Raf kinase inhibitor protein: mechanism of loss of expression and association with genomic instability.

AIMS: Raf kinase inhibitory protein (RKIP; also known as PEBP, for phosphatidylethanolamine-binding protein) is an endogenous inhibitor of the Raf- MAPK kinase (MEK)-MAP kinase pathway. It has emerged as a significant metastasis suppressor in a variety of human cancers including colorectal cancer (CRC) and was recently shown to regulate the spindle checkpoint in cultured cells. This study aims at correlating RKIP expression with chromosomal instability in colorectal cancer samples and identifies possible mechanisms of RKIP loss. METHODS: Chromosomal instability was assessed using metaphase-based comparative genomic hybridisation (CGH) and loss of heterozygosity (LOH) in 65 cases with microsatellite stable CRC and correlated with RKIP expression. Methyl-specific PCR was used on DNA extracted from 82 cases with CRC to determine CpG methylation status at the RKIP promoter and the results correlated with RKIP protein expression. RESULTS: We demonstrate for the first time that in microsatellite stable (MSS) CRC, the number of chromosomal losses is inversely proportional to RKIP expression levels. We also show that methylation of the RKIP promoter is a major mechanism by which RKIP expression is silenced in CRC. CONCLUSIONS: RKIP loss by hypermethylation of its promoter could have a significant influence on colorectal cancer aneuploidy, which might explain its association with metastatic progression.

Metastatic recurrence of early-stage colorectal cancer is linked to loss of heterozygosity on chromosomes 4 and 14q.

OBJECTIVE: To investigate the prognostic value for loss of heterozygosity (LOH) of chromosomes 4 and 14q in early-stage colorectal cancer (CRC). METHODS: A total of 70, largely microsatellite stable, tumours and their corresponding normal mucosa were subjected to microdissection and analysed for LOH at chromosomes 4 and 14q by using 13 highly polymorphic microsatellite markers. LOH was correlated with the survival of the patients, using univariate, multivariate and Kaplan-Meier's survival curves. RESULT: LOH at D4S2935, D4S1579 and D4S1595 on chromosome 4 was significantly associated with metastatic recurrence of early-stage CRC. For chromosome arm 14q, two minimal regions of deletion were associated with metastatic recurrence and mapped to neighbouring markers D14S275/D14S49 at 14q12-13 and D14S65/D14S250 at 14q32. High-level loss (loss of five to eight of the informative microsatellite markers) on both chromosomes 4 and 14q, to be an independent prognostic indicator in early-stage CRC was shown by multivariate analysis. CONCLUSION: Determining the LOH of chromosomes 4 and 14q and their extent in primary tumours of patients with early-stage CRC may constitute a molecular signature of metastatic recurrence. This may be achieved if new finding sheds light on the treatment of this subgroup of patients that have been largely ignored.

Alpha-lipoic acid mitigates insulin resistance in Goto-Kakizaki rats.

Impaired glucose uptake and metabolism by peripheral tissues is a common feature in both type I and type II diabetes mellitus. This phenomenon was examined in the context of oxidative stress and the early events within the insulin signalling pathway using soleus muscles derived from non-obese, insulin-resistant type II diabetic Goto-Kakizaki (GK) rats, a well-known genetic rat model for human type II diabetes. Insulin-stimulated glucose transport was impaired in soleus muscle from GK rats. Oxidative and non-oxidative glucose disposal pathways represented by glucose oxidation and glycogen synthesis in soleus muscles of GK rats appear to be resistant to the action of insulin when compared to their corresponding control values. These diabetes-related abnormalities in glucose disposal were associated with a marked diminution in the insulin-mediated enhancement of protein kinase B (Akt/PKB) and insulin receptor substrate-1 (IRS-1)-associated phosphatidylinostol 3-kinase (PI 3-kinase) activities; these two kinases are key elements in the insulin signalling pathway. Moreover, heightened state of oxidative stress, as indicated by protein bound carbonyl content, was evident in soleus muscle of GK diabetic rats. Chronic administration of the hydrophobic/hydrophilic antioxidant alpha -lipoic-acid (ALA, 100 mg/kg, i.p.) partly ameliorated the diabetes-related deficit in glucose metabolism, protein oxidation as well as the activation by insulin of the various steps of the insulin signalling pathway, including the enzymes Akt/PKB and PI-3 kinase. Overall, the current investigation illuminates the concept that oxidative stress may indeed be involved in the pathogenesis of certain types of insulin resistance. It also harmonizes with the notion of including potent antioxidants such as ALA in the armamentarium of antidiabetic therapy.

Co-administration of etomoxir and RU-486 mitigates insulin resistance in hepatic and muscular tissues of STZ-induced diabetic rats.

Insulin resistance is a condition of central importance in a cluster of clinical disorders including diabetes mellitus, hypertension, dyslipidemia, central obesity and coronary heart disease. Despite its association with numerous health problems, the mechanism responsible for the development of this phenomenon remains to be established. A novel theory has proposed that insulin resistance in diabetes stems, at least in part, from enhanced free fatty acid (FFA) oxidation and/or excessive production of glucocorticoids (GCs). Several key predictions of this premise were subjected to experimental testing using streptozotocin (STZ)-treated rats as a model for insulin-dependent diabetes mellitus and euglycemic-hyperinsulinemic clamp technique for the in vivo measurement of insulin actions. Euglycemic clamp studies with an insulin infusion index of 5 mU/kg/min were used to measure endogenous glucose production (EGP), glucose infusion rate (GIR), glucose disposal rate (GDR) and skeletal muscle glucose utilization index (GUI). Post-absorptive basal EGP and plasma levels of glucose and free fatty acids (FFA) were elevated in the STZ diabetic rats compared to their corresponding control values. In contrast, hypoinsulinemia was evident in these animals. Steady-state GIR and GDR during euglycemic-hyperinsulinemic clamp were markedly decreased in the STZ diabetic rats. Similarly, insulin-mediated suppression of EGP and plasma FFA concentration was also impaired in these animals. GUI, a measure of 2-deoxyglucose (2-DG) uptake, was increased in response to insulin in the order of white gastrocnemus (WG), red gastrocnemus (RG), extensor digitorum longus and soleus muscles. This parallels the percentage of red fibers in these muscles. Diabetes interferes with insulin's ability to increase 2-DG uptake in all of the above muscles with the exception of WG. Nullification of the associated hyperlipidemic and hypercortisolemic states of diabetes with etomoxir (hyperlipidemic) and the glucocorticoid receptor blocker RU-486 (hypercortisolemic) ameliorated the diabetes-related impairment of the in vivo insulin action. Overall these results together with those garnered from the literature support the notion that hypercortisolemia and the enhancement of FFA oxidation are involved, at least in part, in the development of hepatic and skeletal muscle insulin resistance in poorly controlled type I diabetes.

Diabetes attenuates the response of the lumbospinal noradrenergic system to idazoxan.

Allodynia is a common feature of painful diabetic neuropathy. This phenomenon appears to be under endogenous noradrenergic control and can be ameliorated effectively by alpha(2)-adrenoceptor agonists. Accordingly, diabetic lumbospinal noradrenergic dynamics was evaluated using high performance liquid chromatography with electrochemical detector (HPLC-ECD), in vitro ligand binding and RT-PCR-based techniques. Streptozotocin (STZ)-treated and Goto-Kakizaki (GK) diabetic rats were included, respectively, as models for type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus. The data from these studies revealed that lumbospinal norepinephrine (NE) release, as indicated by the 3-methoxy-4-hydroxyphenyl glycol (MHPG)/NE ratio, was decreased as a function of diabetes. Similarly, the binding density of [3H] p-aminoclonidine and the level of expression of mRNA transcripts encoding for the alpha(2A)-adrenoceptor subtype and noradrenergic transporter were also reduced in this disease state. Analogous findings were obtained in non-diabetic Wistar rats rendered hypercortisolemic by the subcutaneous implantation of slow releasing pellets containing a supraphysiological dose of glucocorticoid (GC). Tactile allodynia was consistently observed in STZ- and GC-treated animals. The responsiveness of alpha(2)-adrenoceptors to idazoxan (alpha(2)-adrenoceptor antagonist) indicated a dose-dependent enhancement of noradrenergic transmission in lumbar segments of normal spinal cord. In stark contrast, this neurochemical action of idazoxan was attenuated in diabetic and hypercortisolemic animals. The institution of insulin therapy ameliorated diabetes-related abnormalities in lumbospinal noradrenergic dynamics. Overall, the current finding suggests that diabetic and hypercortisolemic allodynic symptoms may stem from, at least in part, down-regulation of alpha(2)-adrenoceptors in these disease states.

Insulin-like growth factor 1-induced alterations in lumbospinal monoamine dynamics.

A wealth of evidence indicates that insulin-like growth factor (IGF-1) is involved in neurotransmitter release, synaptic plasticity, morphogenesis and regulation of gene expression. RT-PCR and immunocytochemical-based techniques revealed that IGF-1 and its receptor are highly expressed by different neuronal elements of the spinal cord lumbar enlargement. Accordingly, the present study intended to examine lumbospinal monoamine dynamics in the context of the neurotrophic factor IGF-1. Spinal release of norepinephrine (NE) represented by 3-methoxy-4-hydroxyphenylglycol (MHPG)/NE ratio was enhanced by IGF-1. This action of IGF-1 was associated with a similar increase in both tyrosine hydroxylase (TH) immunoreactivity and the level of its mRNA. In contrast, neuronal contents of serotonin and its metabolite 5-hydroxyindoleacetic acid in IGF-1-treated animals remained at control level. Genistein, a tyrosine kinase inhibitor, which by itself had no effect on NE metabolism, abolished the induction effect of IGF-1 on TH and MHPG/NE ratio. Our results suggest that IGF-1 augments the lumbospinal noradrenergic system by an intracellular mechanism involving a receptor-linked tyrosine kinase. The physiological consequences of the IGF-1 actions are discussed in terms of neuroprotection and nociception.

Insulin and glucocorticoid-dependent suppression of the IGF-I system in diabetic wounds.

BACKGROUND: Growth-promoting polypeptides, including insulin-like growth factor-I (IGF-I), orchestrate different biochemical events that culminate in the restoration of functional integrity of wounded skin. The nonhealing cutaneous wound is a well-documented phenomenon in experimental and clinical diabetes. Accordingly, we undertook this study to ascertain whether diabetes impairs the healing process by suppressing the wound microenvironmental IGF-I system (eg, IGF-I; IGF-I receptor [IGF-I R]; and IGF-I binding protein [IGF-BP(3)]). METHODS: The induction of diabetes was achieved by the intravenous injection of streptozotocin at a dose of 55 mg/kg. Subcutaneously implanted polyvinyl alcohol sponge and stainless steel mesh chamber models were used to study wound healing. Nondiabetic and diabetic animals received, respectively, subcutaneous 30-day time-release pellets of glucocorticoid (200 mg) and mifepristone (RU-486, 25 mg). Corresponding control animals received placebo pellets. Polyvinyl alcohol sponge and wound fluid expression of the IGF-I system were evaluated by using ligand blotting, radioimmunoassay, and reverse transcriptase polymerase chain reaction-based techniques. RESULTS: Polyvinyl alcohol sponge contents of messenger RNA (mRNA) transcripts encoding for IGF-I, IGF-I R, and IGF-BP(3) were reduced in diabetic and glucocorticoid-treated control animals. A similar pattern of changes in protein levels of IGF-I and IGF-BP(3) occurred in wound fluid collected from these animals. Partial normalization of the associated hyperglycemic and hypercortisolemic states of diabetes with insulin (hyperglycemia) and the glucocorticoid receptor blocker RU-486 (hypercortisolemia) ameliorated the diabetes-related decrease in the IGF-I system during wound healing. CONCLUSIONS: The current data, together with data garnered from the literature, support the concept that the state of hypercortisolemia in diabetes mellitus impairs the healing process, at least in part, by suppressing the wound microenvironmental IGF-I system. Confirmation regarding this premise awaits further investigation.

Heat-shock protein 72/73 and impaired wound healing in diabetic and hypercortisolemic states.

BACKGROUND: Impaired wound healing is a well-documented phenomenon in experimental and clinical diabetes. Emerging evidence favors the involvement of glucocorticoids (GCs) in the pathogenesis of this diabetic complication. Recent data indicated that a heat-shock protein (HSP) with a molecular weight of about 70 kd is expressed in wound healing and it is under the control of the hypothalamic-pituitary-adrenal axis. In view of these findings, the current study was designed to examine the influence of diabetes and the hypercortisolemic state on the expression of HSP 72/73 during wound healing. METHODS: Induction of diabetes was achieved by the intravenous injection of streptozotocin at a dose of 55 mg/kg. Subcutaneously implanted polyvinyl alcohol (PVA) sponges were used as a wound healing model. Control and diabetic animals received, respectively, subcutaneous 30-day timed-release pellets of GC (200 mg) and RU 486 (25 mg). Corresponding animals received placebo pellets. Expression of HSP 72/73 within the PVA sponges was assayed with use of Western blotting and immunohistochemical techniques. RESULTS: GCs caused a Cushing-like syndrome with weight loss and adrenal atrophy. A pronounced accumulation of constitutive HSP 72/73 was observed in the cytoplasm of various cell types including fibroblasts, macrophages, and endothelium of nondiabetic controls. The PVA sponge contents of HSP 72/73 were decreased as a function of diabetes. A similar phenomenon was seen in control animals receiving high doses of GCs. Partial normalization of the associated hyperglycemic and hypercortisolemic states of diabetes with insulin (hyperglycemia) and the GC receptor block RU 486 (hypercortisolemia) ameliorated the diabetes-related decrease in PVA sponge contents of HSP 72/73. CONCLUSIONS: The current study provides evidence that both diabetes and the hypercortisolemic state are associated with a reduction in PVA sponge content of HSP 72/73. An amelioration of these changes was achieved by the institution of RU 486 therapy. Although our data may point to the possibility that the diabetes-related decrease in HSP 72/73 is mediated at least in part by GCs, a confirmation regarding this premise awaits further investigation.

Spinal cord noradrenergic dynamics in diabetic and hypercortisolaemic states.

Disorders of pain sensation including spontaneous pain, allodynia and hyperalgesia are commonly seen in neuropathic diabetic patients. A wealth of evidence indicates that spinal monoamine systems are implicated in pain modulation but whether abnormalities in these systems underlay such disorders is unclear. The present study was therefore initiated to investigate spinal noradrenergic dynamics during diabetes. Spinal release of norepinephrine (NE) represented by 3-methoxy-4-hydroxyphenylglycol (MHPG)/NE ratio was markedly suppressed in 30-day streptozotocin (STZ)-treated diabetic male and female rats. The density of [3H] p-aminoclonidine binding sites and the level of expression of mRNA encoding for alpha2A-adrenoceptor subtype were also reduced as a function of diabetes. In contrast, an increase in the density of [3H] prazosin binding to spinal synaptosomal membranes was evident in these animals. Clonidine-induced elevation in nociceptive threshold was attenuated in diabetics. Control animals subjected to chronic treatment with a supraphysiological dose of glucocorticoid (GC) exhibited a neurochemical pattern which is similar in many respects to that produced by the diabetic state. Both insulin and the GC receptor blocker, RU 486, restored most of the neurochemical and behavioural abnormalities of diabetes. Overall, the present study supports the concept that a diabetes-related deficit in spinal noradrenergic dynamics may be a reflection of an overactivity of the hypothalamic-pituitary-adrenal axis.

Glucocorticoid-dependent impairment of wound healing in experimental diabetes: amelioration by adrenalectomy and RU 486.

BACKGROUND: Failure of wounds to heal represents one of the major diabetic complications. Emerging evidence favors the involvement of glucocorticoids (GCs) in the pathogenesis of impaired wound healing in diabetes mellitus. OBJECTIVE: The purpose of this study was to examine wound healing potential in diabetics under conditions in which the hypercortisolemic state is normalized. DESIGN AND INTERVENTION: Linear skin incision and polyvinyl alcohol (PVA) sponge were used as wound healing models. Six groups of rats matched with respect to age, sex, and strain were included in this study. Animals in groups 1 and 6 were injected with citrate buffer, whereas rats in groups 2,3,4, and 5 received streptozotocin (STZ, 55 mg/kg iv in citrate buffer). Five days later animals in groups 4,5, and 6 received insulin (group 4) and subcutaneous implantation of slow-releasing pellets containing either the GC receptor blocker RU 486 (group 5) or a high dose of GC (group 6). MAIN OUTCOME MEASUREMENTS: Skin wound tensile strength and PVA sponge collagen metabolism were determined using tensiometric, spectrosphotometric, and polymerase chain reaction-based assays. In addition, cell infiltration and granulation tissue growth were assessed using a well-established histochemical technique. RESULTS: Wound-related parameters including fibroplasia, neovascularization, and inflammatory cell numbers were reduced as a function of diabetes. Similarly, skin wound tensile strength, PVA sponge hydroxyproline content, and the levels of mRNA transcripts for type I and III collagen were also decreased in this disease state. This diabetes-related deficit in wound healing potential was ameliorated by subjecting diabetic animals to insulin treatment or by counteracting the excessive actions of GCs using both pharmacological (RU 486) and endocrinological (ADX) paradigms. CONCLUSION: The current study supports the notion that GCs are implicated in the wound healing deficit of diabetics. Moreover, it illuminates the therapeutic potential of the GC receptor blocker (e. g., RU 486) in promoting wound repair under hypercortisolemic conditions including diabetes and Cushing's syndrome.

Absence of myocardial release of troponin T after coronary bypass surgery on a beating heart.

BACKGROUND: Coronary artery bypass grafting (CABG) operations in connection with cardiopulmonary bypass (CPB) appear to be associated with a number of side effects including trauma, cognitive dysfunction and myocardial damage. Accordingly, a current interest in performing CABG on a beating heart begins to emerge. This study examines the premise that conducting CABG on a beating heart limits the extent of myocardial injury and other complications. METHODS: Forty-five consecutive patients underwent CABG on a beating heart (group A, 12 patients) or in connection with CPB (group B, 33 patients). Inclusion criteria were poor left ventricular function and evolving myocardial ischemia or infarction. Results were assessed primarily on the basis of clinical outcome. In addition, measurement of plasma levels of troponin T (TnT), creatine kinase MB (CK-MB) and lactate dehydrogenase (LD) was conducted in 12 patients of group A preoperatively and 24 h after completion of surgery. These biological data were compared with those from control patients who underwent CABG in connection with CPB within the same time span. RESULTS: All patients in groups A and B survived the CABG procedure and those on a beating heart maintained an excellent perioperative hemodynamic measurements. The mean bypass time was 75 +/- 21 min and the mean cardiac standstill was 40 +/- 17 min. The intensive care unit stay was for group A: 18 +/- 4 h, group B: 48 +/- 12 h; and the total hospital stay was for group A: 6 +/- 1 days, group B: 8 +/- 3 days. Angiographic studies showed good anastomatic patency in both groups. Postoperative low output syndrome as indicated by the need of ionotropic drugs for more than 24 h was demonstrated in 4% and 6% of groups A and B, respectively. Limitation of myocardial injury in group A was demonstrated by the minimal increase in postoperative TnT levels (16.0 +/- 0.9 versus 30 +/- 8.0 pg/ml). A similar pattern of changes was observed with other infarction markers including CK-MB and LD. Contrastingly, the pre- and post-operative values of TnT in group B were 18 +/- 1.6 and 790 +/- 140 pg/ml, respectively. CONCLUSIONS: CABG on a beating heart shares many of the positive features of CPB with a distinct advantage of eliminating the intraoperative myocardial ischemia.

Glucocorticoid dynamics and impaired wound healing in diabetes mellitus.

The aim of the present study was to examine corticosterone dynamics and its role in the pathogenesis of impaired wound healing in diabetes mellitus (DM). The streptozotocin-treated rat was used as an animal model for type I DM. A linear skin incision and subcutaneously implanted polyvinyl alcohol sponge disks were considered as wound-healing models. The data regarding corticosterone dynamics revealed diabetes-related increments in plasma corticosterone concentrations at various time intervals during the diurnal cycle (9:00 a.m., 12:00 noon, and 3:00 p.m.) Moreover, a reduction in the levels of hippocampal glucocorticoid receptors was also evident in this disease state. Immobilization-induced stress elevated plasma corticosterone levels in both control and 30-day diabetic rats. Although the diabetic rat seems capable of appropriately initiating a corticosterone stress response, it is dramatically impaired in its capacity to terminate it. A progressive decrease in collagen deposition on polyvinyl alcohol sponge and wounded skin tensile strength was seen as a function of the duration of diabetes. Similarly, polyvinyl alcohol sponges retrieved from 30-day diabetic rats also showed a marked reduction in the expression of mRNA transcripts for type I and type III collagen. A simulation of the impairment in wound-healing potential in DM was achieved by treating control animals with a supraphysiological dose of hydrocortisone. It is worthy of note that an endocrinological paradigm involving adrenalectomy and replacement therapy with hydrocortisone significantly improved the wound-related parameters, including collagen metabolism and wounded skin tensile strength in the streptozotocin diabetic rats. Overall, our data provide evidence that the diabetic state is associated with hypercortisolemia and that this phenomenon may contribute to impaired wound healing in DM.

Insulin-like growth factor-1 reverses diabetes-induced wound healing impairment in rats.

Impaired wound healing is an enigmatic and debilitating complication of diabetes. A consensus as to the pathogenesis of this disorder has yet to emerge. Recent concepts suggest that IGF-1 is an important regulator of the healing process. The level of this growth factor is reduced in the wound environment of diabetics. We tested the premise that IGF-1 administration may prevent or ameliorate wound healing impairment in streptozotocin (STZ, 55 mg/kg, iv) diabetic rats. IGF-1 (15 micrograms/day) or placebo was infused via mini-osmotic pumps into standardized stainless steel dorsal wound chambers. Wound-related parameters including protein, DNA, hydroxyproline and macrophages were decreased as a function of diabetes. A 14-day treatment with IGF-1 reversed the diabetes effect and increased total hydroxyproline, DNA, protein and macrophage numbers by 48%, 52%, 31% and 40% above vehicle-control values, respectively. The data support the premise that diabetes, related suppression of IGF-1 and/or macrophage function within the wound environment is responsible, at least in part, for the wound healing impairment in this disease state.

The role of catecholamines in the etiology of infertility in diabetes mellitus.

Patients suffering from diabetes mellitus often develop reproductive dysfunction including anovulation, infertility and disrupted pregnancy. The biochemical basis of these phenomena is yet to be provided. The current study utilizes a neuroendocrine paradigm involving an in vitro microdissection technique in conjunction with jugular catheterization to examined the proestrus dynamics of norepinephrine (NE) and the preovulatory luteinizing hormone (LH) surge in streptozotocin (STZ) treated female rats, an animal model for insulin dependent diabetes mellitus. Radioimmunoassays revealed that in control subjects LH was at basal level during the morning of proestrus (900-1200 h); the first significant increase in the level of this pituitary hormone occurred at 1400-1500 h. A maximum peak concentration of LH was attained at 1700 h. In contrast, plasma levels of LH in diabetic subjects showed the first significant increase at 1500 h and peaked at 2000 h. The peak of the LH curve in diabetic rats was reduced by about 65% with a 3 h shift to the right. Alpha-methyl-p-tyrosine-induced blockade of newly synthesized NE-based assay showed that NE turnover rates in several hypothalamic nuclei (e.g. medial preoptic nucleus, MPN; median eminence, ME; suprachiasmatic nucleus, SCN; arcuate nucleus, AN) of control subjects were at basal level during the morning of proestrus (0900-1100 h). However, they increased by the 1200-1400 h interval and remained elevated during the 1500-1700 h. This time dependent increase in hypothalamic NE turnover rates during proestrus was not observed in the STZ diabetic rats. Most of the above metabolic derangements were partially reversed following the institution of insulin replacement therapy. Overall, our data support the concept that the endocrine abnormalities (e.g. infertility, delayed preovulatory LH surge) in diabetes are due, at least in part, to a functional deficit in noradrenergic neurons within the hypothalamus.

Insulin-dependent attenuation in alpha 2-adrenoreceptor-mediated nociception in experimental diabetes.

Diabetes mellitus is associated with abnormalities in central noradrenergic dynamics, a system that appears to be involved in the regulation of nociception in both humans and experimental animals. To this end, we investigated the responsiveness of nociceptive threshold to the actions of clonidine (an alpha 2-adrenoreceptor agonist) and yohimbine (an alpha 2-adrenoreceptor antagonist) during diabetes. The induction of diabetes was achieved by the administration of streptozotocin (STZ) (55 mg/kg, intravenously). Nociceptive threshold, as indicated by the tail-flick latency of the tail immersion test, was progressively elevated as a function of the duration of diabetes. Systemic administration of clonidine and yohimbine respectively produced dose-dependent analgesic and hyperalgesic effects in control animals. Both of these phenomena were impaired in chronically diabetic animals. In contrast, insulin-treated diabetics displayed supersensitivity to clonidine's antinociceptive effect, especially at low doses. Acute hyperglycemia did not interfere with the alpha 2-agonist-mediated elevation in nociceptive threshold. Attenuation in clonidine antinociceptive effect was also observed following its intrathecal administration to diabetic animals. Overall, these data suggest that the impaired responsiveness of diabetic rats might be due to a central alpha 2-adrenoreceptor desensitization and/or biochemical defect in the postreceptor events.

Diabetes-induced suppression of IGF-1 and its receptor mRNA levels in rat superior cervical ganglia.

Insulin-like growth factor-I (IGF-I) is implicated in the development, survival and maintenance of function of sympathetic and sensory neurons. These neurons are affected at an early stage during the course of diabetes. Reverse transcriptase polymerase chain reaction (RT-PCR) based assay revealed that rat superior cervical ganglia (SCG) express mRNA transcripts for IGF-I and its receptor. Moreover, specific membrane protein binding sites for IGF-I within the SCG have also been demonstrated using competition-inhibition and affinity cross-linking techniques. An induction of diabetes with streptozotocin (STZ, 55 mg/kg, i.v.) produced a marked decrease in the SCG levels of mRNA transcripts for IGF-I and its receptor. Concentrations of circulating IGF-I and its receptor protein within the SCG were also reduced in this disease state. Insulin treatment partially prevented diabetes-related alterations in circulating IGF-I and the SCG-IGF-I system. Overall, the data described in this study may be of value in understanding the pathogenetic mechanism(s) responsible for the development of diabetic sympathetic neuropathy.

Socio-demographic features and knowledge of diabetes mellitus among diabetic patients in kuwait.

OBJECTIVE: To determine the socio-demographic profile and assess knowledge about the nature, symptoms, complications, and treatment of diabetes mellitus among diabetic patients in Kuwait. METHODS AND RESULTS: A cross-sectional sample survey of 788 patients attending specialized diabetic clinics was conducted in 1995. Kuwaitis were significantly more represented in this sample than in the general population (52.5% versus 37%). Female to male ratio among Kuwait population was 1.07 and among non-Kuwaitis it was 0.28. Age at diagnosis ranged between sixteen to eighty years with a mean of 48 ± 10.8 years. Overall knowledge was assessed by percentage of correct responses for individual knowledge questions. There was no significant difference in knowledge of diabetes seen among Kuwaitis (66%) and non-Kuwaitis (64%). No sex difference in knowledge was seen. Knowledge about diabetes was highest among diabetic patients with increasing educational achievement but lowest with advantage age. CONCLUSION: Most patients lack a lot of information that could have a significant impact on their motivation and ability to remain healthy. To overcome this growing problem, instructing a standard diabetes education system was found to be one way of improving in morbidity due to diabetes and reduced hospital admission.

Antinociceptive action of intrathecally administered IGF-I and the expression of its receptor in rat spinal cord.

mRNA transcripts for insulin-like growth factor I (IGF-I) and its receptor are expressed in the lumbar region of the spinal cord. Accordingly, we examined the involvement of IGF-I in nociceptive transmission. An intrathecal injection of IGF-I (200-1000 ng) produced a dose-dependent elevation in nociceptive threshold as indicated by tail flick/withdrawal latency. In contrast, comparable doses of insulin had no significant effect. The time-response curve (15-75 min) revealed that the peak for IGF-I's antinociceptive effect is attained at 30 min. Our data provide evidence that the IGF-I system within the spinal cord may serve as a target for novel analgesics.

Transforming growth factor-beta and insulin-like growth factor-I in relation to diabetes-induced impairment of wound healing.

Impaired wound healing is a well-documented phenomenon in diabetes mellitus, yet little is known of the fundamental cause of this pathology. This study examined the effects of streptozotocin (STZ)-induced diabetes on the healing process using three wound models: (i) a linear skin incision (tensile strength), (ii) subcutaneously implanted polyvinyl alcohol sponge PVAs (collagen deposition), and (iii) stainless steel mesh chamber (TGF-beta, IGF-I and its binding proteins, extracellular matrix remodeling enzymes). RIA specific for IGF-I revealed that diabetes induced a 42% (wound fluid) and a 48% (serum) reduction in IGF-I levels. IGF-II western ligand blots found that diabetes produced a marked reduction in the level of a wound fluid 46 kDa IGF binding proteins. A proliferation-based bioassay indicates that TGF-beta level is also reduced in diabetic wound fluid (55%). Diabetes of graded metabolic severity induced by variable doses of STZ (25 mg-200 mg/kg) showed stepwise reduction in wound tensile strength and PVAs collagen deposition. In contrast, zymographic analysis of extracellular matrix proteases revealed that the diabetic wound fluid contains increased levels of 21, 69, and 72 kDa gelatinases. A single dose of TGF-beta (2 micrograms) in a collagen vehicle partially reversed the diabetes-related decrease in the tensile strength of standardized incisions. These data support the premise that wound-healing impairment in diabetes is due, at least in part, to a deficiency in growth factor activity within the wound environment.