Lavenia LaGrone

Stat proteins play a role in tumor necrosis factor alpha gene expression

Trauma produces dysfunction in immunity, which appears to be partially related to alterations in the cytokine response. Signal transducer and activator of transcription proteins (STATs) mediate activation of several cytokine genes. However, the effect of STAT proteins on tumor necrosis factor-alpha (TNFalpha) activation is not fully defined. We identified binding sites for STAT 3 and STAT 5/6 within the promoter region of TNFalpha and hypothesize that alterations in these sites would affect TNFalpha expression. The TNFalpha promoter was inserted into the luciferase reporter vector, and binding sites for STAT 3, STAT 5/6, and activator protein-1 (AP-1) were mutated using site-directed mutagenesis. Murine macrophages were transfected with the resultant plasmids, then incubated with and without lipopolysaccharide (LPS) or IFNalpha. Gene expression was measured by dual luciferase assay. Mutation of the STAT 3 binding site was associated with decreased LPS-inducible activity. Mutation of the AP-1 and STAT 5/6 consensus binding sites alone had no effect on TNFalpha expression. However, combined mutation of both STAT 5/6 and AP-1 was associated with increased LPS-inducible activity. Mutations of the STAT binding sites in the promoter region of TNFalpha affect TNFalpha gene expression. These results suggest a regulatory role for STATs in TNF gene transcription.

Urinary Excretion of N-Acetyl-Beta-D-Glucosaminidase in Children with Type I Diabetes Mellitus

N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, has been shown to be increased in the urine of patients with various glomerulonephritides, tubulointerestitial diseases, renal allograft rejection, toxic renal injury, and diabetes mellitus. Although it has been suggested that urinary NAG may reflect blood glucose control, no studies have correlated this with other measures of metabolic control. Thirty-four children from a diabetic summer camp were found to have urinary NAG to creatinine ratios significantly above those of normal controls of similar age (5.22 ± 1.19 versus 1.51 ± 0.17 U). Urinary NAG was found to positively correlate with an arbitrary control index (r =0.82; P < 0.05) and in seven patients with hemoglobin A1c (r =0.70; P < 0.001). In a closely followed group of 40 clinic patients, urinary NAG to creatinine ratio was again found to be significantly increased over normal controls (7.55 ± 0.70 versus 1.51 ± 0.17 U; P < 0.05). Again, urinary NAG was positively correlated with HbA1c (r =0.62; P < 0.001) and urinary albumin to creatinine ratio (r =0.47; P < 0.01). In neither group was there a correlation with UNAG:UCr and duration of disease. Thus, these data suggest that urinary NAG to creatinine ratio appears to be a reflection of blood sugar control.

Somatostatin limits rise in glomerular filtration rate after a protein meal

To evaluate the glomerular filtration rate (GFR) response to a protein meal in patients with diabetes and to study the role of glucagon and growth hormone, we studied inulin clearance for three 30-minute periods before and 3 hours after an 80 g protein meal in seven healthy volunteers and 10 patients with diabetes. Patients with diabetes were chosen because their renal response to such a meal has been reported to be abnormal. All had an increase in GFR and plasma glucagon levels after the protein meal. The peak rise in GFR occurred from 1 to 2 1/2 hours after the meal (mean +/- SEM, delta 26 +/- 5 mL/min/m2, controls; delta 22 +/- 7 mL/min/m2, patients with diabetes), with the mean time to normal rise in GFR occurring at 2 hours after the meal. Similarly, plasma glucagon values peaked at different times in individual patients (delta 769 +/- 532 pg/mL, controls; delta 267 +/- 69 pg/mL, patients with diabetes), with the mean plasma glucagon rise occurring 1 hours after the meal. Premeal growth hormone levels tended to be higher in the patients with diabetes (7.6 +/- 1.4 vs 2.1 +/- 0.4 ng/mL), and did not change after the meal. To allow study of the contribution of the increased plasma glucagon to the rise in GFR, eight of these patients (five with diabetes) volunteered to undergo a second GFR response test with a simultaneous infusion of somatostatin. The glucagon response was significantly lowered at all time periods during the infusion (P less than 0.05); no significant change in growth hormone occurred. Without somatostatin in these eight patients, peak increase in postmeal GFR average 20.6 +/- 1.5 mL/min/m2; with the somatostatin, peak increase in GFR was 6.0 +/- 1.8 mL/min/m2 (P less than 0.01). Neither metabolic control nor degree of albuminuria was significantly different at the time of the two studies. Thus, as has been shown in animals, somatostatin infusion limits the rise in GFR after a protein meal in humans.

Immunoreactive urinary prostaglandins A and E in neonates, children, and adults

Urinary prostaglandins were measured by radioimmunoassay in neonates (31-35 weeks gestational age), children (4-15 years) and adults (24-36 years). Neonates showed significantly lower levels of both iPGE and iPGA compared to children (p less than 0.01) and adults (p less than 0.01). Children also had significantly lower levels than adults (p less than 0.01). Since urinary prostaglandins reflect intrarenal levels of prostaglandins, the results support other studies that suggest prostaglandins may play a role in controlling renal blood flow in infants.

Response of urinary N-acetyl-β-D-glucosaminidase to rapid decreases in blood glucose

Urinary N-acetyl-beta-D-glucosaminidase (NAG) has been shown to be a sensitive indicator of blood sugar control. Twelve insulin-dependent diabetics whose blood glucoses were being controlled with the artificial pancreas had concurrent urinary NAG activity measured. Blood glucose dropped markedly from 198 +/- 22 mg/dl (x +/- SEM) to 121 +/- 7 mg/dl during the 25 h on the artificial pancreas. For the entire group UNAG: UCr dropped from 14.9 +/- 4.4 to 7.25 +/- 1.68 units. In order to determine if larger decreases in blood glucoses over the course of the study resulted in larger decreases in UNAG: UCr, an arbitrary division at 180 mg/dl was made. Six patients with blood glucoses at or above this level at the start of the study showed a drop in both blood glucoses and UNAG: UCr (261 +/- 24 to 134 +/- 12 mg/dl and 21.1 +/- 6.1 to 7.29 +/- 1.53 U, respectively). Even though the other six patients had blood glucoses below the renal threshold, both blood glucoses and UNAG: UCr declined (136 +/- 4 to 110 +/- 4 mg/dl; 8.89 +/- 2.4 to 6.2 +/- 1.64 U, respectively). Thus not only may urinary NAG activity reflect long-term control and renal complications of diabetes, but this enzyme is also responsive to acute changes in blood glucose.

Urinary N-acetyl-β-D-glucosaminidase in streptozotocin-induced diabetic rats

Excretion of urinary N-acetyl-β-d-glucosaminidase has been found to be elevated in diabetic humans and rats. This urinary glycosidase may reflect blood sugar control over time, since it has been significantly and positively correlated with hemoglobin A1 in children with insulin-dependent diabetes. Other studies have suggested that urinary NAG may predict diabetic nephropathy. In order to more carefully define the relationship between urinary NAG excretion and blood and urine sugars, hemoglobin A1, and microalbuminuria, 48 rats were made diabetic by the use of streptozotocin. All rats were uninephrectomized at 3 weeks. Of these, 23 were treated with daily insulin injections, 25 were untreated, and both groups were compared to 13 control, nondiabetic rats. Urine volume, glucose, albumin, and blood sugar were all significantly (P < 0.05) elevated in the untreated rats compared to the treated and control groups. Urinary NAG:UCr was significantly (P < 0.01) elevated in the untreated group with lower but still elevated levels (P < 0.05) in the treated rats. To further define the time course of the increase in UNAG:UCr 12 rats were followed serially at 12-hr intervals for 92 hr after streptozotocin. Urinary NAG increased significantly (P < 0.05) at 12 hr after streptozotocin injection and reached a plateau at 36 hr while hemoglobin A1 did not rise until 2 weeks after onset of hyperglycemia. Urinary NAG increases more rapidly than hemoglobin A1 after onset of hyperglycemia and glycosuria. Although it does not reflect blood sugar control over as long a time period, it does, however, reflect varying degrees of hyperglycemia and glycosuria.

Resistin and postburn insulin dysfunction.

BACKGROUND Postburn insulin dysfunction is a significant contributor to morbidity and mortality. A satisfactory mechanism for explaining this phenomenon remains elusive; however, resistin has been postulated to be involved. Initially discovered as an insulin antagonist secreted from adipose tissue in murine models, resistins function in humans has been more obscure. Resistin is not expressed significantly in human adipocytes although it has been detected in monocytes. We postulate that mononuclear activation at the site of burn injury affects the release of resistin and contributes to insulin dysfunction. METHODS Plasma from burned and healthy control individuals was characterized for glucose, insulin, and resistin protein levels. Adipose tissue from both groups was analyzed for resistin transcript; levels were found to be somewhat higher in the burned group though not significantly so. Circulating monocyte expression of resistin transcript was assayed in similar fashion. RESULTS In addition to finding that insulin and glucose were elevated postburn, a finding in agreement with past studies, we demonstrate that circulating resistin levels are significantly elevated as well. Insulin resistance was found to increase at a similar rate to resistin expression in the burn population, suggesting a correlation in these events. Adipose tissue from both groups was analyzed for resistin transcript; levels were found to be somewhat higher in the burned group though not significantly so. Circulating monocyte expression of resistin transcript was assayed and found to be profoundly elevated in the burn population. CONCLUSIONS This data suggests that resistin is produced by activated monocytes in the adipose tissue around the periphery of burn wound. We suggest that postburn insulin function is adversely affected by resistin produced as a result of this monocyte activation.

Effect of Indomethacin on the Glomerular Filtration Rate After a Protein Meal in Humans

The renal response to a protein meal has been characterized by increases in glomerular filtration rate (GFR) and renal plasma flow and decrease in renal vascular resistance. Several hormonal mediators of this response have been proposed, including renal prostaglandins (PGs). We studied ten normal subjects before and after ingestion of indomethacin. All subjects had three 30-minute baseline creatinine and iothalamate clearances measured before and three one-hour clearances measured after an 80-g protein meal. The night before the second test, the subjects took 25 mg indomethacin and 150 mg one hour before the test meal. Urine PG excretion decreased significantly during the second test, from 0.60 +/- 0.23 to 0.30 +/- 0.14 ng/min (P less than 0.01). Initial iothalamate clearance increased from 110 +/- 10 to a mean of 122 +/- 15 mL/min/1.73 m2 (average increase of 12 mL/min/1.73 m2) during the second hour after the test meal. After ingestion of indomethacin, the GFR remained unchanged from a baseline of 101 +/- 9 to 101 +/- 7 mL/min/1.73 m2 (average change of -1 mL/min/1.73 m2). Because the time of peak increase after the meal varied from subject to subject, the maximal increase in GFR after the meal was calculated and found to be significantly less during the second test, 13 +/- 3 v 22 +/- 4 mL/min/1.73 m2 (P less than 0.05). Because PGs can stimulate glucagon secretion and because glucagon has been suggested to mediate the protein-stimulate GFR response, we measured plasma glucagon during both tests; these levels were not different.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid antibody production and sample preparation for radioimmunoassay of prostaglandin A.

A dialysis equilibrium method for plasma or tissue preparation for radioimmunoassay of prostaglandins is presented and compared to the silica gel column technique. The plasma or tissue is extracted, dried, redissolved in buffer and dialyzed for further purification before assay. Results comparing both methods shows that dialysis equilibrium greatly simplifies the silica gel column technique yet preserves specificity, sensitivity and reproducibility. In addition a rapid micromethod for the development of a specific antibody to prostaglandin A is detailed.

Alterations in resistin expression after thermal injury.

BACKGROUND Burn injury, it was hypothesized, may induce changes in resistin expression that contribute to postburn metabolic derangements. This study examined resistin gene expression, serum levels of resistin protein, and glucose levels in burned mice. METHODS Ten male Balb-c mice were anesthetized and then given a 30% total burn surface area using heated probes. Burned and sham-burned mice were killed at 2, 4, 24, and 48 hours. The total ribonucleic acid from gonadal fat tissues was isolated for the measurement of resistin gene expression using real-time reverse transcriptase-polymerase chain reaction. Serum levels of resistin, insulin, and glucose were measured. Statistical analysis was performed by two-way analysis of variance using Bonferronis test to find differences between groups. All p values less than 0.05 were considered significant. RESULTS Increases in resistin gene expression and serum resistin levels were detected in the burned animals, and these correlated with relative insulin resistance. CONCLUSION The findings suggest a potential role for resistin in the pathophysiology of the metabolic response to injury.