María Elisa García

Early alterations in vascular contractility associated to changes in fatty acid composition and oxidative stress markers in perivascular adipose tissue

AimTo test the early effect of fructose-induced changes in fatty acid composition and oxidative stress markers in perivascular adipose tissue (PVAT) upon vascular contractility.MethodsAdult male Wistar rats were fed a commercial diet without (CD) or with 10% fructose (FRD) in the drinking water for 3 weeks. We measured plasma metabolic parameters, lipid composition and oxidative stress markers in aortic PVAT. Vascular contractility was measured in aortic rings sequentially, stimulated with serotonin (5-HT) and high K+-induced depolarization using intact and thereafter PVAT-deprived rings.ResultsComparable body weights were recorded in both groups. FRD rats had increased plasma triglyceride and fructosamine levels. Their PVAT had an increased saturated to mono- or poly-unsaturated fatty acid ratio, a significant decrease in total superoxide dismutase and glutathione peroxidase activities and in the total content of glutathione. Conversely, lipid peroxidation (TBARS), nitric oxide content, and gluthathione reductase activity were significantly higher, indicating an increase in oxidative stress. In aortic rings, removal of PVAT increased serotonin-induced contractions, but the effect was significantly lower in rings from FRD rats. This effect was no longer observed when the two contractions were performed in PVAT-deprived rings. PVAT did not affect the contractions triggered by high K+-induced depolarization either in CD or FRD rats.ConclusionsFRD induces multiple metabolic and endocrine systemic alterations which also alter PVAT and the vascular relaxant properties of this tissue. The changes in PVAT would affect its paracrine modulation of vascular function.

Antioxidant treatment prevents the development of fructose-induced abdominal adipose tissue dysfunction

In the present study, we tested the effect of OS (oxidative stress) inhibition in rats fed on an FRD [fructose-rich diet; 10% (w/v) in drinking water] for 3 weeks. Normal adult male rats received a standard CD (commercial diet) or an FRD without or with an inhibitor of NADPH oxidase, APO (apocynin; 5 mM in drinking water; CD-APO and FRD-APO). We thereafter measured plasma OS and metabolic-endocrine markers, AAT (abdominal adipose tissue) mass and cell size, FA (fatty acid) composition (content and release), OS status, LEP (leptin) and IRS (insulin receptor substrate)-1/IRS-2 mRNAs, ROS (reactive oxygen species) production, NADPH oxidase activity and LEP release by isolated AAT adipocytes. FRD-fed rats had larger AAT mass without changes in body weight, and higher plasma levels of TAG (triacylglycerol), FAs, TBARS (thiobarbituric acid-reactive substance) and LEP. Although no significant changes in glucose and insulin plasma levels were observed in these animals, their HOMA-IR (homoeostasis model assessment of insulin resistance) values were significantly higher than those of CD. The AAT from FRD-fed rats had larger adipocytes, higher saturated FA content, higher NADPH oxidase activity, greater ROS production, a distorted FA content/release pattern, lower insulin sensitivity together with higher and lower mRNA content of LEP and IRS-1-/2 respectively, and released a larger amount of LEP. The development of all the clinical, OS, metabolic, endocrine and molecular changes induced by the FRD were significantly prevented by APO co-administration. The fact that APO treatment prevented both changes in NADPH oxidase activity and the development of all the FRD-induced AAT dysfunctions in normal rats strongly suggests that OS plays an important role in the FRD-induced MS (metabolic syndrome) phenotype.

Effect of Pioglitazone on the Fructose-Induced Abdominal Adipose Tissue Dysfunction

Aim. To test the potential role of PPARγ in the endocrine abdominal tissue dysfunction induced by feeding normal rats with a fructose rich diet (FRD) during three weeks. Methodology. Adult normal male rats received a standard commercial diet (CD) or FRD, (10% in drinking water) without or with pioglitazone (PIO) (i.p. 0.25 mg/Kg BW/day; CD-PIO and FRD-PIO). Thereafter, we measured circulating metabolic, endocrine, and oxidative stress (OS) markers, abdominal adipose tissue (AAT) mass, leptin (LEP) and plasminogen activator inhibitor-1 (PAI-1) tissue content/expression, and leptin release by isolated adipocytes incubated with different concentrations of insulin. Results. Plasma glucose, insulin, triglyceride, TBARS, LEP, and PAI-1 levels were higher in FRD rats; PIO coadministration fully prevented all these increments. AAT adipocytes from FRD rats were larger, secreted a higher amount of LEP, and displayed decreased sensitivity to insulin stimulation; these effects were significantly ameliorated by PIO. Whereas AAT LEP and PAI-1 (mRNA) concentrations increased significantly in FRD rats, those of insulin-receptor-substrate- (IRS-) 1 and IRS-2 were reduced. PIO coadministration prevented FRD effects on LEP, PAI-1, and IRS-2 (fully) and IRS-1 (partially) mRNAs in AAT. Conclusion. PPARγ would play a relevant role in the development of the FRD-induced metabolic-endocrine dysfunction.

Tyrosine hydroxylase activity in the endocrine pancreas: changes induced by short-term dietary manipulation.

BackgroundTyrosine hydroxylase (TH) activity and its possible participation in the control of insulin secretion were studied in pancreatic islets of adult Wistar rats fed a standard commercial diet (SD) or carbohydrates alone (CHD) for one week. TH activity, norepinephrine (NE) content, and glucose-induced insulin secretion were assessed. Blood glucose and insulin levels were measured at the time of sacrifice.ResultsCHD rats had significantly higher blood glucose and lower insulin levels than SD rats (114.5 ± 6.7 vs 80.7 ± 7.25 mg/dl, p < 0.001; 20.25 ± 2.45 vs 42.5 ± 4.99 μU/ml, p < 0.01, respectively). Whereas TH activity was significantly higher in CHD isolated islets (600 ± 60 vs 330 ± 40 pmol/mg protein/h; p < 0.001), NE content was significantly lower (18 ± 1 vs 31 ± 5 pmol/mg protein), suggesting that TH activity would be inhibited by the end-products of catecholamines (CAs) biosynthetic pathway. A similar TH activity was found in control and solarectomized rats (330 ± 40 vs 300 ± 80 pmol/mg protein/h), suggesting an endogenous rather than a neural origin of TH activity. CHD islets released significantly less insulin in response to glucose than SD islets (7.4 ± 0.9 vs 11.4 ± 1.1 ng/islet/h; p < 0.02).ConclusionsTH activity is present in islet cells; dietary manipulation simultaneously induces an increase in this activity together with a decrease in glucose-induced insulin secretion in rat islets. TH activity – and the consequent endogenous CAs turnover – would participate in the paracrine control of insulin secretion.

Transcription, expression and tissue binding in vivo of INGAP and INGAP-related peptide in normal hamsters

We studied islet neogenesis-associated protein (INGAP) transcription and its immunocytochemical presence in and binding in vivo of (125)I-tyrosylated INGAP pentadecapeptide ((125)I-T-INGAP-PP) to different normal male hamster tissues. (125)I-T-INGAP-PP was injected intraperitoneally with or without unlabeled T-INGAP-PP (0-1 mg/100 g bw), drawing blood samples at different times after injection; radioactivity was measured in serum, brain, skeletal muscle, dorsal root ganglia, liver, kidney, small intestine and pancreas samples, expressing results as organ:serum ratio. INGAP transcription (RT-PCR) and immunopositive cells were investigated in liver, kidney, brain, small intestine and pancreas. Total serum radioactivity increased progressively as a function of time; whereas 71% of this activity was displaced by unlabeled T-INGAP-PP at 5, 10 and 20 min, only 9% was at 60 min. Only liver, pancreas and small intestine specifically bound (125)I-T-INGAP-PP. The pancreas tissue dose-response curve showed a 50% displacement at 3.9x10(4) ng/100 g bw, suggesting a low binding affinity of its receptor. INGAP-mRNA was only identified in pancreatic islets and exocrine tissue. Our results suggest that INGAP transcription/expression is probably restricted to pancreas cells exerting its effect in a paracrine fashion. INGAP would be released and circulate bound to a serum protein from where it is bound and inactivated by the liver. Tissue binding could also explain INGAPs immunocytochemical presence in small intestine, where it could affect epithelial cell turnover.

Role of phospholipase and calmodulin inhibitors on insulin, arachidonic acid and prostaglandin E2 release

Using several experimental approaches, we have studied simultaneously the effect of glucose upon insulin, arachidonic acid and prostaglandin E2 release by rat pancreatic islets. A 16.6 mmol/l glucose concentration stimulated the release of insulin, arachidonic acid and prostaglandins. All these effects were significantly reduced either by calmodulin and phospholipase A2 inhibitors, or by the omission of calcium in the incubation medium. Phospholipase A2 inhibitors do not modify the glucose-induced net 45Ca2+ uptake by isolated islets. Our results would suggest that activation of phospholipases, particularly A2, is involved in the mechanism by which glucose stimulates insulin release. This activation increases the intracellular concentration of arachidonic acid, prostaglandins and probably phospholipid degradation products, that could act as messengers for the stimulus-secretion coupling of insulin. The calcium-calmodulin complex would take part in this effect. Conversely, the glucose-induced net calcium uptake by the islets might either be preceded by phospholipase activation or not significantly affected by the blockade of its activity.

Effect of the Removal of Different Endocrine Glands upon Insulin Secretion and B-Cell Ultrastructure

Glucose-induced insulin secretion and B-cell ultrastructure were studied in islets obtained from normal, adrenalectomized, radiothyroidectomized, ovariectomized and orchidectomized rats. Both parameters were also studied in the same experimental groups submitted to specific substitutive therapy. Insulin secretion in response to high glucose was significantly diminished in adrenalectomized, hypothyroid and male castrated rats. Conversely, this secretion was enhanced in ovariectomized rats. These abnormal insulin responses were restored to normal range by specific substitutive therapy. B-cell ultrastructure was markedly altered in hypothyroid and in female and male castrated rats. No significant changes were observed in the adrenalectomized rats. No conspicuous alterations were depicted in the other islet cell populations. The features of the morphological alterations were mainly related to changes in the B-granules and the rough endoplasmic reticulum. Modifications of the other B-cell organelles were less frequent. In the castrated rats, a distinctive feature was the appearance of a finely granulated colloid material. These B-cell alterations, consecutive to changes in the circulating levels of a given hormone, seemed to depend on the chemical structure of the hormone itself rather than on the changes induced in the B-cell secretory function. The ultrastructural changes described were reversed, as in the case of insulin release, by specific substitutive therapy. It is concluded that changes in the circulating levels of the hormones studied are followed by specific alterations in both B-cell secretion and ultrastructure.