Interferon-gamma released from omental adipose tissue of insulin-resistant humans alters adipocyte phenotype and impairs response to insulin and adiponectin release.

BACKGROUND: Inflammatory factors derived from adipose tissue have been implicated in mediating insulin resistance in obesity. We sought to identify these using explanted human adipose tissue exposed to innate and adaptive immune stimuli. METHODS: Subcutaneous and omental adipose tissue from obese, insulin-resistant donors was cultured in the presence of macrophage and T-cell stimuli, and the conditioned medium tested for its ability to inhibit insulin-stimulated glucose uptake into human Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes. The nature of the inhibitory factor in conditioned medium was characterized physicochemically, inferred by gene microarray analysis and confirmed by antibody neutralization. RESULTS: Conditioned medium from omental adipose tissue exposed to a combination of macrophage- and T-cell stimuli inhibited insulin action and adiponectin secretion in SGBS adipocytes. This effect was associated with a pronounced change in adipocyte morphology, characterized by a decreased number of lipid droplets of increased size. The bioactivity of conditioned medium was abolished by trypsin treatment and had a molecular weight of 46 kDa by gel filtration. SGBS adipocytes exposed to a bioactive medium expressed multiple gene transcripts regulated by interferon-gamma (IFN-γ). Recombinant human IFN-γ recapitulated the effects of the bioactive medium and neutralizing antibody against IFN-γ but not other candidate factors abrogated medium bioactivity. CONCLUSIONS: IFN-γ released from inflamed omental adipose tissue may contribute to the metabolic abnormalities seen in human obesity.

GM3 ganglioside and phosphatidylethanolamine-containing lipids are adipose tissue markers of insulin resistance in obese women.

AIMS: The association between central obesity and insulin resistance reflects the properties of visceral adipose tissue. Our aim was to gain further insight into this association by analysing the lipid composition of subcutaneous and omental adipose tissue in obese women with and without insulin resistance. METHODS: Subcutaneous and omental adipose tissue and serum were obtained from 29 obese non-diabetic women, 13 of whom were hyperinsulinemic. Histology, lipid and gene profiling were performed. RESULTS: In omental adipose tissue of obese, insulin-resistant women, adipocyte hypertrophy and macrophage infiltration were accompanied by an increase in GM3 ganglioside and its synthesis enzyme ST3GAL5; in addition, phosphatidylethanolamine (PE) lipids were increased and their degradation enzyme, phosphatidylethanolamine methyl transferase (PEMT), decreased. ST3GAL5 was expressed predominantly in adipose stromovascular cells and PEMT in adipocytes. Insulin resistance was also associated with an increase in PE lipids in serum. INTERPRETATION: The relevance of these findings to insulin resistance in humans is supported by published mouse studies, in which adipocyte GM3 ganglioside, increased by the inflammatory cytokine tumour necrosis factor-α, impaired insulin action and PEMT was required for adipocyte lipid storage. Thus in visceral adipose tissue of obese humans, an increase in GM3 ganglioside secondary to inflammation may contribute to insulin resistance and a decrease in PEMT may be a compensatory response to adipocyte hypertrophy.

Maturity-onset diabetes of the young type 5 in a family with diabetes and mild kidney disease diagnosed by whole exome sequencing.

Exome sequencing is being increasingly used to identify disease-associated gene mutations. We used whole exome sequencing to determine the genetic basis of a syndrome of diabetes and renal disease affecting a mother and her son. We identified a mutation in the hepatocyte nuclear factor 1-b (HNF1B) gene that encoded a methionine to valine amino acid change (M160V) in the HNF1B protein. This leads us to the previously unappreciated diagnosis of maturity-onset diabetes of the young type 5 and provided a basis for genetic counselling of other family members.

Ex-vivo analysis of human natural killer T cells demonstrates heterogeneity between tissues and within established CD4(+) and CD4(-) subsets.

Our understanding of human type 1 natural killer T (NKT) cells has been heavily dependent on studies of cells from peripheral blood. These have identified two functionally distinct subsets defined by expression of CD4, although it is widely believed that this underestimates the true number of subsets. Two recent studies supporting this view have provided more detail about diversity of the human NKT cells, but relied on analysis of NKT cells from human blood that had been expanded in vitro prior to analysis. In this study we extend those findings by assessing the heterogeneity of CD4(+) and CD4(-) human NKT cell subsets from peripheral blood, cord blood, thymus and spleen without prior expansion ex vivo, and identifying for the first time cytokines expressed by human NKT cells from spleen and thymus. Our comparative analysis reveals highly heterogeneous expression of surface antigens by CD4(+) and CD4(-) NKT cell subsets and identifies several antigens whose differential expression correlates with the cytokine response. Collectively, our findings reveal that the common classification of NKT cells into CD4(+) and CD4(-) subsets fails to reflect the diversity of this lineage, and that more studies are needed to establish the functional significance of the antigen expression patterns and tissue residency of human NKT cells.

Linkage disequilibrium of a type 1 diabetes susceptibility locus with a regulatory IL12B allele.

Type 1 diabetes (T1D; or insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease with both genetic and environmental components. In addition to the human leukocyte antigen (HLA) complex, the single major genetic contributor of susceptibility, an unknown number of other unidentified genes are required to mediate disease. Although many loci conferring susceptibility to T1D have been mapped, their identification has proven problematic due to the complex nature of this disease. Our strategy for finding T1D susceptibility genes has been to test for human homologues of loci implicated in diabetes-prone NOD (non-obese diabetic) mice, together with application of biologically relevant stratification methods. We report here a new susceptibility locus, IDDM18, located near the interleukin-12 (IL-12)p40 gene, IL12B. Significant bias in transmission of IL12B alleles was observed in affected sibpairs and was confirmed in an independent cohort of simplex families. A single base change in the 3' UTR showed strong linkage disequilibrium with the T1D susceptibility locus. The IL12B 3' UTR alleles showed different levels of expression in cell lines. Variation in IL-12p40 production may influence T-cell responses crucial for either mediating or protecting against this and other autoimmune diseases.

INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells. RESULTS: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells. CONCLUSIONS/INTERPRETATION: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs.

T-cell antigen receptor transmembrane peptides modulate T-cell function and T cell-mediated disease.

This study describes a novel method of inhibiting T-cell function by the use of peptides rationally designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence involved with TCR receptor assembly. The most effective peptide (core peptide, CP) modulating in vitro and in vivo T-cell function contained nine amino acids two of which, lysine and arginine, were hydrophilic and separated by four hydrophobic amino acids. CP without chemical modification or conjugation was able to enter non-T and T cells. Conjugation of CP at the carboxyl terminus with palmitic acid resulted in a greater inhibition of T-cell interleukin-2 (IL-2) production in vitro than peptide alone. When examined for effects in vivo, CP reduced clinical signs of inflammation in three T cell-mediated disease models including adjuvant-induced arthritis, experimental allergic neuritis, and cyclophosphamide-induced diabetes in NOD/Lt(F) mice. This peptide or its analogues has potential as a therapeutic agent in human inflammatory and autoimmune disorders.

Aerosol insulin induces regulatory CD8 gamma delta T cells that prevent murine insulin-dependent diabetes.

Cellular immune hyporesponsiveness can be induced by the presentation of soluble protein antigens to mucosal surfaces. Most studies of mucosa-mediated tolerance have used the oral route of antigen delivery and few have examined autoantigens in natural models of autoimmune disease. Insulin is an autoantigen in humans and nonobese diabetic (NOD) mice with insulin-dependent diabetes mellitus (IDDM). When we administered insulin aerosol to NOD mice after the onset of subclinical disease, pancreatic islet pathology and diabetes incidence were both significantly reduced. Insulin-treated mice had increased circulating antibodies to insulin, absent splenocyte proliferation to the major epitope, insulin B chain amino acids 9-23, which was associated with increased IL-4 and particularly IL-10 secretion, and reduced proliferation to glutamic acid decarboxylase, another islet autoantigen. The ability of splenocytes from insulin-treated mice to suppress the adoptive transfer of diabetes to nondiabetic mice by T cells of diabetic mice was shown to be caused by small numbers of CD8 gamma delta T cells. These findings reveal a novel mechanism for suppressing cell-mediated autoimmune disease. Induction of regulatory CD8 gamma delta T cells by aerosol insulin is a therapeutic strategy with implications for the prevention of human IDDM.

Glutamic acid decarboxylase 67-reactive T cells: a marker of insulin-dependent diabetes.

Glutamic acid decarboxylase (GAD) has been shown to be a target of autoantibodies in insulin-dependent diabetes (IDD). Two forms of GAD, with molecular weights of 67,000 and 65,000, have been cloned from separate genes. As pancreatic islet beta cell destruction DD is an autoimmune process mediated by T cells, we sought to determine if recombinant GAD67 was recognized by T cells in IDD subjects and particularly their first-degree relatives with islet cell antibodies known to be at risk for IDD. The central regions of human islet and brain GAD67 (amino acids 208-404) were cloned as fusion proteins with glutathione-S-transferase (GST). Proliferation of peripheral blood T cells in the presence of recombinant GAD67 was significantly higher in both at-risk relatives and recent-onset IDD subjects than in other autoimmune disease subjects and human histocompatibility leukocyte antigen (HLA)-matched healthy controls. Thus, 12 of 29 (41%) at-risk relatives and 11 of 29 (38%) recent-onset IDD subjects responded to GAD67, compared with 1 of 7 (14%) other autoimmune disease subjects and 1 of 23 (4%) HLA-matched controls. T cell responses to GST alone or to tetanus toxoid were not different between the groups. These findings demonstrate that GAD67 is a target autoantigen of T cells in IDD and suggest the possibility that GAD-reactive T cells may delineate asymptomatic subjects at increased risk for IDD.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice.

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes.

We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which develop a scleroderma-like syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NH2-terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapeptide sequence homologous to that in fusion protein C was identified in the UL70 protein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data suggest that B cell clones producing antitopo autoantibodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease.

Siglec-10 expression is up-regulated in activated human CD4+ T cells.

Most sialic acid-binding immunoglobulin-like lectins (Siglecs) suppress immune cell function but are expressed at low levels on human T cells. We found that soluble CD52 inhibited T cell signalling by ligating Siglec-10, but the presence of Siglec-10 on human T cells has been questioned. To address this concern, we examined the expression of Siglec-10 at the RNA and protein level in human CD4+ T cells. Analysis by RNAseq, qPCR and flow cytometry demonstrated that, in contrast to other Siglecs, after activation of CD4+ T cells Siglec-10 was selectively upregulated in a subset of cells also high for CD52 expression. This observation is consistent with a homeostatic role for Siglec-10 in human CD4+ T cells.

Immune characterization of an individual with an exceptionally high natural killer T cell frequency and her immediate family.

Natural killer T cells (NKT) are a regulatory subset of T lymphocytes whose frequency in peripheral blood is highly variable within the human population. Lower than normal NKT frequencies are associated with increased predisposition to a number of diseases, including type 1 diabetes and some forms of cancer, raising the possibility that an increased frequency may be protective. However, there is little or no understanding of how high NKT frequencies arise or, most importantly, whether the potential exists to boost and maintain NKT levels for therapeutic advantage. Here, we provide a detailed functional and phenotypic characterization of the NKT compartment of a human donor with NKT levels approximately 50 times greater than normal, including an analysis of NKT in her immediate family members. The study focuses upon the characteristics of this donor and her family, but demonstrates more broadly that the size and flexibility of the NKT niche is far greater than envisioned previously. This has important implications for understanding how the human NKT compartment is regulated, and supports the concept that the human NKT compartment might be expanded successfully for therapeutic benefit.

The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.

Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing beta cells. While the role of CD4(+) T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4(+) T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4(+) T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6-A7). Here we describe the analysis of a CD4(+) T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4(+) T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4(+) T cell responses in human T1D.

Autoantibodies to beta 2-adrenergic receptors: a possible cause of adrenergic hyporesponsiveness in allergic rhinitis and asthma.

Autoantibodies to beta 2-adrenergic receptors have been identified in the serum of one patient with allergic rhinitis ("hay fever") and two patients with asthma. The antibodies precipitate solubilized dog lung beta receptors in an indirect immunoprecipitation assay and inhibit the specific binding of iodine-125-labeled iodohydroxybenzylpindolol to membrane-associated receptors from dog lung, calf lung, and human placenta. Ligand binding to canine heart beta 1 receptors is not affected by the antibodies.

Radioimmunoassay of the insulin receptor: a new probe of receptor structure and function.

A sensitive and specific radioimmunoassay for the insulin receptor has been developed employing receptor autoantibodies from the serum of a patient with insulin-resistant diabetes. The assay detects insulin binding sites at concentrations as low as 0.1 nanomolar; distinguishes between receptors originating from human placental membranes, human lymphoblastoid cells, and mouse liver membranes; and measures the receptor independently of its binding function. Down-regulation, or loss of binding after exposure to insulin, is associated with loss of immunoreactive receptor.

Markers on distal chromosome 2q linked to insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is a multigenic autoimmune disease. An IDDM susceptibility gene was mapped to chromosome 2q34. This gene may act early in diabetogenesis, because "preclinical" individuals also showed linkage. Human leukocyte antigen (HLA)-disparate, but not HLA-identical, sibs showed linkage, which was even stronger in families with affected females. The genes encoding insulin-like growth factor-binding proteins 2 and 5 were mapped to a 4-megabase pair interval near this locus. These results indicate the existence of a gene that acts at an early stage in IDDM development, screening for which may identify a specific subset of at-risk individuals.

Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins.

The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor.

Correlation between insulin receptor binding in isolated fat cells and insulin sensitivity in obese human subjects.

This study examined the relationship between receptor binding of insulin in a metabolically significant target tissue in vitro and sensitivity to insulin in vivo in obese human subjects. Specific insulin binding was measured at 24 degrees C in isolated enlarged fat cells obtained from 16 patients, by observing the effect of increasing concentrations of unlabeled insulin on the binding of [125I]insulin. Scratchard plots of the binding data were curvilinear with an upward concavity, similarity shaped, and essentially parallel. Kinetic studies on the dissociation of [125I]insulin from fat cells indicated that these curvilinear Scratchard plots could be explained by the presence of site:site interactions of the negative cooperative type. Differences in binding between individual patients were predominantly due to differences in the numbers of receptor sites whether expressed in relation to cell number, cell volume, or cell surface area. These findings were not accounted for by differences in [125I]insulin degradation. Acute exposure of adipose tissue to insulin in vitro had no significant effect on [125I]insulin binding to isolated cells. The number of receptor sites was directly correlated with insulin sensitivity in vivo, measured as the rate constant (Kitt) for the fall in blood glucose after intravenous insulin, and was inversely correlated with the level of fasting plasma insulin. These findings corroborate those from other studies using human mononuclear leukocytes and various tissues from the obese mouse, which indicate that decreased insulin binding is a characteristic feature of insulin resistance in obesity.

Essential role for interferon-gamma and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/Wehi mice.

Experimental studies in vitro suggest that cytokines are important mediators in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). However, there is little evidence for the role of cytokines in vivo, either in humans or in the spontaneous animal models of IDDM such as the NOD mouse or BB rat. To address this question, we used the model of cyclophosphamide (CYP)-induced autoimmune diabetes in the NOD/Wehi mouse to examine for (a) the production of IFN-gamma and IL-6 from isolated islets, and (b) the effect of anti IFN-gamma or anti IL-6 monoclonal antibodies on the development of diabetes. After cyclophosphamide, the majority of these mice develop of mononuclear cell infiltrate (insulitis) which by 10-14 d is associated with beta cell destruction. IFN-gamma activity at low levels (2.7 +/- 0.3 U/ml) could be detected only in culture supernatants from islets isolated at day 7 post-cyclophosphamide. In contrast, IL-6 activity progressively increased from 457 +/- 44 U/ml at day 0 to 6,020 +/- 777 U/ml at day 10. Culture of islets with anti-CD3 monoclonal antibody resulted in a significant increase in IFN-gamma activity from 41 +/- 7 U/ml at day 0 to 812 +/- 156 U/ml at day 10. Mice given either anti-IFN-gamma or anti-IL-6 antibody had a significantly reduced (P less than 0.001) incidence of diabetes and especially with IFN-gamma, decreased severity of insulitis. We conclude that IFN-gamma and IL-6 have essential roles in the pathogenesis of pancreatic islet beta cell destruction in this model.