Paolo Sbraccia

Mandibuloacral Dysplasia Is Caused by a Mutation in LMNA-Encoding Lamin A/C

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation. The LMNA gene encoding two nuclear envelope proteins (lamins A and C [lamin A/C]) maps to chromosome 1q21 and has been associated with five distinct pathologies, including Dunnigan-type familial partial lipodystrophy, a condition that is characterized by subcutaneous fat loss and is invariably associated with insulin resistance and diabetes. Since patients with MAD frequently have partial lipodystrophy and insulin resistance, we hypothesized that the disease may be caused by mutations in the LMNA gene. We analyzed five consanguineous Italian families and demonstrated linkage of MAD to chromosome 1q21, by use of homozygosity mapping. We then sequenced the LMNA gene and identified a homozygous missense mutation (R527H) that was shared by all affected patients. Patient skin fibroblasts showed nuclei that presented abnormal lamin A/C distribution and a dysmorphic envelope, thus demonstrating the pathogenic effect of the R527H LMNA mutation.

Defects of the insulin receptor substrate (IRS) system in human metabolic disorders

Insulin receptor substrate (IRS) molecules are key mediators in insulin signaling and play a central role in maintaining basic cellular functions such as growth, survival, and metabolism. They act as docking proteins between the insulin receptor and a complex network of intracellular signaling molecules containing Src homology 2 (SH2) domains. Four members (IRS‐1, IRS‐2, IRS‐3, IRS‐4) of this family have been identified that differ as to tissue distribution, subcellu‐lar localization, developmental expression, binding to the insulin receptor, and interaction with SH2 domain‐containing proteins. Results from targeted disruption of the IRS genes in mice have provided important clues to the functional differences among these related molecules, suggesting they play different and specific roles in vivo. The available data are consistent with the notion that IRS‐1 and IRS‐2 are not functionally interchangeable in tissues that are responsible for glucose production (liver), glucose uptake (skeletal muscle and adipose tissue), and insulin production (pancreatic β cells). In fact, IRS‐1 appears to have its major role in skeletal muscle whereas IRS‐2 appears to regulate he‐patic insulin action as well as pancreatic β cell development and survival. By contrast, IRS‐3 and IRS‐4 genes appear to play a redundant role in the IRS signaling system. Defects in muscle IRS‐1 expression and function have been reported in insulin‐resistant states such as obesity and type 2 diabetes. Several polymorphisms in the IRS genes have been identified, but only the Gly→Arg972 substitution of IRS‐1, interacting with environmental factors, seems to have a patho‐genic role in the development of type 2 diabetes. In contrast, polymorphisms of the other IRS genes do not appear to contribute to type 2 diabetes.—Sesti, G., Federici, M., Hribal, M. L., Lauro, D., Sbraccia, P., Lauro, R. Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. FASEB J. 15, 2099–2111 (2001)

Insulin-Dependent Activation of Endothelial Nitric Oxide Synthase Is Impaired by O-Linked Glycosylation Modification of Signaling Proteins in Human Coronary Endothelial Cells

Background—Hyperglycemia impairs functional properties of cytosolic and nuclear proteins via O-linked glycosylation modification (O-GlcNAcylation). We studied the effects of O-GlcNAcylation on insulin signaling in human coronary artery endothelial cells. Methods and Results—O-GlcNAcylation impaired the metabolic branch of insulin signaling, ie, insulin receptor (IR) activation of the IR substrate (IRS)/phosphatidylinositol 3-kinase (PI3-K)/Akt, whereas it enhanced the mitogenic branch, ie, ERK-1/2 and p38 (mitogen-activated protein kinase). Both in vivo and in vitro phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt were reduced by hyperglycemia and hexosamine activation. Insulin-induced eNOS activity in vivo was reduced by hyperglycemia and hexosamine activation, which was coupled to increased activation and expression of matrix metalloproteinase-2 and -9; these phenomena were reversed by inhibition of the hexosamine pathway. Finally, carotid plaques from type 2 diabetic patients showed increased endothelial O-GlcNAcylation with respect to nondiabetics. Conclusions—Our data show that hyperglycemia, through the hexosamine pathway, impairs activation of the IR/IRS/PI3-K/Akt pathway, resulting in deregulation of eNOS activity.

Insulin receptor activation by IGF-II in breast cancers: evidence for a new autocrine/paracrine mechanism

IGF-II, produced by breast cancer epithelial and stromal cells, enhances tumor growth by activating the IGF-I receptor (IGF-I-R) via autocrine and paracrine mechanisms. Previously we found that the insulin receptor (IR), which is related to the IGF-I-R, is overexpressed in breast cancer cells. Herein, we find that, in breast cancer the IR is activated by IGF-II. In eight human breast cancer cell lines studied there was high affinity IGF-II binding to the IR, with subsequent IR activation. In these lines, IGF-II had a potency up to 63% that of insulin. In contrast, in non malignant human breast cells, IGF-II was less than 1% potent as insulin. Via activation of the IR tyrosine kinase IGF-II stimulated breast cancer cell growth. Moreover, IGF-II also activated the IR in breast cancer tissue specimens; IGF-II was 10 – 100% as potent as insulin. The IR occurs in two isoforms generated by alternative splicing of exon 11; these isoforms are IR-A (Ex11−) and IR-B (Ex11+). IR-A was predominantly expressed in breast cancer cells and specimens and the potency of IGF-II was correlated to the expression of this isoform (P<0.0001). These data indicate, therefore, that the IR-A, which binds IGF-II with high affinity, is predominantly expressed in breast cancer cells and represents a new autocrine/paracrine loop involved in tumor biology.

Insulin receptor substrate (IRS) transduction system: distinct and overlapping signaling potential.

Insulin receptor substrate (IRS) proteins play a central role in maintaining basic cellular functions such as growth and metabolism. They act as an interface between multiple growth factor receptors possessing tyrosine kinase activity, such as the insulin receptor, and a complex network of intracellular signalling molecules containing Src homology 2 (SH2) domains. Four members (IRS‐1, IRS‐2, IRS‐3, IRS‐4) of this family have been identified which differ in their subcellular distribution and interaction with SH2 domain proteins. In addition, differential IRS tissue‐ and developmental‐specific expression patterns may contribute to specificity in their signaling potential. Copyright

Timp3 deficiency in insulin receptor–haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-α

Activation of inflammatory pathways may contribute to the beginning and the progression of both atherosclerosis and type 2 diabetes. Here we report a novel interaction between insulin action and control of inflammation, resulting in glucose intolerance and vascular inflammation and amenable to therapeutic modulation. In insulin receptor heterozygous (Insr+/-) mice, we identified the deficiency of tissue inhibitor of metalloproteinase 3 (Timp3, an inhibitor of both TNF-alpha-converting enzyme [TACE] and MMPs) as a common bond between glucose intolerance and vascular inflammation. Among Insr+/- mice, those that develop diabetes have reduced Timp3 and increased TACE activity. Unchecked TACE activity causes an increase in levels of soluble TNF-alpha, which subsequently promotes diabetes and vascular inflammation. Double heterozygous Insr+/-Timp3+/- mice develop mild hyperglycemia and hyperinsulinemia at 3 months and overt glucose intolerance and hyperinsulinemia at 6 months. A therapeutic role for Timp3/TACE modulation is supported by the observation that pharmacological inhibition of TACE led to marked reduction of hyperglycemia and vascular inflammation in Insr+/- diabetic mice, as well as by the observation of increased insulin sensitivity in Tace+/- mice compared with WT mice. Our results suggest that an interplay between reduced insulin action and unchecked TACE activity promotes diabetes and vascular inflammation.

Mice Heterozygous for Tumor Necrosis Factor-α Converting Enzyme Are Protected From Obesity-Induced Insulin Resistance and Diabetes

OBJECTIVE—Tumor necrosis factor (TNF)-α is known to affect insulin sensitivity, glucose, and lipid metabolism through alternative and redundant mechanisms at both translational and post-translational levels. TNF-α exerts its paracrine effects once the membrane-anchored form is shed and released from the cell membrane. TNF-α cleavage is regulated by TNF-α converting enzyme (TACE), which regulates the function of several transmembrane proteins, such as interleukin-6 receptor and epidermal growth factor receptor ligands. The role of TACE in high-fat diet (HFD)-induced obesity and its metabolic complications is unknown. RESEARCH DESIGN AND METHODS—To gain insights into the role of TACE in metabolic disorders, we used Tace+/− mice fed a standard or high-fat diet for 16 weeks. RESULTS—We observed that Tace+/− mice are relatively protected from obesity and insulin resistance compared with wild-type littermates. When fed an HFD, wild-type mice exhibited visceral obesity, increased free fatty acid and monocyte chemoattractant protein (MCP)1 levels, hypoadiponectinemia, glucose intolerance, and insulin resistance compared with Tace+/− mice. Interestingly, Tace+/− mice exhibited increased uncoupling protein-1 and GLUT4 expression in white adipose tissue. CONCLUSIONS—Our results suggest that modulation of TACE activity is a new pathway to be investigated for development of agents acting against obesity and its metabolic complications.

Phosphorylation of GATA2 by Akt Increases Adipose Tissue Differentiation and Reduces Adipose Tissue–Related Inflammation A Novel Pathway Linking Obesity to Atherosclerosis

Background—Obesity-related inflammation is emerging as a major cause of insulin resistance and cardiovascular diseases. GATA2 transcription factor is an inhibitor of adipogenesis and an activator of vascular cells. We hypothesized that GATA2 activity is controlled by insulin during adipogenesis, linking metabolic homeostasis and inflammation. Methods and Results—We show that insulin induces GATA2 phosphorylation on serine 401 in a PI-3K/Akt–dependent manner. Insulin-dependent phosphorylation of serine 401 impairs GATA2 translocation to the nucleus and its DNA binding activity. A GATA2 mutant not phosphorylable by Akt (GATA2S401A) acts similarly to wild-type GATA2. In contrast, a GATA2 mutant that mimics Akt phosphorylation (GATA2S401D) is restrained in the cytoplasm. Cultured preadipocytes bearing GATA2S401A do not convert to adipocytes and express high levels of inflammatory cytokines like monocyte chemotactic protein-1 (MCP-1). On the contrary, GATA2S401D preadipocytes differentiate to adipocytes. When GATA2S401A preadipocytes are injected in mice fed a high-fat diet, they do not differentiate adequately into adipocytes, maintaining the expression of inflammatory markers like MCP-1. In contrast, injection of GATA2S401D preadipocytes in mice fed a high-fat diet results in development of adipocytes and no expression of inflammatory markers. Conclusions—GATA2 could be a new target in the prevention and treatment of obesity-related inflammation and its complications.

Fish oil supplementation improves endothelial function in normoglycemic offspring of patients with type 2 diabetes

OBJECTIVE Offspring of patients with type 2 diabetes (OPDs) exhibits endothelial dysfunction (ED) associated with a chronic inflammatory state. N-3 polyunsaturated fatty acids (n-3 PUFA) may have antioxidant and anti-inflammatory properties that are beneficial for cardiovascular and metabolic health. Therefore, in the present study, we tested the hypothesis that dietary supplementation with fish oil rich in n-3 PUFA may improve ED in otherwise healthy OPDs. METHODS AND DESIGN A double-blind, placebo-controlled trial was conducted with 50 OPDs. Participants were randomized to treatment with either placebo or n-3 PUFA (2g/day) for 12 weeks. Before and after treatment we evaluated endothelial function (using flow-mediated dilation (FMD) of the brachial artery), circulating inflammatory markers (adiponectin, TNF-alpha, and high sensitivity-CRP), and insulin resistance (QUICKI). RESULTS No significant changes were observed in study outcomes in subjects treated with placebo. By contrast, when compared with baseline values, subjects treated with n-3 PUFA had significant improvement in FMD (9.1+/-5.8% vs. 11.7+/-4.4%, p=0.02) that was accompanied by decreased plasma triglycerides (117+/-73mg/dl vs. 86+/-44mg/dl, p=0.001) and TNF-alpha levels (8.9+/-2.3pg/ml vs. 6.8+/-2.7pg/ml, p=0.001), and a trend towards increased plasma adiponectin levels (7.8+/-4.5microg/ml vs. 9.5+/-5.1microg/ml, p=0.09). When data were analyzed by multiple regression analysis, decreased TNF-alpha after treatment with n-3 PUFA predicted increased FMD. CONCLUSION Dietary supplementation with n-3 PUFA significantly improved endothelial function and reduced pro-inflammatory markers in OPDs. Thus, fish oil consumption may have beneficial cardiovascular and metabolic health effects in otherwise healthy subjects predisposed to diabetes and its vascular complications.

Molecular mechanism of insulin resistance in type 2 diabetes mellitus : role of the insulin receptor variant forms

Type 2 diabetes is a heterogeneous and polygenic disorder resulting from interaction of genetic factors with environmental influences. Numerous candidate genes for insulin signaling proteins have been screened, but no single major susceptibility gene for type 2 diabetes has been identified. Due to its pivotal role in insulin action, the insulin receptor was considered a plausible candidate gene. The insulin receptor exists in two isoforms differing by the absence (Ex11−) or presence (Ex11+) of a 12 amino acid sequence in the COOH‐terminus of the α‐subunit, as a consequence of alternative splicing of exon 11. The Ex11− binds insulin with two‐fold higher affinity than the Ex11+. This difference is paralleled by a decreased sensitivity for metabolic actions of insulin. Some, but not all, studies have reported that expression of the low‐affinity Ex11+ is increased in target tissues from type 2 diabetic patients, thus suggesting that alterations in abundance of the two isoforms might contribute to insulin resistance. Insulin and type 1 IGF receptors have been shown to form hybrid receptors in tissues co‐expressing both molecules. Hybrid receptors bind IGF‐I, but not insulin, with high affinity, and behave as IGF‐I holoreceptors, rather than insulin receptors, in terms of receptor autophosphorylation, and hormone internalization. It has been shown that the abundance of hybrid receptors is increased in skeletal muscle and adipose tissue from type 2 diabetic patients, and is negatively correlated with in vivo insulin sensitivity. Mutations in the insulin receptor gene have been identified in studies which examined an appropriately sized population of patients with type 2 diabetes. The prevalence of mutations in the insulin receptor gene ranged from 0.4%–7.8%. This review will focus on the structural and functional heterogeneity of the insulin receptor, and will discuss the pathogenetic role of insulin receptor variant forms and polymorphisms in the development of the common form of type 2 diabetes. Copyright