Insulin resistance and the modulation of rat cardiac K(+) currents

K(+) currents were measured using a whole cell voltage-clamp method in enzymatically isolated rat ventricular myocytes obtained from two hyperinsulinemic, insulin-resistant models. Fructose-fed rats as well as genetically obese rats, both of which are resistant to the metabolic effects of insulin, were used. The normal augmentation of a calcium-independent sustained K(+) current was reduced or abolished in insulin-resistant states. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadyl sulfate (3-4 wk treatment or after 5-6 h in vitro) enhanced the sustained K(+) current. The in vitro effect of vanadyl was blocked by cycloheximide. Insulin resistance of the K(+) current was not reversed by vanadyl sulfate. The results show that insulin resistance is expressed in terms of insulin actions on ion channels, in addition to its actions on metabolism. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadate compounds, which mimic the effects of insulin on metabolism, also mimic the augmenting effects of insulin on a cardiac K(+) current in a manner suggesting synthesis of new channels.     

Genetic effects induced by nickel sulfate in germline and somatic cells of WR mice

We examined the effects of nickel sulfate at doses 0.5 to 5.0 mg/kg (LD50) on the frequency of dominant lethal mutations and two-strand DNA breaks (TSBs) in germline cells and on an increase in frequency in gene mutations W(y) in pigment cells of first-generation mice. The results indicated that spermatogenesis stages most sensitive to nickel sulfate (at a dose of 1.0 mg/kg) are spermatozoids, early spermatids, late spermatocytes, and stem spermatogonia. No statistically significant increase in the total TSB level was detected in spermatozoids 4 weeks after exposure. At the same time, a significant (P < 0.05) increase in percentage of cells with an extremely high level of DNA fragmentation (supposedly apoptotic cells) was observed upon exposure at a dose of 0.5 mg/kg. Nickel sulfate at doses of 5.0 and 1.0 mg/kg induced a marked increase in the c-kit gene expression in pigment cells of heterozygous first-generation WR mice as compared to control (P < 0.001). It was shown that the nonobservable adverse effect level (NOAEL) of nickel sulfate on the dominant lethal mutation frequency and gene mutations was 1/200 LD50, while the lowest observable adverse effect level (LOAEL) was 1/100 LD50.     

Consistency of the disposition index in the face of diet induced insulin resistance: potential role of FFA

Objective

Insulin resistance induces hyperinsulinemic compensation, which in turn maintains almost a constant disposition index. However, the signal that gives rise to the hyperinsulinemic compensation for insulin resistance remains unknown.

Methods

In a dog model of obesity we examined the possibility that potential early-week changes in plasma FFA, glucose, or both could be part of a cascade of signals that lead to compensatory hyperinsulinemia induced by insulin resistance.

Results

Hypercaloric high fat feeding in dogs resulted in modest weight gain, and an increase in adipose tissue with no change in the non-adipose tissue size. To compensate for the drop in insulin sensitivity, there was a significant rise in plasma insulin, which can be attributed in part to a decrease in the metabolic clearance rate of insulin and increased insulin secretion. In this study we observed complete compensation for high fat diet induced insulin resistance as measured by the disposition index. The compensatory hyperinsulinemia was coupled with significant changes in plasma FFAs and no change in plasma glucose.

Conclusions

We postulate that early in the development of diet induced insulin resistance, a change in plasma FFAs may directly, through signaling at the level of β-cell, or indirectly, by decreasing hepatic insulin clearance, result in the observed hyperinsulinemic compensation.     

Blood levels of dehydroepiandrosterone sulfate (DHEA-S) and the risk of breast cancer

In North American women at low or high risk of developing breast cancer, as assessed by an epidemiologic questionnaire, the plasma concentration of dehydroepiandrosterone sulfate shows a statistically significant circannual variation. In adolescents, in all seasons, circulating dehydroepiandrosterone sulfate is a classifier of the risk of developing breast cancer, a relatively low concentration of this hormone being associated with an increased risk.     

Quantitative proteomic analysis of sokotrasterol sulfate-stimulated primary human endothelial cells

The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue. New blood vessel formation, or angiogenesis, is initiated by the activation of endothelial cells and is an important process required for various pathological and physiological situations. This study used cleavable isotope-coded affinity tag reagents combined with mass spectrometry to investigate the molecular basis of a recently discovered angiogenesis-promoting steroid, sokotrasterol sulfate. Changes in the relative abundances of over 1000 proteins within human endothelial cells treated with sokotrasterol sulfate and vehicle-treated cells were identified and quantitated using this technique. A method that examines the entire ensemble of quantitative measurements was developed to identify proteins that showed a statistically significant change in relative abundance resulting from treatment with sokotrasterol sulfate. A total of 93 proteins was significantly up-regulated, and 37 were down-regulated in response to sokotrasterol sulfate stimulation of endothelial cells. Among the up-regulated proteins, several were identified that are novel to endothelial cells and are likely involved in cell communication and morphogenesis. These findings are consistent with a role for sokotrasterol sulfate in endothelial sprouting.     

Potentiation by clofibrate of in-vivo tumor inhibition by hydrazine sulfate and cytotoxic agents, in Walker 256 carcinosarcoma

Clofibrate as a sole agent was found to have statistically perceptible antitumor effects against the in vivo growth of Walker 256 intramuscular carcinosarcoma in rats. Clofibrate was also found to potentiate the antitumor effect of hydrazine sulfate, cytotoxic drugs and the combination of hydrazine sulfate + cytotoxic drug. The results suggest that substances which might directly or indirectly interfere with host lipogenesis and/or lipolysis, may also have adjunctal antitumor potential.     

Failure of chronic hyperinsulinemia to suppress pancreatic glucagon in vivo in the rat.

To determine the effects of chronic hyperinsulinemia on glucagon release, rats were made hyperinsulinemic for 14 days by supplementation of drinking water with sucrose (10%; sucrose-fed) to increase endogenous release or by implantation of osmotic minipumps (subcutaneous, s.c.; or intraperitoneal, i.p.) to deliver exogenous insulin (6 U/day). Both s.c. and i.p. rats also had sucrose in the drinking water to prevent hypoglycemia. Plasma insulin levels were significantly elevated in sucrose-fed, s.c., and i.p. rats. However, glucose levels were significantly elevated in sucrose-fed rats only. Surprisingly, plasma glucagon concentrations were elevated in i.p. and s.c. rats and were not suppressed in sucrose-fed rats. Inverse relationships were found between the plasma levels of insulin and glucose (n = 65; r = -0.42, p less than 0.0001) and between glucose and glucagon (n = 73; r = -0.46, p less than 0.0001). However, unexpectedly, a positive correlation between insulin and glucagon (n = 65; r = 0.47, p less than 0.0001) was established. As suppression of plasma glucagon levels below basal was not observed in any of the hyperinsulinemic or hyperglycemic rats, we wished to establish further whether pancreatic glucagon release could be suppressed below basal levels in the rat by another means. Thus, high doses of somatostatin (50-100 micrograms.kg-1.min-1) were infused for 45 min into normal rats without or with a concomitant hyperinsulinemic, hyperglycemic glucose clamp. Somatostatin fully suppressed insulin, but although plasma glucagon levels were decreased by somatostatin infusion relative to saline-infused animals, there was still no suppression below basal levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skeletal muscle glycogen synthase subcellular localization: effects of insulin and PPAR-alpha agonist (K-111) administration in rhesus monkeys

Insulin covalently and allosterically regulates glycogen synthase (GS) and may also cause the translocation of GS from glycogen-poor to glycogen-rich locations. We examined the possible role of subcellular localization of GS and glycogen in insulin activation of GS in skeletal muscle of six obese monkeys and determined whether 1) insulin stimulation during a hyperinsulinemic euglycemic clamp and/or peroxisome proliferator-activated receptor (PPAR)-alpha agonist treatment (K-111, 3 mg.kg(-1).day(-1); Kowa) induced translocation of GS and 2) translocation of GS was associated with insulin activation of GS. GS and glycogen were present in all fractions obtained by differential centrifugation, except for the cytosolic fraction, under both basal and insulin-stimulated conditions. We found no evidence for translocation of GS by insulin. GS total (GST) activity was strongly associated with glycogen content (r = 0.70, P < 0.001). Six weeks of treatment with K-111 increased GST activity in all fractions, except the cytosolic fraction, and mean GST activity, GS independent activity, and glycogen content were significantly higher in the insulin-stimulated samples compared with basal samples, effects not seen with vehicle. The increase in GST activity was strongly related to the increase in glycogen content during the hyperinsulinemic euglycemic clamp after K-111 administration (r = 0.74, P < 0.001). Neither GS protein expression nor GS gene expression was affected by insulin or by K-111 treatment. We conclude that 1) in vivo insulin does not cause translocation of GS from a glycogen-poor to a glycogen-rich location in primate skeletal muscle and 2) the mechanism of action of K-111 to improve insulin sensitivity includes an increase in GST activity without an increase in GS gene or protein expression.     

Kinetics of Sulfate Reduction in a Coastal Aquifer Contaminated with Petroleum Hydrocarbons

An integrated field and laboratory study was conducted to quantify the effect of environmental determinants on the activity of sulfate reducers in a freshwater aquifer contaminated with petroleum hydrocarbons (PHC). Within the contaminated zone, PHC-supported in␣situ sulfate reduction rates varied from 11.58±3.12 to 636±53 nmol cm⁻³ d⁻¹ and a linear increase (R ²=0.98) in reduction rate was observed with increasing in situ sulfate concentrations suggesting sulfate limitation. Half-saturation concentration (K _s) for sulfate reduction coupled to PHC mineralization was determined for the first time. At two different sites within the␣aquifer, maximum sulfate reduction rate under␣non-limiting conditions (R _max) was 5,000 nmol cm⁻³ d⁻¹, whereas the retrieved K _s values were 3.5 and 7.5 mM, respectively. The K _s values are the highest ever reported from a natural environment. Furthermore, the K _s values were significantly higher than in situ sulfate concentrations confirming sulfate limited growth. On addition of lactate and formate, sulfate reduction rate increased indicating that reactivity and bioavailability of organic substrate may also have played a role in rate inhibition in certain parts of the aquifer. Experiments with sulfide amendments show statistically minor decrease in sulfate reduction rates on addition of sulfide and analogous increase in sulfide toxicity with increasing sulfide concentrations (0.5–10 mM) was not apparent.     

Elevated plasma total homocysteine levels in hyperinsulinemic obese subjects

Homocysteine has been associated with the oxidative stress in the pathogenesis of atherosclerosis. Oxidative stress caused by triglycerides and free fatty acids is known to cause insulin resistance and hyperinsulinemia. On the other hand, insulin resistance may increase homocysteine levels. Since obesity is associated with insulin resistance and hyperinsulinemia, we aimed to study the possible association of homocysteine with hyperinsulinemia in obese subjects. 20 obese male subjects (body mass index >29), aged 33--55 (mean 45 years old) were studied. A fasting blood sample was obtained for the study and the subjects undertook an oral glucose tolerance test with samples taken at 1 and 2 h after glucose. Subjects were divided in two groups according to the fasting insulin levels, < 9 &mgr;U/ml or normoinsulinemic (group 1) and >9 &mgr;U/ml or hyperinsulinemic (group 2). Glucose, insulin, homocysteine, folate, B(12,) total cholesterol, HDL-cholesterol and triglycerides levels were determined in fasting blood samples. In oral glucose tolerance test, glucose, insulin and homocysteine levels were measured. Hyperinsulinemic obese subjects (group 2) had higher levels of insulin and glucose at 1 h and 2 h postglucose, compared with group 1. Fasting total homocysteine and triglyceride levels were also increased in this group, whereas folate and B(12) levels were similar in both groups. Fasting homocysteine significantly correlated with fasting insulin (r = 0.6, p <0.01). Homocysteine levels slightly but significantly decreased after glucose loading in normoinsulinemic but not in hyperinsulinemic obese subjects. These results show that higher homocysteine levels are observed in the hyperinsulinemic obese subjects and suggest that homocysteine could play a role in the higher risk of cardiovascular disease in obesity.     

Glycosaminoglycan synthesis in explants derived from bleomycin-treated fibrotic hamster lungs

Glycosaminoglycan synthesis was studied in explant cultures of hamster lungs 15 and 45 days following intratracheal administration of Bleomycin. At both time points, a statistically significant increase in 35S-sulfate incorporation into glycosaminoglycans was seen in the Bleomycin-treated explants compared with that of the controls. Furthermore, the percentage of label associated with dermatan sulfate was significantly higher in the treated explants than in controls at both 15 and 45 days. Conversely, the percentage of labeled heparin and/or heparan sulfate was significantly lower for the treated explants compared to controls at these times. These results indicate that glycosaminoglycan synthesis is altered from normal in this model of interstitial lung disease. Comparison of these data with previous measurements of glycosaminoglycan synthesis in another model of interstitial lung disease, induced by N-nitroso-N-methylurethane, reveals marked similarity in the changes from normal in 35S-labeling.     

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

Brain region-dependent effects of dexamethasone on counterregulatory responses to hypoglycemia in conscious rats

The aim of this study was to determine whether activation of central type II glucocorticoid receptors can blunt autonomic nervous system counterregulatory responses to subsequent hypoglycemia. Sixty conscious unrestrained Sprague-Dawley rats were studied during 2-day experiments. Day 1 consisted of either two episodes of clamped 2-h hyperinsulinemic (30 pmol x kg(-1) x min(-1)) hypoglycemia (2.8 +/- 0.1 mM; n = 12), hyperinsulinemic euglycemia (6.2 +/- 0.1 mM; n = 12), hyperinsulinemic euglycemia plus simultaneous lateral cerebroventricular infusion of saline (24 microl/h; n = 8), or hyperinsulinemic euglycemia plus either lateral cerebral ventricular infusion (n = 8; LV-DEX group), fourth cerebral ventricular (n = 10; 4V-DEX group), or peripheral (n = 10; P-DEX group) infusion of dexamethasone (5 microg/h), a specific type II glucocorticoid receptor analog. For all groups, day 2 consisted of a 2-h hyperinsulinemic (30 pmol x kg(-1) x min(-1)) or hypoglycemic (2.9 +/- 0.2 mM) clamp. The hypoglycemic group had blunted epinephrine, glucagon, and endogenous glucose production in response to subsequent hypoglycemia. Consequently, the glucose infusion rate to maintain the glucose levels was significantly greater in this group vs. all other groups. The LV-DEX group did not have blunted counterregulatory responses to subsequent hypoglycemia, but the P-DEX and 4V-DEX groups had significantly lower epinephrine and norepinephrine responses to hypoglycemia compared with all other groups. In summary, peripheral and fourth cerebral ventricular but not lateral cerebral ventricular infusion of dexamethasone led to significant blunting of autonomic counterregulatory responses to subsequent hypoglycemia. These data suggest that prior activation of type II glucocorticoid receptors within the hindbrain plays a major role in blunting autonomic nervous system counterregulatory responses to subsequent hypoglycemia in the conscious rat.     

Chlorpromazine exacerbates hepatic insulin sensitivity via attenuating insulin and leptin signaling pathway, while exercise partially reverses the adverse effects

Investigated in this study are the effects and mechanisms of exercise and chlorpromazine (CPZ), a widely used conventional antipsychotic drug, on the hepatic insulin sensitivity of 90% pancreatectomized (Px) male Sprague–Dawley rats. The Px diabetic rats were provided with 0, 5, or 50 mg CPZ per kg of body weight (No-CPZ, LCPZ, or HCPZ) for 8 weeks, and half of each group had regular exercise. LCPZ did not exacerbate hepatic insulin sensitivity through insulin and leptin signaling in diabetic rats. However, HCPZ decreased whole-body glucose infusion rates in hyperinsulinemic clamped states, but not whole-body glucose uptake. This was due to the elevated hepatic glucose output in hyperinsulinemic states. The decreased hepatic insulin sensitivity was associated with insulin receptor substrate-2 (IRS2) protein levels in the liver. Decreased IRS2 levels attenuated hepatic insulin and leptin signaling pathways in hyperinsulinemic states, which elevated glucose production by inducing phosphoenolpyruvate carboxykinase expression. Long-term exercise recovered hepatic insulin sensitivity attenuated by HCPZ to reduce the hepatic glucose output in hyperinsulinemic clamped states. This recovery was related to enhanced insulin and leptin signaling via increased IRS2 gene and protein levels by activating the cAMP responding element-binding protein, but exercise improved only insulin signaling. In conclusion, HCPZ exacerbates hepatic insulin action by attenuating insulin and leptin signaling in type 2 diabetic rats, while regular exercise partially reverses the attenuation of hepatic insulin sensitivity by improving insulin signaling. Enhancement of insulin and leptin signaling through an induction of IRS2 may play an important role in improving hepatic glucose homeostasis.     

Heparan sulfate chains of syndecan-1 regulate ectodomain shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.     

Suitability of cryopreserved samples for pharmacological studies of ciliary activity

The effect of terbutaline sulfate on the ciliary activity of fresh and cryopreserved human nasal epithelium was evaluated. Cryopreservation had no effect on baseline ciliary beat frequency. Both fresh and cryopreserved samples exposed to 10(-4) M terbutaline showed a statistically significant increase in ciliary beat frequency (27 and 25%, respectively). When the percentage changes after drug challenge for fresh and cryopreserved samples were compared no statistical difference emerged. It is concluded that cryopreservation in liquid nitrogen at -196 degrees C does not affect membrane receptors, at least beta-adrenergic receptors, and therefore cryopreserved samples are suitable for pharmacological studies of ciliary activity.