Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates insulin secretion in a glucose-dependent manner. Selective GLP-1 secretagogue would be one of the potential therapeutic targets for type 2 diabetes. Here, we describe a newly identified small molecule compound (compound A) that stimulates secretion of GLP-1 in murine enteroendocrine cell lines, STC-1 and GLUTag cells, and in primary cultured fetal rat intestinal cells (FRIC). The underlying mechanism by which compound A stimulated GLP-1 secretion was also examined. Compound A stimulated GLP-1 secretion from STC-1 cells in a concentration-dependent manner, and also from GLUTag cells and FRIC. The action of compound A was selective against other tested endocrine functions such as secretion of insulin from rat islets, growth hormone from rat pituitary gland cells, and norepinephrine from rat PC-12 cells. In STC-1 cells, the compound A-stimulated GLP-1 secretion was neither due to cyclic AMP production nor to Ca2+ release from intracellular stores, but to extracellular Ca2+ influx. The response was inhibited by the presence of either L-type Ca2+ channel blockers or K+ ionophore. Perforated-patch clamp study revealed that compound A induces membrane depolarization. These results suggest that neither Gαs- nor Gαq-coupled signaling account for the mechanism of action, but depolarization-coupled Ca2+ influx from extracellular space is the primary cause for the GLP-1 secretion stimulated by compound A. Identifying a specific target molecule for compound A will reveal a selective regulatory pathway that leads to depolarization-mediated GLP-1 secretion.
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- Abstract: Diabetes x
- Abstract: Islets x
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- Abstract: BetaCells x
- Abstract: Pancreas x
- Abstract: Obesity x
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- Abstract: Hypoglycemia x
- Abstract: Insulinoma x
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- Abstract: IGF* x
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- Abstract: Type 2 x
Jun-ichi Eiki, Kaori Saeki, Norihiro Nagano, Tomoharu Iino, Mari Yonemoto, Yoko Takayenoki-Iino, Satoru Ito, Teruyuki Nishimura, Yoshiyuki Sato, Makoto Bamba, Hitomi Watanabe, Kaori Sasaki, Sumika Ohyama, Akio Kanatani, Toshio Nagase and Toshihiko Yada
C Y Shan, J H Yang, Y Kong, X Y Wang, M Y Zheng, Y G Xu, Y Wang, H Z Ren, B C Chang and L M Chen
For centuries, Berberine has been used in the treatment of enteritis in China, and it is also known to have anti-hyperglycemic effects in type 2 diabetic patients. However, as Berberine is insoluble and rarely absorbed in gastrointestinal tract, the mechanism by which it works is unclear. We hypothesized that it may act locally by ameliorating intestinal barrier abnormalities and endotoxemia. A high-fat diet combined with low-dose streptozotocin was used to induce type 2 diabetes in male Sprague Dawley rats. Berberine (100 mg/kg) was administered by lavage to diabetic rats for 2 weeks and saline was given to controls. Hyperinsulinemia and insulin resistance improved in the Berberine group, although there was no significant decrease in blood glucose. Berberine treatment also led to a notable restoration of intestinal villi/mucosa structure and less infiltration of inflammatory cells, along with a decrease in plasma lipopolysaccharide (LPS) level. Tight junction protein zonula occludens 1 (ZO1) was also decreased in diabetic rats but was restored by Berberine treatment. Glutamine-induced glucagon-like peptide 2 (GLP2) secretion from ileal tissue decreased dramatically in the diabetic group but was restored by Berberine treatment. Fasting insulin, insulin resistance index, plasma LPS level, and ZO1 expression were significantly correlated with GLP2 level. In type 2 diabetic rats, Berberine treatment not only augments GLP2 secretion and improves diabetes but is also effective in repairing the damaged intestinal mucosa, restoring intestinal permeability, and improving endotoxemia. Whether these effects are mechanistically related will require further studies, but they certainly support the hypothesis that Berberine acts via modulation of intestinal function.
GE Rice, MH Wong, W Farrugia and KF Scott
Although phospholipase A2 (PLA2) enzymatic activities have been implicated in the regulation of phospholipid metabolism and eicosanoid formation in human gestational tissues, the role and contribution made by individual PLA2 isozymes has not been established. The aim of this study, therefore, was to determine the contribution made by Type II PLA2 to PLA2 enzymatic activity present in human term placenta. The experimental paradigm used to establish the contribution made by Type II PLA2 to total in vitro PLA2 enzymatic activity present in placental extracts was to remove Type II PLA2 by immunoaffinity extraction and then to quantify residual PLA2 enzymatic activity. Before immunoaffinity extraction, Type II PLA2 immunoactivity and total PLA2 enzymatic activity present in placental extracts averaged 28.0 +/- 10.0 ng/mg protein and 1040 +/- 367 pmol/h per mg protein (n = 3) respectively. After solid-phase immunoaffinity batch extraction of placental extracts, immunoreactive Type II PLA2 was not detectable by ELISA, and PLA2 enzymatic activity was decreased by 82 +/- 1% (P < 0.001). Residual (i.e. non-Type II) PLA2 enzymatic activity was further characterised by Western blot analysis and enzyme activity assay. The data obtained are consistent with a contribution by both cytosolic PLA2 and other secretory PLA2 isozymes (i.e. non-Type II) to residual PLA2 enzymatic activity. The results obtained in this study support the conclusion that Type II PLA2 is quantitatively the primary PLA2 isozyme that contributes to in vitro PLA2 enzymatic activity present in extracts of human term placenta, accounting for at least 80% of total activity. These data further support the involvement of this extracellularly active isozyme in the regulation of placental phospholipid metabolism and eicosanoid formation during late gestation.
SJ Fisher, ZQ Shi, HL Lickley, S Efendic, M Vranic and A Giacca
At supraphysiological levels, IGF-I bypasses some forms of insulin resistance and has been proposed as a therapeutic agent in the treatment of diabetes. Unfortunately, side effects of high-dose IGF-I (100-250 microg/kg) have precluded its clinical use. Low-dose IGF-I (40-80 microg/kg), however, shows minimal side effects but has not been systematically evaluated. In our previous study under conditions of declining glucose, low-dose IGF-I infusion was more effective in stimulating glucose utilization, but less effective in suppressing glucose production and lipolysis than low-dose insulin. However, under conditions of hyperglycemia, we could not observe any differential effects between high-dose infusions of IGF-I and insulin. To determine whether the differential effects of IGF-I and insulin are dose-related or related to the prevailing glucose level, 3 h glucose clamps were performed in the same animal model as in the previous studies, i.e. the moderately hyperglycemic (175 mg/dl) insulin-infused depancreatized dog, with additional infusions of low-dose IGF-I (67.8 microg/kg, i.e. 29.1 microg/kg bolus plus 0.215 microg/kg( )per min infusion; n=5) or insulin 49.5 mU/kg (9 mU/kg bolus plus 0.45 mU/kg per min; n=7). As in the previous study under conditions of declining glucose, low-dose IGF-I had significant metabolic effects in vivo, in our model of complete absence of endogenous insulin secretion. Glucose production was similarly suppressed with both IGF-I and insulin, by 54+/-3 and 56+/-2% s.e. (P=NS) respectively. Glucose utilization was stimulated to the same extent (IGF-I 5.2+/-0.2, insulin 5.5+/-0.3 mg/kg per min, P=NS). Glucagon, free fatty acid, glycerol, alanine and beta-hydroxybutyrate, were suppressed, while lactate and pyruvate levels were raised, similarly with IGF-I and insulin. We conclude that: (i) differential effects of IGF-I and insulin may be masked under hyperglycemic conditions, independent of the hormone dose; (ii) low-dose IGF-I has no selective advantage over additional insulin in suppressing glucose production and lipolysis, nor in stimulating glucose utilization during hyperglycemia and subbasal insulin infusion when insulin secretion is absent, as in type 1 diabetes mellitus.
R. C. Bonney, S. T. Qizilbash and S. Franks
The inhibition of endometrial phospholipase A2 activity by the non-steroidal anti-inflammatory agents mefenamic acid and indomethacin was studied over the concentration range 1 mmol/l–0·1 μmol/l. Both phospholipase A2 type 1 (a calcium-dependent enzyme) and phospholipase A2 type 2 (a calciumindependent enzyme) were inhibited by mefenamic acid, but the magnitude of the inhibition was dependent on calcium concentration. Phospholipase A2 type 1 was inhibited 50% by 10 μmol mefenamic acid/1 in the presence of 1·25–5 mmol calcium/l, but a concentration of 2·2 mmol mefenamic acid/l was required for 50% inhibition in the absence of calcium. On the other hand, phospholipase A2 type 2 was inhibited 50% by 22 μmol mefenamic acid/1 in the absence of calcium and by 100 μmol mefenamic acid/l in the presence of calcium (2·5 mmol/l). Although indomethacin was a less effective inhibitor of phospholipase A2 activity, a similar relationship with calcium was demonstrated. However, indomethacin also had a stimulatory effect on phospholipase A2 type 1 activity in the absence of calcium. Our findings suggest that the two endometrial enzymes may be inhibited by different mechanisms and that the dependence of the enzyme on calcium for activation may be a contributing factor.
J. Endocr. (1988) 119, 141–145
G Üçkaya, P Delagrange, A Chavanieu, G Grassy, M-F Berthault, A Ktorza, E Cerasi, G Leibowitz and N Kaiser
Glucagon-like peptide 1 (GLP-1) analogues are considered potential drugs for type 2 diabetes. We studied the effect of a novel GLP-1 analogue, S 23521 ([a8-des R36] GLP-1-[7–37]-NH2), on the metabolic state and β-cell function, proliferation and survival in the Psammomys obesus model of diet-induced type 2 diabetes. Animals with marked hyperglycaemia after 6 days of high-energy diet were given twice-daily s.c. injection of 100 μg/kg S 23521 for 15 days. Food intake was significantly decreased in S 23251-treated P. obesus; however, there was no significant difference in body weight from controls. Progressive worsening of hyperglycaemia was noted in controls, as opposed to maintenance of pre-treatment glucose levels in the S 23521 group. Prevention of diabetes progression was associated with reduced mortality. In addition, the treated group had higher serum insulin, insulinogenic index and leptin, whereas plasma triglyceride and non-esterified fatty acid levels were decreased. S 23521 had pronounced effect on pancreatic insulin, which was 5-fold higher than the markedly depleted insulin reserve of control animals. Immunohistochemical analysis showed islet degranulation with disrupted morphology in untreated animals, whereas islets from S 23521-treated animals appeared intact and filled with insulin; β-cell apoptosis was approximately 70% reduced, without a change in β-cell proliferation. S 23521 treatment resulted in a 2-fold increase in relative β-cell volume. Overall, S 23521 prevented the progression of diabetes in P. obesus with marked improvement of the metabolic profile, including increased pancreatic insulin reserve, β-cell viability and mass. These effects are probably due to actions of S 23521 both directly on islets and via reduced food intake, and emphasize the feasibility of preventing blood glucose deterioration over time in type 2 diabetes.
Maaike M Roefs, Françoise Carlotti, Katherine Jones, Hannah Wills, Alexander Hamilton, Michael Verschoor, Joanna M Williams Durkin, Laura Garcia-Perez, Melissa F Brereton, Laura McCulloch, Marten A Engelse, Paul R V Johnson, Barbara C Hansen, Kevin Docherty, Eelco J P de Koning and Anne Clark
Type 2 diabetes (T2DM) is associated with pancreatic islet dysfunction. Loss of β-cell identity has been implicated via dedifferentiation or conversion to other pancreatic endocrine cell types. How these transitions contribute to the onset and progression of T2DM in vivo is unknown. The aims of this study were to determine the degree of epithelial-to-mesenchymal transition occurring in α and β cells in vivo and to relate this to diabetes-associated (patho)physiological conditions. The proportion of islet cells expressing the mesenchymal marker vimentin was determined by immunohistochemistry and quantitative morphometry in specimens of pancreas from human donors with T2DM (n = 28) and without diabetes (ND, n = 38) and in non-human primates at different stages of the diabetic syndrome: normoglycaemic (ND, n = 4), obese, hyperinsulinaemic (HI, n = 4) and hyperglycaemic (DM, n = 8). Vimentin co-localised more frequently with glucagon (α-cells) than with insulin (β-cells) in the human ND group (1.43% total α-cells, 0.98% total β-cells, median; P < 0.05); these proportions were higher in T2DM than ND (median 4.53% α-, 2.53% β-cells; P < 0.05). Vimentin-positive β-cells were not apoptotic, had reduced expression of Nkx6.1 and Pdx1, and were not associated with islet amyloidosis or with bihormonal expression (insulin + glucagon). In non-human primates, vimentin-positive β-cell proportion was larger in the diabetic than the ND group (6.85 vs 0.50%, medians respectively, P < 0.05), but was similar in ND and HI groups. In conclusion, islet cell expression of vimentin indicates a degree of plasticity and dedifferentiation with potential loss of cellular identity in diabetes. This could contribute to α- and β-cell dysfunction in T2DM.
LM Thurston, E Chin, KC Jonas, IJ Bujalska, PM Stewart, DR Abayasekara and AE Michael
In a range of tIssues, cortisol is inter-converted with cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). To date, two isoforms of 11betaHSD have been cloned. Previous studies have shown that human granulosa cells express type 2 11betaHSD mRNA during the follicular phase of the ovarian cycle, switching to type 1 11betaHSD mRNA expression as luteinization occurs. However, it is not known whether protein expression, and 11betaHSD enzyme activities reflect this reported pattern of mRNA expression. Hence, the aims of the current study were to investigate the expression and activities of 11betaHSD proteins in luteinizing human granulosa-lutein (hGL) cells. Luteinizing hGL cells were cultured for up to 3 days with enzyme activities (11beta-dehydrogenase (11betaDH) and 11-ketosteroid reductase (11 KSR)) and protein expression (type 1 and type 2 11betaHSD) assessed on each day of culture. In Western blots, an immunopurified type 1 11betaHSD antibody recognized a band of 38 kDa in hGL cells and in human embryonic kidney (HEK) cells stably transfected with human type 1 11betaHSD. The type 2 11betaHSD antibody recognized a band of 48 kDa in HEK cells transfected with human type 2 11betaHSD cDNA but the type 2 protein was not expressed in hGL cells throughout the 3 days of culture. While the expression of type 1 11betaHSD protein increased progressively by 2.7-fold over 3 days as hGL cells luteinized, both 11betaDH and reductase activities declined (by 52.9% and 34.2%; P<0.05) over this same period. Changes in enzyme expression and activity were unaffected by the suppression of ovarian steroid synthesis.
Akhilesh K Pandey, Wei Li, Xiangling Yin, Douglas M Stocco, Paula Grammas and XingJia Wang
Previous studies have reported the roles of Ca2+ in steroidogenesis. The present study has investigated an inhibitory effect of Ca2+ influx through L-type Ca2+ channels on gene expression of steroidogenic acute regulatory (STAR) protein that regulates the transfer of substrate cholesterol to the inner mitochondrial membrane for steroidogenesis. Blocking Ca2+ influx through L-type Ca2+ channels using the selective Ca2+ channel blocker, nifedipine, markedly enhanced cAMP-induced STAR protein expression and progesterone production in MA-10 mouse Leydig cells. This was confirmed by utilization of different L-type Ca2+ channel blockers. Reverse transcription-PCR analyses of Star mRNA and luciferase assays of Star promoter activity indicated that blocking Ca2+ influx through L-type Ca2+ channels acted at the level of Star gene transcription. Further studies showed that blocking the Ca2+ channel enhanced Star gene transcription by depressing the expression of DAX-1 (NR0B1 as listed in the MGI Database) protein, a transcriptional repressor of Star gene expression. It was also observed that there is a synergistic interaction between nifedipine and cAMP. Normally, sub-threshold levels of cAMP are unable to induce steroidogenesis, but in the presence of the L-type Ca2+ channel blocker, they increased STAR protein and steroid hormone to the maximal levels. However, in the absence of minimal levels of cAMP, none of the L-type Ca2+ channel blockers are able to induce Star gene expression. These observations indicate that Ca2+ influx through L-type Ca2+ channels is involved in an inhibitory effect on Star gene expression. Blocking L-type Ca2+ channel attenuated the inhibition and reduced the threshold of cAMP-induced Star gene expression in Leydig cells.
SG Derman, S Kol, I Ben-Shlomo, CE Resnick, RM Rohan and EY Adashi
Transforming growth factor beta1 (TGFbeta1) acts as an inhibitor of the actions of interleukin-1beta (IL-1beta) in various organ systems. In order better to understand the inter|P-actions between these polypeptides in the ovary, we evaluated the effect of TGFbeta1 co-treatment on various IL-1beta-mediated actions in cultures of whole ovarian dispersates. Treatment with IL-1beta enhanced media accumulation of nitrites (4.8-fold), prostaglandin E2 (PGE2, 3. 9-fold) and lactate (2.0-fold), and enhanced glucose consumption (2. 1-fold). Treatment with TGFbeta1 alone did not significantly affect any of these parameters. However, the addition of TGFbeta1 inhibited IL-1beta-stimulated nitrite (100%), PGE2 (44%) and lactate (78%) accumulation and inhibited IL-1beta-stimulated glucose consumption (74%) in a dose-dependent manner. The addition of TGFbeta1 also suppressed the steady-state levels of IL-1beta-stimulated IL-1beta, type I IL-1 receptor and IL-1 receptor antagonist transcripts (98, 67 and 83% inhibition respectively). These data suggest that TGFbeta1 is capable of inhibiting several IL-1beta-stimulated endpoints. Since IL-1 has been identified as a possible proinflammatory mediator of ovulation and TGFbeta has been implicated as a promotor of fibrosis and healing, we speculate that IL-1 and TGFbeta might play antagonistic roles in the normal ovulatory sequence.