Khalid A. Al-Regaiey

Local expression of GH and IGF-1 in the hippocampus of GH-deficient long-lived mice

Beneficial effects of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) on the development and function of the central nervous system are well documented. In spite of primary deficiency of GH and secondary IGF-1 deficiency, Ames dwarf mice live considerably longer than normal animals, exhibit apparently normal cognitive functions and maintain them into advanced age. In an attempt to reconcile these findings, we have examined local expression of GH and IGF-1 in the hippocampus of normal and Ames dwarf mice. We found that both hippocampal GH and IGF-1 protein levels are increased and the corresponding mRNAs are normal in Ames dwarf as compared with normal mice. Increased phosphorylation of Akt and cyclic AMP responsive element-binding protein (CREB) were detected in the hippocampus of Ames dwarf mice. Our results suggest that increase in hippocampal GH and IGF-1 protein expression and subsequent activation of PI3K/Akt-CREB signal transduction cascade might contribute to the maintenance of cognitive function and is likely to be responsible for the integrity of neuronal structure, and maintenance of youthful levels of cognitive function in these long-lived mice during aging.

Effects of caloric restriction on insulin pathway gene expression in the skeletal muscle and liver of normal and long-lived GHR-KO mice.

Growth hormone receptor/binding protein knockout (GHR-KO) mice are characterized by resistance to growth hormone (GH), reduced insulin like growth factor 1 (IGF1) levels and enhanced insulin sensitivity and markedly increased lifespan. Findings in these and other long-lived mutant mice, and in normal animals subjected to caloric restriction (CR) indicate that insulin signaling is importantly involved in the control of longevity. We have examined the mRNA expression level of genes involved in insulin/IGF1 action in the skeletal muscle and liver of normal and GHR-KO mice fed ad libitum or subjected to long term 30% CR. The levels of IR, IRS1, IRS2, GLUT4 and IGF1 message in the skeletal muscle were reduced by CR in both normal and GHR-KO mice. In the liver, the results indicate that in GHR-KO mice mRNA expression of genes related to early steps of insulin signaling is up-regulated in the liver but not in the muscle. The results also show that improved insulin sensitivity in response to CR is not due to increased mRNA expression of the above genes in either normal or GHR-KO animals.

Effects of dietary restriction on the expression of insulin-signaling-related genes in long-lived mutant mice.

Hypopituitary Ames dwarf mice and growth-hormone-resistant (growth hormone receptor knockout, GHRKO) mice have reduced plasma levels of insulin-like growth factor 1 and insulin, enhanced insulin sensitivity and a remarkably increased life span. This resembles the phenotypic characteristics of genetically normal animals subjected to dietary restriction (DR). Interestingly, DR leads to further increases in insulin sensitivity and longevity in Ames dwarfs but not in GHRKO mice. It was therefore of interest to examine the effects of DR on the expression of insulin-related genes in these two types of long-lived mutant mice. The effects of DR partially overlapped but did not duplicate the effects of Ames dwarfism or GHR deletion on the expression of genes related to insulin signaling and cell responsiveness to insulin. Moreover, the effects of DR on the expression of the examined genes in different insulin target organs were not identical. Some of the insulin-related genes were similarly affected by DR in both GHRKO and normal mice, some were affected only in GHRKO mice and some only in normal animals. This last category is of particular interest since genes affected in normal but not GHRKO mice may be related to mechanisms by which DR extends longevity.

Caloric Restriction and Growth Hormone Receptor Knockout: Effects on Expression of Genes Involved in Insulin Action in the Heart

Blockade of growth hormone (GH), decreased insulin-like growth factor-1 (IGF1) action and increased insulin sensitivity are associated with life extension and an apparent slowing of the aging process. We examined expression of genes involved in insulin action, IR, IRS1, IRS2, IGF1, IGF1R, GLUT4, PPARs and RXRs in the hearts of normal and GHR-/- (KO) mice fed ad libitum or subjected to 30% caloric restriction (CR). CR increased the cardiac expression of IR, IRS1, IGF1, IGF1R and GLUT4 in normal mice and IRS1, GLUT4, PPARalpha and PPARbeta/delta in GHR-KO animals. Expression of IR, IRS1, IRS2, IGF1, GLUT4, PPARgamma and PPARalpha did not differ between GHR-KO and normal mice. These unexpected results suggest that CR may lead to major modifications of insulin action in the heart, but high insulin sensitivity of GHR-KO mice is not associated with alterations in the levels of most of the examined molecules related to intracellular insulin signaling.

Effects of long-term caloric restriction on glucose homeostasis and on the first steps of the insulin signaling system in skeletal muscle of normal and Ames dwarf (Prop1df/Prop1df) mice

Ames dwarf mice are a model of retarded aging and extended longevity and display enhanced insulin sensitivity. Caloric restriction (CR) and the dwarf mutation have additive effects on lifespan. To begin to understand the mechanisms behind this effect, an analysis of the in vivo status of the insulin signaling system was performed in skeletal muscle from Ames dwarf (df/df) and normal mice fed ad libitum or subjected to long-term (over 1 year) CR. The response to CR was different in both groups of animals. In normal animals, CR induced a significant reduction in both circulating insulin and glucose levels, together with an increase in the in vivo insulin-stimulated phosphorylation of the IR, a trend towards an increase in the in vivo insulin-stimulated phosphorylation levels of IR substrate-1, and an increase in the abundance of GLUT4 in muscle. In contrast, CR did not modify none of these parameters in df/df mice. Interestingly, CR induced a reduction in the p85 subunit of phosphatidylinositol 3-kinase abundance in skeletal muscle in both groups of animals. These results suggest that in skeletal muscle, long-term CR induces different effects on the first steps of the insulin signaling system in normal mice than in df/df mice.

Effect of every other day feeding diet on gene expression in normal and in long-lived ames dwarf mice

Ames dwarf mutant mice are long-lived, hypoinsulinemic and hypoglycemic and exhibit enhanced sensitivity to injected insulin. Their phenotypic characteristics show many similarities to animals subjected to caloric restriction (CR) but Ames dwarf mice are not CR mimetics. Reducing daily food intake by 30% prolongs longevity in both normal and Ames dwarf mice. In the present study, the animals were subjected to a different type of CR, every other day feeding (EOD). Using real-time PCR, we have examined the expression of genes related to insulin signaling in the liver of normal and dwarf mice after 9 months of EOD. The results indicate that EOD produces some changes in the insulin and IGF1 signaling pathways, and that these changes are consistent with EOD increasing insulin sensitivity.

Growth hormone abolishes beneficial effects of calorie restriction in long-lived Ames dwarf mice.

Disruption of the growth hormone (GH) axis promotes longevity and delays aging. In contrast, GH over-expression may lead to accelerated aging and shorter life. Calorie restriction (CR) improves insulin sensitivity and may extend lifespan. Long-lived Ames dwarf (df/df) mice have additional extension of longevity when subjected to 30% CR. The aim of the study was to assess effects of CR or GH replacement therapy separately and as a combined (CR+GH) treatment in GH-deficient df/df and normal mice, on selected metabolic parameters (e.g., insulin, glucose, cholesterol), insulin signaling components (e.g., insulin receptor [IR] β-subunit, phosphorylated form of IR [IR pY1158], protein kinase C ζ/λ [p-PKCζ/λ] and mTOR [p-mTOR]), transcription factor p-CREB, and components of the mitogen-activated protein kinase (MAPK) signaling (p-ERK1/2, p-p38), responsible for cell proliferation, differentiation and survival. CR decreased plasma levels of insulin, glucose, cholesterol and leptin, and increased hepatic IR β-subunit and IR pY1158 levels as well as IR, IRS-1 and GLUT-2 gene expression compared to ad libitum feeding, showing a significant beneficial diet intervention effect. Moreover, hepatic protein levels of p-PKCζ/λ, p-mTOR and p-p38 decreased, and p-CREB increased in CR mice. On the contrary, GH increased levels of glucose, cholesterol and leptin in plasma, and p-mTOR or p-p38 in livers, and decreased plasma adiponectin and hepatic IR β-subunit compared to saline treatment. There were no GH effects on adiponectin in N mice. Moreover, GH replacement therapy did not affect IR, IRS-1 and GLUT-2 gene expression. GH treatment abolishes the beneficial effects of CR; it may suggest an important role of GH-IGF1 axis in mediating the CR action. Suppressed somatotrophic signaling seems to predominate over GH replacement therapy in the context of the examined parameters and signaling pathways.

Study of the association of adrenomedullin and basic-fibroblast growth factors with the peripheral arterial blood flow and endothelial dysfunction biomarkers in type 2 diabetic patients with peripheral vascular insufficiency

BackgroundProgressive micro-vascular vaso-degeneration is the major factor in progression of diabetic complications. Adrenomedullin (AM) and basic-Fibroblast growth factor (b-FGF) are strongly correlated with angiogenesis in vascular diseases. This study aims to provide base line data regarding the vascular effects and correlation of AM, and b-FGF with the peripheral blood flow in diabetic patients with peripheral vascular disease (PVD), and their effect on endothelial dysfunction markers. Ninety age- and sex matched females were enrolled in the study: 30 were controls, 30 had diabetes without complications (group II) and 30 had diabetes with PVD (group III) diagnosed by ankle/ brachial index (A/BI). Plasma levels of AM, b-FGF, intercellular adhesion molecule −1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were measured by indirect enzyme immunoassay (ELISA).ResultsThere was a significant increase in plasma AM, VCAM-1and ICAM-1, while a significant decrease in plasma b-FGF in diabetic patients with PVD (p < 0.05). A positive correlation was observed between plasma AM, b-FGF and A/BI and a negative correlation with VCAM −1 and ICAM in diabetic PVD. AM was not a predictor, while b-FG, VCAM-1 and ICAM-1 could be predictors for peripheral blood flow in diabetic PVD.ConclusionThis study elucidates for the first time that AM and b-FGF are correlated and have a direct impact on the peripheral blood flow, the rise of AM in diabetic PVD may be a consecutive and compensatory vasculo-protective effect as its angiogenic and anti-inflammatory properties act to relief the endothelial insult. Down expression of b-FGF may be a predisposing factor for micro-vascular derangement. It is not clear if the rise of AM and the decline of b- FGF levels may be consequences or predisposing factors for VCAM-1 and ICAM-1 elevation as these endothelial dysfunction biomarkers could reduce peripheral blood flow and vascular integrity. It is optimistic to believe that drug intervention through AM and b-FGF administration together with reversing the endothelial inflammatory process by targeting VCAM and ICAM could reduce the prevalence of diabetic vascular complications, reduce the risk of cerebrovascular and cardiovascular morbidity in diabetes through normalizing vascular endothelium function and peripheral blood flow.

The role of serum levels of thioredoxin and thioredoxin-interacting protein in stroke

Aims: Serum level of thioredoxin (TRX), a redox-regulating protein with antioxidant activity, increases under oxidative stress. The present study measured serum levels of TRX and its inhibitor TRX-interacting protein (TXNIP) in patients who experienced first-ever acute ischemic stroke (AIS). Subjects and Methods: We retrospectively enrolled 45 patients who experienced AIS and 33 age- and sex-matched healthy controls. Serum TRX and TXNIP levels in stroke patients and healthy controls were analyzed by performing solid-phase sandwich enzyme-linked immunosorbent assay. Results: Our results showed that mean serum TXNIP levels were significantly higher in stroke patients than in healthy controls (P = 0.044). However, serum TRX levels were not significantly different between stroke patients and healthy controls (P = 0.405). Moreover, we observed a significant positive correlation between TRX and TXNIP levels (R2 = 0.476, P < 0.003). Conclusions: These results suggest that TRX and TXNIP are rapid, inexpensive, and convenient biomarkers of stroke. However, additional studies should be performed to validate these preliminary observations and the role of TRX and TXNIP in AIS.