Molecular mechanisms of cell cycle control in the mouse Y1 adrenal cell line

Y1 adrenocortical tumor cells possess amplified and overexpressed c-Ki-ras proto-oncogene, displaying chronic high levels of the c-Ki-Ras-GTP protein. Despite this oncogenic lesion, we previously reported that Y1 cells retain tight regulatory mechanisms of cell cycle control typified by the mitogenic response triggered by FGF2 in G0/G1-arrested cells. ACTH, on the other hand, elicits cAMP/PKA-mediated antimitogenic mechanisms involving Akt/PKB dephosphorylation/deactivation and c-Myc protein degradation, blocking G1 phase progression stimulated by FGF2. In this paper we report that ACTH does not directly antagonize any of the early or late sequential steps comprising the mitogenic response triggered by FGF2. In effect, ACTH targets deactivation of constitutively phosphorylated-Akt, restraining the potential of c-Ki-Ras-GTP to subvert Y1 cell cycle control. Thus, we can consider ACTH a tumor suppressor rather than an antimitogenic hormone. In addition, we present initial results showing that high constitutive levels of c-Ki-Ras-GTP render Y1 cells susceptible to dye upon FGF2 treatment. This surprising FGF2 death-effect, that is independent of the well known FGF2-mitogenic activity, might involve a natural unsuspected mechanism for restraining oncogene-induced proliferation.     

Cell line-specific differences in the control of cell cycle progression in the absence of mitosis.

This paper reports that there are major differences between mammalian cell lines in the propensity to progress into subsequent cell cycles when mitosis is inhibited with agents that disrupt the assembly of the mitotic spindle apparatus (Colcemid, nocodazole, and taxol). Human HeLa S3 cells, which represent one extreme, remain arrested in mitosis, with elevated levels of cyclin B and p34cdc2 kinase activity. In Chinese hamster ovary cells, at the other extreme, the periodic rise and fall of cyclin B levels and p34cdc2 kinase activity is only transiently inhibited in the absence of mitosis. The cells progress into subsequent cell cycles, without dividing, resulting in serial doublings of cellular DNA content. In general, the propensity to progress into subsequent cell cycles in the absence of mitosis appears to be species related, such that human cell lines remain permanently blocked in a mitotic state, whereas rodent cell lines are only transiently inhibited when spindle assembly is disrupted. We interpret these results to indicate that in mammalian cell lines there exists a checkpoint which serves to couple cell cycle progression to the completion of certain karyokinetic events. Furthermore, either such a checkpoint exists in some cell lines but not in others or the stringency of the control mechanism varies among different cell lines.     

Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function

Acetylcholine (ACh), the major parasympathetic neurotransmitter, is released by intrapancreatic nerve endings during the preabsorptive and absorptive phases of feeding. In beta-cells, ACh binds to muscarinic M(3) receptors and exerts complex effects, which culminate in an increase of glucose (nutrient)-induced insulin secretion. Activation of PLC generates diacylglycerol. Activation of PLA(2) produces arachidonic acid and lysophosphatidylcholine. These phospholipid-derived messengers, particularly diacylglycerol, activate PKC, thereby increasing the efficiency of free cytosolic Ca(2+) concentration ([Ca(2+)](c)) on exocytosis of insulin granules. IP3, also produced by PLC, causes a rapid elevation of [Ca(2+)](c) by mobilizing Ca(2+) from the endoplasmic reticulum; the resulting fall in Ca(2+) in the organelle produces a small capacitative Ca(2+) entry. ACh also depolarizes the plasma membrane of beta-cells by a Na(+)- dependent mechanism. When the plasma membrane is already depolarized by secretagogues such as glucose, this additional depolarization induces a sustained increase in [Ca(2+)](c). Surprisingly, ACh can also inhibit voltage-dependent Ca(2+) channels and stimulate Ca(2+) efflux when [Ca(2+)](c) is elevated. However, under physiological conditions, the net effect of ACh on [Ca(2+)](c) is always positive. The insulinotropic effect of ACh results from two mechanisms: one involves a rise in [Ca(2+)](c) and the other involves a marked, PKC-mediated increase in the efficiency of Ca(2+) on exocytosis. The paper also discusses the mechanisms explaining the glucose dependence of the effects of ACh on insulin release.     

Determinants of pancreatic beta-cell regeneration

Recent studies have revealed a surprising plasticity of pancreatic beta-cell mass. beta-cell mass is now recognized to increase and decrease in response to physiological demand, for example during pregnancy and in insulin-resistant states. Moreover, we and others have shown that mice recover spontaneously from diabetes induced by killing of 70-80% of beta-cells, by beta-cell regeneration. The major cellular source for new beta-cells following specific ablation, as well as during normal homeostatic maintenance of adult beta-cells, is proliferation of differentiated beta-cells. More recently, it was shown that one form of severe pancreatic injury, ligation of the main pancreatic duct, activates a population of embryonic-type endocrine progenitor cells, which can differentiate into new beta-cells. The molecular triggers for enhanced beta-cell proliferation during recovery from diabetes and for activation of embryonic-type endocrine progenitors remain unknown and represent key challenges for future research. Taken together, recent data suggest that regenerative therapy for diabetes may be a realistic goal.     

Impaired development of pancreatic beta-cell mass is a primary event during the progression to diabetes in the GK rat

Summary In the endocrine pancreas of the GK rat, a genetic model of non-insulin-dependent diabetes mellitus (NIDDM), it is not clear whether the histopathological changes reported up to now are related to the pathogenesis of hyperglycaemia or whether they occur secondarily to metabolic alterations. Using GK rats from the Paris colony, our study chronicles for the first time the pathophysiologic changes that occur in the GK pancreas from the late fetal period (day 21.5) until adult age (18 weeks). As compared to Wistar controls, GK fetuses exhibited higher plasma glucose level, lower plasma insulin level and normal plasma glucagon level. Their pancreatic insulin content and the relative volume and the total mass of their beta cells were sharply decreased, representing only 23, 38 and 23 % of control values, respectively. During the period from 4 days to 14 days after birth, GK neonates exhibited normal basal plasma glucose and glucagon levels despite decreased plasma insulin level. Their pancreatic insulin content represented only 31–40 % of values found in the age-related control pancreases and their total beta-cell mass was only 35 % on day 4, 30 % on day 7 and 37 % on day 14. The adult diabetic GK rats exhibited higher basal plasma glucose and insulin levels while their basal plasma glucagon level remained normal. Their pancreatic insulin content and the total beta-cell mass remained decreased, representing only 32 % and 47 % of control values, respectively. Moreover, the adult GK pancreases exhibited noticeable alteration in the architecture of the large islet sub-population which displayed considerable fibrosis with clusters of beta cells widely separated from each other by strands of connective tissue. Concerning the development of alpha cells in the GK rats, their relative volume was found to be normal during fetal and early neonatal periods. It was found to be moderately decreased (representing 64–67 % of corresponding control values) in 14-day-old neonates and adult GK rats. Our findings demonstrate that in the GK rat, the deficit of total beta-cell mass as observed in the adult animal is related to impaired beta-cell development. The restriction of the beta-cell mass must be considered as a primary and crucial event in the sequence leading to overt diabetes in this NIDDM model. [Diabetologia (1997) 40: 916–925] Received: 6 February 1997 and in revised form: 21 April 1997     

Novel aspects on signal-transduction in the pancreatic beta-cell

The glucose-stimulus/insulin-secretion-coupling by the pancreatic beta-cell, which guarantees the maintenance of glucose homeostasis in man, is regulated by a sophisticated interplay between glucose and a plethora of additional factors. Besides other nutrients, incretins, nerval innervation, systemic growth factors as well as autocrine and paracrine regulatory loops within the islet of Langerhans modulate the function of the insulin-producing beta-cell. Although the modulatory role of these factors is well appreciated, the underlying molecular mechanisms involved remain poorly understood. However, in most cases beta-cell membrane receptors coupled primarily to either G-proteins or tyrosine kinases, which subsequently activate respective second messenger cascades, are involved. In the present mini-review we will discuss the role of signaling through some of these receptor-operated effector systems in the light of pancreatic beta-cell signal-transduction.     

胰腺星状细胞凋亡研究进展

胰腺纤维化是各类慢性胰腺炎的共同特征,胰腺星状细胞(PSC)在胰腺纤维化进程中起重要作用。诱导胰腺星状细胞发生凋亡不仅可抑制纤维化的进展,而且还能为慢性胰腺炎的治疗提供可能的理想途径。但目前胰腺星状细胞凋亡的具体机制尚未完全明了,国内尚无相关报道。     

Physiologic relevance of heterogeneity in the pancreatic beta-cell population

Diabetologia - In vitro studies on purified rat beta cells have indicated a functional diversity among insulincontaining cells. Intercellular differences were found in the rates of glucose-induced...     

Physiologic relevance of heterogeneity in the pancreatic beta-cell population

In vitro studies on purified rat beta cells have indicated a functional diversity among insulincontaining cells. Intercellular differences were found in the rates of glucose-induced insulin synthesis and release. They are attributed to differences in cellular thresholds for glucose utilization and oxidation, as can be caused by varying activities in rate limiting steps such as glucokinase-dependent phosphorylation. The percent of functionally active beta cells increases dose-dependently with the glucose concentration, making cellular heterogeneity and its regulation by glucose major determinants for the dose-response curves of the total beta-cell population. Beta cells which are already responsive to low glucose concentrations are characterized by a higher content in pale immature granules; their activated biosynthetic and secretory activity accounts for preferential release of newly-formed hormone by the total beta-cell population. At any glucose level, the amplitude of insulin release depends on the percent glucose-activated cells and their cyclic AMP content, an integrator of (neuro)hormonal influences. The in vitro described heterogeneity in beta-cell functions may bear physiological relevance as several of its characteristics are also detectable in intact pancreatic tissue; furthermore, in vitro signs of heterogeneity can be altered by prior in vivo treatment indicating that they express properties of the cells in their in situ configuration. Elevated basal levels of (pro)insulin may reflect the existence of an increased number of beta cells that are activated at low physiologic glucose concentrations. Reductions in stimulated insulin levels can be caused by decreased numbers of beta cells that are activated at the prevailing glucose concentration or by insufficient cyclic AMP levels in beta cells, possibly as a result of inadequate signalling from hormones of local or distal origin. Only few markers are currently available with which to explore these mechanisms in vivo. Additional markers and tests should help assess the possible role of variations in beta-cell heterogeneity in the pathogenesis of diabetes mellitus.     

Parallel signaling pathways of melatonin in the pancreatic beta-cell

Previous results demonstrated that melatonin inhibits cAMP production and stimulates IP(3) liberation in rat insulinoma INS1 cells, a model for the pancreatic beta-cell. This study addresses the impact of melatonin on insulin release. Insulin, cAMP and IP(3) levels of INS1 cells in a superfusion system were measured. Initially, forskolin was used to stimulate cAMP and subsequently insulin release. Incubation of forskolin (5 micromol/L)-stimulated cells with melatonin (100 nmol/L) inhibited cAMP and insulin levels (down to 60% of insulin and cAMP release). The G(i)alpha-protein-inhibitor pertussis toxin (PTX) was used to distinguish between the G(i)alpha-dependent cAMP pathway and the G(i)alpha-independent IP(3) pathway. In our experiments we employed a specific stimulation pattern to prove proper inhibition of G(i)alpha-proteins by PTX. In INS1 cells incubated with 250 ng/mL PTX for 24 hr, melatonin was no longer able to inhibit the forskolin-induced cAMP and insulin release. In a study, carbachol was used to stimulate IP(3) and subsequently insulin release. Surprisingly, incubation of carbachol (300 micromol/L)-stimulated cells with melatonin (100 nmol/L) inhibited insulin release (down to 75% of insulin release). Finally, in PTX-incubated INS1 cells, melatonin (100 nmol/L) increased carbachol (300 micromol/L)-induced insulin release (up to 124% of insulin release). In conclusion, we found that the melatonin MT(1)-receptor on pancreatic beta-cells is coupled to parallel signaling pathways, with opposite influences on insulin secretion. The cAMP- and subsequently insulin-inhibiting signaling pathway involves PTX-sensitive G(i)alpha-proteins and is predominant in terms of insulin release.     

Effect of creatine on the pancreatic beta-cell.

We report on the stimulatory effect of creatine on insulin secretion and ATP concentration in MIN-6 beta-cells. The addition of creatine (5 mM) to MIN-6 cells in the presence of glucose (1-10 mM) elicited a significant (p<0.001) increase in insulin secretion, but no effect was demonstrated in the absence of glucose. The lack of effect of creatine in the absence of glucose suggests that creatine may act as a potentiator of insulin secretion rather than as an initiator. The potentiatory effect of creatine is specific for glucose since no effect was found in the presence of other known initiators of insulin secretion (K(+), 2-ketoisocaproic acid and tolbutamide). Cellular ATP content was markedly increased by glucose (1-15 mM). Creatine (5 and 10 mM) further increased the ATP level at all glucose concentrations, and the effect was observed even in the absence of glucose. The results from this study demonstrate the ability of creatine to increase insulin secretion only in the presence of glucose, while its effect on increased cellular ATP was independent of the presence of glucose. The mechanism whereby creatine potentiates insulin release is yet to be investigated. However, our data suggest possible unique interactions between creatine and the glucose-dependent insulin secretory pathway.     

Transformed mammalian cells are deficient in kinase-mediated control of progression through the G1 phase of the cell cycle.

To investigate the role of kinase-mediated mechanisms in regulating mammalian cell proliferation, we determined the effects of the general protein kinase inhibitor staurosporine on the proliferation of a series of nontransformed and transformed cultured rodent and human cells. Levels of staurosporine as low as 1 ng/ml prevented nontransformed cells from entering S phase (i.e., induced G1 arrest), indicating that kinase-mediated processes are essential for commitment to DNA replication in normal cells. At higher concentrations of staurosporine (50-75 ng/ml), nontransformed mammalian cells were arrested in both G1 and G2. The period of sensitivity of nontransformed human diploid fibroblasts to low levels of the drug commenced 3 hr later than the G0/G1 boundary and extended through the G1/S boundary. Interference with activity of the G1-essential kinase(s) caused nontransformed human cells traversing mid-to-late G1 at the time of staurosporine addition to be "set back" to the initial staurosporine block point, suggesting the existence of a kinase-dependent "G1 clock" mechanism that must function continuously throughout the early cycle in normal cells. The initial staurosporine block point at 3 hr into G1 corresponds to neither the serum nor the amino acid restriction point. In marked contrast to the behavior of nontransformed cells, neither low nor high concentrations of staurosporine affected G1 progression in transformed cultures; high drug concentrations caused transformed cells to be arrested solely in G2. These results indicate that kinase-mediated regulation of DNA replication is lost as the result of neoplastic transformation, but the G2-arrest mechanism remains intact.     

Forskolin-induced desensitization of pancreatic beta-cell insulin secretory responsiveness: possible involvement of impaired information flow in the inositol-lipid cycle.

Isolated rat islets of Langerhans were incubated for 2 h in a myo-[2-3H]inositol-containing solution to label their phosphoinositides. Also included during this labeling period was forskolin (0.1-5 microM), a compound established to elevate islet cAMP levels. These islets were subsequently perifused, and their insulin secretory responses to 20 mM glucose or 1 microM of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) were assessed. Determined in parallel with secretion were [3H] inositol efflux patterns and, at the termination of the perifusion, labeled inositol phosphate accumulation. The following major observations were made. 1) Forskolin had no deleterious effect on the total amount of [3H]inositol incorporated by the islets during the labeling period. 2) However, labeling in forskolin resulted in subsequent dose-dependent decreases in 20 mM glucose-induced insulin secretion, [3H]inositol efflux and inositol phosphate accumulation. 3) Inclusion of the diacylglycerol (DAG) kinase inhibitor monooleoylglycerol (50 microM) restored to a significant degree glucose-induced release from forskolin-desensitized islets. 4) Pretreatment with 5 microM forskolin had no deleterious effect on TPA-induced insulin release. 5) Prior exposure to forskolin also impaired phosphoinositide hydrolysis in response to cholecystokinin stimulation. 6) Similar to forskolin, labeling in isobutylmethylxanthine (1 mM) reduced in a parallel fashion islet [3H]inositol efflux and insulin secretion in response to 20 mM glucose stimulation. These findings demonstrate that prior chronic elevation of islet cAMP levels suppresses the activation of phospholipase-C in response to subsequent stimulation. Defective insulin secretory responsiveness of these islets appears to be the result of impaired generation of phosphoinositide-derived second messenger molecules, particularly DAG. By substituting for DAG, however, TPA circumvents this biochemical lesion and evokes a normal insulin secretory response from forskolin-pretreated islets.     

Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2

Summary Cytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1, tumour necrosis factor-, and interferon-) induced apoptotic cell death in the mouse pancreatic beta-cell line TC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line TC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in TC1 cells. The abundance of endogenous Bcl-2 in TC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in TC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.Abbreviations IDDM Insulin-dependent diabetes mellitus - IL interleukin - TNF tumour necrosis factor - IFN interferon - FBS fetal bovine serum - ATA aurintricarboxylic acid - CHX cycloheximide - PI propidium iodide     

Growth factor-dependent proliferation of the pancreatic beta-cell line betaTC-tet: an assay for beta-cell mitogenic factors

The ability to expand normal pancreatic islet beta cells in culture would significantly advance the prospects of cell therapy for diabetes. A number of growth factors can stimulate limited islet cell replication, however other factors may exist which are more effective beta-cell-specific mitogens. The search for novel beta-cell growth factors has been hampered by the lack of a beta-cell-specific proliferation assay. We developed a simple and sensitive assay for beta-cell growth factors based on a conditionally-transformed mouse beta-cell line (betaTC-tet). These cells express the SV40 T antigen (Tag) oncoprotein under control of the tetracycline (Tc) operon regulatory system. In the presence of Tc, Tag expression is tightly shut off and the cells undergo complete growth arrest. Here we show that the growth-arrested cells can proliferate in response to growth factors in the absence of Tag. Using this assay, a number of growth factors previously shown to be mitogenic to a mixed islet cell population were found to induce proliferation of pure beta cells. We conclude that growth-arrested betaTC-tet cells can be employed in a survey of factors from various sources for identifying novel factors with beta-cell mitogenic activity.     

Regulation of cell cycle progression by calcium/calmodulin-dependent pathways

Many hormones, growth factors, and cytokines regulate proliferation of their target cells. Perhaps the most universal signaling cascades required for proliferative responses are those initiated by transient rises in intracellular calcium (Ca(2+)). The major intracellular receptor for Ca(2+) is calmodulin (CaM). CaM is a small protein that contains four EF-hand Ca(2+) binding sites and is highly conserved among eukaryotes. In all organisms in which the CaM gene has been deleted, it is essential. Although Ca(2+)/CaM is required for proliferation in both unicellular and multicellular eukaryotes, the essential targets of Ca(2+)/CaM-dependent pathways required for cell proliferation remain elusive. Potential Ca(2+)/CaM-dependent targets include the serine/threonine phosphatase calcineurin and the family of multifunctional Ca(2+)/CaM-dependent protein kinases. Whereas these enzymes are essential in Aspergillus nidulans, they are not required under normal growth conditions in yeast. However, in mammalian cells, studies demonstrate that both types of enzymes contribute to the regulation of cell cycle progression. Unfortunately, the mechanism by which Ca(2+)/CaM and its downstream targets, particularly calcineurin and the Ca(2+)/CaM-dependent protein kinases, regulate key cell cycle-regulatory proteins, remains enigmatic. By understanding how Ca(2+)/CaM regulates cell cycle progression in normal mammalian cells, we may gain insight into how hormones control cell division and how cancer cells subvert the need for Ca(2+) and its downstream targets to proliferate.     

胰腺星状细胞对高糖诱导的胰岛β细胞凋亡的影响

目的 观察胰腺星状细胞对高糖诱导的大鼠胰岛β细胞株Ins-1细胞活力及凋亡的影响,探索胰腺星状细胞在糖尿病胰岛功能衰竭进程中的作用.方法 构建Ins-1及胰腺星状细胞共培养系统,细胞分为Ins-1对照组、Ins-1高糖组(25 mmol/L葡萄糖)、Ins-1高渗组(25 mmol/L甘露醇)、共培养对照组、共培养高糖组(25 mmol/L葡萄糖)、共培养高渗组(25 mmol/L甘露醇).24 h后采用流式细胞法分析Ins-1细胞早期凋亡水平,48 h后分别采用嚷唑蓝(MTT)法和4,6-二氨基-2-苯基吲哚(DAPI)染色法检测细胞活力及凋亡细胞形态.使用单因素方差分析进行数据统计.结果 Ins-1高糖组与Ins-1对照组比较,Ins-1细胞凋亡率显著增加(分别为7.93%±0.41%、3.73%±0.35%,F=55.68,P<0.05);共培养高糖组与共培养对照组比较,Ins-1细胞凋亡率显著增加(分别为11.73%±1.20%、5.03%±0.41%,F=55.68,P<0.05).Ins-1高糖组与Ins-1对照组比较,细胞活力明显下降(分别为2.28±0.13、2.85±0.31,F=97.75,P<0.05);共培养高糖组与共培养对照组比较,细胞活力明显下降(分别为0.62±0.06、1.29±0.19,F=97.75,P<0.05).共培养对照组、共培养高糖组、共培养高渗组与Ins-1对照组、Ins-1高糖组、Ins-1高渗组比较,Ins-1细胞凋亡率显著增加(分别为5.03%±0.41%、3.73%±0.35%;11.73%±1.20%、7.93%±0.41%;7.60%±0.72%、5.60%±0.40%;F=55.68,P<0.05),细胞活力明显下降(分别为1.29±0.19、2.85±0.31;0.62±0.06、2.28±0.13;0.65±0.07、2.35±0.12;F=97.75,P<0.05),并可见凋亡细胞典型形态特征.结论 高糖、胰腺星状细胞可致大鼠胰岛β细胞Ins-1活力下降,凋亡增加;胰腺星状细胞可促进高糖诱导的胰岛β细胞凋亡.