Diabetology & Metabolic Syndrome BioMed Central

Insulin signalling pathways were investigated in a 33 year-old woman with immunologic insulin resistance. Her past medical history was remarkable for intermittent use of insulin and allergic reactions to several drugs, and measure of plasma anti-insulin antibodies level corroborated the clinical suspicion of immune mediated insulin resistance (8074 nU/ml - RIA - Ref value: <60). Treatment with several immunosuppressive regimens was tried, however the results were disappointing. Possible subcellular mechanisms of insulin resistance were investigated by performing analysis of insulin receptor and post receptor signaling in skeletal muscle biopsy. The expression of insulin receptor (IR), insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT-4) was evaluated in total extract from muscle tissue by Western blotting. Basal IR, IRS-1 and GLUT-4 expression was detected, however receptor autophosphorylation was not observed. A study of translocation of GLUT-4 to plasma membrane showed that tissue presented low levels of membrane-associated GLUT-4. When in vitro stimulation was undertaken, tissue was capable to be responsive to insulin. Our results suggest that even though IR expression was normally occurring, IR β-subunit tyrosine kinase activity in muscle was down-regulated leading to alterations in insulin post receptor signaling. Consistent with normal insulin receptor and post receptor signaling, our results were compatible with decreased insulin binding to IR probably due to neutralization by anti-insulin antibodies. In conclusion, this patient has immunologic insulin resistance and treatment should be based on immunosuppressive drugs as tolerated.

Diabetes is one of the major risk factors for development of micro-and macrovascular disorders. Microangiopathy in type 1 diabetes is characterized by a generalized precapillary vasodilation and increased microvascular blood flow [1], resulting in capillary hypertension and endothelial dysfunction, which in turn lead to vascular complications [2]. Using intravital videomicroscopy, we recently demonstrated that capillary recruitment is impaired in patients with type 1 diabetes who do not display clinical complications [3]. The present study was designed to study skin capillary density and recruitment in patients with type 1 diabetes with vascular complications.

Methods
This cross-sectional observational study was performed on a group of 88 outpatients consecutively attending the diabetes clinic of the State University of Rio de Janeiro and also included 47 control subjects without familiar history for diabetes and without diabetes (fasting blood glucose < 100 mg/dl), matched for age, gender and smoking habit ( Table 1). The inclusion criteria were patients with diabetes diagnosed before 30 years of age who had only used insulin since the diagnosis, without symptoms of diabetes decompensation or acute infection. In females, all measurements were performed during the follicular phase of the menstrual cycle. Among diabetic patients, 26 (29.5%) had complications (microalbuminuria, retinopathy or coronary artery disease). The study was approved by the local Ethics Committee and patients and controls or their parents gave written informed consent.
Intravital video microscopy was carried out in the morning using a standardized and well-validated technique [4][5][6][7] in a temperature-controlled room (21-24°C). Subjects were seated and the skin of the dorsum of the middle phalanx of the left hand and proximal phalanx of the great toe was examined. Capillary density was defined as the total number of spontaneously perfused capillaries per mm 2 of skin. Percentage capillary recruitment was assessed by post-occlusive reactive hyperemia (PORH) after stopping arterial blood flow to the forearm and hand or to the foot by inflating a sphygmomanometer cuff over 3 min to 200 mmHg. Ten minutes after PORH, maximization of skin capillaries was obtained with 2 minutes of venous occlusion inflating the cuff to 60 mmHg (upper limb) or 90 mmHg (lower limb).
Data were analyzed by SPSS 13.0 and the Student's t test, Mann-Whitney, Wilcoxon and X 2 tests were used when indicated. p values < 0.05 were considered statistically significant. Figure 1 shows a difference in the mean capillary density (MCD) at baseline in feet, but not hands, of controls and patients without complications. Baseline MCD was significantly higher in hands and feet of patients with complications than in healthy controls or patients without complications. During PORH, MCD was also significantly higher in hands and feet of patients with complications than in controls or patients without complications. Capillary recruitment during PORH (% increase of MCD, Figure 2) was significantly higher in controls than in patients without or with complications (both in hands and feet). During venous occlusion, the MCD increase was also higher in controls than in patients without or with complications (both hands and feet). Finally, MCD during venous occlusion was also higher in patients with compli- Mean skin capillary density before (baseline) and during post-occlusive reactive hyperemia (PORH) or venous occlusion (VO) in the hands and feet of control subjects and patients with diabetes with or without vascular complications

Discussion
Microvascular disease is a major feature of type 1 diabetes that results from chronic functional and structural alterations [1]. Patients display generalized microvascular vasodilation, mainly due to altered levels of vasoactive substances, chronic plasma volume expansion and tissue hypoxia [8]. The absence of capillary recruitment in both extremities during PORH, which is related to endothelium-dependent vasodilation at the pre-capillary level, in the nutritive skin microcirculation of diabetic patients without complications has been demonstrated previously by our group [3] and was confirmed in the present study. Patients either with or without vascular complications were not able to increase capillary density during reactive hyperemia. Microvascular endothelial dysfunction associated with blunted myogenic responsiveness in type 1 diabetes may account for the pressure-induced glomerular damage in the renal pre-glomerular arterioles [9].
The new finding of the present study is that increased functional as well as structural capillary density in both extremities is observed only in diabetic patients presenting with vascular complications. Not surprisingly, capillary density was positively correlated with age and disease duration, which were significantly higher in patients with complications. Functional density corresponds to values obtained during PORH, while maximal skin capillary density (structural density) was evaluated using venous congestion, which is known to increase the red blood cell content inside capillaries and expose capillaries non-perfused at resting conditions [5]. The absence of capillary recruitment in these patients suggests that diabetic capillaries at rest are already recruited maximally as well as a loss of microvascular autoregulatory capacity. Increased capillary density at baseline (spontaneously perfused capillaries) was observed in the feet, but not in the hands of patients without complications, suggesting that microvascular alterations in type 1 diabetes initiate in the lower extremities and could contribute to complications such as chronic foot ulcers [10]. Accordingly, these microvascular alterations in lower extremities observed in our study could be considered as an early marker for foot complications. It is also worth noting that different growth factors, including vascular endothelial growth factor, are involved in the pathophysiology of diabetic kidney microvascular disease [11]. For that reason, the increased structural skin capillary density observed in our patients presenting with vascular complications could have resulted from increased levels of circulating growth factors. Taken together, our results suggest that besides microvascular vasodilation and loss of autoregulatory capacity, patients with type 1 diabetes with vascular complications present microvascular growth, probably angiogenesis, as a compensatory response to the disease process.