J Clin Endocrinol Metab. 2016 Mar;101(3):1274-81. 

HMGB1 Is Increased by CFTR Loss of Function, Is Lowered by Insulin, and Increases In Vivo at Onset of CFRD. 

Montanini L, Cirillo F, Smerieri A, Pisi G, Giardino I, d’Apolito M, Spaggiari C, Bernasconi S, Amarri S, Street ME.

Department of Pediatrics (L.M., A.S., G.P., C.S., S.B.), University Hospital of Parma, 43126 Parma, Italy; Department of Pediatrics (F.C., S.A., M.E.S.), Istituto di Ricovero e Cura a Carattere Scientifico-Arcispedale Santa Maria Nuova, 42123 Reggio Emilia, Italy; and Department of Clinical and Experimental Medicine (I.G, M.d.), University of Foggia, University Hospital of Foggia, 71122, Foggia, Italy.

 

Abstract  

Cystic Fibrosis-Related Diabetes (CFRD) is associated with worsening of inflammation and infections, and the beginning of insulin treatment is debated.

To verify HMGB1 levels in CF patients according to glucose tolerance state, and analyze relationships with insulin secretion and resistance. To verify, in an in vitro model, whether HMGB-1 gene expression and protein content were affected by insulin administration, and whether these changes were dependent on CFTR loss of function.

Fourty-three patients in stable clinical conditions and 35 age- and sex matched controls were enrolled.

Glucose tolerance was established in patients based on a five point OGTT. Fasting glucose to insulin ratio (FGIR), HOMA-IR index, WBISI, and the areas under the curve for glucose and insulin (AUCI) were calculated. HMGB1 was assayed in serum, in cell lysates and conditioned media using a specific ELISA kit. For the in vitro study we used CFBE41o- cells, homozygous for the F508del mutation, and 16HBE14o- as non-CF control. HMGB1 gene expression was studied by real time RT-PCR. Cells were stimulated with insulin at 2.5 and 5 ng/ml. The CFTR inhibitor 172 and CFTR gene silencing were used to induce CFTR loss of function in 16HBE14o- cells.

HMGB1 levels were increased at onset of CFRD (5.04± 1.2 vs 2.7± 0.3 ng/ml in controls, p<0.05), and correlated with FGIR (R:+0.43;P:0.038), and AUCI (R:+0.43;P:0.013). CFTR loss of function in the 16HBE14o- cells increased HMGB1, and was lowered by insulin.

HMGB1 was increased in CF patients with deranging glucose metabolism, and showed relationships with indexes of glucose metabolism. The increase in HMGB1 was related to CFTR loss of function, and insulin lowered HMGB1. Further research is required to verify whether HMGB1 could potentially be a candidate marker of onset of CFRD and to establish when to start insulin treatment. 

PMID:26760176; DOI:10.1210/jc.2015-3730

Supplement: 

High Mobility Group Box-1 (HMGB-1) is a chromatin-linked non-histonic small protein with cytokine activity that has nuclear, cytosolic and extracellular actions.  

It is described to be located in different tissues, such as lymphoid tissue, testis, ovary, neurons, and hepatocytes.  

In the nucleus, HMGB1 binds chromosomal DNA contributing to nucleosomal structure, regulation of gene expression, DNA recombination, repair, and replication, and interacts with many proteins such as transcriptional factors.  

In the cytosol, HMGB1 promotes autophagy and acts as a cell-membrane form.

In the presence of inflammation, HMGB1 is actively secreted in the extracellular space, following either active secretion by innate immune or passive release by necrotic cells, and is responsible for autocrine and paracrine effects.

Under inflammatory stimuli, HMGB-1 traslocates from nucleus to cytosol and it is actively secreted into the extracellular space being responsible for autocrine and paracrine effects.

HMGB-1 signals through RAGEs (receptor for advanced glycation end products) and through TLR2 and TLR4. Signaling through these pathways ultimately leads to the activation of Nuclear Factor-kappaB (NF-kB), inducing the up-regulation of leukocyte adhesion molecules, production of pro-inflammatory cytokines and angiogenic factors in both hematopoietic and endothelial cells, thereby promoting inflammation (Figure 1). 

HMGB-1 has been proposed to be a crucial mediator in the pathogenesis of many diseases including sepsis, arthritis, cancer, autoimmunity diseases and diabetes.

Interestingly, in recent years HMGB-1 has been shown to be altered in chronic inflammatory conditions characterized by IR, and it has been shown to be a stimulatory factor of insulin secretion in beta-pancreatic cells (1,2).  

Hagiwara et al. demonstrated that hyperglycemia is associated with higher HMGB1 levels and lung damage in sepsis; insulin therapy significantly reduced lung damage, suggesting that management of hyperglycemia with insulin might decrease HMGB1 levels in the serum and lung tissue (3). 

Recently, Ni et al. described that hyperinsulinemia increased HMGB1,  promoting apoptosis of rat ovarian granulosa cells, providing a new possible role of this protein in the PCOS pathogenesis (4).  

There are several patents proposed for controlling the production, secretion and neutralization of HMGB-1. In particular, six groups based on mechanism of action have been described: 1) inhibition of HMGB-1 using anti-HMGB-1 antibodies; 2) inhibition of HMGB-1 release from the nucleus into the extracellular space; 3) HMGB-A box as a competitive antagonist of HMGB-1; 4) blockage of RAGE-HMGB-1 signaling using RAGE antagonists (i.e. angiotensin receptor blockers-ARBs); 5) blockage of TLR-HMGB-1 signaling using anti-TLR2 antibodies and 6) other molecules that modulate HMGB-1 activity using e.g. human soluble thrombomodulin. 

In the last years several relatively small molecules – derived either 1) from natural sources (e.g. glycyrrhizin, tanshinone IIA, (−)-epigallocatechin-3-gallate, quercetin, lycopene), mainly from Chinese medical herbs [Danshen (Salvia miltiorrhiza), Gancao (licorice)], or 2) from chemical synthesis (nafamostat, sivelestat, atorvastatin, simvastatin, gabexate mesilate, methotrexate) – were explored for their ability to inhibit HMGB1 activity. 

The aim of our study was first, to investigate HMGB1 serum concentrations in CF patients with normal, impaired glucose tolerance, and at the onset of CFRD. Second, to correlate HMGB1 serum levels with the glucose tolerance state, and indexes of insulin secretion and resistance.

Furthermore, in a CF in vitro model (Figure 2), we verified if HMGB-1 gene expression and protein content were affected by insulin administration, and whether these changes were dependent on CFTR loss of function. 

In order to investigate a possible relation between circulating HMGB1 levels and glucose metabolism, we analysed, first, the HMGB1 serum concentrations in CF patients with normal, impaired glucose tolerance, and at the onset of CFRD. Second, we correlated HMGB1 serum levels with the glucose tolerance state, and indexes of insulin secretion and resistance.

Furthermore, in a CF in vitro model, we verified if HMGB1 gene expression and protein content were affected by insulin administration, and whether these changes were dependent on CFTR loss of function 

For the first time we showed that circulating HMGB1 increases in  serum of CF patients  along with deranging glucose metabolism, and it correlates with the amount of insulin being released during an oral glucose tolerance test.

The in vitro data showed that the increase in HMGB1 was due to CFTR loss of function, and that insulin stimulation lowered HMGB1 (Figure 3). 

HMGB1 is thus, tightly correlated with inflammation and glucose metabolism/insulin secretion.

Furthermore, it could potentially be a candidate marker of onset of CFRD, help to establish when to start insulin treatment, and help to monitor treatment in patients with CFRD.

 

 

 

Figure 1. Structure and molecular functions of HMGB1 

A. HMGB1 protein is structurally composed of three different functional domains: two homologous DNA-binding domains (HMG-box A and HMG-box B) and a C-terminal negatively charged tail.

Two Nuclear Localization Signals (NLS1 and NLS2) control nuclear transport of HMGB1. Three redox-sensitive cysteine residues (Cys-23, Cys-45, and Cys-106) are important for HMGB1 activity. All-reduced- and disulfide-HMGB1 has proinflammatory role recruiting leukoytes by chemoattraction and inducing cytokines activity;  whereas  all-oxidized HMGB1 has a non-immune activity. Residues 89 to 108 are responsible for binding to Toll-like receptors (TLRs), whereas residues 150 to 183 are responsible for binding to Receptor for Advanced-Glycation-End (RAGE).

B. Under resting conditions, HMGB1 is localized in the nucleus, where it plays an important role in chromatin structure and gene expression by interaction between HMG-boxes and DNA minor groove.  

C. During inflammation, HMGB1 can be secreted actively by a caspase-1 mediated vesicular pathway, or passively by dead cells. In the extracellular compartment, it interacts with Toll Like or RAGE receptors leading to NF-kB transcription causing the release of proinflammatory cytokines from endothelial cells.

 

 

 

Figure 2. Airway epithelial cells used in the in vitro experiments

 

 

Figure 3 Schematic overview on the in vivo and in vitro conditions underlying HMGB1 increase

 

References:

1.    Kenia Pedrosa Nunes, Eric Guisbert, Theodora Szasz and Clinton Webb (2015). The Innate Immune System via Toll-Like Receptors (TLRs) in Type 1 Diabetes. Mechanistic Insights, Major Topics in Type 1 Diabetes, Dr. Kenia Nunes (Ed.), InTech, DOI: 10.5772/61925.

2.    Guzmán-Ruiz R, Ortega F, Rodríguez A, Vázquez-Martínez R, Díaz-Ruiz A, Garcia-Navarro S, Giralt M, Garcia-Rios A, Cobo-Padilla D, Tinahones FJ, López-Miranda J5, Villarroya F, Frühbeck G, Fernández-Real JM, Malagón MM. Alarmin high-mobility group B1 (HMGB1) is regulated in human adipocytes in insulin resistance and influences insulin secretion in β-cells. Int J Obes (Lond). 2014 Dec;38(12):1545-54.

3.    Hagiwara S, Iwasaka H, Hasegawa A, Koga H, Noguchi T. Effects of hyperglycemia and insulin therapy on high mobility group box 1 in endotoxin-induced acute lung injury in a rat model. Crit Care Med. 2008 Aug;36(8):2407-13.

4.    Ni XR, Sun ZJ, Hu GH, Wang RH. High concentration of insulin promotes apoptosis of primary cultured rat ovarian granulosa cells via its increase in extracellular HMGB1. Reprod Sci. 2015 Mar;22(3):271-7.

 

Aknowledgements

The authors wish to thank Dr. P. Lazzeroni, Dr C. Sartori and Dr. C. Catellani for her help in preparing this manuscript and figures.

 

Contacts: 

Dott.ssa Maria E. Street

Head  Division of Paediatric Endocrinology and Diabetology

Dept. of Paediatrics

Arcispedale S. Maria Nuova – IRCCS

Viale Risorgimento, 80

42123 Reggio Emilia

Tel. 0522- 296194/6936

Fax. 0522-295909

 

 

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