Gerd Larsson-Nyrén

Diabetes reduces β-cell mitochondria and induces distinct morphological abnormalities, which are reproducible by high glucose in vitro with attendant dysfunction.

We investigated the impact of a diabetic state with hyperglycemia on morphometry of β cell mitochondria and modifying influence of a K+-ATP channel opener and we related in vivo findings with glucose effects in vitro. For in vivo experiments islets from syngeneic rats were transplanted under the kidney capsule to neonatally streptozotocin-diabetic or non-diabetic recipients. Diabetic recipients received vehicle, or tifenazoxide (NN414), intragastrically for 9 weeks. Non-diabetic rats received vehicle. Transplants were excised 7 d after cessation of treatment (wash-out) and prepared for electron microscopy. Morphological parameters were measured from approx. 25,000 mitochondria. Rat islets were cultured in vitro for 2–3 weeks at 27 or 11 (control) mmol/l glucose. Transplants to diabetic rats displayed decreased numbers of mitochondria (-31%, p < 0.05), increased mitochondrial volume and increased mitochondrial outer surface area, p < 0.001. Diabetes increased variability in mitochondrial size with frequent appearance of mega-mitochondria. Tifenazoxide partly normalized diabetes-induced effects, and mega-mitochondria disappeared. Long-term culture of islets at 27 mmol/l glucose reproduced the in vivo morphological abnormalities. High-glucose culture was also associated with reduced ATP and ADP contents, reduced oxygen consumption, reduced signaling by MitoTracker Red and reduction of mitochondrial proteins (complexes I–IV), OPA 1 and glucose-induced insulin release. We conclude that (1) a long-term diabetic state leads to a reduced number of mitochondria and to distinct morphological abnormalities which are replicated by high glucose in vitro; (2) the morphological abnormalities are coupled to dysfunction; (3) K+-ATP channel openers may have potential to partly reverse glucose-induced effects.

Interaction between perchlorate and nifedipine on insulin secretion from mouse pancreastic islets

In order to elucidate the mechanisms responsible for the stimulatory effect of perchlorate (ClO4−) on insulin secretion, we have investigated the interaction between this chaotropic anion and the organic calcium antagonist nifedipine. This drug, known as a blocker of L-type calcium channels, was chosen as a tool to test the idea that ClO4− acts on insulin secretion by stimulating the gating of voltage-controlled Ca2+ channels. ClO4− amplified the stimulatory effect of D-glucose on insulin release from perfused pancreas (first and second phases) as well as from isolated islets incubated in static incubations for 60 min. This indicates that ClO4− amplifies physiologically regulated insulin secretion. Nifedipine reduced D-glucose-induced (20 mM) insulin release in a dose-dependent manner with half-maximum effect at about 0.8 μM and apparent maximum effect at 5 μM nifedipine. In the presence of 20 mM D-glucose, the inhibitory effects of 0.5, 1 or 5 μM nifedipine were only slightly, if at all, counteracted by perchlorate. When 12 mM ClO4− and 20 mM D-glucose were combined, calculation of the specific effect of ClO4− revealed that nifedipine produced almost maximum inhibition already at 0.05 μM. Thus, the perchlorate-induced amplification of D-glucose-stimulated insulin release shows higher sensitivity to nifedipine than the D-glucose-effect as such. This supports the hypothesis that perchlorate primarily affects the voltage-sensitive L-type calcium channel in the β-cell.

Phospholipase A2 is important for glucose induction of rhythmic Ca2+ signals in pancreatic beta cells.

Objectives: Pancreatic &bgr; cells respond to glucose stimulation with pulses of insulin release generated by oscillatory rises of the cytoplasmic Ca2+ concentration ([Ca2+]i). The observation that exposure to external ATP and other activators of cytoplasmic phospholipase A2 (cPLA2) rapidly induces rises of [Ca2+]i similar to ordinary oscillations made it important to analyze whether suppression of the cPLA2 activity affects glucose-induced [Ca2+]i rhythmicity in pancreatic &bgr; cells. Methods: Ratiometric fura-2 technique was used for measuring [Ca2+]i in single &bgr; cells and small aggregates prepared from ob/ob mouse islets. Results: Testing the effects of different inhibitors of cPLA2 in the presence of 20 mM glucose, it was found that N-(p-amylcinnamoyl)anthranilic acid (ACA) removed the oscillations at a concentration of 25 &mgr;M, arachidonyl trifluoromethyl ketone (AACOCF3) at 10 &mgr;M, and bromoenol lactone (BEL) at 10 to 15 &mgr;M. Withdrawal of ACA and BEL resulted in reappearance of the oscillations. Suppression of the arachidonic acid production by addition of 5 &mgr;M of the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267) effectively removed the [Ca2+]i oscillations, an effect reversed by removal of the inhibitor or addition of 100 &mgr;M tolbutamide. Suppression of the arachidonic acid production had a restrictive influence also on the transients of [Ca2+]i supposed to synchronize the &bgr;-cell rhythmicity. Although less sensitive than the oscillations, most transients disappeared during exposure to 50 &mgr;M ACA or 35 &mgr;M RHC 80267. Conclusions: The results support the idea that cyclic variations of cPLA2 activity are important for the generation and synchronization of the &bgr;-cell [Ca2+]i oscillations responsible for pulsatile release of insulin.

Pancreatic beta cells from db/db mice show cell-specific [Ca2+]i and NADH responses to glucose but not to alpha-ketoisocaproic acid.

Objective: We recently showed that timing and magnitude of the glucose-induced cytoplasmic calcium [Ca2+]i response are reproducible and specific for the individual β cell. We now wanted to identify which step(s) of stimulus-secretion coupling determine the cell specificity of the [Ca2+]i response and whether cell specificity is lost in β-cells from diabetic animals. Besides glucose, we studied the effects of glyceraldehyde, a glycolytic intermediate, and α-ketoisocaproic acid (KIC), a mitochondrial substrate. Methods: Early [Ca2+]i changes were studied stimulations in fura-2-labeled dispersed β cells from lean, ob/ob, and db/db mice. Lag time and peak height were compared during 2 consecutive stimulations with the same stimulator. Nicotinamide adenine dinucleotide (NADH) responses to glucose and KIC were studied as a measure of metabolic flux. Results: Both glyceraldehyde and KIC induced cell-specific temporal responses in lean mouse β cells with a correlation between lag times for [Ca2+]i rise during the first and second stimulation. β Cells from ob/ob and db/db mice showed cell-specific temporal [Ca2+]i responses to glucose and glyceraldehyde but not to KIC. Glucose induced cell-specific NADH responses in all 3 models, but KIC did so only in lean mouse β cells. Conclusions: A cell-specific response may be induced at several steps of β-cell stimulus-secretion coupling. Mitochondrial metabolism generates a cell-specific response in normal β cells but not in db/db and ob/ob mouse β cells.

Anion-selective amplification of glucose-induced insulin secretion

Abstract The functional roles of anions on glucose-induced insulin secretion are poorly understood. We investigated the effects of the monovalent anions thiocyanate, iodide, bromide, nitrate and chloride on the dynamics of insulin secretion in isolated pancreatic islets from non-inbred Umeåob/ob mice. All anion species (12 mM), except Cl−, significantly amplified glucose-induced (20 mM) first- and second-phase insulin secretion (selectivity sequence: SCN−>NO3−>I−>Br−>Cl−). Simultaneously, the anions reduced the lag-time prior to the initiation of the secretion (SCN−=I−=NO3−>Br−>Cl−). The results indicate that pancreatic β-cell activation can be initiated and amplified by an anion-selective mechanism showing increasing degrees of activation in the order of the anion series of Hofmeister. On the basis of the strikingly similar anion selectivity of amplified secretion and shortened lag-phase, we suggest that both types of anion effects are caused by action at a single site on the β-cell.