Showing papers by "Andreas Pfeiffer published in 1997"

Growth factor alterations in advanced diabetic retinopathy: a possible role of blood retina barrier breakdown

Abstract: Chronic hyperglycemia may cause growth factor alterations that are likely to participate in tissue remodeling typical for diabetic late complications. However, few details of such events are known. The ocular vitreous fluid allows studies of growth factor levels in human eyes (after vitrectomy). The vitreous is highly inert and protected by the blood-retina barrier and thus probably reflects growth factor production by the normal retina. Vitreous from patients with proliferative diabetic retinopathy (PDR) was compared with vitreous obtained from patients with nonproliferative eye disease and with vitreous from patients without diabetes but with marked neovascular proliferations due to ischemia. This design permits us to distinguish diabetes-related from non-diabetes-related alterations. Insulin-like growth factor I (IGF-I), IGF-II, IGF binding protein 2 (IGFBP-2), and IGFBP-3 were elevated 3-to 13-fold in nondiabetic retinal ischemia and 1.5- to 3-fold in PDR, indicating that the changes were not restricted to diabetes. These changes may partially be explained by leakage of serum into the vitreous, since IGFs and IGFBPs are 20- to 50-fold higher in serum than in vitreous, and vitreous protein content was 1.5-fold elevated in PDR subjects and 5-fold in ischemia patients compared with control subjects. TGF-β is a proposed antiangiogenic factor in the eye. TGF-β 2 was the predominant subtype in vitreous, and its total amount was not altered in PDR patients. More importantly, the active fraction of TGF-β was decreased by 30 and 70% in PDR and nondiabetic retinal ischemia patients, respectively. Since plasmin may control TGF-β activation, the serum protein α 2 -antiplasmin was measured and found to be significantly elevated to 150 and 250% of control values in PDR and ischemia patients, respectively. Thus, influx of serum proteins due to microvascular disturbances and hypoxia is proposed as a possible cause for vitreous alterations of IGF-I and of active TGF-β. These changes seem to occur late in the sequence of events leading to PDR and are not specific for diabetes, but they were also observed in other diseases characterized by retinal hypoxia.

Circulating tumor necrosis factor alpha is elevated in male but not in female patients with type II diabetes mellitus.

Abstract: The cytokine tumor necrosis factor alpha (TNF alpha) was proposed to mediate obesity related insulin resistance upon production in fat cells and to participate in tissue remodelling leading to vascular complications upon being released by macrophages. To assess its putative role in diabetes we determined plasma levels of TNF alpha in 105 adult humans. Male nondiabetic subjects had significantly lower TNF alpha levels than female controls (4.4 +/- 0.3, n = 17 vs. 6.6 +/- 1.0 pg/ml, n = 13; p = 0.049). Men with NIDDM had elevated TNF alpha (6.7 +/- 0.6 pg/ml, n = 34) compared to nondiabetic subjects (4.4 +/- 0.3 pg/ml, n = 17; p = 0.012). Such a difference was not apparent in women. Levels of TNF alpha were correlated with serum triglyceride levels in male controls (r2 = 0.64; p = 0.007) but not in NIDDM. Neither body mass index nor glycosylated hemoglobin correlated with TNF alpha in any of the groups. The presence of retinopathy (p = 0.046) but not of neuropathy or nephropathy or macroangiopathy was associated with significantly elevated plasma TNF alpha. We conclude that plasma levels of TNF alpha are sex-dependent and that increased TNF alpha occurs in male but not female NIDDM and may participate in the development of diabetic complications.

Deficiency of phosphofructo-1-kinase/muscle subtype in humans impairs insulin secretion and causes insulin resistance.

Abstract: Non-insulin-dependent diabetes mellitus (NIDDM) is caused by peripheral insulin resistance and impaired beta cell function. Phosphofructo-1-kinase (PFK1) is a rate-limiting enzyme in glycolysis, and its muscle subtype (PFK1-M) deficiency leads to the autosomal recessively inherited glycogenosis type VII Tarui's disease. It was evaluated whether PFK1-M deficiency leads to alterations in insulin action or secretion in humans. A core family of four members was evaluated for PFK1-M deficiency by DNA and enzyme-activity analyses. All members underwent oral and intravenous glucose tolerance tests (oGTT and ivGTT) and an insulin-sensitivity test (IST) using octreotide. Enzyme activity determinations in red blood cells showed that the father (46 yr, body mass index [BMI] 22. 4 kg/m2) and older son (19 yr, BMI 17.8 kg/m2) had a homozygous, while the mother (47 yr, BMI 28.4 kg/m2) and younger son (13 yr, BMI 16.5 kg/m2) had a heterozygous PFK1-M deficiency. DNA analyses revealed an exon 5 missense mutation causing missplicing of one allele in all four family members, and an exon 22 frameshift mutation of the other allele of the two homozygously affected individuals. The father showed impaired glucose tolerance, and the mother showed NIDDM. By ivGTT, both parents and the older son had decreased first-phase insulin secretion and a diminished glucose disappearance rate. The IST showed marked insulin resistance in both parents and the older, homozygous son, and moderate resistance in the younger son. PFK1-M deficiency causes impaired insulin secretion in response to glucose, demonstrating its participation in islet glucose metabolism, and peripheral insulin resistance. These combined metabolic sequelae of PFK-1 deficiency identify it as a candidate gene predisposing to NIDDM.

Insulinoma cells contain an isoform of Ca2+/calmodulin-dependent protein kinase II delta associated with insulin secretion vesicles.

Abstract: The Ca2+/calmodulin dependent protein kinase II (CaM kinase II) is thought to play an important part in glucose-stimulated insulin secretion To determine which of the known subtypes (α, β, γ,δ ) occur in insulin-secreting cells, we amplified all types of CaM kinase II by RT-PCR and found the β3-, γ-,δ 2- and δ6-subtypes in RINm5F insulinoma cells None of the other 8 δ-subtypes was present Antibodies generated against the bacterially expressed association domain of theδ 2-subtype recognized the recombinant γ andδ -subtypes In INS-1 and RINm5F cells, as well as freshly isolated rat islets, only a 55-kDa protein corresponding in size to theδ 2-subtype expressed in NIH3T3 fibroblasts was detected The δ2-subtype therefore appears to represent the predominant subtype of CaM kinase II present in insulin secreting cells The enzyme was primarily associated with cytoskeletal structures, and very little was present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells An analysis

mRNA expression of ligands of the epidermal-growth-factor-receptor in the uterus.

Abstract: Six different ligands of the epidermal-growth-factor receptor (EGFR) have been identified in the past. In some cervical squamous-cell carcinomas, an increased amount of proteins binding to the EGFR has been reported. In order to identify the mRNA of EGFR ligands (EGFRL), which might be overexpressed in cervical and endometrial cancers, we performed semi-quantitative reverse-transcription/polymerase chain reactions (RT-PCR) for all 6 EGFRL in RNA extracts of normal and malignant tissue samples of the human uterus. PCR products from RNA extracts of 83 patients were quantitated relative to the housekeeping gene and internal standard pyruvate dehydrogenase by analyzing the PCR kinetics of product synthesis. In extracts of normal cervix, the level of mRNA expression of the EGFRL was significantly higher than in endometrium. No significant difference was detected between normal cervix and cervical carcinomas. However, both in cervical and in endometrial cancers, mRNA expression was non-parametrically distributed and in some cervical cancers overexpression of transforming growth factor alpha (TGF-alpha), amphiregulin or EGF was observed. In endometrial cancers, mRNA levels of all EGFRL were higher than in normal endometrium. This increase was significant (p < 0.005) for TGF-alpha and amphiregulin. Thus, TGF-alpha mRNA is overexpressed in approximately 10% of cervical cancers and in the majority of endometrial cancers. Since TGF-alpha anti-sense therapy might represent a future strategy in such cancers, we also determined the absolute level of TGF-alpha mRNA expression by quantitative PCR using a cloned standard.

Analysis of a protein kinase C-alpha mutation in human pituitary tumours.

Abstract: It is generally accepted that protein kinase C-alpha (PKC-alpha) is an important enzyme in the cellular regulation of growth and differentiation by phosphorylating proteins. Recent studies have described a point mutation of PKC-alpha (position 908 of the genetic sequence, codon GAC becoming GGC) in invasive human pituitary tumours which leads to an exchange of amino acids in the protein. We investigated 11 human pituitary tumours to evaluate the data obtained previously. cDNA was subcloned and up to ten individual clones were sequenced from each tumour, resulting in 85 clones analyzed in total. All of the pituitary adenomas showed a normal wild-type sequence of PKC-alpha DNA. Even if the tumour was 'invasive' (infiltration of the dura mater) no mutation at position 908 of the sequence was found. Moreover, using Western blot analyses we did not observe any differences in PKC-alpha protein expression in invasive as compared with noninvasive pituitary adenomas. Until now we have been unable to confirm the data of other investigators, suggesting that mutated PKC-alpha is an inconsistent feature of invasive pituitary tumours.

Update Diabetes 1997

Abstract: Die DCCT-S tud ie hat belegt, da/] der mittlere Blutzucker eine dominante Gr6l]e Rir das Auftreten von Komplikationen ist [7]. Je niedriger andererseits der nfittlere Blutzucker ist, desto h6her ist das Risiko von Hypoglyk~imien. l)ie Gefahr von Komplikationen nimmt mit steigendem H b A l c tiberproportional zu, so daB tats~ichlich im Bereich zwischen 6 und 8\"/,, H b A l c cine geringere absolute Zunahme des Komplikationsrisikos erfolgt als zwischen 8 und 10% HbAlc . Krolewski et al. beobachteten 1995 in einer retrospektiven Studie keine Verringerung des Nephropathie[211 und l~etinopathierisikos bei HbAIc -Wer t en unter 8\"/,,, wohl aber cine Zunahme von Hypoglyk:dmien und schlugen deshalb einen H b A l c Wert von 8'%, als g(instigsten Kompromil3 zwischen dem E.isiko von Komplikationen und Hypoglykiimien vor. l)ie l )CCT-Stud ie 17] erlaubt wegen der h5ufig durchgeRihrten Komplikationsund HbAlc-Kont ro l len cine pr?izise Analyse dieser Frage. Tats~ichlich findet sich hier eine hochsignifikante proportionale Risikoreduktion tiir I