Luigi Laviola

The GH/IGF1 axis and signaling pathways in the muscle and bone: mechanisms underlying age-related skeletal muscle wasting and osteoporosis

The widespread increase in life expectancy is accompanied by an increased prevalence of features of physical frailty. Signs and symptoms may include sarcopenia and osteopenia, reduced exercise capacity, and diminished sense of well-being. The pathogenesis of age-associated sarcopenia and osteopenia is multifactorial, and hormonal decline may be a contributing factor. Aging is associated with a progressive decrease in GH secretion, and more than 30% of elderly people have circulating IGF1 levels below the normal range found in the young. GH acts directly on target tissues, including skeletal muscle and bone among many others, but many effects are mediated indirectly by circulating (liver-derived) or locally produced IGF1. Aging is also associated with reduced insulin sensitivity which, in turn, may contribute to the impairment of IGF1 action. Recent experimental evidence suggests that besides the age-dependent decline in GH and IGF1 serum levels, the dysregulation of GH and IGF1 actions due to impairment of the post-receptor signaling machinery may contribute to the loss of muscle mass and osteopenia. This article will focus on the molecular mechanisms of impaired GH and IGF1 signaling and action in aging, and their role in the pathogenesis of sarcopenia and osteoporosis.

The IGF-I Signaling Pathway

The insulin-like growth factor (IGF)-I is implicated in the regulation of protein turnover and exerts potent mitogenic and differentiating effects on most cell types. IGF-I biological actions are mediated by the IGF-I receptor, comprised of two extra-cellular alpha-subunits, containing hormone binding sites, and two membrane-spanning beta-subunits, encoding an intracellular tyrosine kinase. Hormone binding activates the receptor kinase, leading to receptor autophosphorylation and tyrosine phosphorylation of multiple substrates, including the IRS and Shc proteins. Through these initial tyrosine phosphorylation reactions, IGF-I signals are transduced to a complex network of intracellular lipid and serine/threonine kinases that are ultimately responsible for cell proliferation, modulation of tissue differentiation, and protection from apoptosis. This review will focus on the IGF-I receptor structure and function, its intracellular signaling pathways, and some important implications of the activation of the IGF-I signal transduction system in specific tissues.

Clinical significance of nonalbuminuric renal impairment in type 2 diabetes.

Objective In type 2 diabetes, prevalence of nonalbuminuric renal impairment is increasing worldwide, though its clinical significance remains unclear. This large-cohort study aimed at evaluating the association of this phenotype with cardiovascular risk factors and other complications. Methods Type 2 diabetic patients from the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study (n = 15 773), visiting consecutively 19 hospital-based Diabetes Clinics in years 2007–2008, were examined. Serum creatinine was assessed by the Jaffe method; albuminuria was measured by immunonephelometry or immunoturbidimetry. Results Of patients with renal impairment, as identified by an estimated glomerular filtration rate (eGFR) less than 60 ml/min per 1.73 m2, 56.6% were normoalbuminuric, 30.8% were microalbuminuric, and 12.6% were macroalbuminuric. Percentages were similar when GFR was estimated using the more accurate Chronic Kidney Disease Epidemiology Collaboration equation instead of the simplified Modification of Diet in Renal Disease formula, and were independent of age, thus indicating that the increasing prevalence of this phenotype does not reflects misclassification of elderly patients. Nonalbuminuric renal impairment was not associated with HbA1c and correlated less strongly with retinopathy and hypertension than albuminuria, either alone or associated with reduced eGFR. It was associated with a higher prevalence of cardiovascular disease (CVD) than albuminuria alone, but lower than albuminuric renal impairment. Female sex correlated with nonalbuminuric renal impairment and male sex with the albuminuric forms. Conclusions These data show that type 2 diabetic patients with nonalbuminuric renal impairment exhibit distinct clinical features, suggesting predominance of macroangiopathy as underlying renal pathology, and that this phenotype is associated with significant CVD burden.

Fat depot-related differences in gene expression, adiponectin secretion, and insulin action and signalling in human adipocytes differentiated in vitro from precursor stromal cells

Aim/hypothesisThe distinct metabolic properties of visceral and subcutaneous adipocytes may be due to inherent characteristics of the cells that are resident in each fat depot. To test this hypothesis, human adipocytes were differentiated in vitro from precursor stromal cells obtained from visceral and subcutaneous fat depots and analysed for genetic, biochemical and metabolic endpoints.MethodsStromal cells were isolated from adipose tissue depots of nondiabetic individuals. mRNA levels of adipocyte-specific proteins were determined by real-time RT-PCR. Insulin signalling was evaluated by immunoblotting with specific antibodies. Glucose transport was measured by a 2-deoxy-glucose uptake assay. Adiponectin secretion in the adipocyte-conditioned medium was determined by a specific RIA.ResultsWith cell differentiation, mRNA levels of PPARG, C/EBPα (also known as CEBPA), AP2 (also known as GTF3A), GLUT4 (also known as SLC2A4) were markedly upregulated, whereas GLUT1 (also known as SLC2A1) mRNA did not change. However, expression of C/EBPα, AP2 and adiponectin was higher in subcutaneous than in visceral adipocytes. By contrast, adiponectin was secreted at threefold higher rates by visceral than by subcutaneous adipocytes while visceral adipocytes also showed two- to threefold higher insulin-stimulated glucose uptake. Insulin-induced phosphorylation of the insulin receptor, IRS proteins, Akt and extracellular signal-regulated kinase-1/2 was more rapid and tended to decrease at earlier time-points in visceral than in subcutaneous adipocytes.Conclusions/interpretationSubcutaneous and visceral adipocytes, also when differentiated in vitro from precursor stromal cells, retain differences in gene expression, adiponectin secretion, and insulin action and signalling. Thus, the precursor cells that reside in the visceral and subcutaneous fat depots may already possess inherent and specific metabolic characteristics that will be expressed upon completion of the differentiation programme.

The adapter protein Grb10 associates preferentially with the insulin receptor as compared with the IGF-I receptor in mouse fibroblasts.

To identify receptor-associated proteins that may contribute to the specificity of insulin and IGF-I signaling responses, a mouse embryo library was screened using the yeast two-hybrid system. Multiple receptor-interactive clones encoding the SH2 domain of the adapter protein Grb10 were isolated. Subsequent cloning of the full-length Grb10 sequence from a mouse fat cDNA library defined a previously unknown Grb10 variant, that appears to be the predominant isoform in mouse tissues. Receptor-deficient R- cells (fibroblasts from mice with homologous disruption of the IGF-I receptor gene) and transfected R- cells expressing either insulin receptors (R-IR cells) or IGF-I receptors (R+ cells) were used to investigate the specificity of Grb10 interaction with the two related receptors. Hormone-activated insulin receptors in R-IR cells coprecipitated with three species, all recognized as Grb10 isoforms by specific Grb10 antibody. Under the same conditions, Grb10 was essentially undetectable in IGF-I receptor immunoprecipitates from stimulated R+ cells. Grb10 association with insulin receptors was maximal at 10 nM insulin stimulation and sustained from 5-10 min after hormone stimulation in R-IR cells. In conclusion, Grb10 interacts preferentially with insulin vs. IGF-I receptors in intact cells and, thus, may have a role in mediating insulin receptor-specific cellular responses.

Interaction between the Grb10 SH2 Domain and the Insulin Receptor Carboxyl Terminus

Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor β-subunit using the yeast two-hybrid system. The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin-dependent manner with insulin receptors from cell lysates and with purified insulin receptors. Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1. These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.

HbA1c Variability as an Independent Correlate of Nephropathy, but Not Retinopathy, in Patients With Type 2 Diabetes: The Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study

OBJECTIVE To examine the association of hemoglobin (Hb) A1c variability with microvascular complications in the large cohort of subjects with type 2 diabetes from the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study. RESEARCH DESIGN AND METHODS Serial (3–5) HbA1c values collected in a 2-year period before enrollment were available from 8,260 subjects from 9 centers (of 15,773 patients from 19 centers). HbA1c variability was measured as the intraindividual SD of 4.52 ± 0.76 values. Diabetic retinopathy (DR) was assessed by dilated funduscopy. Chronic kidney disease (CKD) was defined based on albuminuria, as measured by immunonephelometry or immunoturbidimetry, and estimated glomerular filtration rate (eGFR) was calculated from serum creatinine. RESULTS Median and interquartile range of average HbA1c (HbA1c-MEAN) and HbA1c-SD were 7.57% (6.86–8.38) and 0.46% (0.29–0.74), respectively. The highest prevalence of microalbuminuria, macroalbuminuria, reduced eGFR, albuminuric CKD phenotypes, and advanced DR was observed when both HbA1c parameters were above the median and the lowest when both were below the median. Logistic regression analyses showed that HbA1c-SD adds to HbA1c-MEAN as an independent correlate of microalbuminuria and stages 1–2 CKD and is an independent predictor of macroalbuminuria, reduced eGFR, and stages 3–5 albuminuric CKD, whereas HbA1c-MEAN is not. The opposite was found for DR, whereas neither HbA1c-MEAN nor HbA1c-SD affected nonalbuminuric CKD. CONCLUSIONS In patients with type 2 diabetes, HbA1c variability affects (albuminuric) CKD more than average HbA1c, whereas only the latter parameter affects DR, thus suggesting a variable effect of these measures on microvascular complications.

Gender differences in cardiovascular disease risk factors, treatments and complications in patients with type 2 diabetes: the RIACE Italian multicentre study

Poorer control of risk factors for cardiovascular disease (CVD) has been reported in diabetic women, as compared with diabetic men. It has been proposed that this finding is due to gender disparities in treatment intensity. We investigated this hypothesis in a large contemporary cohort of subjects with type 2 diabetes.

Cardiovascular disease and glycemic control in type 2 diabetes: now that the dust is settling from large clinical trials.

The relationship between glucose control and cardiovascular outcomes in type 2 diabetes has been a matter of controversy over the years. Although epidemiological evidence exists in favor of an adverse role of poor glucose control on cardiovascular events, intervention trials have been less conclusive. The Action to Control Cardiovascular Risk in Diabetes (ACCORD) study, the Action in Diabetes and Vascular Disease (ADVANCE) study, and the Veterans Affairs Diabetes Trial (VADT) have shown no beneficial effect of intensive glucose control on primary cardiovascular endpoints in type 2 diabetes. However, subgroup analysis has provided evidence suggesting that the potential beneficial effect largely depends on patients’ characteristics, including age, diabetes duration, previous glucose control, presence of cardiovascular disease, and risk of hypoglycemia. The benefit of strict glucose control on cardiovascular outcomes and mortality may be indeed hampered by the extent and frequency of hypoglycemic events and could be enhanced if glucose‐lowering medications, capable of exerting favorable effects on the cardiovascular system, were used. This review examines the relationship between intensive glucose control and cardiovascular outcomes in type 2 diabetes, addressing the need for individualization of glucose targets and careful consideration of the benefit/risk profile of antidiabetes medications.

Cross-Talk between PPARγ and Insulin Signaling and Modulation of Insulin Sensitivity

PPARγ activation in type 2 diabetic patients results in a marked improvement in insulin and glucose parameters, resulting from an improvement of whole-body insulin sensitivity. Adipose tissue is the major mediator of PPARγ action on insulin sensitivity. PPARγ activation in mature adipocytes induces the expression of a number of genes involved in the insulin signaling cascade, thereby improving insulin sensitivity. PPARγ is the master regulator of adipogenesis, thereby stimulating the production of small insulin-sensitive adipocytes. In addition to its importance in adipogenesis, PPARγ plays an important role in regulating lipid, metabolism in mature adipocytes by increasing fatty acid trapping. Finally, adipose tissue produces several cytokines that regulate energy homeostasis, lipid and glucose metabolism. Disturbances in the production of these factors may contribute to metabolic abnormalities, and PPARγ activation is also associated with beneficial effects on expression and secretion of a whole range of cytokines.