Biomedical subjects

Per-Olof Berggren

Publications and source records attributed to Per-Olof Berggren.

48 records · Page 3Linked to original sources

PubMed2003

On-line monitoring of apoptosis in insulin-secreting cells.

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate beta-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the beta-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 +/- 23 min (n = 9) and 257 +/- 59 min (n = 4; mean +/- SE) after activation of apoptosis with staurosporine (6 micromol/l), showing that this method worked in insulin-producing cells.

Animals↗

PubMed2002

Insulin feedback action on pancreatic beta-cell function.

Pancreatic beta-cell function is essential for the regulation of glucose homeostasis and its impairment leads to diabetes mellitus. Besides glucose, the major nutrient factor, inputs from neural and humoral components and intraislet cell-cell communication act together to guarantee an appropriate pancreatic beta-cell function. Data obtained over the last 5 years in several laboratories have revitalized a controversial concept, namely the autocrine feedback action of secreted insulin on beta-cell function. While, historically, insulin was suggested to exert a negative effect on beta-cells, recent data provide evidence for a positive role of insulin in transcription, translation, ion flux, insulin secretion and beta-cell survival.

Animals↗

PubMed2002

Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase in response to dopamine regulates its catalytic activity in intact cells. Because fission of clathrin-coated pits requires dynamin, we examined the mechanisms by which dopamine receptor signals promote dynamin-2 recruitment and assembly at the site of Na(+),K(+)-ATPase endocytosis. Western blotting revealed that dopamine increased the association of dynamin-2 with the plasma membrane and with phosphatidylinositol 3-kinase. Dopamine inhibited Na(+),K(+)-ATPase activity in OK cells and in those overexpressing wild type dynamin-2 but not in cells expressing a dominant-negative mutant. Dephosphorylation of dynamin is important for its assembly. Dopamine increased protein phosphatase 2A activity and dephosphorylated dynamin-2. In cells expressing a dominant-negative mutant of protein phosphatase 2A, dopamine failed to dephosphorylate dynamin-2 and to reduce Na(+),K(+)-ATPase activity. Dynamin-2 is phosphorylated at Ser(848), and expression of the S848A mutant significantly blocked the inhibitory effect of dopamine. These results demonstrate a distinct signaling network originating from the dopamine receptor that regulates the state of dynamin-2 phosphorylation and that promotes its location (by interaction with phosphatidylinositol 3-kinase) at the site of Na(+),K(+)-ATPase endocytosis.

Animals↗

PubMed2002

Sulfonylurea receptor type 1 knock-out mice have intact feeding-stimulated insulin secretion despite marked impairment in their response to glucose.

The ATP-sensitive potassium channel is a key molecular complex for glucose-stimulated insulin secretion in pancreatic beta cells. In humans, mutations in either of the two subunits for this channel, the sulfonylurea type 1 receptor (Sur1) or Kir6.2, cause persistent hyperinsulinemic hypoglycemia of infancy. We have generated and characterized Sur1 null mice. Interestingly, these animals remain euglycemic for a large portion of their life despite constant depolarization of membrane, elevated cytoplasmic free Ca(2+) concentrations, and intact sensitivity of the exocytotic machinery to Ca(2+). A comparison of glucose- and meal-stimulated insulin secretion showed that, although Sur1 null mice do not secrete insulin in response to glucose, they secrete nearly normal amounts of insulin in response to feeding. Because Sur1 null mice lack an insulin secretory response to GLP-1, even though their islets exhibit a normal rise in cAMP by GLP-1, we tested their response to cholinergic stimulation. We found that perfused Sur1 null pancreata secreted insulin in response to the cholinergic agonist carbachol in a glucose-dependent manner. Together, these findings suggest that cholinergic stimulation is one of the mechanisms that compensate for the severely impaired response to glucose and GLP-1 brought on by the absence of Sur1, thereby allowing euglycemia to be maintained.

ATP-Binding Cassette Transporters↗

PubMed2002

Inositol hexakisphosphate promotes dynamin I- mediated endocytosis.

Membrane homeostasis is maintained by exocytosis and endocytosis. The molecular mechanisms regulating the interplay between these two processes are not clear. Inositol hexakisphosphate (InsP(6)) is under metabolic control and serves as a signal in the pancreatic beta cell stimulus-secretion coupling by increasing Ca(2+)-channel activity and insulin exocytosis. We now show that InsP(6) also promotes dynamin I-mediated endocytosis in the pancreatic beta cell. This effect of InsP(6) depends on calcineurin-induced dephosphorylation and is accounted for by both activation of protein kinase C and inhibition of the phosphoinositide phosphatase synaptojanin and thereby formation of phosphatidylinositol 4,5-bisphosphate. In regulating both exocytosis and endocytosis, InsP(6) thus may have an essential integral role in membrane trafficking.

Animals↗

PubMed2002

Down-regulated expression of exocytotic proteins in pancreatic islets of diabetic GK rats.

Exocytosis is regulated by exocytotic proteins, which are present in insulin-secreting beta-cells and play regulatory roles in insulin secretion. Non-insulin dependent diabetes mellitus (type 2 diabetes) is a disease characterized by impaired insulin secretion and insulin resistance. Exocytotic protein immunoreactivities were studied in pancreatic islets of type 2 diabetic Goto-Kakizaki (GK) rats using immunofluorescence histochemistry. The immunoreactivities for vesicle-associated membrane protein-2 (VAMP-2), synaptotagmin III, cysteine string protein (CSP), mammalian homologue of the unc-18 gene (Munc-18), alpha-soluble N-ethylmaleimide-sensitive attachment protein (alpha-SNAP), N-ethylmaleimide-sensitive factor (NSF) and synaptosomal-associated protein of 25 kDa (SNAP-25) exhibited weaker immunofluorescence intensity in islets of GK rats as compared to control Wistar rats. Insulin immunoreactivity was also decreased in GK rat beta-cells, whereas no detectable alterations in the expression of actin immunoreactivity could be detected. The data suggest that reduced expression of exocytotic proteins and decreased insulin content may contribute to the diabetic syndrome in the GK rat.

Actins↗

PubMed2002

Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis.

In renal epithelial cells endocytosis of Na(+),K(+)-ATPase molecules is initiated by phosphorylation of its alpha(1)-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na(+),K(+)-ATPase alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na(+),K(+)-ATPase alpha(1)-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent alpha-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased alpha(1)-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na(+),K(+)-ATPase activity. Because phorbol esters increase Na(+),K(+)-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na(+),K(+)-ATPase endocytosis.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Insulinotropic activity of the imidazoline derivative RX871024 in the diabetic GK rat.

The insulinotropic activity of the imidazoline derivative RX871024 was compared in pancreatic islets from nondiabetic Wistar rats and spontaneously diabetic Goto-Kakizaki (GK) rats. RX871024 significantly stimulated insulin secretion in islets from both animal groups. The insulinotropic activity of RX871024 was higher than that of the sulfonylurea glibenclamide. This difference was more pronounced in islets from GK rats compared with Wistar rat islets. More importantly, RX871024 substantially improved glucose sensitivity in diabetic beta-cells, whereas glibenclamide stimulated insulin secretion about twofold over a broad range of glucose concentrations in nondiabetic and diabetic rats. RX871024 induced a faster increase in cytosolic free Ca(2+) concentration and faster inhibition of ATP-dependent K(+) channel activity in GK rat islets compared with Wistar rat islets. RX871024 also induced a more pronounced increase in diacylglycerol concentration in GK rat islets. These data support the idea that imidazoline compounds can form the basis for the development of novel drugs for treatment of type 2 diabetes, which can restore glucose sensitivity in diabetic beta-cells.

Adenosine Triphosphate↗

PubMed2002

Phosphorylated inositol compounds in beta -cell stimulus-response coupling.

Pancreatic beta-cell function is essential for the regulation of glucose homeostasis in humans, and its impairment leads to the development of type 2 diabetes. Inputs from glucose and cell surface receptors act together to initiate the beta-cell stimulus-response coupling that ultimately leads to the release of insulin. Phosphorylated inositol compounds have recently emerged as key players at all levels of the stimulus-secretion coupling process. In this current review, we seek to highlight recent advances in beta-cell phosphoinositide research by dividing our examination into two sections. The first involves the events that lead to insulin secretion. This includes both new roles for inositol polyphosphates, particularly inositol hexakisphosphate, and both conventional and 3-phosphorylated inositol lipids. In the second section, we deal with the more novel concept of the autocrine role of insulin. Here, released insulin initiates signal transduction cascades, principally through the activity of phosphatidylinositol 3-kinase. This new round of signal transduction has been established to activate key beta-cell genes, particularly the insulin gene itself. More controversially, this insulin feedback has also been suggested to either terminate or enhance insulin secretion events.

Animals↗

PubMed2002

Glucose metabolites inhibit protein phosphatases and directly promote insulin exocytosis in pancreatic beta-cells.

In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion is an early pathogenetic event. Glucose is the cardinal physiological stimulator of insulin secretion from the pancreatic beta-cell, but the mechanisms involved in glucose sensing are not fully understood. Specific ser/thr protein phosphatase (PPase) inactivation by okadaic acid promotes Ca(2+) entry and insulin exocytosis in the beta-cell. We now show that glycolytic and Krebs cycle intermediates, whose concentrations increase upon glucose stimulation, not only dose dependently inhibit ser/thr PPase enzymatic activities, but also directly promote insulin exocytosis from permeabilized beta-cells. Thus, fructose-1,6-bisphosphate, phosphoenolpyruvate, 3-phosphoglycerate, citrate, and oxaloacetate inhibit PPases and significantly enhance insulin exocytosis, nonadditive to that of okadaic acid, at micromolar Ca2+ concentrations. In contrast, the effect of GTP is potentiated by okadaic acid, suggesting that the action of GTP does not require PPase inactivation. We conclude that specific glucose metabolites and GTP inhibit beta-cell PPase activities and directly stimulate Ca2+-independent insulin exocytosis. The glucose metabolites, but not GTP, seem to require PPase inactivation for their stimulatory effect on exocytosis. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by metabolic intermediates, may be a novel regulatory mechanism linking glucose sensing to insulin exocytosis in the beta-cell.

Animals↗

PubMed2002

Two generations of insulinotropic imidazoline compounds.

The imidazoline RX871024 increased basal- and glucose-stimulated insulin release in vitro and in vivo. The compound inhibited activity of ATP-sensitive K(+) channels as well as voltage-gated K(+) channels, which led to membrane depolarization, an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)), and insulin release. Importantly, RX871024 also enhanced the insulinotropic effect of glucose in cells with clamped [Ca(2+)](i) but in the presence of high ATP and Ca(2+)concentration inside the cell. We believe that the latter effect on insulin exocytosis was at least in part mediated by a rise in diacylglycerol, which then activated protein kinase C (PKC) and increased the generation of arachidonic acid (AA) metabolites. Activation of both the PKC and AA pathways resulted in potentiation of glucose effects on insulin secretion. Unlike RX871024, the novel imidazoline BL11282 did not block ATP-dependent K(+) channels, but similarly to RX871024, it stimulated insulin secretion in depolarized or permeabilized islets. Accordingly, BL11282 did not influence glucose and insulin levels under basal conditions either in vitro or in vivo, but it markedly enhanced the insulinotropic effects of glucose. BL11282 restored the impaired insulin response to glucose in islets from spontaneously diabetic GK rats. We conclude that BL11282 belongs to a new class of insulinotropic compounds that demonstrate a strong glucose-dependent effect on insulin exocytosis.

Animals↗

PubMed2002

Modulation of L-type Ca(2+) channels by distinct domains within SNAP-25.

Cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are now known to associate the secretory vesicle with both the target plasma membrane and Ca(2+) channels in order to mediate the sequence of events leading to exocytosis in neurons and neuroendocrine cells. Neuroendocrine cells, particularly insulin-secreting islet beta-cells, t-SNARE proteins, 25-kDa synaptosomal-associated protein (SNAP-25), and syntaxin 1A, independently inhibit the L-type Ca(2+) channel (L(Ca)). However, when both are present, they actually exhibit stimulatory actions on the L(Ca). This suggests that the positive regulation of the L(Ca) is conferred by a multi-SNARE protein complex. We hypothesized an alternate explanation, which is that each of these SNARE proteins possess distinct inhibitory and stimulatory domains that act on the L(Ca). These SNARE proteins were recently shown to bind the Lc(753-893) domain corresponding to the II and III intracellular loop of the alpha1C subunit of the L(Ca). In this study, using patch-clamp methods on primary pancreatic beta-cells and insulinoma HIT-T15 cells, we examined the functional interactions of the botulinum neurotoxin A (BoNT/A) cleavage products of SNAP-25, including NH(2)-terminal (1-197 amino acids) and COOH-terminal (amino acid 198-206) domains, on the L(Ca), particularly at the Lc(753-893) domain. Intracellular application of SNAP-25(1-206) in primary beta-cells decreased L(Ca) currents by approximately 15%. The reduction in L(Ca) currents was counteracted by coapplication of Lc(753-893). Overexpression or injection of wild-type SNAP-25 in HIT cells reduced L(Ca) currents by approximately 30%, and this inhibition was also blocked by the recombinant Lc(753-893) peptide. Expression of BoNT/A surprisingly caused an even greater reduction of L(Ca) currents (by 41%), suggesting that the BoNT/A cleavage products of SNAP-25 might possess distinct inhibitory and positive regulatory domains. Indeed, expression of SNAP-25(1-197) increased L(Ca) currents (by 19% at 10 mV), and these effects were blocked by the Lc(753-893) peptide. In contrast, injection of SNAP-25(198-206) peptide into untransfected cells inhibited L(Ca) currents (by 47%), and more remarkably, these inhibitory effects dominated over the stimulatory effects of SNAP-25(1-197) overexpression (by 34%). Therefore, the SNARE protein SNAP-25 possesses distinct inhibitory and stimulatory domains that act on the L(Ca). The COOH-terminal 197-206 domain of SNAP-25, whose inhibitory actions dominate over the opposing stimulatory NH(2)-terminal domain, likely confers the inhibitory actions of SNAP-25 on the L(Ca). We postulate that the eventual accelerated proteolysis of SNAP-25 brought about by BoNT/A cleavage allows the relatively intact NH(2)-terminal SNAP-25 domain to assert its stimulatory action on the L(Ca) to increase Ca(2+) influx, and this could in part explain the observed weak or inconsistent inhibitory effects of BoNT/A on insulin secretion. The present study suggests that distinct domains within SNAP-25 modulate L(C) subtype Ca(2+) channel activity in both primary beta-cells and insulinoma HIT-T15 cells.

Animals↗