Bo Hellman

STUDIES IN OBESE‐HYPERGLYCEMIC MICE*

Spontaneous development of obesity and hyperglycemia in mice is generally referred to as the obese-hyperglycemic syndrome. The American variety was first discovered as a new mutant by Ingalls, Dickie and Snell’ and found to be homozygous for a recessive gene. Another recessive and fully penetrant gene causing obesity in mice was later described by Falconer and Isaacson in Edinburgh.’ While the American and Scottish homozygotes are indistinguishable in external appearance, the two genes are not allelomorphic. The hyperglycemic hereditary form of obesity in mice, originally described from New Zealand by Bielschowsky and Biel~chowsky,~ differs from that of the American mice in several important Mice with a yellow coat colour and obesity have been studied since the beginning of this century, and it is generally agreed that the gene for yellow fur is identical with tha t for obesity.’ The fact tha t a line of these mice display considerably increased blood glucose levels”’ means tha t a t present there may exist four different varieties of the obese-hyperglycemic syndrome in mice. In the present paper different aspects of the American variety of the obesehyperglycemic syndrome in mice will be presented in the light of the findings which have been made in our laboratory.

Glucose-Induced Amplitude Regulation of Pulsatile Insulin Secretion From Individual Pancreatic Islets

The insulin secretory response to glucose was studied in single pancreatic islets isolated from ob/ob mice and rats. The perfusate from an individual islet was collected during 18-s periods and analyzed for insulin with an ELISA technique. Increase of the glucose concentration from 3 to ≥ 5.5 mM resulted in pulses of insulin release often originating from the basal level and having a frequency of 0.4/min. Glucose regulation of insulin release from the individual islet was manifested by alterations of the amplitudes of the pulses but not of their frequency. It is concluded that the large amplitude oscillations of cytoplasmic Ca2+ known to occur in the pancreatic β-cells have their counterpart in pulses of insulin release and that glucose stimulation of the secretory activity may be the result of recruitment of more β-cells into an oscillatory state.

Cytoplasmic Ca2+ oscillations in pancreatic ß-cells

In the last 15 years it has been a growing interest in the cyclic variations of circulating insulin [46]. After the suggestion that this phenomenon may be due to oscillations of the beta-cell membrane potential [8,39], it was demonstrated that [Ca2+]i oscillates in the glucose-stimulated beta-cell with a similar frequency to that of pulsatile insulin release. The present review describes four types of [Ca2+]i oscillations in the pancreatic beta-cell. The slow sinusoidal oscillations, referred to as type-a, are those which most closely correspond to pulsatile insulin release. Although not affecting the properties of the type-a oscillations in individual beta-cells, the concentration of glucose is a determinant for their generation and further transformation into a sustained increase. Accordingly, cytoplasmic Ca2+ is regulated by sudden transitions between oscillatory and steady-state levels at threshold concentrations of glucose, which are characteristic for the individual beta-cell. This behaviour explains the observation of a gradual recruitment of previously non-secreting cells with increase of the extracellular glucose concentration [44]. However, it still remains to be elucidated how the sudden transitions between these three states translate into the co-ordinated slow oscillations of [Ca2+]i in the intact islet. Cyclic variations of circulating insulin require a synchronization of the [Ca2+]i cycles also among the islets in the pancreas. It is still an open question by which means the millions of islets communicate mutually to establish a pattern of pulsatile insulin release from the whole pancreas. The discovery that the beta-cell is not only the functional unit for insulin synthesis but also generates the [Ca2+]i oscillations required for pulsatile insulin release has both physiological and clinical implications. The fact that minor damage to the beta-cells prevents the type-a oscillations with maintenance of a glucose response in terms of raised [Ca2+]i reinforces previous arguments [54] that loss of insulin oscillations is an early indicator of type-2 diabetes. Further analyses of the [Ca2+]i oscillations in the beta-cells should include not only the mechanisms for their generation and subsequent propagation within or among the islets but also how modulation of their frequency affects the insulin sensitivity of various target cells. The latter approach may be important in the attempts to maintain normoglycemia under conditions minimizing the vascular effects of insulin supposed to precipitate hypertonia and atherosclerosis [70,71,77].

Propagation of cytoplasmic Ca2+ oscillations in clusters of pancreatic β-cells exposed to glucose

Digital image analysis was employed for resolving the temporal and spatial variations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells loaded with the Ca(2+)-indicator Fura-2. Glucose-stimulated individual beta-cells exhibited large amplitude oscillations of [Ca2+]i with a mean frequency of 0.33 min-1. When Ca2+ diffusion was restricted by increasing the Ca2+ buffering capacity, the sugar-induced rise of [Ca2+]i preferentially affected the peripheral cytoplasm. When glucagon was present glucose also caused less prominent oscillations with about a 10-fold higher frequency superimposed on an elevated [Ca2+]i. In small clusters of 6-14 cells the average frequency of the large amplitude oscillations increased to 0.60 min-1. The clusters were found to contain micro-domains of electrically coupled cells with synchronized oscillations. After increasing the glucose concentration, adjacent domains became functionally coupled. The oscillations originated from different cells in the cluster. Also the fast glucagon-dependent oscillations were synchronized between cells and had different origins. The results indicate that coupling of beta-cells leads to an increased frequency of the large amplitude oscillations, and that the oscillatory characteristics are determined collectively among electrically coupled beta-cells rather than by particular pacemaker cells. In the light of these data it is necessary to reconsider the previous ideas that glucose-induced oscillations of membrane potential and [Ca2+]i require coupling between many beta-cells, and that the peak [Ca2+]i values reached during oscillations should increase with the size of the coupled cluster.

The islets of Langerhans in ducks and chickens with special reference to the argyrophil reaction.

SummaryThe pancreas of birds is a suitable object for studying the A and B cells separately, since the two cell systems are topografically almost entirely segregated in the form of light (= B cells) and dark (= A cells) islets of Langerhans.On the whole in the chicken and duck the actual distribution of the light islets into different size classes followed the same regular pattern previously found in the rat and man. In the body of the pancreas, containing the great majority of islets, the volume distribution curves thus appeared symmetrical.With the silver impregnation method used a distinct argyrophil reaction in both types of islets was obtained on paraffin sections of the pancreas. According to the presence or absence of blackening, the cells of the dark islets could be divided into two distinct fractions. Especially in the duck the silver-positive cells were grouped in a characteristic way along the walls of the capillaries. Ducks and chickens are not the only animals in which it is possible to identify an argyrophil fraction in what the usual granule stains had shown to be A cells. Parallel studies of various mammals are in complete agreement with these observations. It is, however, still uncertain whether we are here dealing with differences in function, age etc. in one and the same type of cell or with two completely different kinds. No correlation between the argyrophil reaction in the dark islet cells and their content of SH and SS groups or tryptophane could be established.

Evidence for mediated transport of glucose in mammalian pancreatic β-cells

Abstract Uptake of glucose by microdissected pancreatic islets of obese-hyperglycemic mice was studied at 8°. The use of a double-label procedure permitted correction for label in the extracellular space. The following observations and interpretations were made: 1. 1. l -Glucose was restricted to the sucrose space, whereas d -glucose was uniformly equilibrated over the β-cell membrane. 2. 2. l -Glucose (5–40 mM) had no effect on the uptake of d -glucose (1 mM). 3. 3. The uptake of d -glucose was saturable with a v max of about 400 mmoles/h per kg dry islet, and with a K m around 50 mM. 4. 4. At a medium concentration of 5 mM d -glucose, the uptake of this sugar was almost completely blocked by 10 mM phlorizin. Under similar conditions, 20 mM mannoheptulose had no significant effect on d -glucose uptake. The result contradict the previous hypothesis that the β-cell membrane is freely permeable to d -glucose. It is suggested that the uptake of glucose by these cells is mediated by a membrane-located transport molecule with stereospecificity for d -glucose. Renewed attention should therefore be given to the β-cell membrane as a possible locus for the triggering of insulin release by d -glucose.

Glucose-induced oscillations of cytoplasmic Ca2+ in the pancreatic β-cell

The cytoplasmic calcium concentration (Ca2+i) was measured in individual mouse pancreatic β-cells loaded with fura-2 by recording the 340/380 nm fluorescence excitation ratio. An increase of the glucose concentration from 3 to 20 mM, caused initial lowering of Ca2+i followed by a rise with a peak preceding constant elevation at an intermediary level. However, at 11 mM glucose there were large Ca2+i oscillations with a frequency of 1 cycle per 2–6 min. The results indicate that both first and second phase secretion depend on elevated Ca2+i, and that many electrically coupled cells collectively determine the pace of rhythmic depolarization.

Effects of glucose on 45Ca2+ uptake by pancreatic islets as studied with the lanthanum method.

1. Fluxes of 45Ca2+ were studied in pancreatic islets from non‐inbred ob/ob‐mice. Because La3+ blocked the transmembrane fluxes of 45Ca2+ in islet cells, incubations aimed at measuring glucose‐induced changes of the intracellular Ca2+ were ended by washing the islets with 2 mM‐La3+ for 60 min. 2. Uptake of 45Ca2+ progressed for 2 hr; the intracellular concentration of exchangable Ca2+ was about 7 m‐mole/kg dry wt., as estimated from the isotope distribution at apparent equilibrium in islets exposed to 3 mM D‐glucose. Raising the D‐glucose concentration to 20 mM enhanced the 45 Ca2+ uptake whether or not the islets had first been equilibrated with the isotope. The stimulatory effect of D‐glucose was observed in Tris buffer containing no anions but Cl‐ as well as in polyanionic bicarbonate buffer. The effect could not be reproduced with equimolar L‐glucose. 3. The rate of 45Ca2+ release was the same whether the islets had been pre‐loaded in the presence of 3 or 20 mM D‐glucose. Thus the 45Ca2+ that had been taken up in response to 20 mM D‐glucose appeared to be released much more slowly than the bulk of intracellular 45Ca2+. The release of 45Ca2+ was not significantly influenced by D‐glucose during the release period. Incubation for 30 min was require for half of the radioactivity to be released. 4. The rates of insulin secretion were about the same in uni‐anionic Tris buffer as in polyanionic bicarbonate buffer. A marked insulin secretory response to 20 mM D‐glucose was observed in either buffer. 5. It is concluded that 20 mM D‐glucose causes a net uptake of Ca2+ from the extracellular fluid into the interior of the beta‐cells. This uptake is probably not regulated at the level of the plasma membrane but more likely reflects an increased affinity of some intracellular phase or compartment for the ion. Because the observed uptake and release of intracellular 45Ca2+ are slow processes in comparison with the rapid effects of extracellular Ca2+ on insulin secretion, insulin secretion may also depend on a more superficial and La3+‐displacable Ca2+ pool.

Origin of slow and fast oscillations of Ca2+ in mouse pancreatic islets.

1 Pancreatic islets exposed to 11 mM glucose exhibited complex variations of cytoplasmic Ca2+ concentration ([Ca2+]i) with slow (0.3‐0.9 min−1) or fast (2‐7 min−1) oscillations or with a mixed pattern. 2 Using digital imaging and confocal microscopy we demonstrated that the mixed pattern with slow and superimposed fast oscillations was due to separate cell populations with the respective responses. 3 In islets with mixed [Ca2+]i oscillations, exposure to the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (DTBHQ) resulted in a selective disappearance of the fast pattern and amplification of the slow pattern. 4 In addition, the protein kinase A inhibitor RP‐cyclic adenosine 3′,5′‐monophosphorothioate sodium salt transformed the mixed [Ca2+]i oscillations into slow oscillations with larger amplitude. 5 Islets exhibiting only slow oscillations reacted to low concentrations of glucagon with induction of the fast or the mixed pattern. In this case the fast oscillations were also counteracted by DTBHQ. 6 The spontaneously occurring fast oscillations seemed to require the presence of cAMP‐elevating glucagon, since they were more common in large islets and suppressed during culture. 7 Image analysis revealed [Ca2+]i spikes occurring irregularly in time and space within an islet. These spikes were preferentially observed together with fast [Ca2+]i oscillations, and they became more common after exposure to glucagon. 8 Both the slow and fast oscillations of [Ca2+]i in pancreatic islets rely on periodic entry of Ca2+. However, the fast oscillations also depend in some way on paracrine factors promoting mobilization of Ca2+ from intracellular stores. It is proposed that such a mobilization in different cells within a tightly coupled islet syncytium generates spikes which co‐ordinate the regular bursts of action potentials underlying the fast oscillations.

GLUCOSE INDUCES OSCILLATORY CA2+ SIGNALLING AND INSULIN RELEASE IN HUMAN PANCREATIC BETA CELLS

SummaryMechanisms of pulsatile insulin release in man were explored by studying the induction of oscillatory Ca2+ signals in individual beta cells and islets isolated from the human pancreas. Evidence was provided for a glucose-induced closure of ATP-regulated K+ channels, resulting in voltage-dependent entry of Ca2+. The observation of step-wise increases of capacitance in response to depolarizing pulses suggests that an enhanced influx of Ca2+ is an effective means of stimulating the secretory activity of the isolated human beta cell. Activation of muscarinic receptors (1–10 μmol/l carbachol) and of purinergic P2 receptors (0.01–1 μmol/l ATP) resulted in repetitive transients followed by sustained elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i). Periodic mobilisation of intracellular calcium was seen also when injecting 100 μmol/l GTP-γ-S into beta cells hyperpolarized to −70 mV. Individual beta cells responded to glucose and tolbutamide with increases of [Ca2+]i, manifested either as large amplitude oscillations (frequency 0.1–0.5/min) or as a sustained elevation. Glucose regulation was based on sudden transitions between the basal and the two alternative states of raised [Ca2+]i at threshold concentrations of the sugar characteristic for the individual beta cells. The oscillatory characteristics of coupled cells were determined collectively rather than by particular pacemaker cells. In intact pancreatic islets the glucose induction of well-synchronized [Ca2+]i oscillations had its counterpart in 2–5 min pulses of insulin. Each of these pulses could be resolved into regularly occurring short insulin transients. It is concluded that glucose stimulation of insulin release in man is determined by the number of beta cells entering into a state with Ca2+-induced secretory pulses.