Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog.

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.

Identification and characterization of a benzothiophene inhibitor of herpes simplex virus type 1 replication which acts at the immediate early stage of infection.

Analysis of a large compound library in a high throughput virus infection assay screen identified the benzothiophene PD146626 as a potent and specific inhibitor of herpes simplex virus type 1 (HSV-1) replication. PD146626 possessed an EC(50) and EC(90) against HSV-1 of 0.1 and 1 microM, respectively, and mediated no detectable cytotoxicity in cells at concentrations up to 1 microM. Western blot analyses and time of addition experiments demonstrated that in the presence of PD146626 HSV-1 underwent a specific block in viral gene expression at the immediate early stage. However, several observations indicated that a cellular function rather than a viral immediate early transactivator protein represented the molecular target for PD146626, including the lack of resistance of VP16 and ICP0 mutant viruses to the compound, the inability to select resistant strains of HSV-1 following exhaustive serial passaging of virus in the presence of the compound, and the sensitivity of human cytomegalovirus, which lacks VP16 and ICP0 homologs, to the compound. Moreover, kinetic studies suggested an unusual pattern of responsiveness of the host cell to PD146626, in that the compound could induce an extended antiviral state in cells after only a brief exposure. Together these results suggest that PD146626 targets a novel cellular function that is critical for the expression of HSV-1 immediate early genes but not host cell genes.

Cross-reactivity of the anti-PML antibody PG-M3 with the herpes simplex virus type 1 immediate early protein ICP4.

The PML protein is one of the components of ND10, nuclear matrix-associated structures which undergo rapid disintegration at the onset of herpes simplex virus type 1 (HSV-1) infection. This disruption event has been frequently visualized in immunofluorescence assays using the anti-PML mouse monoclonal antibody PG-M3. This antibody was surprisingly found to also stain nuclear virus replication compartments when employed at higher concentrations. This was shown to be due to an unexpected cross-reactivity of the PG-M3 antibody with the HSV-1 immediate early protein ICP4, a known component of replication compartments. The sequences of ICP4 recognized by PG-M3 were found to map to the extreme amino-terminal end of the protein, which includes a 21 amino acid segment that is partially homologous to the peptide of PML that was used to make PG-M3. These results suggest that PG-M3 may no longer represent an appropriate antibody for use in visualizing the fate of PML and ND10 during HSV-1 infection.

Decreased insulin sensitivity and compensatory hyperinsulinemia after hormone treatment in children with short stature.

To assess the effects of GH treatment on carbohydrate and protein metabolism, we studied eight patients with short stature before and after the commencement of GH treatment. The hyperglycemic clamp procedure was employed to produce a hyperglycemic stimulus of 50 mg/dL above fasting levels for 120 min. These patients were then treated with 0.3 mg/kg. week GH for 6 months, after which they were restudied. Patients were compared to eight healthy control children matched for age, sex, and Tanner stage. Fasting plasma glucose did not change significantly, but fasting plasma insulin levels were higher after GH therapy (P < 0.005). Despite identical glucose increments during the glucose clamp procedure, both first, and second phase insulin responses were markedly greater after instituting GH treatment. Even in the face of higher mean plasma insulin levels after GH treatment, the rate of insulin-stimulated glucose metabolism did not differ during the last 60 min of both studies. Hence, the rate of insulin-stimulated glucose metabolism/mean plasma insulin ratio (an index of insulin sensitivity) was sharply reduced after GH treatment (P < 0.01). During the clamp, the fall in circulating branched chain amino acid levels was significantly greater after GH therapy (P < 0.02). We conclude that glucose-stimulated insulin responses are increased in short children treated with GH and that such hyperinsulinemic responses compensate for reductions in insulin sensitivity. The compensatory hyperinsulinemic responses induced by GH therapy may serve a beneficial role by augmenting insulin's anabolic effects on protein metabolism.

Case report. An unusual case of precocious puberty.

An almost 3-year-old boy was evaluated for pubic hair and unilateral testicular enlargement 1 week after resection of an astrocytoma of the posterior fossa. Rather than the expected diagnosis of central precocity due to increased intracranial pressure, a diagnosis of congenital adrenal hyperplasia (21-hydroxylase deficiency) with testicular adrenal rest cells was made. The differential diagnosis, laboratory evaluation, and currently accepted medical management of congenital adrenal hyperplasia are described.

Resistance to neuroglycopenia: an adaptive response during intensive insulin treatment of diabetes.

Counterregulation and awareness of hypoglycemia begins at lower plasma glucose levels in insulin-dependent diabetes mellitus (IDDM) subjects given intensive insulin treatment. To determine whether these changes are associated with an alteration in the susceptibility of the brain to mild hypoglycemia, we compared central nervous system responses to hypoglycemia in 8 intensively treated (hemoglobin A1, 8.3 +/- 0.2%; normal, <8%) and 11 conventionally treated IDDM patients (hemoglobin A1, 14.6 +/- 1.3%) with those in 10 healthy subjects. Plasma glucose was lowered from approximately 4.6 mmol/L in 0.5-0.6 steps using the clamp technique. Glucose levels triggering hormonal responses and perception of hypoglycemic symptoms were significantly lower in intensively treated patients compared to their poorly controlled counterparts (P < 0.05), and hormonal responses were suppressed compared to those in healthy controls. Similarly directed changes occurred in the level of circulating glucose required to alter cortical evoked potentials during hypoglycemia. A greater reduction in plasma glucose was required to alter P300 event-related potentials in the intensively treated patients (2.2 mmol/L) compared to those in the conventionally treated and nondiabetic groups (approximately 3.5 and approximately 3.0 mmol/L, respectively). We conclude that intensively treated IDDM patients are resistant to changes in cortical evoked potentials induced by mild hypoglycemia. This may explain why intensively treated IDDM counterregulate and experience hypoglycemic symptoms at a lower glucose level than conventionally treated patients.

1H NMR studies of glucose transport in the human brain.

The difference between 1H nuclear magnetic resonance (NMR) spectra obtained from the human brain during euglycemia and during hyperglycemia is depicted as well-resolved glucose peaks. The time course of these brain glucose changes during a rapid increase in plasma glucose was measured in four healthy subjects, aged 18-22 years, in five studies. Results demonstrated a significant lag in the rise of glucose with respect to plasma glucose. The fit of the integrated symmetric Michaelis-Menten model to the time course of relative glucose signals yielded an estimated plasma glucose concentration for half maximal transport, Kt, of 4.8 +/- 2.4 mM (mean +/- SD), a maximal transport rate, Tmax, of 0.80 +/- 0.45 micromol g-1 min-1, and a cerebral metabolic glucose consumption rate (CMR)glc of 0.32 +/- 0.16 micromol g-1 min-1. Assuming cerebral glucose concentration to be 1.0 micromol/g at euglycemia as measured by 13CMR, the fit of the same model to the time course of brain glucose concentrations resulted in Kt = 3.9 +/- 0.82 mM, Tmax = 1.16 +/- 0.29 micromol g-1 min-1, and CMRglc = 0.35 +/- 0.10 micromol g-1 min-1. In both cases, the resulting time course equaled that predicted from the determination of the steady-state glucose concentration by 13C NMR spectroscopy within the experimental scatter. The agreement between the two methods of determining transport kinetics suggests that glucose is distributed throughout the entire aqueous phase of the human brain, implying substantial intracellular concentration.

Enhanced adrenomedullary response and increased susceptibility to neuroglycopenia: mechanisms underlying the adverse effects of sugar ingestion in healthy children.

OBJECTIVE: Eating simple sugars has been suggested as having adverse behavioral and cognitive effects in children, but a physiologic mechanism has not been established. This study was performed to address this issue. DESIGN: Metabolic, hormonal, and symptomatic responses to a standard oral glucose load (1.75 gm/kg; maximum, 120 gm) were compared in 25 healthy children and 23 young adults, and the hypoglycemic clamp, together with measurements of P300 auditory evoked potentials, was used to assess whether children are more vulnerable than adults to neuroglycopenia. SETTING: Children's Clinical Research Center, Yale University School of Medicine. RESULTS: Baseline and oral glucose-stimulated plasma glucose and insulin levels were similar in both groups, including the nadir glucose level 3 to 5 hours after oral administration of glucose (3.4 +/- 0.1 mmol/L (61 +/- 1.8 mg/dl) in children and 3.5 +/- 0.1 mmol/L (63 +/- 1.8 mg/dl) in adults). The late glucose decrease stimulated a rise in plasma epinephrine levels that was twofold higher in children than in adults (2260 +/- 289 vs 1031 +/- 147 pmol/L (407 +/- 52 vs 186 +/- 26 pg/ml), p < 0.01) and a significant increase in hypoglycemic symptom scores in children (p < 0.01), but not in adults. During control experiments, in which six of the healthy children ingested a sugar-free drink, there were no significant changes in plasma glucose levels, hormone concentrations, or hypoglycemic symptom scores. During the hypoglycemic clamp, P300 potentials did not change in any of eight adult subjects until the plasma glucose concentration was lowered to 3.0 mmol/L (54 mg/dl), whereas similar changes in P300 potentials were observed in six of seven children at glucose levels 3.6 to 4.2 mmol/L (65 to 75 mg/dl). CONCLUSION: Enhanced adrenomedullary responses to modest reductions in plasma glucose concentration and increased susceptibility to neuroglycopenia may be important contributing factors to adverse behavioral and cognitive effects after sugar ingestion in healthy children.

Localized 13C NMR spectroscopy in the human brain of amino acid labeling from D-[1-13C]glucose.

Cerebral metabolism of D[1-13C]glucose was studied with localized 13C NMR spectroscopy during intravenous infusion of enriched [1-13C]glucose in four healthy subjects. The use of three-dimensional localization resulted in the complete elimination of triacylglycerol resonance that originated in scalp and subcutaneous fat. The sensitivity and resolution were sufficient to allow 4 min of time-resolved observation of label incorporation into the C3 and C4 resonances of glutamate and C4 of glutamine, as well as C3 of aspartate with lower time resolution. [4-13C]Glutamate labeled rapidly reaching close to maximum labeling at 60 min. The label flow into [3-13C]glutamate clearly lagged behind that of [4-13C]-glutamate and peaked at t = 110-140 min. Multiplets due to homonuclear 13C-13C coupling between the C3 and C4 peaks of the glutamate molecule were observed in vivo. Isotopomer analysis of spectra acquired between 120 and 180 min yielded a 13C isotopic fraction at C4 glutamate of 27 +/- 2% (n = 4), which was slightly less than one-half the enrichment of the C1 position of plasma glucose (63 +/- 1%), p < 0.05. By comparison with an external standard the total amount of [4-13C]glutamate was directly quantified to be 2.4 +/- 0.1 mumol/ml-brain. Together with the isotopomer data this gave a calculated brain glutamate concentration of 9.1 +/- 0.7 mumol/ml, which agrees with previous estimates of total brain glutamate concentrations. The agreement suggests that essentially all of the brain glutamate is derived from glucose in health human brain.

Effects of puberty and diabetes on metabolism of insulin-sensitive fuels.

Insulin's ability to stimulate glucose metabolism is reduced during normal puberty; these changes are exaggerated in adolescents with insulin-dependent diabetes mellitus (IDDM). Because the effects of puberty and IDDM on the other actions of insulin have not been established, we studied leucine kinetics (using [1-13C]leucine) and fat metabolism during euglycemic hyperinsulinemia (20 mU.m2.min-1) for 3 h in eight healthy and nine IDDM (HbA1 14 +/- 2%) adolescents and six healthy young adult controls. IDDM subjects received overnight low-dose insulin infusion to normalize fasting glucose. Basal and steady-state insulin values (approximately 240 pM) during the study were similar in all three groups. Insulin-stimulated glucose metabolism was reduced by 40% in healthy adolescents vs. adults (P < 0.05) and by an additional 40% in poorly controlled IDDM (P < 0.05 vs, normal adolescents). Although basal glucose and lipid oxidation rates (measured by indirect calorimetry) were similar in all three groups, when insulin was infused, glucose oxidation increased and lipid oxidation decreased only in the two nondiabetic groups. Similarly, insulin significantly reduced plasma free fatty acid levels only in the nondiabetics. Basal leucine flux (an index of protein degradation) was similar in healthy controls but was markedly increased in IDDM adolescents. Despite similar increments in plasma insulin during the clamp, leucine flux remained higher in IDDM adolescents than in healthy controls. Basal leucine oxidation rates were also increased in IDDM subjects compared with nondiabetic groups and declined to a lesser extent during insulin infusion. We conclude that insulin resistance of puberty is selective for glucose metabolism, sparing amino acid/protein metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the metabolic effects of recombinant human insulin-like growth factor-I and insulin. Dose-response relationships in healthy young and middle-aged adults.

The actions of recombinant human insulin-like growth factor-I (rhIGF-I) and insulin were compared in 21 healthy young (24 +/- 1 yr) and 14 healthy middle-aged (48 +/- 2 yr) subjects during 3-h paired euglycemic clamp studies using one of three doses (rhIGF-I 0.2, 0.4, and 0.8 micrograms/kg.min and insulin 0.2, 0.4, and 0.8 mU/kg.min, doses chosen to produce equivalent increases in glucose uptake). In younger subjects, rhIGF-I infusions suppressed insulin by 19-33%, C-peptide by 47-59% and glucagon by 33-47% (all, P < 0.02). The suppression of C-peptide was less pronounced with insulin than with rhIGF-I (P < 0.007). The metabolic responses to rhIGF-I and insulin were remarkably similar: not only did both hormones increase glucose uptake and oxidation in a nearly identical fashion, but they also produced similar suppression of glucose production, free fatty acid levels, and fat oxidation rates. In contrast, rhIGF-I had a more pronounced amino acid-lowering effect than did insulin (P < 0.004). In middle-aged subjects, basal IGF-I levels were 44% lower (P < 0.0001) whereas basal insulin and C-peptide were 20-25% higher than in younger subjects. Age did not alter the response to rhIGF-I. However, insulin-induced stimulation of glucose uptake was blunted in older subjects (P = 0.05). Our data suggest that absolute IGF-I and relative insulin deficiency contribute to adverse metabolic changes seen in middle age.

Metabolic effects of insulin-like growth factor I in normal humans.

Since the development of recombinant DNA technology, there has been a rapid expansion of interest in the use of human insulin-like growth factor I (IGF-I) synthesized by recombinant DNA technology for the treatment of clinical disorders. This article reviews recent studies of the metabolic effects of recombinant human IGF-I in normal humans. These studies demonstrated that under euglycemic conditions, IGF-I had potent effects on glucose (hepatic and peripheral), lipid and amino acid metabolism that closely resemble those of insulin, despite a concomitant inhibitory effect on insulin secretion. Hypoglycemia produced by IGF-I infusions (free-fall study and glucose clamps) had a different effect on counterregulatory responses compared with insulin. The glucagon response was absent, growth hormone (GH) release was attenuated, while norepinephrine levels were increased. Suppression of glucagon release during hypoglycemia impaired glucose recovery. Paradoxically, awareness of hypoglycemia was enhanced with IGF-I, partly due to stimulation of sympathetic activity. Studies performed under hyperglycemic conditions showed that IGF-I inhibited glucose-stimulated insulin secretion, but that this inhibitory effect was partially overcome by increasing the hyperglycemic stimulus. Moreover, despite the decrease in insulin secretion, glucose disposal was accelerated by IGF-I. These observations imply that IGF-I might be effective in human diabetes. In particular, normalization of the decreased basal IGF-I levels, which are characteristic of poorly controlled patients with insulin-dependent diabetes mellitus (IDDM), in pubertal patients might lower glucagon and GH levels and improve cellular metabolism in muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Correction of hyperinsulinemia by glyburide treatment in nondiabetic patients with thalassemia major.

Hyperinsulinemia and insulin resistance precede the development of diabetes in patients with thalassemia major on hypertransfusion/desferoxamine therapy. To examine whether these early metabolic defects could be reversed, seven nondiabetic patients with thalassemia (17 +/- 4 y) were studied for 12 mo before and during 12 mo of low-dose treatment with glyburide (1.25 to 3.75 mg/d), a second-generation oral hypoglycemic agent. Plasma glucose responses to oral glucose (1.75 g/kg body weight) were normal before and after glyburide. Plasma insulin responses were markedly increased before glyburide therapy (area under insulin response curve 86 +/- 15 and 96 +/- 15 versus 40 +/- 5 nmol/min/L in normal controls, p < 0.001). However, insulin responses to glucose fell significantly after 3 mo of glyburide (to 52 +/- 7 nmol/min/L, p < 0.05 versus pretreatment) and were normalized after 12 mo (42 +/- 7 nmol/min/L, p = NS versus controls). The rate of insulin-stimulated glucose metabolism during euglycemic insulin clamps (40 mU/m2/min) was low in the patients before treatment (163 +/- 10 versus 215 +/- 17 mg/m2/min in controls, p < 0.05) and increased to 205 +/- 30 mg/m2/min after 3 mo of glyburide. The treatment was well tolerated. In conclusion, in nondiabetic, hyperinsulinemic, thalassemic patients, chronic glyburide therapy normalizes insulin responses to oral glucose. To the extent that insulin hypersecretion contributes to the development of diabetes in thalassemia, glyburide therapy may provide a means of postponing this complication of the disease.

Direct measurement of brain glucose concentrations in humans by 13C NMR spectroscopy.

Glucose is the main fuel for energy metabolism in the normal human brain. It is generally assumed that glucose transport into the brain is not rate-limiting for metabolism. Since brain glucose concentrations cannot be determined directly by radiotracer techniques, we used 13C NMR spectroscopy after infusing enriched D-[1-13C]glucose to measure brain glucose concentrations at euglycemia and at hyperglycemia (range, 4.5-12.1 mM) in six healthy children (13-16 years old). Brain glucose concentrations averaged 1.0 +/- 0.1 mumol/ml at euglycemia (4.7 +/- 0.3 mM plasma) and 1.8-2.7 mumol/ml at hyperglycemia (7.3-12.1 mM plasma). Michaelis-Menten parameters of transport were calculated to be Kt = 6.2 +/- 1.7 mM and Tmax = 1.2 +/- 0.1 mumol/g.min from the relationship between plasma and brain glucose concentrations. The brain glucose concentrations and transport constants are consistent with transport not being rate-limiting for resting brain metabolism at plasma levels greater than 3 mM.

Effect of growth hormone treatment on hyperinsulinemia associated with Turner syndrome.

To determine whether the insulin resistance in patients with Turner syndrome, which may be exaggerated by treatment with human growth hormone, leads to excessive insulin secretion, we applied the hyperglycemic glucose-clamp technique to produce a standard hyperglycemic stimulus (6.9 mmol/L, or 125 mg/dl, greater than fasting plasma glucose level for 120 minutes) in seven patients with Turner syndrome and in seven healthy children. These studies were repeated in the patients after 6 to 12 months of therapy with growth hormone. Fasting plasma levels of insulin were comparable in control subjects and patients before therapy but increased significantly in the patients after 6 to 12 months of treatment with growth hormone. Despite identical glucose increments in the two groups during the glucose-clamp procedure, both first- and second-phase insulin responses were significantly greater in the patients than in the control subjects. Moreover, the hyperinsulinemic responses to glucose were markedly exaggerated in the patients after their treatment with growth hormone, reaching values (first phase 474 +/- 100 pmol and second phase 826 +/- 100 pmol; p less than 0.02 vs pretreatment values) that were almost threefold greater than those in control subjects (p less than 0.001). Nevertheless, the rate of insulin-stimulated glucose metabolism during the last 60 minutes of the clamp procedure was similar in all three groups of studies. Glycosylated hemoglobin, total cholesterol level, and blood pressure remained normal in patients after therapy with growth hormone. We conclude that glucose-stimulated insulin response is increased in patients with Turner syndrome and that these alterations are further exaggerated by treatment with growth hormone. These hyperinsulinemic responses appear to compensate for reductions in insulin sensitivity.

Diverse effects of insulin-like growth factor I on glucose, lipid, and amino acid metabolism.

The metabolic effects of recombinant human insulin-like growth factor I (rhIGF-I) on glucose, amino acid, and free fatty acid (FFA) metabolism were examined in nine healthy nonobese subjects. Each received a 3-h primed continuous infusion of rhIGF-I (20 micrograms/kg bolus, 0.4 microgram.kg-1.min-1) while maintaining euglycemia using an exogenous glucose infusion. Total IGF-I levels increased from 125 +/- 11 to 444 +/- 22 ng/ml, and free IGF-I levels rose from undetectable to 73 +/- 5 ng/ml. Insulin levels fell from 95 +/- 9 to 64 +/- 8 pM, and C-peptide fell from 453 +/- 48 to 206 +/- 29 pM; circulating glucagon levels also declined from 72 +/- 9 to 42 +/- 4 pg/ml, rhIGF-I produced a two- to threefold increase in glucose uptake as measured by [3H] glucose (from 10.3 +/- 0.6 to 27.4 +/- 3 mumol.kg-1.m-1), and, despite the fall in insulin secretion, there was a marked 60-70% inhibition of hepatic glucose production. Furthermore, FFA and branched-chain amino acids declined by 40-60% (411 +/- 58 to 165 +/- 36 and 406 +/- 23 to 219 +/- 14 microM, respectively). Our data demonstrate that rhIGF-I has potent effects on glucose (hepatic and peripheral), lipid, and amino acid metabolism in normal humans. The scope of the actions of rhIGF-I closely resemble those of insulin, despite a concomitant inhibitory effect on insulin secretion.

Does insulin species modify counterregulatory hormone response to hypoglycemia?

OBJECTIVE: To examine whether pork and human insulin induce different counterregulatory responses to hypoglycemia. RESEARCH DESIGN AND METHODS: The responses to a mild hypoglycemic stimulus were determined in 35 healthy young adults with the glucose-clamp technique to ensure standardization of glucose and insulin levels. Either pork (n = 15) or human (n = 20) regular insulin was infused (0.8 mU.kg-1.min-1) to lower plasma glucose from 4.7 +/- 0.07 to 3.3 +/- 0.04 mM (both groups) over approximately 40 min. Plasma glucose was maintained at that level (with variable rate glucose infusion) for an additional 60 min. RESULTS: Steady-state insulin levels were similar in both groups (316 +/- 50 vs. 280 +/- 29 pM, pork vs. human). Before insulin administration, basal counterregulatory hormone levels were indistinguishable. Most importantly, after plasma glucose was lowered, hormonal responses were nearly identical. No significant differences in peak values of epinephrine (1769 +/- 404 vs. 1775 +/- 311 pM, pork vs. human), norepinephrine (1.64 +/- 0.23 vs. 1.87 +/- 0.20 nM, pork vs. human), glucagon (163 +/- 29 vs. 175 +/- 20 ng/L, pork vs. human), growth hormone (14 +/- 3 vs. 17 +/- 3 micrograms/L, pork vs. human), or cortisol (543 +/- 83 vs. 458 +/- 28 nM, pork vs. human) occurred. CONCLUSIONS: Our data suggest that pork and human insulin produce a comparable and robust hormonal response in healthy adults under conditions of controlled hypoglycemia.