The A98V single nucleotide polymorphism (SNP) in hepatic nuclear factor 1 alpha (HNF-1alpha) is associated with insulin sensitivity and beta-cell function.

OBJECTIVE: Mutations in the hepatic nuclear factor-1-alpha ( HNF-1alpha) gene is considered as a candidate for the aetiology of type 2 diabetes. The aims of the study was to determine whether two single nucleotide polymorphisms (SNPs) ile27-to-leu and ala98-to-val in the HNF-1alpha gene associate with diabetes, insulin sensitivity as well as beta-cell function. RESEARCH DESIGN AND METHODS: 1 479 subjects of a volunteer sample with increased risk of type 2 diabetes were investigated. They underwent a 75 g oral glucose tolerance test (OGTT) with measurements of plasma glucose, insulin and C-peptide at fasting and at 30, 60, 90 and 120 minutes after the glucose challenge. The HNF-1alpha SNPs, I27L and A98V were genotyped. RESULT: Patients harbouring the V98 allele exhibited higher serum insulin and C-peptide levels. The heterozygote variant was also associated with decrease in beta-cell function but better insulin sensitivity. No significant differences of any clinical parameters were found for I27L gene variants. CONCLUSION: Significant associations between the heterozygote A98V genotype and clinical parameters of insulin metabolism were reported but no relationship with type 2 diabetes was obtained. This may be explained by a balancing negative effect on insulin secretion and concomitant positive effect on insulin resistance in Val allele carriers.

Heparin-induced Extracorporal LDL precipitation (H.E.L.P) in diabetic foot syndrome - preventive and regenerative potential?

Peripheral arterial disease is more aggressive in concomitant diabetes posing an increased risk for critical limb ischemia and subsequent limb loss. The majority of therapies available are not effective to prevent amputation in patients with severe disease. The current observational study reports the effect of the heparin-induced extracorporal LDL-precipitation (H.E.L.P.) as a novel therapeutic approach in patients with severe diabetic foot syndrome. Seventeen diabetic patients with septic foot lesions recruited from the diabetic outpatient clinic underwent H.E.L.P. apheresis regularly until fibrinogen levels were stabilized at 3 g/l or infection was controllable as evidenced by alleviation of necrosis. Patients were subsequently followed up for 2 to 73 months. Fibrinogen levels were reduced by 68% after H.E.L.P. treatment. No severe complications were noted. Necrosis could be confined in sixteen patients. Minor amputations were indicated in twelve patients. Three patients underwent major amputations of the lower limb and two patients received surgical reconstruction. In conclusion, H.E.L.P. apheresis may offer an alternative therapeutic option to diabetic patients with critically ischemic feet and appears to have a beneficial major/minor amputation ratio.

Mutations and intronic variants in the HNF-1 beta gene in a group of German and Czech Caucasians with type 2 diabetes mellitus and progressive diabetic nephropathy.

Mutations in the hepatocyte nuclear factor - 1 beta (HNF-1 beta) gene cause maturity onset diabetes of the young type 5 (MODY 5). A clinical feature of the resulting phenotype besides impaired glucose tolerance is a variety of renal abnormalities, ranging from renal cysts to end-stage renal failure. Using a candidate gene approach we investigated the prevalence of mutations in the HNF-1 beta gene in a group of 63 patients from two different European populations (33 Germans, 30 Czechs) with type 2 diabetes mellitus and diabetic nephropathy diagnosed by increased albuminuria (39 patients) or end-stage renal failure (24 patients). No mutations were found in any of the 9 exons or in a minimal promoter region. Three intronic variants (single nucleotide polymorphisms - SNPs) were detected. The frequencies of these variants showed no difference between the two studied populations and were comparable to data reported from healthy subjects. No association between SNPs or formed haplotypes and any clinical parameters (like age of disease onset, BMI and severity of renal failure) was found. The results confirm that the genetic variations in the HNF-1 beta gene would be a very uncommon cause of progressive nephropathy in patients with type 2 diabetes mellitus.

Measurement of serum exon 3-retaining growth hormone-binding protein in children and adolescents by radioimmunoassay.

Recently described assays for the determination of growth hormone-binding protein (GHBP) show a wide variety of normal ranges. Their results depend on the assay design and in the case of ligand-immunofunctional assay (LIFA), probably also on the binding characteristics, i.e. epitope specificity and affinity of the employed antibody. These facts underline the necessity to look for more accurate and specific assays. In this report we describe an accurate and simple radioimmunoassay (RIA) which allows the specific quantitation of the exon 3-retaining GHBP isoform (E3-GHBP). Data of the E3-GHBP RIA were compared to those of a LIFA measuring undifferentiated functional forms of GHBP. Our results demonstrate significant relationships between GHBP and age, BMI and IGF-I as determined by RIA and by LIFA in normal children and adolescents (n = 115, p < 0.001). Moreover, BMI is the only regulating factor of GHBP for both methods as shown by multiple regression analysis (p < 0.001). All our data suggest a qualitatively paralleled regulation of E3-GHBP and undifferentiated functional GHBP forms. This finding was confirmed by a good correlation between RIA and LIFA data (r = 0.74, p < 0.001). Children with idiopathic short stature (ISS, n = 47) had significantly lower GHBP levels than normal controls (n = 58) measured by the E3-GHBP RIA (p < 0.0001) and by LIFA (p < 0.01). We conclude that (1) ISS children may have a structural or quantitative defect at the level of the GHR, and (2) the highly specific assay for E3-GHBP immunoreactivity provides a sensitive diagnostic tool in conditions with partial GH insensitivity.

Transformed alpha 2-macroglobulin as a low-affinity growth hormone-binding protein.

Human growth hormone (GH) forms complexes with the purified proteinase inhibitor, alpha 2-macroglobulin (alpha 2-M). This inhibitor occurs in two different forms in serum, known as native and transformed alpha 2-M. It has been clearly demonstrated, using chromatography and electrophoresis combined with autoradiography, that human GH binds specifically to the transformed inhibitor and not to the native protein. The binding was characterized as being mainly non-covalent and involved specific binding sites present only in the transformed inhibitor molecule. Binding analysis, using an immunoprecipitation technique, revealed that GH possesses two different types of binding sites, with dissociation constants of 0.49 +/- 0.12 mumol/l and 61 +/- 8 mumol/l for the high- and low-affinity binding site, respectively. Distribution analysis of 125I-labelled GH in whole plasma suggests that the hormone is bound to two different proteins: first, to the high-affinity GH-binding protein (GHBP) and, second, to the low-affinity GHBP, identified as transformed alpha 2-macroglobulin.

Growth hormone-binding protein related immunoreactivity is regulated by the degree of insulinopenia in diabetes mellitus.

OBJECTIVE: Derangements in the GH/IGF axis are common in patients with diabetes mellitus. In insulin-dependent diabetes mellitus (IDDM), these disturbances seem to be due to a partial defect in GH action on its own receptor or via a post-receptor defect. In non-insulin-dependent diabetes mellitus (NIDDM), data are limited, and the regulation of the GH receptor (GHR) remains unclear. However, animal studies with diabetic rats demonstrated that the GHR density may be influenced by insulin disposal at the hepatocyte. With respect to this hypothesis we studied the relation between peripheral insulin status and the serum GH-binding protein (GHBP), which reflects indirectly the GHR density in the tissues. Patients with IDDM were compared to a NIDDM group as well as to a group of healthy subjects. DESIGN AND PATIENTS: Basal blood samples for the determination of serum GHBP, GH, and IGF-I were obtained from patients with IDDM (n = 27), subjects with NIDDM (n = 112) and healthy controle (n = 42). Insulin, proinsulin, C-peptide and IGF-binding protein 1 (IGFBP-1) serum levels were used to estimate the insulin status in diabetic patients. RESULTS: GHBP serum levels were significantly lower in patients with IDDM than in either NIDDM or controls (P < 0.001). Conversely, the IGF-I levels were reduced in both groups of diabetics. A subgroup of hypoinsulinaemic NIDDM patients showed significantly decreased GHBP concentrations (P < 0.05) compared to the NIDDM sub-group with hyperinsulinaemia. Furthermore, GHBP levels were significantly decreased in insulin-treated patients with NIDDM compared to either non-insulin-requiring subjects or normal controls (P < 0.05). A significant direct relation was found between levels of GHBP and total insulin dose (P < 0.01) in patients with IDDM. In the NIDDM group, GHBP was correlated with proinsulin (P < 0.001), C-peptide (P < 0.01), Insulin (P < 0.05) and inversely with IGFBP-1 (P < 0.001). Multiple linear regression analysis indicated a significant contribution of proinsulin and IGFBP-1 to the variation of GHBP. CONCLUSIONS: Decreased GHBP levels in IDDM as well as in NIDDM correlate with insulinopenia. Since the degree of insulinopenia depends on the capability of the beta-cells to secrete proinsulin, C-peptide and insulin, we hypothesize that these hormones at least partially influence the serum level of GHBP. Low GHBP levels may reflect a reduced GH receptor density and a concomitant GH insensitivity, which leads to an impaired IGF generation in insulin-deficient patients.

Growth hormone-binding protein-related immunoreactivity in the serum of patients with acromegaly is regulated inversely by growth hormone concentration.

In this report we describe a newly developed radioimmunoassay (RIA) for the determination of the high-affinity growth hormone-binding protein (GHBP) in human blood. Using this RIA for the measurement of GHBP in serum of 29 patients with acromegaly, decreased concentrations were found compared to the normal range, depending on the activity of the disease. Growth hormone-binding protein was correlated inversely to log GH (r = -0.7, p < 0.001). A weaker relationship was shown between the GHBP activity determined in a functional assay based on charcoal separation and log GH (r = -0.51, p < 0.01). While insulin-like growth factor I (IGF-I) and IGF binding protein 3 (IGFBP-3) were correlated directly to log GH (r = 0.77 and r = 0.66, p < 0.001), an inverse and weaker relationship was evident between GHBP measured by RIA and IGF-I or IGFBP-3 (r = -0.61 and r = -0.57, p < 0.01). In contrast, no correlation could be detected between data of the functional GHBP assay and IGF-I or IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of transformed alpha 2-macroglobulin as a growth hormone-binding protein in human blood.

We have found that the human GH forms complexes with the purified proteinase inhibitor, alpha 2-macroglobulin. This inhibitor occurs in two different forms in human blood, known as native and transformed alpha 2-macroglobulin. It could be clearly demonstrated by chromatography and electrophoresis combined with autoradiography that the GH binds specifically to the transformed inhibitor, but not to the native protein. The binding was characterized as being mainly noncovalent to specific binding sites present only in the transformed inhibitor molecule. Binding analysis using antibody precipitation technique revealed two different classes of binding sites with dissociation constants of 0.49 +/- 0.12 and 61 +/- 8 mumol/L for high and low affinity binding sites, respectively. Distribution analysis of 125I-labeled GH in whole plasma suggested that the hormone is bound to different proteins: 1) to the high affinity GH-binding protein, and 2) to the low affinity GH-binding protein identified as transformed alpha 2-macroglobulin.