Walter S. Zawalich

Effect of chronic hyperglycemia on in vivo insulin secretion in partially pancreatectomized rats.

We have examined the effect of chronic (4 wk) hyperglycemia on insulin secretion in vivo in an awake, unstressed rat model. Three groups of animals were examined: control, partial (90%) pancreatectomy, and partial pancreatectomy plus phlorizin, in order to normalize plasma glucose levels. Insulin secretion in response to arginine (2 mM), hyperglycemia (+100 mg/dl), and arginine plus hyperglycemia was evaluated. In diabetic compared with control animals three specific alterations were observed: (a) a deficient insulin response, in both first and second phases, to hyperglycemia; (b) an augmented insulin response to the potentiating effect of arginine under basal glycemic conditions; and (c) an inability of hyperglycemia to augment the potentiating effect of arginine above that observed under basal glycemic conditions. Normalization of the plasma glucose profile by phlorizin treatment in diabetic rats completely corrected all three beta cell abnormalities. These results indicate that chronic hyperglycemia can lead to a defect in in vivo insulin secretion which is reversible when normoglycemia is restored.

Insulin secretion: Combined effects of phorbol ester and A23187

The effect of the ionophore, A23187, and/or the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on insulin secretion were compared with those of glucose. Glucose induces a biphasic pattern of insulin secretion; A23187 a comparable initial spike but no second phase; and TPA a slowly progressive increase. Combined A23187 and TPA evoke a pattern similar to that induced by glucose. Forskolin enhances both phases of glucose- induced and of TPA-A23187-induced insulin secretion. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system, the calmodulin branch and the C-kinase branch, control, respectively, the initial and sustained phases of insulin secretion.

Overexpression of Parathyroid Hormone-related Protein in the Pancreatic Islets of Transgenic Mice Causes Islet Hyperplasia, Hyperinsulinemia, and Hypoglycemia

Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the post-prandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIP-PTHrP mice than in their normal littermates. PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion.

Physiology and Pathophysiology of Insulin Secretion

Mechanisms by which various classes of extracellular signals regulate insulin secretion are discussed regarding their cellular and molecular actions. Under physiological circumstances, the small postprandial changes in plasma glucose concentrations (∼4.4–6.6 mM) primarily serve as a conditional modifier of insulin secretion and dramatically alter the responsiveness of islets to a combination of neurohumoral agonists. These agonists have two functions. Cholecystokinin (CCK) and acetylcholine activate the hydrolysis of polyphosphoinositides, and gastric inhibitory polypeptide (GIP) and glucagonlike peptide 1 activate adenylate cyclase. These two functional classes of neurohumoral agonists act synergistically to enhance insulin secretion when plasma glucose is >6.0 mM but not when it is ≤4 mM. On the other hand, an increase in plasma glucose concentration to 8–10 mM induces an increase in insulin secretory rate in the absence of any of the neurohumoral agonists. Remarkably, high glucose leads to an increase in the same intracellular signals, as does a combination of acetylcholine and GIP. On the basis of these data, a model of how insulin secretion is regulated under physiological circumstances is proposed. This model emphasizes that the regulation of insulin secretion occurs in three stages: cephalic, early enteric, and later enteric. In this view, the crucial event occurring during the first two phases is the agonistinduced translocation of protein kinase C (PKC) to the plasma membrane under conditions in which an increase in Ca2+ influx does not occur. PKC is now in a cellular location and a Ca2+-sensitive conformation such that an increase in Ca2+ influx rate occurring during the third phase leads to its immediate activation and an enhanced rate of insulin secretion. Furthermore, under physiological circumstances, an optimal insulin secretory response is dependent on a correct temporal pattern of signals arising from neural and enteric sources. If this pattern is deranged, an abnormal pattern of insulin secretion is observed. An important new insight is provided by the observation that agonists (e.g., CCK or acetylcholine) that act to stimulate the hydrolysis of phosphatidylinositides, when acting for a short period (10–20 min), induce an enhanced responsiveness of islets to glucose, i.e., proemial sensitization. However, when acting unopposed for several hours, these agonists will induce a time-dependent suppression of responsiveness to glucose and other agonists. The latter observation implies that optimal insulin secretion is dependent on periodic rather than continuous exposure to the correct pattern of extracellular signals. The clinical implications of these new observations are discussed regarding glucose toxicity, the possible role of interleukin 1 in the pathogensis of insulin-dependent diabetes, sulfonylurea therapy, and the abnormalities of insulin secretion seen in non-insulin-dependent diabetes.

Switching of G-protein Usage by the Calcium-sensing Receptor Reverses Its Effect on Parathyroid Hormone-related Protein Secretion in Normal Versus Malignant Breast Cells

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Gαi in MMECs but coupled to Gαs in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer.

Dephosphorylation of β2-syntrophin and Ca2+/μ-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion

Islet cell autoantigen (ICA) 512 is a receptor‐tyrosine phosphatase‐like protein associated with the secretory granules of neuroendocrine cells, including pancreatic β‐cells. Binding of its cytoplasmic tail to β2‐syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS‐1 cells triggers the biosynthesis of pro‐ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+‐dependent proteolysis of ICA512. In vitro μ‐calpain cleaves ICA512 between a putative PEST domain and the β2‐syntrophin binding site, whereas binding of ICA512 to β2‐syntrophin protects the former from cleavage. β2‐syntrophin and its F‐actin‐binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho‐β2‐syntrophin and stimulation of insulin secretion induces the Ca2+‐dependent, okadaic acid‐sensitive dephosphorylation of β2‐syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from β2‐syntrophin–utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by μ‐calpain.

Regulation of Glucose Metabolism in Pancreatic Islets

We evaluated the possible role of islet glucokinase in controlling the rate of islet glucose metabolism, and thereby the rate of glucose-induced insulin release. The activities of glucokinase, hexokinase, P-fructokinase, and glyceraldehyde-P dehydrogenase were quantitated in sonicated or isotonically homogenized islet preparations using pyridine nucleotide-dependent fluorometric assays. In sonicates, about ¼ of the islet glucose phosphorylating activity was due to an enzyme with kinetic properties similar to glucokinase; % of the activity was due to hexokinase. The procedure for determining islet glucokinase activity was improved by centrifuging isotonic islet homogenates at 12,000 × g. The supernatant fraction was enriched for glucokinase. About ½ of the glucose phosphorylating activity in this fraction was due to glucokinase and ½ was due to hexokinase. The glucokinase activity in islet homogenates was ⅓ of the activity of hexokinase, V40 of the activity of P-fructokinase, and 1/400 of the activity of glyceraldehyde-P dehydrogenase. Detailed concentration dependency curves of glucose and mannose utilization were also obtained with intact isolated pancreatic rat islets. Glucose and mannose usage in islets was governed by two superimposed hyperbolic systems differing in Km and Vmax. A high Km system (Km for glucose 11 mM and for mannose 21 mM) predominated. A low Km system (Km for glucose 215 and for mannose 530 μM) contributed about 15% to the total activity. The available data with intact islets could be rationalized by the existence of two distinct hexose phosphorylating enzymes with differing capacities and kinetic properties. These enzymes, tentatively identified as glucokinase and hexokinase, could coexist in the same cell or could be distributed among different cell types. The possible physiologic significance of these results is discussed,emphasizing the idea of dual control of glycolysis and insulin release by glucokinase and hexokinase. An earlier proposal that glucokinase serves as glucoreceptor of β-cells [J. Biol. Chem. 243:2730 (1968] is greatly strengthened by the present studies.

Interactions of Cholecystokinin and Glucose in Rat Pancreatic Islets

The effects of sulfated cholecystokinin (CCK-8S) and glucose on insulin secretion and polyphosphoinositide (PPI) metabolism were studied in isolated rat islets. Both agonists stimulate PPI hydrolysis, inositol phosphate accumulation, 3H efflux from [3H]inositol-prelabeled tissue, and 45Ca efflux from prelabeled cells. However, the effects ofCCK-8S on PPI metabolism are considerably greater than those of glucose. Furthermore, the effectsof CCK-8S on PPI and Ca2+ metabolism are observed whether islets are incubated in either 2.75 or 7 mM glucose, but CCK-8S only stimulates insulin secretion (a biphasic response) when the higher glucose concentration is present. Addition of 1 μM forskolin to islets incubated in media containing 2.75 mM glucose does not influence basal insulin secretion but sensitizes the islets to the action of CCK-8S. In the presence of forskolin, CCK-8S induces a very marked first phase but no second phase of insulin secretion. We postulate that CCK-8S acts in this tissue via receptor-linked PPI hydrolysis, leading to an inositol trisphosphate-induced Ca2+ efflux. These receptor- mediated effects of CCK-8S are not altered either by the ambient glucose concentration or the cAMP content of the islets, but these two factors determine the responsiveness of the islets (in terms of insulin secretion) to a given CCK-8S signal.

Phosphoinositide Hydrolysis and Insulin Release From Isolated Perifused Rat Islets: Studies With Glucose

The ability of glucose to promote the hydrolysis of pre I a be led [2-3H]inositol-containing phosphoinositides (PI) was assessed by measuring the efflux of 3H in response to glucose and the accumulation of labeled inositol phosphates. The inclusion of nonradioactive inositol (1 mM) in the perifusion medium dramatically improved our ability to monitor glucose-induced increases in 3H efflux. Efflux studies with this method revealed the following. 1) 3H efflux is significantly greater at 7 than at 2.75 mM glucose, and this parallels a small but significant increase in insulin secretion. 2) D-manno-Heptulose reduces 3H efflux with 7 mM glucose to a level approximating that seen in the presence of 2.75 mM glucose and has no effect on 3H efflux with 2.75 mM glucose. 3) In the presence of 20 mM glucose plus 1 mM inositol, 3H efflux is rapid and biphasic, a response that parallels the timing and amplitude of the biphasic pattern of insulin secretion. Direct measurements of labeled inositol and inositol phosphate levels in islets revealed the following. 4) After 50 min of perifusion with 2.75 or 7 mM glucose, labeled inositol phosphates were significantly greater with 7 mM glucose. 5) In response to 20 mM glucose alone, islet levels of free inositol, inositol monophosphate (IP,), and inositol bisphosphate (IP2) increased. 6) In response to 20 mM glucose plus 1 mM cold inositol, islet levels of free inositol increased, whereas islet levels of IP1, IP2, and inositol trisphosphate (IP3) were reduced compared with values obtained with 20 mM glucose alone. 7) In response to perifusion with 20 mM glucose, IP3 accumulation was biphasic in nature, and this response precedes the biphasic pattern of both insulin output and 3H efflux by several minutes. These results suggest that PI hydrolysis in islets is tightly regulated by the ambient glucose level and that second-messenger signals generated by activation of this pathway may contribute to the biphasic pattern of glucose-induced insulin secretion.

Physiological Role of Cholecystokinin in Meal-Induced Insulin Secretion in Conscious Rats Studies With L 364718, A Specific Inhibitor of CCK-Receptor Binding

It has been suggested that the gut hormone cholecystokinin (CCK), by modulating insulin output from pancreatic β-cells, plays an important role in the enteroinsular axis. To investigate this hypothesis, eight rats were studied on two different occasions: after injection of L 364718, a specific antagonist of CCK binding to its membrane receptor, and after vehicle injection. In both studies a mixture of casein (11%) and glucose (9%) was infused through a chronic indwelling intraduodenal catheter to evoke CCK secretion. Plasma was analyzed for insulin, glucose, glucagon, and tyrosine many times during the procedure. Prior administration of the CCK antagonist significantly attenuated the increase in plasma insulin and glucagon after casein infusion. These results support the concept that cholecystokinin plays an important physiologic role in the in vivo regulation of postprandial plasma insulin and glucagon concentrations after protein ingestion.