Higher plasma motilin levels in obese patients decrease after Roux-en-Y gastric bypass surgery and regulate hunger.

OBJECTIVE: Motilin-induced phase III contractions of the migrating motor complex (MMC) signal hunger in healthy volunteers. The current aim was to study the role of motilin as a hunger-inducing factor in obese patients and to evaluate the effect of Roux-en-Y gastric bypass (RYGB) surgery on plasma motilin levels and hunger scores. DESIGN: Motilin and ghrelin plasma levels were determined during a complete MMC cycle in controls and obese patients selected for RYGB before, 6 months and 1 year after surgery. 20 min after the end of the second phase III, obese patients received an intravenous infusion of 40 mg erythromycin. Hunger was scored every 5 min. Hedonic hunger was assessed in obese patients with the Power of Food Scale questionnaire. RESULTS: Obesity caused a switch in the origin of phase III from antrum to duodenum. Obese patients had significantly higher motilin levels compared with controls during the MMC but tended to lack the motilin peak prior to phase III necessary to trigger hunger. Hunger scores during phase III were significantly lower in obese patients, but could be restored to control levels through the administration of a low dose of the motilin agonist, erythromycin. After RYGB surgery motilin, but not ghrelin, levels decreased in parallel with hedonic hunger scores. CONCLUSIONS: Motilin may be an important regulator involved in the pathogenesis of obesity.

Low cytochrome oxidase 4I1 links mitochondrial dysfunction to obesity and type 2 diabetes in humans and mice.

OBJECTIVES: Cytochrome oxidase (COX) dysfunction is associated with mitochondrial oxidative stress. We determined the association between COX expression, obesity and type 2 diabetes. SUBJECTS/METHODS: COX4I1 and COX10 genes were measured in monocytes of 24 lean controls, 31 glucose-tolerant and 67 diabetic obese patients, and 17 morbidly obese patients before and after bariatric surgery. We investigated the effect of caloric restriction and peroxisome proliferator-activated receptor (PPAR) agonist treatment on Cox in obese diabetic mice, and that of diet-induced insulin resistance in Streptozotocin-treated mice. RESULTS: Low COX4I1 was associated with type 2 diabetes in obese patients, adjusting for age, gender, smoking, interleukin-6 and high-sensitivity C-reactive protein, all related to metabolic syndrome (MetS; odds ratio: 6.1, 95% confidence interval: 2.3-16). In contrast, COX10 was low in glucose-tolerant and diabetic obese patients. In morbidly obese patients, COX4I1 was lower in visceral adipose tissue collected at bariatric surgery. In their monocytes, COX4I1 decreased after bariatric surgery, and low COX4I1 at 4 months was associated with MetS at 7 years. In leptin-deficient obese diabetic mice, Cox4i1 was low in white visceral adipose tissue (n=13; P<0.001) compared with age-matched lean mice (n=10). PPARγ-agonist treatment (n=13), but not caloric restriction (n=11), increased Cox4i1 (P<0.001). Increase in Cox4i1 depended on the increase in glucose transporter 4 (Glut4) expression and insulin sensitivity, independent of the increase in blood adiponectin. In streptozotocin-treated mice (three groups of seven mice, diet-induced insulin resistance decreased Cox4i1 and Glut4 (P<0.001 for both). CONCLUSION: COX4I1 depression is related to insulin resistance and type 2 diabetes in obesity. In peripheral blood monocytes, it may be a diagnostically useful biomarker.

Age-dependent decline of ß-cell function in type 1 diabetes after diagnosis: a multi-centre longitudinal study.

AIMS: C-peptide secretion is currently the only available clinical biomarker to measure residual ß-cell function in type 1 diabetes. However, the natural history of C-peptide decline after diagnosis can vary considerably dependent upon several variables. We investigated the shape of C-peptide decline over time from type 1 diabetes onset in relation to age at diagnosis, haemoglobin A1c (HbA1c) levels and insulin dose. METHODS: We analysed data from 3929 type 1 diabetes patients recruited from seven European centres representing all age groups at disease onset (childhood, adolescence and adulthood). The influence of the age at onset on ß-cell function was investigated in a longitudinal analysis at diagnosis and up to 5-years follow-up. RESULTS: Fasting C-peptide (FCP) data at diagnosis were available in 3668 patients stratified according to age at diagnosis in four groups (<5 years, n = 344; >5 years < 10 years, n = 668; >10 years < 18 years, n = 991; >18 years, n = 1655). FCP levels were positively correlated with age (p < 0.001); the subsequent decline in FCP over time was log-linear with a greater decline rate in younger age groups (p < 0.0001). CONCLUSIONS: This study reveals a positive correlation between age at diagnosis of type 1 diabetes and FCP with a more rapid decline of ß-cell function in the very young patients. These data can inform the design of clinical trials using C-peptide values as an end-point for the effect of a given treatment.

Prevalence of persistent lipid abnormalities in statin-treated patients: Belgian results of the Dyslipidaemia International Study (DYSIS).

AIM: A substantial number of cardiovascular events are not prevented by statin therapy, which is still regarded as the first-line therapy for hyperlipidaemia. Insights into the prevalence of lipid abnormalities of statin-treated patients in Belgium are lacking and may shed light on an unmet medical need for optimal use of current lipid-lowering therapies. This study aims to assess the prevalence and types of persistent lipid abnormalities in patients receiving statin therapy in a real-life primary care setting in Belgium. METHODS: This cross-sectional cohort study was designed to estimate the prevalence of specific lipid abnormalities in statin-treated patients in Belgium. Total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides were recorded from the patients' medical record. Patient's total cardiovascular risk and corresponding lipid treatment goals were defined based on the recent European Society of Cardiology/European Atherosclerosis Society recommendations. RESULTS: Overall, 56.2% of the statin-treated patients were not at goal for LDL-C. Low HDL-C (< 40 mg dl(-1) in men, < 45 mg dl(-1) in women) and elevated triglycerides (> 150 mg dl(-1) ) were seen in 16.3% and 29.0% of patients, respectively. Very high-risk patients were more likely to have LDL-C not at goal (71.4% of them), while 60.0% of high-risk patients and 34.1% of moderate-risk patients were not at goal for LDL-C. Use of ezetimibe (10 mg) was strongly associated with meeting LDL-C goals (OR 16.9, p < 0.0001). CONCLUSION: In Belgium, lipid abnormalities remained highly prevalent despite statin treatment, with more than half of all patients not reaching their LDL-C treatment goal. This finding clearly indicates that more aggressive lipid-lowering treatment is required in clinical daily practice to achieve the goals of the current guidelines.

A retrospective study of people with familial hypercholesterolaemia in a Belgian lipid clinic.

BACKGROUND: Familial hypercholesterolaemia (FH) is a genetic disease characterised by hypercholesterolaemia and premature cardiovascular events. Early diagnosis and treatment can reduce the cardiovascular burden. We describe the characteristics of patients with heterozygous FH followed in a tertiary hospital in Belgium. METHODS: We retrospectively studied a population of 321 patients with definite heterozygous FH who visited the UZ Leuven lipid clinic at least once between 1 January 2016 and 31 December 2020. Data are represented as mean ± SD. RESULTS: The age at time of diagnosis of FH was 39 ± 18 years. Patients with atherosclerotic disease (secondary prevention) were older (p < .001), more often male (p < .001), had a higher body mass index (p < .001), prevalence of (pre)diabetes (p < .001) and hypertension (p < .001) and had lower levels of low-density lipoprotein-cholesterol (LDL-C) (p < .001) than individuals without atherosclerotic disease (primary prevention). The average LDL-C in both primary (109 ± 53 mg/dL) and secondary (81 ± 63 mg/dL) prevention did not meet the targets of LDL-C as proposed by the 2019 ESC/EAS guidelines for the management of dyslipidaemias. However, LDL-C levels in the subgroup of patients treated with PCSK9 inhibition therapy, and especially in the triple therapy group (combination of statin, ezetimibe and PCSK9 inhibitor), were markedly lower (p < .001). CONCLUSIONS: In this Belgian population, people with heterozygous FH remain undertreated. Reaching treatment targets in FH seems possible, although this requires combination treatment (with PCSK9-targeted therapy) in most patients. Earlier diagnosis of FH, more extensive lipid-lowering treatment and reimbursement options and a more holistic approach are needed to lower LDL-C and cardiovascular risk in patients with FH.

Gastric bypass surgery, but not caloric restriction, decreases dipeptidyl peptidase-4 activity in obese patients with type 2 diabetes.

The mechanism by which incretins and their effect on insulin secretion increase markedly following gastric bypass (GBP) surgery is not fully elucidated. We hypothesized that a decrease in the activity of dipeptidyl peptidase-4 (DPP-4), the enzyme which inactivates incretins, may explain the rise in incretin levels post-GBP. Fasting plasma DPP-4 activity was measured after 10-kg equivalent weight loss by GBP (n = 16) or by caloric restriction (CR,n = 14) in obese patients with type 2 diabetes. DPP-4 activity decreased after GBP by 11.6% (p = 0.01), but not after CR. The increased peak glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) response to oral glucose after GBP did not correlate with DPP-4 activity. The decrease in fasting plasma DPP-4 activity after GBP occurred by a mechanism independent of weight loss and did not relate to change in incretin concentrations. Whether the change in DPP-4 activity contributes to improved diabetes control after GBP remains therefore to be determined.

Effect of obesity on gastrointestinal transit, pressure and pH using a wireless motility capsule.

BACKGROUND: Despite the increasing prevalence and medical burden of obesity, the understanding of gastrointestinal physiology in obesity is scarce, which hampers drug development. AIM: To investigate the effect of obesity and food intake on gastrointestinal transit, pressure and pH. MATERIAL AND METHODS: An exploratory cross-sectional study using a wireless motility capsule (SmartPill©) was performed in 11 participants with obesity and 11 age- and gender-matched participants with normal weight (group) in fasted and fed state (visit). During the first visit, the capsule was ingested after an overnight fast. During a second visit, the capsule was ingested after a nutritional drink to simulate fed state. Linear mixed models were constructed to compare segmental gastrointestinal transit, pressure and pH between groups (obesity or control) and within every group (fasted or fed). RESULTS: Food intake slowed gastric emptying in both groups (both P < 0.0001), though food-induced gastric contractility was higher in participants with obesity compared to controls (P = 0.02). In the small intestine, a higher contractility (P = 0.001), shorter transit (P = 0.04) and lower median pH (P = 0.002) was observed in participants with obesity compared to controls. No differences were observed for colonic measurements. CONCLUSION: Obesity has a profound impact on gastrointestinal physiology, which should be taken into account for drug development.

Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content.

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, we investigated the polarity of the expression of the cell surface heparan sulfate proteoglycans in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identified glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this proteoglycan was expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicated that sorting mechanisms, rather than differential turnover, accounted for this bipolar expression of glypican. Chlorate treatment did not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells expressed wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein resulted in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms had intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Alternatively, inverse glycanation-apical sorting relationships in glypican may insure near constant deliveries of HS to the apical compartment, or "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains.

Molecular cloning of amphiglycan, a novel integral membrane heparan sulfate proteoglycan expressed by epithelial and fibroblastic cells.

We have synthesized an antisense oligonucleotide primer that matches a supposedly conserved sequence in messages for heparan sulfate proteoglycans with transmembrane orientations. With the aid of this primer we have amplified partial and selected full-length copies of a message from human lung fibroblasts that codes for a novel integral membrane heparan sulfate proteoglycan. The encoded protein is 198 amino-acids long, with discrete cytoplasmic, transmembrane, and amino-terminal extracellular domains. Except for the sequences that represent putative heparan sulfate chain attachment sites, the extracellular domain of this protein has a unique structure. The transmembrane and cytoplasmic domains, in contrast, are highly similar to the corresponding domains of fibroglycan and syndecan, the two cell surface proteoglycans that figured as models for the design of the antisense primer. This similarity includes the conservation of four tyrosine residues, one immediately in front of the stop transfer sequence and three in the cytoplasmic segment, and of the most proximal and most distal cytoplasmic sequences. The cDNA detects a single 2.6-kb message in cultured human lung fibroblasts and in a variety of human epithelial and fibroblastic cell lines. Polyclonal and monoclonal antibodies raised against the encoded peptide after expression as a beta-galactosidase fusion protein react with the 35-kD coreprotein of a cell surface heparan sulfate proteoglycan of human lung fibroblasts and decorate the surface of many cell types. We propose to name this proteoglycan "amphiglycan" (from the Greek words amphi, "around, on both sides of" and amphoo, "both") referring to its domain structure which extends on both sides of the plasmamembrane, and to its localization around cells of both epithelial and fibroblastic origin.

Developmental changes in heparan sulfate expression: in situ detection with mAbs.

Two mAbs that are specific for heparan sulfate-related epitopes have been raised and used to analyze the cellular and tissular distribution of this glycosaminoglycan during development. mAb 10E4 reacts with an epitope that occurs in native heparan sulfate chains and that is destroyed by N-desulfation of the glycosaminoglycan. The antibody does not react with hyaluronate, chondroitin sulfate, or DNA, and reacts only poorly with heparin. The reactivity of proteoglycan extracts or tissue sections with the 10E4 antibody is completely abolished by heparitinase, but is only partially affected by heparinase. mAb 3G10, in contrast, reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections. The 3G10 epitope is destroyed by treatment with mercuric acetate, which indicates that the desaturated uronate generated by the lyase is essential for the reactivity of the antibody. The 3G10 epitope is not generated by treating heparan sulfate proteoglycans with heparinase or chondroitin sulfate proteoglycans with chondroitin sulfate lyases, which indicates that the 3G10 antibody recognizes desaturated uronates that occur in specific structural contexts. The antibody 10E4 and, after heparitinase treatment, the antibody 3G10 decorate the surfaces of many cell types and the extracellular matrix in proximity of the cells, in particular, the basement membranes. The analysis of embryonic and adult tissues reveals important temporal and regional differences in the abundance of the 10E4 and 3G10 epitopes at these sites. Moreover, the staining pattern of the two antibodies is not always superimposable, which is indicative of regional differences in the exposure or structure of the tissular heparan sulfates. As a whole the results suggest that heparan sulfate abounds at sites of active morphogenesis and that the expression of this glycosaminoglycan is developmentally regulated.

Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts.

Cultured human fetal lung fibroblasts produce some chondroitin sulfate proteoglycans that are extracted as an aggregate in chaotropic buffers containing 4 M guanidinium chloride. The aggregated proteoglycans are excluded from Sepharose CL4B and 2B, but become included, eluting with a Kav value of 0.53 from Sepharose CL4B, when Triton X-100 is included in the buffer. Conversely, some of the detergent-extractable chondroitin sulfate proteoglycans can be incorporated into liposomes, suggesting the existence of a hydrophobic membrane-intercalated chondroitin sulfate proteoglycan fraction. Purified preparations of hydrophobic chondroitin sulfate proteoglycans contain two major core protein forms of 90 and 52 kD. A monoclonal antibody (F58-7D8) obtained from the fusion of myeloma cells with spleen cells of BALB/c mice that were immunized with hydrophobic proteoglycans recognized the 90- but not the 52-kD core protein. The epitope that is recognized by the antibody is exposed at the surface of cultured human lung fibroblasts and at the surface of several stromal cells in vivo, but also at the surface of Kupffer cells and of epidermal cells. The core proteins of these small membrane-associated chondroitin sulfate proteoglycans are probably distinct from those previously identified in human fibroblasts by biochemical, immunological, and molecular biological approaches.

Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes.

Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.

Does the unfavorable pharmacokinetic and pharmacodynamic profile of the iNOS inhibitor GW273629 lead to inefficacy in acute migraine?

The objective of this study was to investigate the pharmacodynamics and pharmacokinetics of a single dose of GW273629, a selective iNOS inhibitor, given during and outside a migraine attack. GW273629 1500 mg was administered to 15 migraine patients both ictally and interictally. Nasal and exhaled nitric oxide (NO), plasma 3-nitrotyrosine, and nitrates were measured to assess systemic NO production. In addition, pharmacokinetics and treatment response were assessed. Data are mean (95% confidence interval [CI]). Plasma 3-nitrotyrosine was higher ictally: 11.96 (8.22, 15.71) ictally versus 2.74 (2.24, 3.24) ng/10 mg interictally (P < .0001). Exhaled and nasal NO showed a similar trend: 12.5 (6.5, 18.6) and 62.2 (41.5, 82.8) ppb ictally versus 9.9 (6.3, 13.4) ppb and 52.5 (38.5, 66.0) ppb interictally, respectively. Early absorption of GW273629 (AUC(0-2) [90% CI]) was reduced by 41 (22, 55)% during an attack. There was no improvement of headache or associated symptoms. Migraine headache is associated with reduced early absorption of GW273629 and excess NO production. In this open-label study, GW273629 was ineffective in the treatment of acute migraine.

Calcitonin gene-related peptide8-37 antagonizes capsaicin-induced vasodilation in the skin: evaluation of a human in vivo pharmacodynamic model.

The purpose of this study was to identify the mediators involved in capsaicin-induced vasodilation in the human skin and to evaluate a pharmacodynamic model for the early clinical evaluation of calcitonin gene-related peptide (CGRP) receptor antagonists. Dermal blood flow (DBF) response of the forearm skin to topically applied capsaicin was measured using laser Doppler perfusion imaging in 22 subjects. The effect of intra-arterially administered CGRP(8-37) (1200 ng . min(-1) . dl(-1) forearm), indomethacin (5 mug . min(-1) . dl(-1) forearm), and N(G)-monomethyl-l-arginine (l-NMMA; 0.2 mg . min(-1) dl(-1) forearm), and orally administered aprepitant (375 mg) on capsaicin-induced dermal vasodilation was assessed. Furthermore, the diurnal variation of the DBF response to capsaicin was studied. CGRP(8-37) inhibited the capsaicin-induced DBF increase: 217(145, 290)% in infused versus 370 (254, 486)% in the noninfused arm [mean (95% CI); p = 0.004]. In contrast, indomethacin, l-NMMA, aprepitant, and the time of assessment did not affect the DBF response to capsaicin. Thus, capsaicin-induced vasodilation in the human forearm skin is largely mediated by CGRP, but not by vasodilating prostaglandins, nitric oxide, or substance P. The response to capsaicin does not display a circadian rhythm. A pharmacodynamic model is proposed to evaluate CGRP receptor antagonists in humans in vivo.

Basal lamina formation by normal and transformed mouse mammary epithelial cells duplicated in vitro.

Cells from low-passage (LP) cultures of a mouse mammary epithelial line (NMuMG cells) form a basal lamina when they are cultured on a type I collagen gel substratum. A high-passage (HP) strain of this line maintained the morphologic, serologic, and karyologic properties of the LP cells. For the determination of whether transformation of the NMuMG cells might lead to defects in the basal lamina, cells from LP cultures were compared in vivo and in vitro with cells of HP cultures for tumorigenicity, growth characteristics, and ability to form a lamina. The LP NMuMG cells had a typical epithelial morphology and showed no cytologic evidence of cancer. They formed an ultrastructurally normal continuous basal lamina in vivo when they were injected into athymic nude mice. In contrast, the HP cells were pleomorphic and highly invasive when injected into nude mice where they showed frequent and large basal lamina defects. These cells also accumulated only traces of lamina-like materials when cultured on a collagen gel, indicating that neoplastic transformation had markedly reduced the ability of NMuMG cells to form a basal lamina both in vivo and in vitro. Because the collagen gel culture system duplicated the in vivo situation with regard to basal lamina integrity, the basis for this lack of in vitro basal lamina formation may be physiologically relevant for the mechanism of malignant invasion.

Changes in bone macro- and microstructure in diabetic obese mice revealed by high resolution microfocus X-ray computed tomography.

High resolution microfocus X-ray computed tomography (HR-microCT) was employed to characterize the structural alterations of the cortical and trabecular bone in a mouse model of obesity-driven type 2 diabetes (T2DM). C57Bl/6J mice were randomly assigned for 14 weeks to either a control diet-fed (CTRL) or a high fat diet (HFD)-fed group developing obesity, hyperglycaemia and insulin resistance. The HFD group showed an increased trabecular thickness and a decreased trabecular number compared to CTRL animals. Midshaft tibia intracortical porosity was assessed at two spatial image resolutions. At 2 µm scale, no change was observed in the intracortical structure. At 1 µm scale, a decrease in the cortical vascular porosity of the HFD bone was evidenced. The study of a group of 8 week old animals corresponding to animals at the start of the diet challenge revealed that the decreased vascular porosity was T2DM-dependant and not related to the ageing process. Our results offer an unprecedented ultra-characterization of the T2DM compromised skeletal micro-architecture and highlight an unrevealed T2DM-related decrease in the cortical vascular porosity, potentially affecting the bone health and fragility. Additionally, it provides some insights into the technical challenge facing the assessment of the rodent bone structure using HR-microCT imaging.

Cough as the presenting symptom of large vessel vasculitis.

We report the case of a 57-year-old woman presenting with persistent cough, weight loss, and fever. An extensive work-up revealed laboratory signs of inflammation and a mild thickening of the aortic wall on computed tomographic scan of the thorax. These findings raised the suspicion of large vessel vasculitis that was elegantly confirmed by fluorodeoxyglucose positron emission tomography. Persistent cough as the inaugural symptom and involvement of large vessels in Horton's disease are also discussed.

BMI is an important driver of ß-cell loss in type 1 diabetes upon diagnosis in 10 to 18-year-old children.

OBJECTIVE: Body weight-related insulin resistance probably plays a role in progression to type 1 diabetes, but has an uncertain impact following diagnosis. In this study, we investigated whether BMI measured at diagnosis was an independent predictor of C-peptide decline 1-year post-diagnosis. DESIGN: Multicentre longitudinal study carried out at diagnosis and up to 1-year follow-up. METHODS: Data on C-peptide were collected from seven diabetes centres in Europe. Patients were grouped according to age at diagnosis (<5 years, n=126; >5 years <10 years, n=295; >10 years <18 years, n=421; >18 years, n=410). Linear regression was used to investigate whether BMI was an independent predictor of change in fasting C-peptide over 1 year. Models were additionally adjusted for baseline insulin dose and HbA1c. RESULTS: In individuals diagnosed between 0 and 5 years, 5 and 10 years and those diagnosed >18 years, we found no association between BMI and C-peptide decline. In patients aged 10-18 years, higher BMI at baseline was associated with a greater decline in fasting C-peptide over 1 year with a decrease (ß 95% CI; P value) of 0.025 (0.010, 0.041) nM/kg per m(2) higher baseline BMI (P=0.001). This association remained significant after adjusting for gender and differences in HbA1c and insulin dose (ß=0.026, 95% CI=0.0097, 0.042; P=0.002). CONCLUSIONS: These observations indicate that increased body weight and increased insulin demand are associated with more rapid disease progression after diagnosis of type 1 diabetes in an age group 10-18 years. This should be considered in studies of ß-cell function in type 1 diabetes.

Morphological characteristics of epithelial and fibroblastic cells growing out from biopsies of human skin.

Skin biopsies are routinely used to establish fibroblast cultures in vitro, but the ultrastructural properties of the cells during this outgrowth have not received much attention. In the present study, the rate and the nature of the outgrowth of skin biopsy explants were examined in a quantitative and qualitative fashion. Attention was particularly focused on the morphogenetic properties of the various cell types present in the initial outgrowth. The rate of success in obtaining an outgrowth from the explants was slightly dependent on the composition of the medium or the serum. In more than 95% of the cases an initial epithelial outgrowth was obtained. At a later stage the outgrowth became fibroblastic. Remarkable differences in the behavior and ultrastructural properties of epithelial and fibroblastic cells were observed. Incorporation of 3H-thymidine occurred almost simultaneously with the epithelial outgrowth but preceded the migration of the fibroblasts. Under scanning and transmission electron microscopy, the cells of the outermost layers of the epithelium remained flat and polygonal and were covered with small villi during migration. The cells were closely apposed. The germinative cells had a spiky surface and were separated by large intercellular spaces. No basal lamina was formed by the migrating cells. The fibroblasts maintained their shape and smooth surface and reached the substratum under the epithelial outgrowth. They accumulated microfilaments and microtubules in their cytoplasm and were characterized by surface-associated extracellular material. On the substratum, the fibroblasts spreadout. The behavior and ultrastructural properties of these fibroblasts resemble closely those observed on fibroblasts growing-out from aggregates.