Anatomy, ultrastructure and secretion of Hibiscus pernambucensis Arruda (Malvaceae) extrafloral nectary

This paper reports on the extrafloral nectary (EFN) of Hibiscus pernambucensis, a native shrub species occurring in mangrove and restinga along Brazil's coastline. EFNs occur as furrows with a protuberant border on the abaxial surface veins of the leaf blade. Each nectary consists of numerous secretory multicellular trichomes, epidermal cells in palisade-like arrangements and non-vascularized parenchyma tissue. Nectar secretion is prolonged, since secretion starts in very young leaves and remains up to completely expanded leaves. Reduced sugars, lipids, and proteins were histochemically detected in all the nectary cells; phenolic substances were detected in the vacuoles of the epidermal palisade cells and in some secretory trichome cells. The secretory cells that constitute the body of trichomes have large nuclei, dense cytoplasm with numerous mitochondria, dictyosomes, scattered lipid droplets and plastids with different inclusions: protein, lipid droplets or starch grains; vacuoles with different sizes have membranous material, phenolic and lipophilic substances. The palisade cells show thick periclinal walls, reduced cytoplasm with voluminous lipid drops and developed vacuoles. The nectary parenchyma cells contain abundant plasmodesmata and cytoplasm with scattered lipid droplets, mitochondria, plastids with starch grains and endoplasmic reticulum. Mucilage idioblasts are common in the inner nectary parenchyma. Protoderm and ground meristem participate in the formation of EFN. Our data indicate that all nectary regions are involved in nectar production and secretion, constituting a functional unit. Longevity of the extrafloral nectaries is likely associated with the presence of mucilage idioblasts, which increases the capacity of the nectary parenchyma to store water.

Effect of FSH and insulin on lipogenesis in cultures of Sertoli cells from immature rats

Follicle-stimulating hormone (FSH) and insulin regulate glycide metabolism in Sertoli cells, thus stimulating lactate production. These stimulatory effects of FSH and insulin do not require protein synthesis, suggesting a modulation of enzyme activity and/or regulation of glucose transport. The present investigation was performed to characterize the hormonal control of lipid metabolism in Sertoli cells. The data indicate that FSH and insulin have a regulatory effect on lipid metabolism in Sertoli cells. After 8 h of preincubation with insulin (5 µg/ml), the activity of the enzyme ATP-citrate lyase in cultured Sertoli cells was increased from 0.19 to 0.34 nmol NAD+ formed µg protein-1 min-1. FSH (100 ng/ml) had no effect on this enzyme. Glycerol phosphate dehydrogenase activity was not affected by any of the hormones tested. When Sertoli cells from 19-day old rats were incubated with [1,2­14C]acetate for 90 or 360 min, the [14C] label was present predominantly in triglyceride and phospholipid fractions with minor amounts in other lipids. In Sertoli cells pretreated for 16 h with insulin and FSH, an increase in acetate incorporation into lipids was observed. Most of the label was in esterified lipids and this percentage increased with the time of treatment; this increase was remarkable in triglycerides of control cells (18.8% to 30.6%). Since Sertoli cell triglycerides participate in the control of spermatogenesis, the present data suggest that the hormonal control of lipid metabolism in Sertoli cells is important not only for maintaining the energy of the cell itself, but also for the control of the spermatogenesis process.

Monosodium glutamate (MSG)-obese rats develop glucose intolerance and insulin resistance to peripheral glucose uptake

Different levels of insulin sensitivity have been described in several animal models of obesity as well as in humans. Monosodium glutamate (MSG)-obese mice were considered not to be insulin resistant from data obtained in oral glucose tolerance tests. To reevaluate insulin resistance by the intravenous glucose tolerance test (IVGTT) and by the clamp technique, newborn male Wistar rats (N = 20) were injected 5 times, every other day, with 4 g/kg MSG (N = 10) or saline (control; N = 10) during the first 10 days of age. At 3 months, the IVGTT was performed by injecting glucose (0.75 g/kg) through the jugular vein into freely moving rats. During euglycemic clamping plasma insulin levels were increased by infusing 3 mU . kg-1 . min-1 of regular insulin until a steady-state plateau was achieved. The basal blood glucose concentration did not differ between the two experimental groups. After the glucose load, increased values of glycemia (P<0.001) in MSG-obese rats occurred at minute 4 and from minute 16 to minute 32. These results indicate impaired glucose tolerance. Basal plasma insulin levels were 39.9 ± 4 µU/ml in control and 66.4 ± 5.3 µU/ml in MSG-obese rats. The mean post-glucose area increase of insulin was 111% higher in MSG-obese than in control rats. When insulinemia was clamped at 102 or 133 µU/ml in control and MSG rats, respectively, the corresponding glucose infusion rate necessary to maintain euglycemia was 17.3 ± 0.8 mg . kg-1 . min-1 for control rats while 2.1 ± 0.3 mg . kg-1 . min-1 was sufficient for MSG-obese rats. The 2-h integrated area for total glucose metabolized, in mg . min . dl-1, was 13.7 ± 2.3 vs 3.3 ± 0.5 for control and MSG rats, respectively. These data demonstrate that MSG-obese rats develop insulin resistance to peripheral glucose uptake

Time course of changes in heart rate and blood pressure variability in streptozotocin-induced diabetic rats treated with insulin

Autonomic neuropathy is a frequent complication of diabetes associated with higher morbidity and mortality in symptomatic patients, possibly because it affects autonomic regulation of the sinus node, reducing heart rate (HR) variability which predisposes to fatal arrhythmias. We evaluated the time course of arterial pressure and HR and indirectly of autonomic function (by evaluation of mean arterial pressure (MAP) variability) in rats (164.5 ± 1.7 g) 7, 14, 30 and 120 days after streptozotocin (STZ) injection, treated with insulin, using measurements of arterial pressure, HR and MAP variability. HR variability was evaluated by the standard deviation of RR intervals (SDNN) and root mean square of successive difference of RR intervals (RMSSD). MAP variability was evaluated by the standard deviation of the mean of MAP and by 4 indices (P1, P2, P3 and MN) derived from the three-dimensional return map constructed by plotting MAPn x [(MAPn+1) - (MAPn)] x density. The indices represent the maximum concentration of points (P1), the longitudinal axis (P2), and the transversal axis (P3) and MN represents P1 x P2 x P3 x 10-3. STZ induced increased urinary glucose in diabetic (D) rats compared to controls (C). Seven days after STZ, diabetes reduced resting HR from 380.6 ± 12.9 to 319.2 ± 19.8 bpm, increased HR variability, as demonstrated by increased SDNN, from 11.77 ± 1.67 to 19.87 ± 2.60 ms, did not change MAP, and reduced P1 from 61.0 ± 5.3 to 51.5 ± 1.8 arbitrary units (AU), P2 from 41.3 ± 0.3 to 29.0 ± 1.8 AU, and MN from 171.1 ± 30.2 to 77.2 ± 9.6 AU of MAP. These indices, as well as HR and MAP, were similar for D and C animals 14, 30 and 120 days after STZ. Seven-day rats showed a negative correlation of urinary glucose with resting HR (r = -0.76, P = 0.03) as well as with the MN index (r = -0.83, P = 0.01). We conclude that rats with short-term diabetes mellitus induced by STZ presented modified autonomic control of HR and MAP which was reversible. The metabolic control may influence these results, suggesting that insulin treatment and a better metabolic control in this model may modify arterial pressure, HR and MAP variability

Dose-dependent effects of 17-ß-estradiol on pituitary thyrotropin content and secretion in vitro

We studied the basal and thyrotropin-releasing hormone (TRH) (50 nM) induced thyrotropin (TSH) release in isolated hemipituitaries of ovariectomized rats treated with near-physiological or high doses of 17-ß-estradiol benzoate (EB; sc, daily for 10 days) or with vehicle (untreated control rats, OVX). One group was sham-operated (normal control). The anterior pituitary glands were incubated in Krebs-Ringer bicarbonate medium, pH 7.4, at 37oC in an atmosphere of 95% O2/5% CO2. Medium and pituitary TSH was measured by specific RIA (NIDDK-RP-3). Ovariectomy induced a decrease (P<0.05) in basal TSH release (normal control = 44.1 ± 7.2; OVX = 14.7 ± 3.0 ng/ml) and tended to reduce TRH-stimulated TSH release (normal control = 33.0 ± 8.1; OVX = 16.6 ± 2.4 ng/ml). The lowest dose of EB (0.7 µg/100 g body weight) did not reverse this alteration, but markedly increased the pituitary TSH content (0.6 ± 0.06 µg/hemipituitary; P<0.05) above that of OVX (0.4 ± 0.03 µg/hemipituitary) and normal rats (0.46 ± 0.03 µg/hemipituitary). The intermediate EB dose (1.4 µg/100 g body weight) induced a nonsignificant tendency to a higher TSH response to TRH compared to OVX and a lower response compared to normal rats. Conversely, in the rats treated with the highest dose (14 µg/100 g body weight), serum 17-ß-estradiol was 17 times higher than normal, and the basal and TRH-stimulated TSH release, as well as the pituitary TSH content, was significantly (P<0.05) reduced compared to normal rats and tended to be even lower than the values observed for the vehicle-treated OVX group, suggesting an inhibitory effect of hyperestrogenism. In conclusion, while reinforcing the concept of a positive physiological regulatory role of estradiol on the TSH response to TRH and on the pituitary stores of the hormone, the present results suggest an inhibitory effect of high levels of estrogen on these responses

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC

Expression and cytokine secretion in the states of immune reactivation in leprosy

Leprosy is a chronic inflammatory disease caused by Mycobacterium leprae. The human response to this pathogen exhibits intriguing aspects which are up to now not well understood. The present study discusses the probable mechanisms involved in T cell-specific unresponsiveness observed in lepromatous patients. Analysis of the cytokine profile either in blood leukocytes or in skin specimens taken from leprosy lesions indicates that some parameters of Th1 immune response are present in lepromatous patients under reactional states

Radioactive fucose as a tool for studying glycoprotein secretion

The efficiency and reliability of radioactive fucose as a specific label for newly synthesized glycoproteins were investigated. Young adult male rabbits were injected intravitreally with [3H]-fucose, [3H]-galactose, [3H]-mannose, N-acetyl-[3H]-glucosamine or N-acetyl-[3H]-mannosamine, and killed 40 h after injection. In another series of experiments rabbits were injected with either [3H]-fucose or several tritiated amino acids and the specific activity of the vitreous proteins was determined. Vitreous samples were also processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and histological sections of retina, ciliary body and lens (the eye components around the vitreous body) were processed for radioautography. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always much higher than that of the proteins labeled with any of the other monosaccharides or any of the amino acids. There was a good correlation between the specific activity of the proteins labeled by any of the above precursors and the density of the vitreous protein bands detected by fluorography. This was also true for the silver grain density on the radioautographs of the histological sections of retina, ciliary body and lens. The contribution of radioautography (after [3H]-fucose administration) to the elucidation of the biogenesis of lysosomal and membrane glycoproteins and to the determination of the intracellular process of protein secretion was reviewed. Radioactive fucose is the precursor of choice for studying glycoprotein secretion because it is specific, efficient and practical for this purpose

Histologic distribution of insulin and glucagon receptors

Insulin and glucagon are the hormonal polypeptides secreted by the B and A cells of the endocrine pancreas, respectively. Their major physiologic effects are regulation of carbohydrate metabolism, but they have opposite effects. Insulin and glucagon have various physiologic roles, in addition to the regulation of carbohydrate metabolism. The physiologic effects of insulin and glucagon on the cell are initiated by the binding of each hormone to receptors on the target cells. Morphologic studies may be useful for relating biochemical, physiologic, and pharmacologic information on the receptors to an anatomic background. Receptor radioautography techniques using radioligands to label specific insulin and glucagon receptors have been successfully applied to many tissues and organs. In this review, current knowledge of the histologic distribution of insulin and glucagon receptors is presented with a brief description of receptor radioautography techniques

Distribution of HLA-DRB1 alleles in a mixed population with insulin-dependent diabetes mellitus from the Southeast of Brazil

HLA class II genes are strongly associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). The present study reports the HLA-DRB1 genotyping of 41 IDDM patients and 99 healthy subjects from the Southeast of Brazil (Campinas region). Both groups consisted of an ethnic mixture of Caucasian, African Negro and Amerindian origin. HLA-DRB1*03 and *04 alleles were found at significantly higher frequencies among IDDM patients compared to the controls (DRB1*03: 48.8% vs 18.2%, P<0.005, RR = 4.27; DRB1*04: 43.9% vs 15.1%, P<0.008, RR = 4.37) and were associated with a susceptibility to the disease. DRB1*03/*04 heterozygosity conferred a strong IDDM risk (RR = 5.44). In contrast, the HLA-DRB1*11 allele frequency was lower among IDDM patients (7.3% vs 26.3% in controls), but the difference was not significant. These data agree with those described for other populations and allow genetic characterization of IDDM in Brazil

Abnormal nocturnal blood pressure fall in normotensive adolescents with insulin-dependent diabetes is ameliorated following glycemic improvement

Lack of the physiological nocturnal fall in blood pressure (BP) has been found in diabetics and it seems to be related to the presence of diabetic complications. The present study examined the changes in the nocturnal BP pattern of 8 normotensive insulin-dependent diabetic adolescents without nephropathy following improvement in glycemic control induced by an 8-day program of adequate diet and exercise. The same number of age- and sex-matched control subjects were studied. During the first and eighth nights of the program, BP was obtained by ambulatory BP monitoring. After a 10-min rest, 3 BP and heart rate (HR) recordings were taken and the mean values were considered to represent their awake values. The monitor was programmed to cuff insufflation every 20 min from 10:00 p.m. to 7:00 a.m. The glycemic control of diabetics improved since glycemia (212.0 ± 91.5 to 140.2 ± 69.1 mg/dl, P<0.03), urine glucose (12.7 ± 11.8 to 8.6 ± 6.4 g/24 h, P = 0.08) and insulin dose (31.1 ± 7.7 to 16.1 ± 9.7 U/day, P<0.01) were reduced on the last day. The mean BP of control subjects markedly decreased during the sleeping hours of night 1 (92.3 ± 6.4 to 78.1 ± 5.0 mmHg, P<0.001) and night 8 (87.3 ± 6.7 to 76.9 ± 3.6 mmHg, P<0.001). Diabetic patients showed a slight decrease in mean BP during the first night. However, the fall in BP during the nocturnal period increased significantly on the eighth night. The average awake-sleep BP variation was significantly higher at the end of the study (4.2 vs 10.3%, P<0.05) and this ratio turned out to be similar to that found in the control group (10.3 vs 16.3%). HR variation also increased on the eighth night in the diabetics. Following the metabolic improvement obtained at the end of the period, the nocturnal BP variation of diabetics was close to the normal pattern. We suggest that amelioration of glycemic control may influence the awake-sleep BP and HR differences. This effect may be due at least in part to an attenuated insulin stimulation of sympathetic activity

The insulin receptor substrate 1 associates with phosphotyrosine phosphatase SHPTP2 in liver and muscle of rats

Insulin stimulates the tyrosine kinase activity of its receptor resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate-1 (IRS-1) which, in turn, associates with proteins containing SH2 domains. It has been shown that IRS-1 associates with the tyrosine phosphatase SHPTP2 in cell cultures. While the effect of the IRS-1/SHPTP2 association on insulin signal transduction is not completely known, this association may dephosphorylate IRS-1 and may play a critical role in the mitogenic actions of insulin. However, there is no physiological demonstration of this pathway of insulin action in animal tissues. In the present study we investigated the ability of insulin to induce association between IRS-1 and SHPTP2 in liver and muscle of intact rats, by co-immunoprecipitation with anti-IRS-1 antibody and anti-SHPTP2 antibody. In both tissues there was an increase in IRS-1 association with SHPTP2 after insulin stimulation. This association occurred when IRS-1 had the highest level of tyrosine phosphorylation and the decrease in this association was more rapid than the decrease in IRS-1 phosphorylation levels. The data provide evidence against the participation of SHPTP2 in IRS-1 dephosphorylation in rat tissues, and suggest that the insulin signal transduction pathway in rat tissues is related mainly to the mitogenic effects of the hormone.

Insulin receptor has tyrosine kinase activity toward Shc in rat liver

Insulin induces tyrosine phosphorylation of Shc in cell cultures and in insulin-sensitive tissues of the intact rat. However, the ability of insulin receptor (IR) tyrosine kinase to phosphorylate Shc has not been previously demonstrated. In the present study, we investigated insulin-induced IR tyrosine kinase activity towards Shc. Insulin receptor was immunoprecipitated from liver extracts, before and after a very low dose of insulin into the portal vein, and incubated with immunopurified Shc from liver of untreated rats. The kinase assay was performed in vitro in the presence of exogenous ATP and the phosphorylation level was quantified by immunoblotting with antiphosphotyrosine antibody. The results demonstrate that Shc interacted with insulin receptor after infusion of insulin, and, more important, there was insulin receptor kinase activity towards immunopurified Shc. The description of this pathway in animal tissue may have an important role in insulin receptor tyrosine kinase activity toward mitogenic transduction pathways.

Low levels of sex hormone-binding globulin and hyperproinsulinemia as markers of increased pancreatic ß-cell demand in men

Low levels of sex hormone-binding globulin (SHBG) are considered to be an indirect index of hyperinsulinemia, predicting the later onset of diabetes mellitus type 2. In the insulin resistance state and in the presence of an increased pancreatic ß-cell demand (e.g. obesity) both absolute and relative increases in proinsulin secretion occur. In the present study we investigated the correlation between SHBG and pancreatic ß-cell secretion in men with different body compositions. Eighteen young men (30.0 ± 2.4 years) with normal glucose tolerance and body mass indexes (BMI) ranging from 22.6 to 43.2 kg/m2 were submitted to an oral glucose tolerance test (75 g) and baseline and 120-min blood samples were used to determine insulin, proinsulin and C-peptide by specific immunoassays. Baseline SHBG values were significantly correlated with baseline insulin (r = -0.58, P<0.05), proinsulin (r = -0.47, P<0.05), C-peptide (r = -0.55, P<0.05) and also with proinsulin at 120 min after glucose load (r = -0.58, P<0.05). Stepwise regression analysis revealed that proinsulin values at 120 min were the strongest predictor of SHBG (r = -0.58, P<0.05). When subjects were divided into obese (BMI >28 kg/m2, N = 8) and nonobese (BMI £25 kg/m2, N = 10) groups, significantly lower levels of SHBG were found in the obese subjects. The obese group had significantly higher baseline proinsulin, C-peptide and 120-min proinsulin and insulin levels. For the first time using a specific assay for insulin determination, a strong inverse correlation between insulinemia and SHBG levels was confirmed. The finding of a strong negative correlation between SHBG levels and pancreatic ß-cell secretion, mainly for the 120-min post-glucose load proinsulin levels, reinforces the concept that low SHBG levels are a suitable marker of increased pancreatic ß-cell demand.

Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6%) presented hypogonadism (which was central in 45 cases and primary in 2), 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV) of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01) and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02). Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01) as well as free testosterone levels (r = -0.53, P<0.01). In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.