Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart.

This protocol describes the isolation of endogenous c-Kit (also known as CD117)-positive (c-Kit(+)), CD45-negative (CD45(-)) cardiac stem cells (eCSCs) from whole adult mouse and rat hearts. The heart is enzymatically digested via retrograde perfusion of the coronary circulation, resulting in rapid and extensive breakdown of the whole heart. Next, the tissue is mechanically dissociated further and cell fractions are separated by centrifugation. The c-Kit(+)CD45(-) eCSC population is isolated by magnetic-activated cell sorting technology and purity and cell numbers are assessed by flow cytometry. This process takes ∼4 h for mouse eCSCs or 4.5 h for rat eCSCs. We also describe how to characterize c-Kit(+)CD45(-) eCSCs. The c-Kit(+)CD45(-) eCSCs exhibit the defining characteristics of stem cells: they are self-renewing, clonogenic and multipotent. This protocol also describes how to differentiate eCSCs into three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle, and endothelial cells. These processes take 17-20 d.

A polymorphism of HMGA1 is associated with increased risk of metabolic syndrome and related components.

The metabolic syndrome (MetS) is a common disorder, where systemic insulin-resistance is associated with increased risk for type 2 diabetes (T2D) and cardiovascular disease. Identifying genetic traits influencing risk and progression of MetS is important. We and others previously reported a functional HMGA1 gene variant, rs146052672, predisposing to T2D. Here we investigated the association of rs146052672 variant with MetS and related components. In a case-control study from Italy and Turkey, increased risk of MetS was seen among carriers of the HMGA1 variant. In the larger Italian cohort, this variant positively correlated with BMI, hyperglycemia and insulin-resistance, and negatively correlated with serum HDL-cholesterol. Association between rs146052672 variant and MetS occurred independently of T2D, indicating that HMGA1 gene defects play a pathogenetic role in MetS and other insulin-resistance-related conditions. Overall, our results indicate that the rs146052672 variant represents an early predictive marker of MetS, as well as a predictive tool for therapy.

The HMGA1-IGF-I/IGFBP system: a novel pathway for modulating glucose uptake.

We previously showed that loss of the high mobility group A1 (HMGA1) protein expression, induced in mice by disrupting the Hmga1 gene, considerably decreased insulin receptor expression in the major target tissues of insulin action, causing a type 2-like diabetic phenotype, in which, however, glucose intolerance was paradoxically associated with increased peripheral insulin sensitivity. Insulin hypersensitivity despite impairment of insulin action supports the existence of molecular adaptation mechanisms promoting glucose disposal via insulin-independent processes. Herein, we provide support for these compensatory pathways/circuits of glucose uptake in vivo, the activation of which under certain adverse metabolic conditions may protect against hyperglycemia. Using chromatin immunoprecipitation combined with protein-protein interaction studies of nuclear proteins in vivo, and transient transcription assays in living cells, we show that HMGA1 is required for gene activation of the IGF-binding proteins 1 (IGFBP1) and 3 (IGFBP3), two major members of the IGF-binding protein superfamily. Furthermore, by using positron emission tomography with (18)F-labeled 2-fluoro-2-deoxy-d-glucose, in combination with the euglycemic clamp with IGF-I, we demonstrated that IGF-I's bioactivity was increased in Hmga1-knockout mice, in which both skeletal muscle Glut4 protein expression and glucose uptake were enhanced compared with wild-type littermates. We propose that, by affecting the expression of both IGFBP protein species, HMGA1 can serve as a modulator of IGF-I activity, thus representing an important novel mediator of glucose disposal.

Insulin resistance and cancer risk: an overview of the pathogenetic mechanisms.

Insulin resistance is common in individuals with obesity or type 2 diabetes (T2D), in which circulating insulin levels are frequently increased. Recent epidemiological and clinical evidence points to a link between insulin resistance and cancer. The mechanisms for this association are unknown, but hyperinsulinaemia (a hallmark of insulin resistance) and the increase in bioavailable insulin-like growth factor I (IGF-I) appear to have a role in tumor initiation and progression in insulin-resistant patients. Insulin and IGF-I inhibit the hepatic synthesis of sex-hormone binding globulin (SHBG), whereas both hormones stimulate the ovarian synthesis of sex steroids, whose effects, in breast epithelium and endometrium, can promote cellular proliferation and inhibit apoptosis. Furthermore, an increased risk of cancer among insulin-resistant patients can be due to overproduction of reactive oxygen species (ROS) that can damage DNA contributing to mutagenesis and carcinogenesis. On the other hand, it is possible that the abundance of inflammatory cells in adipose tissue of obese and diabetic patients may promote systemic inflammation which can result in a protumorigenic environment. Here, we summarize recent progress on insulin resistance and cancer, focusing on various implicated mechanisms that have been described recently, and discuss how these mechanisms may contribute to cancer initiation and progression.

HMGA1 is a novel downstream nuclear target of the insulin receptor signaling pathway.

High-mobility group AT-hook 1 (HMGA1) protein is an important nuclear factor that activates gene transcription by binding to AT-rich sequences in the promoter region of DNA. We previously demonstrated that HMGA1 is a key regulator of the insulin receptor (INSR) gene and individuals with defects in HMGA1 have decreased INSR expression and increased susceptibility to type 2 diabetes mellitus. In addition, there is evidence that intracellular regulatory molecules that are employed by the INSR signaling system are involved in post-translational modifications of HMGA1, including protein phosphorylation. It is known that phosphorylation of HMGA1 reduces DNA-binding affinity and transcriptional activation. In the present study, we investigated whether activation of the INSR by insulin affected HMGA1 protein phosphorylation and its regulation of gene transcription. Collectively, our findings indicate that HMGA1 is a novel downstream target of the INSR signaling pathway, thus representing a new critical nuclear mediator of insulin action and function.