulin levels induces a downregulation of the PI3K/Akt/eNOS axis. Such impairment of insulin signalling in states of prolonged hyperinsulinemia pontially contributes to detrimental effects on atherogenesis in insulin resistance states, such as the metabolic syndrome and type 2 diabetes.

Insulin regulation of growth hormone receptor gene expression: Evidence for a transcriptional mechanism of down-regulation in rat hepatoma cells

The role of insulin in regulating responsiveness to growth hormone (GH) remains unclear. Continuous insulin treatment reduces GH binding, which suggests that insulin may effect growth hormone receptor (GHR) levels. The present study used rat hepatoma cells to examine the effects of insulin and GH on GHR gene expression. Prolonged insulin treatment (greater than 3 h) significantly reduced GHR mRNA, and removal of insulin led to a gradual recovery. This effect of insulin occurred at physiologic concentrations, occurred many hours before the insulin-regulated decrease in GHR protein, and was mediated by reduction of GHR transcription. GH treatment dramatically reduced GHR protein, but caused only a modest reduction in GHR mRNA. These findings indicate that the heterologous reduction of GHR by insulin occurs via transcriptional downregulation, and the homologous reduction of GHR by GH occurs via a different mechanism. Furthermore, with insulin, extended time of exposure may be necessary for appreciable reduction of GHR.

Insulin Causes a Transient Induction of Proliferation via Activation of the PI3-Kinase Pathway in Airway Smooth Muscle Cells

Insulin regulates glucose metabolism and activates cell growth. As the respiratory system is an alternative route for insulin administration in patients with diabetes mellitus, we studied the effect of insulin on rabbit tracheal airway smooth muscle (ASM) cell proliferation. We show that treatment of quiescent ASM cells with insulin for 24 h increased cell number compared to control cells. This increase was similar to the increase caused by the addition of fetal bovine serum (FBS). Moreover, in contrast to cells treated with FBS, exposure of ASM cells with insulin for longer periods (48 and 72 h) did not have any further influence on cell proliferation. In ASM cells, insulin activated the phosphatidylinositol 3-kinase (PI3 K) pathway, while FBS activated both PI3 K and the mitogen-activated protein kinase (MAPK) pathway. The PI3K pathway inhibitors LY294002 and wortmannin abolished the stimulation of cell proliferation by insulin, indicating a role for this pathway in the cellular response to insulin. These results show that while insulin has a mitogenic effect on ASM cells, prolonged insulin treatment does not lead to excessive ASM cell proliferation and suggest that the use of aerosolized insulin does not represent a potential hazard for airway remodeling.

Insulin Regulation of Cholesterol 7α-Hydroxylase Expression in Human Hepatocytes: ROLES OF FORKHEAD BOX O1 AND STEROL REGULATORY ELEMENT-BINDING PROTEIN 1c

Bile acid synthesis and pool size increases in diabetes, whereas insulin inhibits bile acid synthesis. The objective of this study is to elucidate the mechanism of insulin regulation of cholesterol 7alpha-hydroxylase gene expression in human hepatocytes. Real-time PCR assays showed that physiological concentrations of insulin rapidly stimulated cholesterol 7alpha-hydroxylase (CYP7A1) mRNA expression in primary human hepatocytes but inhibited CYP7A1 expression after extended treatment. The insulin-regulated forkhead box O1 (FoxO1) and steroid regulatory element-binding protein-1c (SREBP-1c) strongly inhibited hepatocyte nuclear factor 4alpha and peroxisome proliferator-activated receptor gamma coactivator-1alpha trans-activation of the CYP7A1 gene. FoxO1 binds to an insulin response element in the rat CYP7A1 promoter, which is not present in the human CYP7A1 gene. Insulin rapidly phosphorylates and inactivates FoxO1, whereas insulin induces nuclear SREBP-1c expression in human primary hepatocytes. Chromatin immunoprecipitation assay shows that insulin reduced FoxO1 and peroxisome proliferators-activated receptor gamma-coactivator-1alpha but increased SREBP-1c recruitment to CYP7A1 chromatin. We conclude that insulin has dual effects on human CYP7A1 gene transcription; physiological concentrations of insulin rapidly inhibit FoxO1 activity leading to stimulation of the human CYP7A1 gene, whereas prolonged insulin treatment induces SREBP-1c, which inhibits human CYP7A1 gene transcription. Insulin may play a major role in the regulation of bile acid synthesis and dyslipidemia in diabetes.

Grb10 mediates insulin-stimulated degradation of the insulin receptor: a mechanism of negative regulation

Growth factor receptor-bound protein 10 (Grb10) is an adapter protein that interacts with a number of tyrosine-phosphorylated growth factor receptors, including the insulin receptor (IR). To investigate the role of Grb10 in insulin signaling, we generated cell lines in which the expression levels of Grb10 are either overexpressed by stable transfection or suppressed by RNA interference. We found that suppressing endogenous Grb10 expression led to increased IR protein levels, whereas overexpression of Grb10 led to reduced IR protein levels. Altering Grb10 expression levels had no effect on the mRNA levels of IR, suggesting that the modulation occurs at the protein level. Reduced IR levels were also observed in cells with prolonged insulin treatment, and this reduction was inhibited in Grb10-deficient cells. The insulin-induced IR reduction was greatly reversed by MG-132, a proteasomal inhibitor, but not by chloroquine, a lysosomal inhibitor. IR underwent insulin-stimulated ubiquitination in cells, and this ubiquitination was inhibited in the Grb10-suppressed cell line. Together, our results suggest that, in addition to inhibiting IR kinase activity by directly binding to the IR, Grb10 also negatively regulates insulin signaling by mediating insulin-stimulated degradation of the receptor.

Expression of Inducible 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase/PFKFB3 Isoforms in Adipocytes and Their Potential Role in Glycolytic Regulation

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (F2,6BP), which is a powerful activator of 6-phosphofructo-1-kinase, the rate-limiting enzyme of glycolysis. Four genes encode PFK-2/FBPase (PFKFB1-4), and an inducible isoform (iPFK-2/PFKFB3) has been found to mediate F2,6BP production in proliferating cells. We have investigated the role of iPFK-2/PFKFB3 and related isoforms in the regulation of glycolysis in adipocytes. Human visceral fat cells express PFKFB3 mRNA, and three alternatively spliced isoforms of iPFK-2/PFKFB3 are expressed in the epididymal fat pad of the mouse. Forced expression of the iPFK-2/PFKFB3 in COS-7 cells resulted in increased glucose uptake and cellular F2,6BP content. Prolonged insulin treatment of 3T3-L1 adipocytes led to reduced PFKFB3 mRNA expression, and epididymal fat pads from db/db mice also showed decreased expression of PFKFB3 mRNA. Finally, anti-phospho-iPFK-2(Ser461) Western blotting revealed strong reactivity in insulin-treated 3T3-L1 adipocyte, suggesting that insulin induces the phosphorylation of PFKFB3 protein. These data expand the role of these structurally unique iPFK-2/PFKFB3 isoforms in the metabolic regulation of adipocytes.

Stereology and ultrastructure of the salivary glands of diabetic Nod mice submitted to long‐term insulin treatment

Insulin-dependent diabetes mellitus compromises the salivary glands, altering their morphology and the mechanisms of salivation, which are fundamental for oral health. Thus, the aim of the present study was to determine the effects of prolonged insulin treatment on the morphology of the salivary glands in Nod mice. Forty-five female mice were divided into five groups: nine positive diabetic Nod mice for 10 days (group 1), nine positive diabetic Nod mice for 20 days (group 2), nine diabetic Nod mice for 10 days (group 3), nine diabetic Nod mice for 20 days (group 4), and nine nondiabetic BALB/c mice (group 5). Animals of groups 3 and 4 received 4-5 U of insulin daily, whereas animals of groups 1, 2, and 5 received the same dose of physiological saline simulating the experimental conditions. Samples of the salivary glands were analyzed by light, transmission, and scanning electron microscopies. The results showed intense alterations in diabetic animals characterized by nuclear and cytoplasmic atrophy, biomembrane disorganization, an increase in fibrillar components of the extracellular matrix, and the presence of inflammatory cells. Insulin treatment exerted positive effects on the recovery of the changes resulting from the diabetic state in both parotid and submandibular glands but the pattern continued to be altered. It can be concluded that, in addition to compromising the processes of tissue maintenance and renewal, tissue destructuring leads to alterations in functional mechanisms in both diabetic animals and animals submitted to glycemic control.

Prolonged insulin treatment inhibits GH signaling via STAT3 and STAT1

Growth hormone (GH) and insulin are important regulators of cellular and whole body metabolism as well as somatic growth and body composition. Studies have indicated complex feedback effects of GH on insulin action and of insulin on GH signaling pathways. Previous studies in our laboratory have shown that GH induction of signal transducers and activators of transcription (STAT)5B tyrosine phosphorylation is inhibited by prolonged insulin treatment, probably via downregulation of GHR. Here, we find that in rat H4IIE hepatoma cells GH-induced tyrosine phosphorylation of two other STATs (STAT3 and STAT1) was also greatly reduced following prolonged insulin pretreatment compared with that induced by GH alone. In the present work, total STAT5B and STAT1 protein levels were not altered by prolonged insulin treatment. However, prolonged insulin treatment (16 h; 10 or 100 nM) resulted in a 30-40% reduction of total STAT3 protein, with little change at 0.1 and 1.0 nM insulin. Thus, there is a selective reduction of total STAT3 protein levels by insulin, but only at high concentration of insulin. Basal tyrosine phosphorylated (PY)-STAT3 was also significantly reduced by prolonged insulin treatment, and to a greater extent than total STAT3 protein levels. The inhibitory effect of insulin on total STAT3 protein and basal PY-STAT3 levels was dependent on activation of the MEK-ERK pathway, rather than the PI3K pathway. In contrast, the MEK-ERK pathway did not play a major role in insulin's inhibition of GH-induced PY-STAT3 and PY-STAT1. The present studies indicate that prolonged hyperinsulinemia, such as that found in some obese patients or patients with Type 2 diabetes mellitus, may have profound effects on GH signaling via STAT3 and STAT1.

Irs–2 Mediates the Antiapoptotic Effect of Insulin in Neonatal Hepatocytes

To assess the role of insulin action and inaction in the liver, immortalized hepatocyte cell lines have been generated from insulin receptor substrate (IRS)-2(-/-) and wild-type mice. Using this model, we have recently demonstrated that the lack of IRS-2 in neonatal hepatocytes resulted in insulin resistance. In the current study, we show that immortalized neonatal hepatocytes undergo apoptosis on serum withdrawal, with caspase-3 activation and DNA laddering occurring earlier in the absence of IRS-2. Insulin rescued wild-type hepatocytes from serum withdrawal-induced caspase-3 activation and DNA fragmentation in a dose-dependent manner, but it failed to rescue hepatocytes lacking IRS-2. In IRS-2(-/-) cells, insulin failed to phosphorylate Bad. Furthermore, in these cells, insulin was unable to translocate Foxo1 from the nucleus to the cytosol. Adenoviral infection of wild-type cells with constitutively active Foxo1 (ADA) induced caspase-8 and caspase-3 activities, proapoptotic gene expression, DNA laddering and apoptosis. Dominant negative Foxo1 regulated the whole pathway in an opposite manner. Prolonged insulin treatment (24 hours) increased expression of antiapoptotic genes (Bcl-xL), downregulated proapoptotic genes (Bim and nuclear Foxo1), and decreased caspase-3 activity in wild-type hepatocytes but not in IRS-2(-/-) cells. Infection of IRS-2(-/-) hepatocytes with adenovirus encoding IRS-2 reconstituted phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/Foxo1 signaling, restored pro- and antiapoptotic gene expression, and decreased caspase-3 activity in response to insulin, thereby blocking apoptosis. In conclusion, IRS-2 signaling is specifically required through PIP3 generation to mediate the survival effects of insulin. Epidermal growth factor, via PIP3/Akt/Foxo1 phosphorylation, was able to rescue IRS-2(-/-) hepatocytes from serum withdrawal-induced apoptosis, modulating pro- and anti-apoptotic gene expression and downregulating caspase-3 activity.