Enhanced Insulin Signaling Research Articles

Functional Roles of Proline/Arginine-Rich End Leucine-Rich Repeat Protein (PRELP) in Adipocytes

Adipose tissue fibrosis is recognized as a hallmark of adipose tissue dysfunction. We have previously identified PRELP as a novel regulator of adipogenesis by using lentiviral short hairpin RNA libraries targeting murine genomes. PRELP belongs to the family of leucine-rich repeat proteins, which are characterized by a series of adjacent leucine-rich motifs flanked by disulfide-bounded domains. Functionally, PRELP anchors basement membranes to connective tissues. However, roles of PRELP in adipocytes remain unclear. In the present study, we show that PRELP expression is up-regulated both in adipocytes of high-fat diet induced obesity mice and ob/ob mice. PRELP reduction in 3T3-L1 adipocytes leads to enhanced insulin signaling and insulin-stimulated glucose uptake. To determine functions of PRELP in the development of metabolic phenotypes, we generated adipocyte-specific PRELP transgenic mice. Adipose-specific overexpression of PRELP results in the development of significant adipose tissue fibrosis and insulin resistance on high fat diet without difference of body weight and adiposity. This phenotype is associated with increased expression of inflammation genes and decreased insulin signaling in adipose tissue of PRELP transgenic mice. Furthermore, we find that global Prelp deficient mice are protected from adipose tissue fibrosis and insulin resistance associated with high fat feeding. These data suggest that PRELP might play a key role in the development of adipose tissue remodeling in obesity. Disclosure J. Eguchi: None. J. Wada: None.

The response of serum Glypican-4 levels and its potential regulatory mechanism to endurance training and chamomile flowers' hydroethanolic extract in streptozotocin-nicotinamide-induced diabetic rats.

Glypican-4 (GPC-4) is a novel adipomyokine that enhances insulin signaling. Glycosylphosphatidylinositol-specific phospholipase D (GPLD1) is thought to release GPC-4 and is itself an insulin-regulated enzyme. Beneficial effects of exercise training and chamomile flowers extract (CFE) are shown through activation of PPARγ, which is a promising drug target in diabetes and associated with GPC-4 synthesis. This study investigated the effects of 14-week treadmill running and CFE on serum GPC-4, GPLD1, and insulin levels in streptozotocin-nicotinamide (STZ-NA)-induced diabetic rats. Thirty-two STZ-NA-induced diabetic male Wistar rats were randomly assigned to four groups: control (C), training (T), CFE treatment (CFE), and training plus CFE treatment (TCFE) groups. The training groups were exercised on treadmill 5days/week and the treating groups were fed with 200mg/kg/day CFE in drinking water for 14weeks. Finally, serum GPC-4, GPLD1, and insulin levels were analyzed via sandwich ELISA. Compared to the control group, serum insulin levels were significantly higher in the T, CFE, and TCFE groups (p < 0.05, p < 0.05, p < 0.01, respectively), while OGTT and serum GPLD1 levels were significantly lower in the T, CFE, and TCFE groups (all p < 0.001). Changes in serum GPC-4 levels were not significant. Serum GPLD1 levels were negatively correlated with insulin levels and HOMA-IS (both p < 0.001). This study suggests that endurance training and CFE may downregulate serum GPLD1 levels in STZ-NA-induced diabetic rats, which associate with the serum insulin profile. However, the results show that endurance training and CFE may not cause serum GPC-4 adaptation in STZ-NA-induced diabetic rats.

Autophagy Differentially Regulates Insulin Production and Insulin Sensitivity

SUMMARYAutophagy, a stress-induced lysosomal degradative pathway, has been assumed to exert similar metabolic effects in different organs. Here, we establish a model where autophagy plays different roles in insulin-producing β cells versus insulin-responsive cells, utilizing knockin (Becn1F121A) mice manifesting constitutively active autophagy. With a high-fat-diet challenge, the autophagy-hyperactive mice unexpectedly show impaired glucose tolerance, but improved insulin sensitivity, compared to mice with normal autophagy. Autophagy hyperactivation enhances insulin signaling, via suppressing ER stress in insulin-responsive cells, but decreases insulin secretion by selectively sequestrating and degrading insulin granule vesicles in β cells, a process we term “vesicophagy.” The reduction in insulin storage, insulin secretion, and glucose tolerance is reversed by transient treatment of autophagy inhibitors. Thus, β cells and insulin-responsive tissues require different autophagy levels for optimal function. To improve insulin sensitivity without hampering secretion, acute or intermittent, rather than chronic, activation of autophagy should be considered in diabetic therapy development.

Neuroprotective effects of protein tyrosine phosphatase 1B inhibitor on cerebral ischemia/reperfusion in mice

Akt (Protein kinase B, PKB), a serine/threonine kinase, plays a critical role in cell development, growth, and survival. Akt phosphorylation mediates a neuroprotective effect against ischemic injury. Recently, a protein-tyrosine phosphatase-1B (PTP1B) inhibitor (KY-226) was developed to elicit anti-diabetic and anti-obesity effects via enhancement of insulin signaling. Previously, we reported that the nonselective PTP1B inhibitor, sodium orthovanadate, rescued neurons from delayed neuronal death during brain ischemia. In this study, we confirmed the ameliorative effects of KY-226 on ischemia/reperfusion (I/R) injury using a murine model of middle cerebral artery occlusion (MCAO). ICR mice were subjected to MCAO for 2 h followed by reperfusion. Although KY-226 permeability was poor through the blood-brain barrier (BBB) of normal mice, it could penetrate through the BBB of mice after I/R insult. Intraperitoneal KY-226 administration elicited dose-dependent reductions in infarcted brain areas and improved neurological deficits. The neuroprotective effects of KY-266 were obtained when administered within 0.5 h after reperfusion. KY-226 (10 mg/kg) also restored reduced Akt phosphorylation and eNOS phosphorylation (Ser-1177) levels following I/R insult. Moreover, 10 mg/kg of KY-226 improved I/R-induced decreased extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, KY-226 attenuated the generation of reactive oxygen species (ROS) in mouse cortex. These results suggest that KY-226 may act as a novel therapeutic candidate for ischemic stroke. Activation of Akt and ERK possibly underlie the neuroprotective mechanism of KY-226.

The beta secretase BACE1 regulates the expression of insulin receptor in the liver

Insulin receptor (IR) plays a key role in the control of glucose homeostasis; however, the regulation of its cellular expression remains poorly understood. Here we show that the amount of biologically active IR is regulated by the cleavage of its ectodomain, by the β-site amyloid precursor protein cleaving enzyme 1 (BACE1), in a glucose concentration-dependent manner. In vivo studies demonstrate that BACE1 regulates the amount of IR and insulin signaling in the liver. During diabetes, BACE1-dependent cleavage of IR is increased and the amount of IR in the liver is reduced, whereas infusion of a BACE1 inhibitor partially restores liver IR. We suggest the potential use of BACE1 inhibitors to enhance insulin signaling during diabetes. Additionally, we show that plasma levels of cleaved IR reflect IR isoform A expression levels in liver tumors, which prompts us to propose that the measurement of circulating cleaved IR may assist hepatic cancer detection and management.

An Extract of Russian Tarragon Prevents Obesity‐Related Ectopic Lipid Accumulation

The primary disorder underlying metabolic syndrome is insulin resistance due to excess body weight and abdominal visceral fat accumulation. In this study, it is asked if dietary intake of an ethanolic extract from Russian tarragon (Artemisia dracunculus L., termed PMI5011), shown to improve glucose utilization by enhancing insulin signaling in skeletal muscle, could prevent obesity-induced insulin resistance, skeletal muscle metabolic inflexibility, and ectopic lipid accumulation in the skeletal muscle and liver. Male wild-type mice are fed a high-fat diet alone or supplemented with PMI5011 (1% w/w) over 3 months. Dietary intake of PMI5011 improved fatty acid oxidation and metabolic flexibility in the skeletal muscle, reduced insulin levels, and enhanced insulin signaling in the skeletal muscle and liver independent of robust changes in expression of factors that control fatty acid oxidation. This corresponds with significantly reduced lipid accumulation in the skeletal muscle and liver, although body weight gain is comparable to a high-fat diet alone. Previous studies showed that PMI5011 enhances insulin sensitivity in the setting of established obesity-induced insulin resistance. The current study demonstrates that dietary intake of PMI5011 prevents high-fat diet-induced insulin resistance, metabolic dysfunction, and ectopic lipid accumulation in the skeletal muscle and liver without reducing body weight.

Peroxiredoxin 2 mediates insulin sensitivity of skeletal muscles through regulation of protein tyrosine phosphatase oxidation.

Insulin signaling is essential for regulating glucose homeostasis. Numerous studies have demonstrated that reactive oxygen species (ROS) affect insulin signaling, and low ROS levels can act as a signal to regulate cellular function. Peroxiredoxins (Prxs) are highly abundant and widely expressed antioxidant enzymes. However, it is unclear whether antioxidant enzymes, such as Prx2, mediate insulin signaling. The aim of our study was to investigate the influence of Prx2 deficiency on insulin signaling. Our western blot results showed that Prx2 deficiency enhanced insulin signaling and increased oxidation of protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homologue (PTEN) in mouse embryonic fibroblasts (MEFs) treated with insulin. In addition, we assessed ROS levels with a Cytosol-HyPer H2O2 sensor. As a result, increased ROS levels and Akt activation were decreased by N-acetyl-cysteine (Nac), which acted as an antioxidant in Prx2-deficient MEFs. Body weight measurements and glucose tolerance test (GTT) revealed significant body weight reduction and increase in glucose clearance in Prx2-/- mice fed a high-fat diet. Interestingly, glucose transporter type 4 (GLUT4) was significantly higher in Prx2-/- mice than in wild-type mice according to western blotting results. Western blotting also revealed that Akt phosphorylation was higher in Prx2-/- MEFs and muscle tissue than in wild-type. Together, our findings indicate that increased ROS due to Prx2 deficiency promotes insulin sensitivity and glucose clearance in skeletal muscles by increasing protein tyrosine phosphatase (PTPs) oxidation. These results provide novel insights into the fundamental mechanisms of insulin signaling induced by Prx2 deficiency and suggest that ROS-based therapeutic strategies can be used to suppress insulin resistance.

Absence of ANGPTL4 in adipose tissue improves glucose tolerance and attenuates atherogenesis.

Alterations in ectopic lipid deposition and circulating lipids are major risk factors for developing cardiometabolic diseases. Angiopoietin-like protein 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL), controls fatty acid (FA) uptake in adipose and oxidative tissues and regulates circulating triacylglycerol-rich (TAG-rich) lipoproteins. Unfortunately, global depletion of ANGPTL4 results in severe metabolic abnormalities, inflammation, and fibrosis when mice are fed a high-fat diet (HFD), limiting our understanding of the contribution of ANGPTL4 in metabolic disorders. Here, we demonstrate that genetic ablation of ANGPTL4 in adipose tissue (AT) results in enhanced LPL activity, rapid clearance of circulating TAGs, increased AT lipolysis and FA oxidation, and decreased FA synthesis in AT. Most importantly, we found that absence of ANGPTL4 in AT prevents excessive ectopic lipid deposition in the liver and muscle, reducing novel PKC (nPKC) membrane translocation and enhancing insulin signaling. As a result, we observed a remarkable improvement in glucose tolerance in short-term HFD-fed AT-specific Angptl4-KO mice. Finally, lack of ANGPTL4 in AT enhances the clearance of proatherogenic lipoproteins, attenuates inflammation, and reduces atherosclerosis. Together, these findings uncovered an essential role of AT ANGPTL4 in regulating peripheral lipid deposition, influencing whole-body lipid and glucose metabolism and the progression of atherosclerosis.

Ononitol monohydrate enhances PRDM16 & UCP-1 expression, mitochondrial biogenesis and insulin sensitivity via STAT6 and LTB4R in maturing adipocytes

Ononitol monohydrate (OMH), a glycoside was originally isolated from Cassia tora (Linn.). Glycosides regulate lipid metabolism but scientific validation desired. Hence, we aimed to evaluate the effect of OMH on enhancing mitochondrial potential, mitochondrial biogenesis, upregulate the expression of brown adipogenesis specific genes in maturing adipocytes. In addition, we observed the inter-relation between adipocyte and T-lymphocyte; whether, OMH treated adipocyte-condition medium stimulate T-cell chemokine linked with insulin resistance. In a dose dependent manner OMH treated to preadipocyte significantly inhibited maturation and enhanced mitochondrial biogenesis, it was confirmed by Oil red ‘O and Nile red stain without inducing cytotoxicity. The mRNA levels of adipocyte browning related genes such as, PR domain containing 16 (PRDM16), peroxisome proliferator activated receptor gamma coactivator 1 alpha (PPARγC1α) and uncoupling protein-1 (UCP-1) have been significantly upregulated. In addition, adipogenic transcription factors [such as proliferator activated receptor γ (PPARγ), CCAAT/enhancer binding protein (C/EBPα) and sterol regulatory element binding protein-1c (SREBP-1c)] and adipogenic genes were significantly down-regulated by treatment with OMH when compared to control cells. Protein expression levels of adiponectin have been increased; leptin, C/EBPα and leukotriene B4 receptor (LTB4R) were down regulated by OMH in mature adipocytes. In addition, adipocyte condition medium and OMH treated T-lymphocyte, significantly increased insulin signaling pathway related mRNAs, such as interlukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT6) and decreased leukotriene B4 (LTB4). The present findings suggest that OMH increased browning factors in differentiating and maturing preadipocyte also decreased adipose tissue inflammation as well as the enhanced insulin signaling.

Unique pharmacology of a novel allosteric agonist/sensitizer insulin receptor monoclonal antibody

ObjectiveInsulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D. MethodsA novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice. ResultsIn vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge. ConclusionCollectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology.

Metformin and blood cancers

Type 2 diabetes mellitus and cancer are correlated with changes in insulin signaling, a pathway that is frequently upregulated in neoplastic tissue but impaired in tissues that are classically targeted by insulin in type 2 diabetes mellitus. Many antidiabetes treatments, particularly metformin, enhance insulin signaling, but this pathway can be inhibited by specific cancer treatments. The modulation of cancer growth by metformin and of insulin sensitivity by anticancer drugs is so common that this phenomenon is being studied in hundreds of clinical trials on cancer.Many meta-analyses have consistently shown a moderate but direct effect of body mass index on the incidence of multiple myeloma and lymphoma and the elevated risk of leukemia in adults. Moreover, new epidemiological and preclinical studies indicate metformin as a therapeutic agent in patients with leukemia, lymphomas, and multiple myeloma. In this article, we review current findings on the anticancer activities of metformin and the underlying mechanisms from preclinical and ongoing studies in hematologic malignancies.

Palmatine Enhances Insulin Signalling Pathway in Muscle of High Fat STZ Induced Diabetic Rat Model

Palmatine Enhances Insulin Signalling Pathway in Muscle of High Fat STZ Induced Diabetic Rat Model

Plant-derived compounds strigolactone GR24 and pinosylvin activate SIRT1 and enhance glucose uptake in rat skeletal muscle cells

Insulin resistance is a characteristic finding in hyperglycaemia and type 2 diabetes. SIRT1 is a NAD+ dependent deacetylase that plays a central role in glucose homeostasis and energy metabolism. SIRT1 activators, including plant polyphenols such as resveratrol, improve insulin sensitivity in skeletal muscle tissue. We hypothesised that the novel plant-derived compounds, strigolactone and pinosylvin, beneficially enhance SIRT1 function, insulin signalling, glucose uptake, and mitochondrial biogenesis in skeletal muscle cells. Rat L6 skeletal muscle myotubes were treated with strigolactone analogue GR24 and pinosylvin. Resveratrol was included in experiments as a reference compound. We measured the effects of these compounds on SIRT1 function, insulin signalling, glucose uptake, mitochondrial biogenesis and gene expression profiles. Strigolactone GR24 upregulated and activated SIRT1 without activating AMPK, enhanced insulin signalling, glucose uptake, GLUT4 translocation and mitochondrial biogenesis. Pinosylvin activated SIRT1 in vitro and stimulated glucose uptake through the activation of AMPK. The regulation of SIRT1 by strigolactone GR24 and the activation of AMPK by pinosylvin may offer novel therapeutic approaches in the treatment of insulin resistance in skeletal muscle.

Induction of glucose uptake in skeletal muscle by central leptin is mediated by muscle \u03b22-adrenergic receptor but not by AMPK

Leptin increases glucose uptake and fatty acid oxidation (FAO) in red-type skeletal muscle. However, the mechanism remains unknown. We have investigated the role of β2-adrenergic receptor (AR), the major β-AR isoform in skeletal muscle, and AMPK in leptin-induced muscle glucose uptake of mice. Leptin injection into the ventromedial hypothalamus (VMH) increased 2-deoxy-D-glucose (2DG) uptake in red-type skeletal muscle in wild-type (WT) mice accompanied with increased phosphorylation of the insulin receptor (IR) and Akt as well as of norepinephrine (NE) turnover in the muscle. Leptin-induced 2DG uptake was not observed in β-AR-deficient (β-less) mice despite that AMPK phosphorylation was increased in the muscle. Forced expression of β2-AR in the unilateral hind limb of β-less mice restored leptin-induced glucose uptake and enhancement of insulin signalling in red-type skeletal muscle. Leptin increased 2DG uptake and enhanced insulin signalling in red-type skeletal muscle of mice expressing a dominant negative form of AMPK (DN-AMPK) in skeletal muscle. Thus, leptin increases glucose uptake and enhances insulin signalling in red-type skeletal muscle via activation of sympathetic nerves and β2-AR in muscle and in a manner independent of muscle AMPK.

Nigella sativa Oil and Chromium Picolinate Ameliorate Fructose-Induced Hyperinsulinemia by Enhancing Insulin Signaling and Suppressing Insulin-Degrading Enzyme in Male Rats.

In vivo and in vitro studies suggested that chromium enhances insulin sensitivity by promoting insulin receptor signaling. However, its effect on insulin clearance has not been yet identified. Nigella sativa, a widely used spice, possesses an antidiabetic activity. We, therefore, hypothesized that chromium picolinate may alter insulin clearance by modulating insulin-degrading enzyme (IDE) in insulin-resistant rats. We evaluated also the effect of Nigella sativa oil on insulin signaling and degradation with respect to chromium picolinate. To assess these hypotheses, insulin resistance was induced in 30 male Wistar albino rats through daily oral administration of high-fructose water (HFW, 20% w/v) for 45days. These rats were then divided into three groups (n=10/group). They were given either no treatment (control group) or Nigella sativa oil (500mg/kgbw/day) or chromium picoloinate (200μg/kgbw/day) orally along with HFW (20% w/v) for 45days. Nigella sativa oil or chromium picolinate concurrent administration with HFW significantly decreased body weight, serum lipids, glucagon, insulin resistance, and hepatic IDE level but increased its mRNA expression and insulin receptor phosphorlyation as well as high-density lipoprotein cholesterol (HDL-C) level as compared to control group values, suggesting their potential as modulators for insulin signaling and clearance. However, Nigella sativa oil exerted better improvement in feeding efficacy ratio as well as the levels of glucagon, insulin, insulin resistance, hepatic IDE level and insulin receptor phosphorylation than chromium picolinate, suggesting its greater insulin sensitizing capacity. Our data, for the first time, prove that Nigella sativa oil and chromium picolinate monotherapy can reduce fructose-induced insulin resistance by reduction of hepatic IDE protein and activation of insulin receptor signaling.

Histone deacetylase regulates insulin signaling via two pathways in pancreatic β cells.

Recent studies demonstrated that insulin signaling plays important roles in the regulation of pancreatic β cell mass, the reduction of which is known to be involved in the development of diabetes. However, the mechanism underlying the alteration of insulin signaling in pancreatic β cells remains unclear. The involvement of epigenetic control in the onset of diabetes has also been reported. Thus, we analyzed the epigenetic control of insulin receptor substrate 2 (IRS2) expression in the MIN6 mouse insulinoma cell line. We found concomitant IRS2 up-regulation and enhanced insulin signaling in MIN6 cells, which resulted in an increase in cell proliferation. The H3K9 acetylation status of the Irs2 promoter was positively associated with IRS2 expression. Treatment of MIN6 cells with histone deacetylase inhibitors led to increased IRS2 expression, but this occurred in concert with low insulin signaling. We observed increased IRS2 lysine acetylation as a consequence of histone deacetylase inhibition, a modification that was coupled with a decrease in IRS2 tyrosine phosphorylation. These results suggest that insulin signaling in pancreatic β cells is regulated by histone deacetylases through two novel pathways affecting IRS2: the epigenetic control of IRS2 expression by H3K9 promoter acetylation, and the regulation of IRS2 activity through protein modification. The identification of the histone deacetylase isoform(s) involved in these mechanisms would be a valuable approach for the treatment of type 2 diabetes.

Protein tyrosine phosphatase 1B deficiency in podocytes mitigates hyperglycemia-induced renal injury

ObjectiveDiabetic nephropathy is one of the most devastating complications of diabetes, and growing evidence implicates podocyte dysfunction in disease pathogenesis. The objective of this study was to investigate the contribution of protein tyrosine phosphatase 1B (PTP1B) in podocytes to hyperglycemia-induced renal injury. MethodsTo determine the in vivo function of PTP1B in podocytes we generated mice with podocyte-specific PTP1B disruption (hereafter termed pod-PTP1B KO). Kidney functions were determined in control and pod-PTP1B KO mice under normoglycemia and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia. ResultsPTP1B expression increased in murine kidneys following HFD and STZ challenges. Under normoglycemia control and pod-PTP1B KO mice exhibited comparable renal functions. However, podocyte PTP1B disruption attenuated hyperglycemia-induced albuminuria and renal injury and preserved glucose control. Also, podocyte PTP1B disruption was accompanied with improved renal insulin signaling and enhanced autophagy with decreased inflammation and fibrosis. Moreover, the beneficial effects of podocyte PTP1B disruption in vivo were recapitulated in E11 murine podocytes with lentiviral-mediated PTP1B knockdown. Reconstitution of PTP1B in knockdown podocytes reversed the enhanced insulin signaling and autophagy suggesting that they were likely a consequence of PTP1B deficiency. Further, pharmacological attenuation of autophagy in PTP1B knockdown podocytes mitigated the protective effects of PTP1B deficiency. ConclusionsThese findings demonstrate that podocyte PTP1B deficiency attenuates hyperglycemia-induced renal damage and suggest that PTP1B may present a therapeutic target in renal injury.

An ALPHA7 Nicotinic Acetylcholine Receptor Agonist (GTS-21) Promotes C2C12 Myonuclear Accretion in Association with Release of Interleukin-6 (IL-6) and Improves Survival in Burned Mice.

The role of interleukin-6 (IL-6) in physiological processes and disease is poorly understood. The hypothesis tested in this study was that selective alpha7 acetylcholine receptor (α7AChR) agonist, GTS-21, releases IL-6 in association with myonuclear accretion and enhances insulin signaling in muscle cells, and improves survival of burn injured (BI) mice. The in vitro effects of GTS-21 were determined in C2C12 myoblasts and 7-day differentiated myotubes (myotubes). The in vivo effects of GTS-21 were tested in BI wild-type (WT) and IL-6 knockout (IL6KO) mice. GTS-21 dose-dependently (0 μM, 100 μM, and 200 μM) significantly increased IL-6 levels in myoblasts and myotubes at 6 and 9 h. GTS-21-induced IL-6 release in myotubes was attenuated by methyllycaconitine (α7AChR antagonist), and by Stat-3 or Stat-5 inhibitors. GTS-21 increased MyoD and Pax7 protein expression, myonuclear accretion, and insulin-induced phosphorylation of Akt, GSK-3β, and Glut4 in myotubes. The glucose levels of burned IL6KO mice receiving GTS-21 decreased significantly compared with sham-burn IL6KO mice. Superimposition of BI on IL6KO mice caused 100% mortality; GTS-21 reduced mortality to 75% in the IL6KO mice. The 75% mortality in burned WT mice was reduced to 0% with GTS-21. The in vitro findings suggest that GTS-21-induced IL-6 release from muscle is mediated via α7AChRs upstream of Stat-3 and -5 pathways and is associated with myonuclear accretion, possibly via MyoD and Pax7 expression. GTS-21 in vivo improves survival in burned WT mice and IL6KO mice, suggesting a potential therapeutic application of α7AChR agonists in the treatment of BI.

Exendin-4 reduces food intake via the PI3K/AKT signaling pathway in the hypothalamus

Exendin-4 (EX-4), a glucagon-like peptide-1 (GLP-1) receptor agonist, has been shown to reduce food intake and to increase proopiomelanocortin (POMC) gene expression in the hypothalamus. In this study, we examined the potential neural mechanisms by which these effects occur. Male Sprague Dawley rats were implanted with a cannula in the third ventricle of the brain through which an inhibitor of phosphatidylinositol-3 kinase (PI3K) (wortmannin) was administered, and EX-4 or vehicle was administered via intraperitoneal (IP) injection. The activity of PI3K/protein kinase B (AKT) and insulin receptor substrate-1 (IRS-1) in the hypothalamic arcuate was determined. We found that EX-4 treatment significantly decreased food intake and body weight. However, there were almost no changes in food intake and body weight when wortmannin injection (into the third ventricle) occurred prior to EX-4 IP injection. EX-4 not only increased the activity of PI3K/AKT, but it also increased IRS-1 activity. These results show that EX-4 likely suppresses food intake due to its ability to enhance insulin signaling.

Role of insulin receptor substrates in the progression of hepatocellular carcinoma

Several cellular signaling pathways, including insulin/IGF signaling, are known to be activated in hepatocellular carcinoma (HCC). Here, we investigated the roles of insulin receptor substrate (Irs) 1 and Irs2, both of which are the major molecules to be responsible for transducing insulin/IGF signaling in the liver, in the development of HCC by inducing chemical carcinogenesis using diethylnitrosamine (DEN) in mice. The Irs1 mRNA and protein expressions were upregulated in the tumors, along with enhanced insulin signaling. Liver-specific Irs1-knockout (LIrs1KO) mice exhibited suppression of DEN-induced HCC development, accompanied by reduced cancer cell proliferative activity and reduced activation of Akt. Gene expression analyses revealed that the tumors in the DEN-treated LIrs1KO mice showed modest metabolic alterations during hepatocarcinogenesis as well as decreased inflammation and invasion potentials. On the other hand, liver-specific Irs2-knockout (LIrs2KO) mice showed a similar pattern of HCC development to the DEN-treated control wild-type mice. Based on the knowledge that Wnt/β-catenin signaling is activated in HCC, we focused on Wnt/β-catenin signaling and demonstrated that Irs1 expression was induced by Wnt3a stimulation in the primary hepatocytes, associated with insulin-stimulated Akt activation. These data suggest that upregulated Irs1 by Wnt/β-catenin signaling plays a crucial role in the progression of HCC.