Dermal neurovascular dysfunction in type 2 diabetes.

OBJECTIVE: To review evidence for a relationship between dermal neurovascular dysfunction and other components of the metabolic syndrome of type 2 diabetes. RESEARCH DESIGN AND METHODS: We review and present data supporting concepts relating dermal neurovascular function to prediabetes and the metabolic syndrome. Skin blood flow can be easily measured by laser Doppler techniques. RESULTS: Heat and gravity have been shown to have specific neural, nitrergic, and independent mediators to regulate skin blood flow. We describe data showing that this new tool identifies dermal neurovascular dysfunction in the majority of type 2 diabetic patients. The defect in skin vasodilation is detectable before the development of diabetes and is partially correctable with insulin sensitizers. This defect is associated with C-fiber dysfunction (i.e., the dermal neurovascular unit) and coexists with variables of the insulin resistance syndrome. The defect most likely results from an imbalance among the endogenous vasodilator compound nitric oxide, the vasodilator neuropeptides substance P and calcitonin gene-related peptide, and the vasoconstrictors angiotensin II and endothelin. Hypertension per se increases skin vasodilation and does not impair the responses to gravity, which is opposite to that of diabetes, suggesting that the effects of diabetes override and counteract those of hypertension. CONCLUSIONS: These observations suggest that dermal neurovascular function is largely regulated by peripheral C-fiber neurons and that dysregulation may be a component of the metabolic syndrome associated with type 2 diabetes.

Platelet dysfunction in type 2 diabetes.

Insulin resistance is a uniform finding in type 2 diabetes, as are abnormalities in the microvascular and macrovascular circulations. These complications are associated with dysfunction of platelets and the neurovascular unit. Platelets are essential for hemostasis, and knowledge of their function is basic to understanding the pathophysiology of vascular disease in diabetes. Intact healthy vascular endothelium is central to the normal functioning of smooth muscle contractility as well as its normal interaction with platelets. What is not clear is the role of hyperglycemia in the functional and organic microvascular deficiencies and platelet hyperactivity in individuals with diabetes. The entire coagulation cascade is dysfunctional in diabetes. Increased levels of fibrinogen and plasminogen activator inhibitor 1 favor both thrombosis and defective dissolution of clots once formed. Platelets in type 2 diabetic individuals adhere to vascular endothelium and aggregate more readily than those in healthy people. Loss of sensitivity to the normal restraints exercised by prostacyclin (PGI(2)) and nitric oxide (NO) generated by the vascular endothelium presents as the major defect in platelet function. Insulin is a natural antagonist of platelet hyperactivity. It sensitizes the platelet to PGI(2) and enhances endothelial generation of PGI(2) and NO. Thus, the defects in insulin action in diabetes create a milieu of disordered platelet activity conducive to macrovascular and microvascular events.

Small fiber neuropathy and neurovascular disturbances in diabetes mellitus.

Functional and organic abnormalities in small unmyelinated C fibers are the hallmark of type 2 diabetes. These may be silent clinically or present with burning feet, neurovascular abnormalities, wherein warm, cold, and heat pain thresholds are disturbed in association with impairment in skin blood flow and loss of PGP 9.5 immunostaining nerves in the skin. There is a dysfunctional phase preceding organic structural damage to the neurovascular unit. It coexists with elements of the metabolic syndrome, particularly insulin resistance (IR), elevated systolic blood pressure, and diabetic dyslipidemia i.e. dysfunction of the neurovascular unit may contribute to IR due to compromised blood flow with decreased delivery of fuels to their target tissues. If this proves to be the case, it will become important to re-focus energies on the defective neuropeptidergic regulation of blood flow as an approach to ameliorating diabetes. Because there is a functional phase that precedes structural damage, reversibility of the defect is achievable.

Diabetic neuropathies.

Diabetic neuropathy is a common complication of diabetes that is often associated both with considerable morbidity and mortality. The epidemiology and natural course of diabetic neuropathy is clouded with uncertainty, largely due to confusion regarding the definition and measurement of this disorder. The recent resurgence of interest in the vascular hypothesis, oxidative stress, the neurotrophic hypothesis and the possibility of the role of autoimmunity have opened up new avenues of investigation for therapeutic intervention. Paralleling our increased understanding of the pathogenesis of diabetic neuropathy, there must be refinements in our ability to measure quantitatively the different types of defects that occur in this disorder, so that appropriate therapies can be targeted to specific fibre types. These tests must be validated and standardised to allow comparability between studies and a more meaningful interpretation of study results. Our ability to manage successfully the many different manifestations of diabetic neuropathy depends ultimately on our success in uncovering the pathogenic processes underlying this disorder.

Possible relationship between changes in islet neogenesis and islet neogenesis-associated protein-positive cell mass induced by sucrose administration to normal hamsters.

The possible relationship between changes in islet cell mass and in islet neogenesis-associated protein (INGAP)-cell mass induced by sucrose administration to normal hamsters was investigated. Normal hamsters were given sucrose (10% in drinking water) for 5 (S8) or 21 (S24) weeks and compared with control (C) fed hamsters. Serum glucose and insulin levels were measured and quantitative immunocytochemistry of the endocrine pancreas was performed. Serum glucose levels were comparable among the groups, while insulin levels were higher in S hamsters. There was a significant increase in beta-cell mass (P<0.02) and in beta-cell 5-bromo-2'-deoxyuridine index (P<0.01), and a significant decrease in islet volume (P<0.01) only in S8 vs C8 hamsters. Cytokeratin (CK)-labelled cells were detected only in S8 hamsters. INGAP-positive cell mass was significantly larger only in S8 vs C8 hamsters. Endocrine INGAP-positive cells were located at the islet periphery ( approximately 96%), spread within the exocrine pancreas ( approximately 3%), and in ductal cells (<1%) in all groups. INGAP positivity and glucagon co-localization varied according to topographic location and type of treatment. In C8 hamsters, 49.1+/-6. 9% cells were INGAP- and glucagon-positive in the islets, while this percentage decreased by almost half in endocrine extra-insular and ductal cells. In S8 animals, co-expression increased in endocrine extra-insular cells to 36.3+/-9.5%, with similar figures in the islets, decreasing to 19.7+/-6.9% in ductal cells. INGAP-positive cells located at the islet periphery also co-expressed CK. In conclusion, a significant increase of INGAP-positive cell mass was only observed at 8 weeks when neogenesis was present, suggesting that this peptide might participate in the control of islet neogenesis. Thus, INGAP could be a potentially useful tool to treat conditions in which there is a decrease in beta-cell mass.

Neurotrophic role of interleukin-6 and soluble interleukin-6 receptors in N1E-115 neuroblastoma cells.

Interleukin 6 (IL-6) plays a role in physiological and pathophysiological processes in neuronal cells. We studied whether IL-6 plays a role in neuroblastoma cells in culture. These studies demonstrate that N1E-115 cells constitutively express IL-6 but not IL-6R. Exogenous IL-6 stimulated neuronal proliferation in a dose-dependent manner. Under serum-free conditions soluble IL-6 receptors (sIL-6R) alone or in combination with IL-6 exerted significant proliferative effects, while IL-6 alone failed to promote cell proliferation. Neutralizing anti-IL-6 antibody caused a 30-40% reduction in IL-6 mediated proliferation. Our results suggest the importance of IL-6/sIL-6R for proliferation and survival of N1E-115 adrenergic neuroblastoma cells.

Specific fiber deficits in sensorimotor diabetic polyneuropathy correspond to cytotoxicity against neuroblastoma cells of sera from patients with diabetes.

OBJECTIVE: Neuropathy is the most common complication of diabetes, and toxic serum factors may contribute to its genesis. RESEARCH DESIGN AND METHODS: We assessed neurotoxicity in the serum of 39 diabetic patients and correlated it with clinical measures of somatic and autonomic nerve fiber damage. Sera were applied to N1E-115 and VSC4.1 neuroblastoma cells in vitro as models of sensory/autonomic (S/A) and motor neurons, respectively. Neurotoxicity was measured as either complete or near-complete cell death (highly toxic), inhibited cell growth (moderately toxic), or normal cell proliferation (nontoxic) compared with pooled human serum controls during culture over 4 days. RESULTS: There was an inverse correlation between neurotoxicity and vibration perception threshold (P < 0.01). Age (P < 0.02), duration of diabetes (P < 0.02), and HbA1c (P < 0.03) correlated with neurotoxicity, suggesting that glycation may contribute to cytotoxicity in this model. S/A neurotoxicity occurred more frequently in the sera of patients with type 1 (19 of 25) than type 2 (5 of 14) diabetes (P < 0.02). None of the sera from either type 1 or type 2 diabetic patients displayed neurotoxicity on VSC4.1 cells, whereas sera from patients with motor neuropathy were highly toxic. CONCLUSIONS: These studies indicate that there is a relationship between the specific nerve fiber dysfunction in the patient and the type of neuronal cell killed, not only for diabetic neuropathy but also for known forms of autoimmune neuropathies. Such toxic factors may contribute to diabetic neuropathy by acting in concert with hyperglycemia to damage sensory/autonomic neurons.

The apoptotic death of neuroblastoma cells caused by serum from patients with insulin-dependent diabetes and neuropathy may be Fas-mediated.

Immunoglobulins from patients with diabetic neuropathy are toxic to neuroblastoma cells. The cell death has characteristics of apoptosis: condensed chromatin, shrunken cytoplasm, elevation of [Ca2+]i and DNA fragmentation. N1E-115 cell membranes contain Fas, a regulator of apoptosis that recently has been shown to be involved in pancreatic beta-cell destruction leading to diabetes. Fas-specific antibodies bind to the surface of N1E-115 cells and induce apoptosis. Serum from patients with diabetic neuropathy block Fas-antibody binding. We conclude that sera from patients with diabetic neuropathy contain an activator of Fas-regulated apoptosis that may contribute to the pathogenesis of diabetic neuropathy.

Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters.

Induction of islet neogenesis by cellophane wrapping (CW) reverses streptozotocin-induced (STZ) diabetes. Administration of Ilotropin, a protein extract isolated from CW pancreata, causes recapitulation of normal islet ontogeny and reverses STZ diabetes, reducing mortality by 50%. We investigated the hypothesis that a novel gene encoding a constituent of Ilotropin was expressed in the hamster pancreas undergoing islet neogenesis. Islet neogenesis associated protein (INGAP) is a product of a novel gene expressed in regenerating hamster pancreas. Northern blot analysis showed a strong single transcript of 850 bp at 1 and 2 d after CW that disappeared by the 6th day and was absent from untreated control pancreata. INGAP gene is expressed in acinar cells, but not in islets. Western blot analysis demonstrated the presence of INGAP in Ilotropin but not in extracts from control pancreata. A synthetic pentadecapeptide, corresponding to a region unique to INGAP, stimulated a 2.4-fold increase in [3H]thymidine incorporation into hamster duct epithelium in primary culture and a rat pancreatic duct cell line but had no effect on a hamster insulinoma tumor cell line. A portion of human INGAP gene was cloned and appears to be highly homologous to the hamster gene. This data suggests that the INGAP gene is a novel pancreatic gene expressed during islet neogenesis whose protein product is a constituent of Ilotropin and is capable of initiating duct cell proliferation, a prerequisite for islet neogenesis.

Islet-cell regeneration in the diabetic hamster pancreas with restoration of normoglycaemia can be induced by a local growth factor(s).

Partial pancreatic duct obstruction in the hamster leads to the induction of endocrine-cell differentiation and new islet formation. We prepared cytosolic extracts from the partially obstructed pancreas and identified one, which when administered i.p., produced significant increases in the incorporation of tritiated thymidine by ductular and islet cells, as well as a corresponding increase in islet mass. In this study, we evaluate the ability of this extract to reverse streptozotocin diabetes mellitus. Hamsters were treated i.p. twice daily for 7 weeks with either 0.9% NaCl (saline) (n = 10) or a cytosol extract (n = 10) prepared previously from partially obstructed hamster pancreata. All animals in the cytosol group survived vs only 60% of the saline group (p = 0.02). Random blood glucose levels were greater than 22.2 mmol/l in 90% of the saline group vs 40% in the cytosol group (p < 0.05). Pancreatic tissue from the surviving saline animals and from persistently hyperglycaemic cytosol-treated animals, showed intra-cytoplasmic vacuolation of islet cells, a characteristic lesion of sustained hyperglycaemic states. Vacuolation was not observed in normoglycaemic extract treated animals. Islets in hyperglycaemic animals demonstrated a profound decrease or absence of immunoreactive insulin, compared to an abundance of immunoreactive beta cells in cytosol-treated animals that reverted to normoglycaemia. In this group, single cells or nests of cells stained for insulin on glucagon cells were identified in ductal epithelium in association with cells budding from the duct. Morphometric analysis of pancreata in reverted cytosol-treated animals showed a new population of small islets compared with saline controls and an increased islet mass. In summary, streptozotocin diabetes can be reversed by new islet formation induced by local pancreatic growth factors, the exact nature of which remains to be determined.

Phospholipid and glutamic acid decarboxylase autoantibodies in diabetic neuropathy.

OBJECTIVE: To determine the prevalence and significance of phospholipid autoantibodies (PLAs) and glutamic acid decarboxylase (GAD) autoantibodies in the circulation of normal patients and diabetic patients with and without neuropathy. RESEARCH DESIGN AND METHODS: We measured PLAs in a total of 78 patients (a diabetic group with somatic or autonomic neuropathy [n = 40] another group without neuropathy [n = 38]), and GAD autoantibodies in a subset of 22 patients. RESULTS: PLAs are found in 2% of the general population. We found PLAs in 32% of the diabetic population without neuropathy, in 88% of those with neuropathy, in 55% of those with retinopathy, and in 25% of those with established nephropathy. The frequencies of immunoglobulins in the neuropathic group were: IgG = 78%, IgM = 33%, and IgA = 23%. There was no correlation between PLAs and microalbuminuria, macrovascular disease, fibrinogen, duration of diabetes, or neuropathy, but there was a strong correlation with total neuropathy score. Sera with high PLA IgG titers bound to the surface of neuroblastoma cells and inhibited cell growth. Antibodies to GAD65 were present in 32% and to GAD67 in 0% of patients. No titers of GAD65, GAD67, or the GAD65 ratio were associated with the degree of neuropathy of the presence of PLAs. CONCLUSIONS: PLAs occur frequently in the sera of patients with diabetes and correlate with the extent of neuropathy, suggesting a role for PLAs in the etiology thereof. The measurement of PLAs may constitute a marker for ongoing damage to nerves.

The neuronal toxic factor in serum of type 1 diabetic patients is a complement-fixing autoantibody.

Type 1 diabetes is an autoimmune disease resulting in destruction of pancreatic beta cells. Many of the pancreatic beta cell autoantigens are also neuronal cell components. Using adrenergic neuroblastoma cells, we have previously demonstrated that humoral mechanisms may contribute to the development of diabetic neuropathy in Type 1 patients. We hypothesize that the toxic factor in Type 1 diabetic serum is an immunoglobulin. When neuroblastoma cells were exposed to immunoglobulins precipitated from serum of Type 1 diabetes patients with neuropathy, cell growth was significantly inhibited by day 5 (3.8 +/- 2.4 x 10(5) cells) compared to cells cultured with immunoglobulins from control (8.2 +/- 2.3 x 10(5) cells) or Type 2 diabetic serum (7.0 +/- 3.0 x 10(5) cells). The inhibitory effect (3.2 +/- 0.9 x 10(5) cells) could be removed from Type 1 diabetic serum by affinity precipitation with protein A-agarose (8.0 +/- 0.8 x 10(5) cells). Mild heat denaturing of the serum reversed the inhibitory effect (3.8 +/- 0.9 vs 1.4 +/- 1.4 x 10(5) cells), indicating a requirement for complement. Immunofluorescent labelling with anti-IgG secondary antibody of cells exposed to Type 1 diabetic serum indicated recognition of a membrane-bound antigen. The studies in this report support the hypothesis that autoimmune neuronal destruction may contribute to the development of diabetic autonomic neuropathy in patients with Type 1 diabetes.

Role of growth factors in pancreatic endocrine cells. Growth and differentiation.

Although previously thought to arise from neural ectoderm, the endocrine cells of the gastrointestinal system are now believed to derive from endoderm. The role of different growth factors in initiation of endocrine cell growth, commitment of the cells to a particular growth pathway, and the initiation and maintenance of tumor growth and remission remains unclear. This article briefly reviews factors controlling cell growth and differentiation of gastrointestinal endocrine cells and presents data on new candidate growth factors pertinent to the authors' model of induction of endocrine cell differentiation in the pancreas.

The toxic effects of serum from patients with type 1 diabetes mellitus on mouse neuroblastoma cells: a new mechanism for development of diabetic autonomic neuropathy.

The pathogenesis of diabetic neuropathy is incompletely understood. The possibility that humoral neurotoxic factors contribute as a cause of diabetic neuropathy was tested by application of serum from patients with Type 1 and Type 2 diabetes to mouse neuroblastoma cells, which have the characteristics of adrenergic neurons in culture. Serum from patients with Type 1 diabetes and somatic neuropathy significantly inhibited both proliferation and differentiation of neuroblastoma cells, while serum from patients with Type 1 diabetes but no symptoms of neuropathy and patients with Type 2 diabetes and neuropathy had no effect on proliferation, and serum from Type 2 patients only marginally inhibited differentiation. The effects of Type 1 diabetic serum could be reversed by pre-absorption of the serum to neuroblastoma cells, and were independent of glucose levels. Immunoglobulins precipitated from the sera mimicked the effects of whole sera. These results suggest that Type 1 diabetes mellitus causes a change in serum composition, possibly related to autoimmunity, that is capable of contributing to adrenergic autonomic neuropathy in diabetic patients.

The low molecular weight heat shock protein (hsp27) in rat Sertoli cells: evidence for identity of hsp27 with a germ cell-responsive phosphoprotein.

The low mol wt heat shock protein (hsp27) exists as three forms. hsp27a is the most basic and is not phosphorylated. The other two forms are phosphorylated, with hsp27b of intermediate isoelectric point and hsp27c as the most acidic form. We hypothesized that a Sertoli cell protein, previously discovered as being responsive to germ cells and referred to as GC1, is hsp27c. To investigate this hypothesis, cultured Sertoli cells were treated with CdCl2 or were heat shocked and then labeled with H3(32)PO4, [35S]methionine, or 3H-labeled amino acids. Sertoli cell proteins were then prepared for two-dimensional polyacrylamide gel electrophoresis and autoradiography or fluorography. The 27K protein did not incorporate [35S] methionine. After CdCl2 or heat shock treatments, hsp27b and hsp27c (GC1) showed increasing 32P incorporation with time. To test if this result was due to increased synthesis of hsp27 Sertoli cell proteins were labeled with 3H-labeled amino acids. Heat shock and CdCl2 resulted in time-dependent increases in 3H label incorporation into all three forms of hsp27. Shortly after heat shock, a greater proportion of hsp27b and hsp27c was observed in the pellet fraction of Sertoli cell homogenates, with more of these hsp27 forms being found in the supernatant fraction after 4 h recovery from heat shock. Treatment of CdCl2-exposed Sertoli cells with germ cells increased 32P labeling of hsp27c (GC1). The location on two-dimensional gels of the most acidic form of hsp27, hsp27c, was exactly coincident with the location of GC1. Taken together, the results support the conclusion that GC1 in Sertoli cells is hsp27c.

The partial isolation and characterization of ilotropin, a novel islet-specific growth factor.

In this series of studies, we have presented evidence for a novel, pancreatic islet-specific growth factor, which we call ilotropin. Ilotropin is acid stable, heat stable, ethanol-precipitable, and sensitive to trypsin digestion. It appears to have a molecular weight between 29 - 44,000, and preliminary data not presented here suggests that it has a relatively basic pI. Unlike many other growth factors, ilotropin does not bind to heparin. Ilotropin is distinguishable from most of the known growth factors on the basis of at least one of the characteristics established in these studies. The apparent molecular weight of 29 - 44,000 eliminates all but the larger growth factors such as PDGF and hepatic growth factor. The fact that ilotropin is acid stable rules out identity with hepatic growth factor, and its lack of binding to heparin and apparent basic pI rules out identity with PDGF. Thus, the combination of characteristics described in these studies eliminates most of the known growth factors as candidates for the role of ilotropin. Certain growth factor precursor molecules (e.g. TGF-a) and several interleukins and cytokines (e.g. pro-IL-1 and melanocyte growth factor) also fall into this molecular weight range. Whether these proteins might be related to ilotropin or play a role in its biological activity remains to be determined. Current studies of ilotropin include further purification to homogeneity, determination of the peptide sequence of ilotropin, and development of an in vitro bioassay using trophic responses of primary cultures of pancreatic duct cells as an indicator of ilotropin activity. With purified material we ought to be able to identify the cells of origin and the target cells for the action of ilotropin, and establish assays to determine the relationship to failure of beta-cell regeneration that accompanies diabetes. Ultimately we hope that ilotropin may lead to new ways of approaching aspects of the problems presented in pancreatic beta-cell failure.