Prolactin and growth hormone synthesis and thymidine incorporation in dissociated rat pituitary tumor cells

The effect of in vivo diethylstilbestrol (DES) treatment on the MtT/W15 transplantable pituitary tumor was examined in dissociated pituitary cells by measuring the rate of incorporation of [3H]thymidine into DNA and the synthesis of prolactin (PRL) and growth hormone (GH) as assessed by the rate of incorporation of [3H]leucine. MtT/W15 transplantable pituitary tumors from rats treated for 3 weeks with DES showed significant reduction in the extent of [3H]thymidine incorporation compared with tumor cells from untreated rats (2231 +/- 182 vs 172 +/- 17 dpm/10(5) cells; n = 3). In addition, tumor cells from DES-treated rats showed a significant increase in GH synthesis compared with tumor cells from untreated rats. In contrast to these findings, dissociated pituitary cells from non-tumor-bearing rats given 10 mg DES in Silastic tubing for 3 weeks showed a three-fold increase in PRL synthesis compared to cells from untreated control rats (29.3 +/- 1.5 vs 10.0 +/- 0.9% of total radioactivity in gel; n = 3. There was also a four-fold increase in the rate of [3H]thymidine incorporation after DES-treatment in non-tumor-bearing rats (695 +/- 114 vs 178 +/- 13.9 dpm/10(5) cells; n = 3). These results indicate that DES inhibits MtT/W15 pituitary tumor cell proliferation, while stimulating synthesis of GH.     

Hypertriglyceridemic serum from magnesium-deficient rats induces proliferation and lipid accumulation in cultured vascular smooth muscle cells

An important characteristic of hyperlipemia associated with magnesium deficiency in rats is the postprandial accumulation of triglyceride-rich lipoproteins. The present investigation was performed to determine the effect of serum from magnesium-deficient animals on cultured vascular smooth muscle cells (VSMC). Sera were obtained from control and magnesium-deficient rats fed adequate or deficient diets for 8 days. Magnesium-deficient animals were hypertriglyceridemic compared with control rats, but their total cholesterolemia was not significantly modified. Pooled sera from control and magnesium-deficient animals were added to the culture medium at various concentrations. The maximum of proliferation for both control and magnesium-deficient sera was reached when they were added at 6% to the culture medium and on day 4 after the begining of incubation. Medium containing serum from magnesium-deficient rats stimulated the cell proliferation as monitored by cell count and [³H]thymidine incorporation. Staining of VSMC with Oil red O and measuring lipids have shown a marked lipid accumulation (triglycerides) in cells incubated with serum obtained from magnesium-deficient animals compared with serum from control rats. These results indicate that serum from magnesium-deficient rats contains factors that stimulate proliferation of arterial medial cells and that hyperlipemia associated with magnesium-deficiency may cause lipid accumulation in vascular cells.     

An aldose reductase inhibitor prevents the glucose-induced increase in PDGF-beta receptor in cultured rat aortic smooth muscle cells.

To examine the role of platelet-derived growth factor (PDGF) and the polyol pathway in the growth activity of smooth muscle cells (SMCs), [(3)H]-thymidine incorporation, [(125)I]-PDGF-BB binding and expression of PDGF-beta receptor protein were measured in rat aortic SMCs cultured with 5.5 or 20 mM glucose with or without anti-PDGF antibody or an aldose reductase inhibitor, epalrestat. SMCs cultured with 20 mM glucose demonstrated an accelerated thymidine incorporation compared with SMCs cultured with 5.5 mM glucose, which was prevented by anti-PDGF antibody. This acceleration of growth activity by 20 mM glucose was accompanied by an increase in PDGF-BB binding, which was due to the increased number of PDGF-beta receptors and the overexpression of PDGF-beta receptor protein. Epalrestat prevented all these abnormalities. These observations suggest that polyol pathway hyperactivity plays an important role in the proliferation of SMCs which may be mediated through the accelerated expression of PDGF-beta receptor protein.     

Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function

Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.     

Antiproliferative effect of UTP on human arterial and venous smooth muscle cells

We have investigated the hypothesis that responses associated with proliferation are regulated by extracellular nucleotides such as ATP and UTP in cultured human vascular smooth muscle cells (VSMC) derived from internal mammary artery (IMA) and saphenous vein (SV). Platelet-derived growth factor (PDGF), ATP, and UTP each generated an increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in both IMA- and SV-derived cells in the absence of detectable inositol 1,4,5-trisphosphate production. ATP alone had no effect on [(3)H]thymidine incorporation into DNA, but with a submaximal concentration of PDGF it raised [(3)H]thymidine incorporation in SV- but not IMA-derived cells. UTP alone also was without effect on [(3)H]thymidine incorporation or cell number. However, in both SV- and IMA-derived cells, UTP reduced the PDGF-stimulated [(3)H]thymidine response and PDGF-stimulated cell proliferation. This cannot be explained by an inhibitory effect on the p42/p44 mitogen-activated protein kinase (MAPK) cascade, since this response to PDGF was not attenuated by UTP. We conclude that, in human VSMC of both arterial and venous origin, UTP acts as an anti-proliferative regulator.     

Pentose phosphate pathway,glutathione-dependent enzymes and antioxidant defense during oxidative stress in diabetic rodent brain and peripheral organs: effects of stobadine and vitamin E

The aim of the present study was to investigate the effects of treatment with antioxidant stobadine (ST) on the activities of enzymes related with pentose phosphate pathway and glutathione-dependent metabolism and the other markers of oxidative stress in brain and peripheral organs of diabetic rats, and to compare the effects of ST treatment alone with the effects of treatments with another antioxidant vitamin E and ST plus vitamin E. Rats were made diabetic by the injection of streptozotocin (STZ; 55 mg/kg IP), and, 2 days later, some control and diabetic rats were left untreated or treated with ST (24.7 mg/kg/day, orally), vitamin E (400–500 U/kg/day, orally), or both substances together. In the brain, although 6-phosphogluconate dehydrogenase activity (6-PGD) did not change, glucose-6-phosphate dehydrogenase activity (G-6PD) was markedly increased in diabetic rats compared with controls; only combined treatment with ST and vitamin E produced a partial prevention on this alteration. The aorta G-6PD and 6-PGD of diabetic rats were 52% and 36% of control values, respectively. Neither single treatments with each antioxidant nor their combination altered the G-6PD and 6-PGD in aorta of diabetic rats. Glutathione peroxidase (GSHPx) activity was increased by STZ-diabetes in brain, heart, and kidney. In diabetic brain, vitamin E alone or combination with ST kept GSHPx at normal levels. Diabetes-induced stimulation in GSHPx did not decrease in response to the treatment with vitamin E in heart and kidney, but was greatly prevented by ST alone. The activity of glutathione reductase (GR) was decreased in brain and heart of diabetic rats. The treatment with each antioxidant or with a combination of both agents completely prevented this deficiency and resulted in further activation of GR in diabetic tissues. Glutathione S-transferase (GST) activity did not significantly change in diabetic brain and aorta. GST was stimulated by all treatment protocols in the brain of diabetic rats and was depressed in aorta of control rats. Catalase (CAT) was activated in diabetic heart but depressed in diabetic kidney. Diabetes-induced abnormalities in CAT activity did not respond to vitamin E alone in heart, was moderately ameliorated by the treatment with this vitamin in kidney, and was completely prevented by ST alone in both tissues. Superoxide dismutase (SOD) activity of brain and heart was unchanged by the diabetes but inhibited in diabetic kidney after the treatment ST alone or ST plus vitamin E. The lipid peroxidation (MDA) was increased in diabetic brain and heart. ST or vitamin E alone partly prevented diabetes-induced increase in MDA in brain and heart; however, antioxidant combination achieved a completely amelioration in MDA of these tissues of diabetic rats. Kidney MDA levels were similar in control and untreated diabetic animals. ST and vitamin E treatments, when applied separately or together, significantly reduced kidney MDA in both control and diabetic rats; and the combined effect of antioxidants was greater than that of each alone. These results are consistent with the degenerative role of hyperglycemia on cellular reducing equivalent homeostasis and antioxidant defense, and provide further evidence that pharmacological intervention of different antioxidants may have significant implications in the prevention of the prooxidant feature of diabetes and protects redox status of the cells.     

Effects of vitamin E on microsomal Ca(2+) -ATPase activity and calcium levels in streptozotocin-induced diabetic rat kidney

Vitamin E treatment has been found to be beneficial in preventing or reducing diabetic nephropathy. Increased tissue calcium and abnormal microsomal Ca(2+)-ATPase activity have been suggested as contributing factors in the development of diabetic nephropathy. This study was undertaken to test the hypothesis that vitamin E reduces lipid peroxidation and can prevent the abnormalities in microsomal Ca(2+)-ATPase activity and calcium levels in kidney of streptozotocin (STZ)-induced diabetic rats. Male rats were rendered diabetic by a single STZ injection (55 mg x kg(-1) i.p.). After diabetes was verified, diabetic and age-matched control rats were untreated or treated with vitamin E (400-500 IU kg(-1) x day(-1), orally) for 10 weeks. Ca(2+)-ATPase activity and lipid peroxidation (MDA) were determined spectrophotometrically. Blood glucose levels increased approximately five-fold (> 500 mg x dl(-1)) in untreated-diabetic rats but decreased to 340+/-27 mg x dl(-1) in the vitamin E treated-diabetic group. Kidney MDA levels did not significantly change in the diabetic state. However, vitamin E treatment markedly inhibited MDA levels in both control and diabetic animals. Ca(2+)-ATPase activity was 0.483+/-0.008 U l(-1) in the control group and significantly increased to 0.754+/-0.010 U l(-1) in the STZ-diabetic group (p < 0.001). Vitamin E treatment completely prevented the diabetes-induced increase in Ca(2+)-ATPase activity (0.307+/-0.025 U l(-1), p < 0.001) and also reduced the enzyme activity in normal control rats. STZ-diabetes resulted in approximately two-fold increase in total calcium content of kidney. Vitamin E treatment led to a significant reduction in kidney calcium levels of both control and diabetic animals (p < 0.001). Thus, vitamin E treatment can lower blood glucose and lipid peroxidation, which in turn prevents the abnormalities in kidney calcium metabolism of diabetic rats. This study describes a potential biochemical mechanism by which vitamin E supplementation may delay or inhibit the development of cellular damage and nephropathy in diabetes.     

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-beta

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.     

Utility of the modified ATP III defined metabolic syndrome and severe obesity as predictors of insulin resistance in overweight children and adolescents: a cross-sectional study

Background

Inhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [³H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [³H]-thymidine incorporation.

Methods

VSMC proliferation was investigated by [³H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [³H]-thymidine uptake. Cell cycle events were analyzed by FACS.

Results

VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [³H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [³H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [³H]-thymidine incorporation or cell numbers.

Conclusion

TZDs but not sulfonylureas nor biguanides (except phenformin at high concentrations) show favorable vascular actions assessed as inhibition of vSMC proliferation. The activity of rosiglitazone and pioglitazone is enhanced under high glucose conditions. These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases. However, the plethora of potentially beneficial actions of TZDs in cell and animal models have not been reflected in the results of major clinical trials and a greater understanding of these complex drugs is required to delineate their ultimate clinical utility in preventing macrovascular disease in diabetes.     

Synchronization of Cultured Vascular Smooth Muscle Cells Following Reversal of Quiescence Induced by Treatment with the AntioxidantN-Acetylcysteine

Smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. Recently we demonstrated the thiol antioxidantN-acetylcysteine (NAC) inhibits constitutive NF-κB/Rel activity and growth of vascular SMCs. Here we show that treatment of human and bovine aortic SMC with the thiol antioxidant NAC causes cells to exit the cell cycle and remain quiescent as determined by a greatly reduced incorporation of [³H]thymidine and G₀/G₁DNA content. Removal of NAC from the culture medium stimulates SMCs to synchronously reenter the cell cycle as judged by induction of cyclin D1 and B-mybgene expression during mid and late G₁phase, respectively, and induction of histone gene expression and [³H]thymidine incorporation during S phase. The time course of cyclin D1, B-myb,and histone gene expression after NAC removal was similar to that of serum-deprived cells induced to resume cell cycle progression by the addition of fetal bovine serum to the culture medium. Taken together, these results indicate that NAC treatment causes SMCs to enter a reversible G₀quiescent, growth-arrested state. Thus, NAC provides an important new method for synchronizing SMCs in culture.     

Hyperosmolality-induced abnormal patterns of calcium mobilization in smooth muscle cells from non-diabetic and diabetic rats

Hyperglycemia and/or hyperosmolality may disturb calcium homeostasis in vascular smooth muscle cells (SMCs), leading to altered vascular contractility in diabetes. To test this hypothesis, the KCl induced increases in [Ca²⁺]_i in primarily cultured vascular SMCs exposed to different concentrations of glucose were examined. With glucose concentration in solutions kept at 5.5 mM, KCl induced a fast increase in [Ca²⁺]_i which then slowly declined (type 1 response) in 83% of SMCs from non-diabetic rats. In 9% of non-diabetic SMCs KCl induced a slow increase in [Ca²⁺]_i (type 2 response). Interestingly, under the same culture conditions KCl induced type 1 and type 2 responses in 47 and 35% of SMCs from diabetic rats. When SMCs from non-diabetic or diabetic rats were cultured in 36 mM glucose, KCl induced a fast increase in [Ca²⁺]_i which, however, maintained at a high level (type 3 response). The sustained level of [Ca²⁺]_i in the presence of KCl was significantly higher in cells cultured with 36 mM glucose than that in non-diabetic cells cultured with 5.5 mM glucose. Furthermore, the hyperglycemia-induced alterations in calcium mobilization were similarly observed in cells cultured in high concentration of mannitol (30.5 mM) or L-glucose, indicating that hyperosmolality was mainly responsible for the abnormal calcium mobilization in diabetic SMCs.     

Perlecan mediates the antiproliferative effect of apolipoprotein E on smooth muscle cells. An underlying mechanism for the modulation of smooth muscle cell growth?

Apolipoprotein E (apoE) is known to inhibit cell proliferation; however, the mechanism of this inhibition is not clear. We recently showed that apoE stimulates endothelial production of heparan sulfate (HS) enriched in heparin-like sequences. Because heparin and HS are potent inhibitors of smooth muscle cell (SMC) proliferation, in this study we determined apoE effects on SMC HS production and cell growth. In confluent SMCs, apoE (10 microg/ml) increased (35)SO(4) incorporation into PG in media by 25-30%. The increase in the medium was exclusively due to an increase in HSPGs (2.2-fold), and apoE did not alter chondroitin and dermatan sulfate proteoglycans. In proliferating SMCs, apoE inhibited [(3)H]thymidine incorporation into DNA by 50%; however, despite decreasing cell number, apoE increased the ratio of (35)SO(4) to [(3)H]thymidine from 2 to 3.6, suggesting increased HS per cell. Purified HSPGs from apoE-stimulated cells inhibited cell proliferation in the absence of apoE. ApoE did not inhibit proliferation of endothelial cells, which are resistant to heparin inhibition. Analysis of the conditioned medium from apoE-stimulated cells revealed that the HSPG increase was in perlecan and that apoE also stimulated perlecan mRNA expression by >2-fold. The ability of apoE isoforms to inhibit cell proliferation correlated with their ability to stimulate perlecan expression. An anti-perlecan antibody completely abrogated the antiproliferative effect of apoE. Thus, these data show that perlecan is a potent inhibitor of SMC proliferation and is required to mediate the antiproliferative effect of apoE. Because other growth modulators also regulate perlecan expression, this may be a key pathway in the regulation of SMC growth.     

Effect of diabetes and Sorbinil treatment on phospholipid metabolism in rat glomeruli

To explore the hypothesis that changes in membrane phospholipids accompany tissue myo-inositol depletion and reduced (Na+ + K+)-ATPase activity in diabetes, we examined phospholipid concentrations in glomeruli isolated from control and streptozotocin-diabetic rats and the effect of diabetes on myo-[3H]inositol incorporation in vitro into glomerular phosphatidylinositol. Since the aldose reductase inhibitor, Sorbinil, prevents the fall in myo-inositol and the decrease in (Na+ + K+)-ATPase activity associated with diabetes, phospholipid and phosphatidylinositol content were also examined in glomeruli isolated from Sorbinil-treated diabetic rats. Total phospholipids (microgram phosphorus/mg dry weight) did not differ in the three groups of animals. The concentration of phosphatidylcholine was elevated in preparations from diabetic rats, both untreated and Sorbinil-treated. Phosphatidylethanolamine was reduced in glomeruli from Sorbinil-treated rats. Neither acute experimental diabetes nor Sorbinil treatment produced detectable changes in the glomerular concentration of phosphatidylinositol. In vitro incubations with glomeruli isolated from control and diabetic animals resulted in increased levels of incorporation of myo-[3H]inositol into phospholipids of diabetic glomeruli. The specific activity of [3H]phosphatidylinositol in glomeruli from diabetic rats was significantly greater than that in control samples. The findings do not support the postulate invoking correspondent changes in myo-inositol and phosphatidylinositol contents as contributory to diminished glomerular (Na+ + K+)-ATPase activity in diabetes, but are compatible with depletion of glomerular intracellular myo-inositol in diabetes.     

Beneficial effect of vitamin E on the metabolic parameters of diabetic rats

The role of vitamin E in the pathogenesis of diabetes mellitus is unknown. The purpose of this study was to examine the effect of oral administration of vitamin E on some of the metabolic parameters of experimental diabetic rats. Diabetes was induced by intraperitoneal injection of streptozotocin (60 mg/kg body weight at 12 weeks of age). Vitamin E (0.2, 0.4, 0.8 mg/kg body weight) was administered orally for a period of 3 weeks to normal and diabetic Wistar rats. In some experiments, Vitamin E was given either before or after the induction of diabetes mellitus. Blood glucose level and weight were recorded for each rat in different groups on a weekly basis. Oral glucose tolerance test (OGTT) was performed on fasted normal, diabetic and vitamin E treated rats at the end of the experiment. Vitamin E significantly (p < 0.01) reduced blood glucose levels in experimental diabetes mellitus at all doses as compared to untreated rats. Vitamin E induced weight loss in normal as well as in diabetic rats. The beneficial effect of vitamin E on the hyperglycaemia of diabetic rats was dose-dependent. Moreover, vitamin E also improved OGTT in diabetic rats compared to untreated diabetics. In conclusion, vitamin E may play a role in glucose metabolism and thus be a useful adjuvant therapy in type I diabetes. (Mol Cell Biochem 261: 35–42, 2004)     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Synergistic action of growth hormone and insulin-like growth factor I (IGF-I) on proliferation and estradiol secretion in porcine granulosa and theca cells cultured alone or in coculture

The effect of growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion and the effects of GH and IGF-I on [(3)H] thymidine incorporation and estradiol (E2) secretion by theca interna (Tc) and granulosa cells (Gc) cultured alone and in coculture were studied in cultured porcine follicular cells, prepared from small (SF), medium (MF) and large (LF) preovulatory follicles. We demonstrated that both Tc and Gc secrete IGF-I and that GH had no effect on IGF-I secretion by Tc but, increased IGF-I secretion by Gc isolated from SF and cultured alone or in coculture. IGF-I stimulated secretion of E2 by all cells, except in Tc derived from SF in which the effect was not statistically significant. The only stimulatory effect of concurrent treatment with GH on E2 secretion was noted in Tc derived from MF. IGF-I increased the [(3)H] thymidine incorporation in all Tc cells but GH did not augment this effect. In Gc, IGF-I stimulated [(3)H] thymidine incorporation in cells derived from SF and MF but not from LF. GH had no stimulatory effect except on Gc derived from LF and grown alone. The highest stimulatory effect was observed in SF. This was smaller in MF and no effect was noted in LF. In conclusion, our work shows that both Gc and Tc are sites of IGF-I production and for the first time shows the stimulatory role of IGF-I in proliferation of Tc cells derived from all types of follicles and augmentation of E2 secretion in Tc derived from MF and LF. The promotion of the mitogenic activity in Tc by IGF-I during all stages of follicular development suggests an important role for theca cells in follicular growth.     

Involvement of osteopontin upregulation on mesangial cells growth and collagen synthesis induced by intermittent high glucose

Glucose fluctuations are strong predictor of diabetic vascular complications. We explored the effects of constant and intermittent high glucose on the proliferation and collagen synthesis of cultured rat mesangial cells. Furthermore, the possible involvement of osteopontin (OPN) was assessed. In rat mesangial cells cultured in 5, 25, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, β3 integrin receptor and β5 integrin receptor, the cell proliferation, collagen synthesis, and the expression of OPN and type IV collagen were assessed. In cultured mesangial cells, treatment with constant or intermittent high glucose significantly increased [³H]thymidine incorporation in a time‐dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Treatment with constant high glucose for 48 h resulted in significant increases in [³H]thymidine incorporation, cell number, [³H]proline incorporation, mRNA, and protein levels of type IV collagen and OPN compared with mesangial cells treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose medium. In addition, neutralizing antibodies to either OPN or its receptor β3 integrin but not neutralizing antibodies to β5 integrin can effectively prevented proliferation and collagen synthesis of mesangial cells induced by constant or intermittent high glucose. Intermittent high glucose exacerbates mesangial cells growth and collagen synthesis by upregulation of OPN expression, indicating that glycemic variability have important pathological effects on the development of diabetic nephropathy, which is mediated by the stimulation of OPN expression and synthesis. J. Cell. Biochem. 109: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

Insulin-like growth factor-1 increases endothelin receptor A levels and action in cultured rat aortic smooth muscle cells

Insulin is known to cause an increase in endothelin-1 (ET-1) receptors in vascular smooth muscle cells (SMCs), but the effect of insulin-like growth factor 1 (IGF-1) on ET-1 receptor expression is not known. We therefore carried out the present study to determine the effect of IGF-1 on the binding of ET-1 to, and ET type A receptor (ETAR) expression and ET-1-induced 3H-thymidine incorporation in, vascular SMCs. In serum-free medium, IGF-1 treatment increased the binding of 125I-ET-1 to SMC cell surface ET receptors from a specific binding of 20.1%+/-3.1% per mg of protein in control cells to 45.1%+/-8.6% per mg of protein in cells treated with IGF-1 (10 nM). The effect of IGF-1 was dose-related, with a significant effect (1.4-fold) being seen at 1 nM. The minimal time for IGF-1 treatment to be effective was 30 min and the maximal effect was reached at 6 h. Immunoblotting analysis showed that ETAR expression in IGF-1-treated cells was increased by 1.7-fold compared to controls. Levels of ETAR mRNA measured by the RT-PCR method and Northern blotting were also increased by 2-fold in IGF-1-treated SMCs. These effects of IGF-1 were abolished by cycloheximide or genistein. Finally, ET-1-stimulated thymidine uptake and cell proliferation were enhanced by IGF-1 treatment, with a maximal increase of 3.2-fold compared to controls. In conclusion, in vascular SMCs, IGF-1 increases the expression of the ET-1 receptor in a dose- and time-related manner. This effect is associated with increased thymidine uptake and involves tyrosine kinase activation and new protein synthesis. These findings support the role of IGF-1 in the development of atherosclerotic, hypertensive, and diabetic vascular complications.     

Antihyperglycemic and antioxidant effects of a flavanone, naringenin, in streptozotocin–nicotinamide-induced experimental diabetic rats

In the present study, the putative antihyperglycemic and antioxidant effects of a flavanone, naringenin, were evaluated in comparison with those of glyclazide, a standard drug for therapy of diabetes mellitus. Diabetes was induced experimentally in 12-h-fasted rats by intraperitoneal injections of first streptozotocin (50 mg/kg b.w.) and then of nicotinamide (110 mg/kg b.w.) after a 15-min interval. Untreated diabetic rats revealed the following in comparison with normal rats: significantly higher mean levels of blood glucose and glycosylated hemoglobin, significantly lower mean levels of serum insulin, significantly lower mean activities of pancreatic antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase), significantly lower mean levels of plasma non-enzymatic antioxidants (reduced glutathione, vitamin C , vitamin E), significantly elevated mean levels of pancreatic malondialdehyde (MDA) and significantly elevated mean activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). Following oral administration of naringenin (50 mg/kg b.w./day) to diabetic rats for 21 days, the following observations were made in comparison with untreated diabetic rats: significantly lower mean levels of fasting blood glucose and glycosylated hemoglobin, significantly elevated serum insulin levels, significantly higher mean activities of pancreatic enzymatic antioxidants, significantly higher mean levels of plasma non-enzymatic antioxidants, lower mean pancreatic tissue levels of MDA and lower mean activities of ALT, AST, ALP and LDH in serum. The values obtained in the naringenin-treated animals approximated those observed in glyclazide-treated animals. Histopathological studies appeared to suggest a protective effect of naringenin on the pancreatic tissue in diabetic rats. These results suggest that naringenin exhibits antihyperglycemic and antioxidant effects in experimental diabetic rats.     

Inhibition by diethylstilboestrol of proliferative potential of follicles of different sizes in immature rat ovaries

Intact immature female rats were treated with 1, 2, 3 or 4 subcutaneous injections of 2 mg diethylstilboestrol (DES)/rat at intervals of 24 h and then killed. Ovaries were collected, cleaned, enzymically digested and serially filtered through Teflon sieves to yield follicles of diameter less than 200 microns (small), 200-400 microns (medium) and greater than 400 microns (large). Follicular supernatant was collected and granulosa cells were extracted from these isolated follicles. There was a general increase in [3H]thymidine incorporation in all sizes of follicles after 1 or 2 DES injections, the increase in the medium and large follicles being significant after 2 doses. With 3 and 4 injections of DES, there was a sudden decrease in the rates of [3H]thymidine incorporation, particularly in the medium-sized follicles, which also had higher concentrations of follicular supernatant protein. Protein contents in small and large follicles did not change significantly. The follicular supernatant protein had a specific and dose-dependent inhibitory effect on [3H]thymidine incorporation when added to cultures of rapidly dividing granulosa cells. Addition of the same amounts of bovine serum albumin (BSA) to the cultures had no effect. Heat-denaturing did not abolish the inhibition by the protein. Removal of the protein from the cultures after the first 48 h resulted in a rebound increase in [3H]thymidine incorporation during the following 48 h, showing that the inhibitory effects were reversible. Though aromatase activity after 1 or 2 DES injections abruptly decreased after 3 and 4 injections, follicular supernatant protein had no effect on steroidogenesis in cultured granulosa cells. Taken together, these findings suggest that oestrogen can inhibit follicular development, depending on the duration of exposure. We propose that the inhibitory effects of DES on cell proliferation are mediated via the synthesis of a specific peptide factor which is produced in high amounts in the medium-sized follicles only, on prolonged exposure to the oestrogen. This factor may be autocrine or paracrine, serving as an in-built autoregulatory control mechanism for follicle development, particularly at pro-oestrus, when oestrogen concentrations are highest.