Spatiotemporal Modeling of Triggering and Amplifying Pathways in GLP-1 Secreting Intestinal L Cells

Glucagon-like peptide 1 (GLP-1) is secreted by intestinal L-cells, and augments glucose-induced insulin secretion, thus playing an important role in glucose control. The stimulus-secretion pathway in L-cells is still incompletely understood and a topic of debate. It is known that GLP-1 secreting cells can sense glucose to promote electrical activity either by the electrogenic sodium-glucose cotransporter SGLT1, or by closure of ATP-sensitive potassium channels after glucose metabolism. Glucose also has an effect on GLP-1 secretion downstream of electrical activity. An important aspect to take into account is the spatial organization of the cell. Indeed, the glucose transporter GLUT2 is located at the basolateral, vascular side, while SGLT1 is exposed to luminal glucose at the apical side of the cell, suggesting that the two types of transporters play different roles in glucose sensing. Here, we extend our recent model of electrical activity in primary L-cells to include spatiotemporal glucose and Ca²⁺ dynamics, and GLP-1 secretion. The model confirmed that glucose transportation into the cell through SGLT1 cotransporters can induce Ca²⁺ influx and release of GLP-1 as a result of electrical activity, while glucose metabolism alone is insufficient to depolarize the cell and evoke GLP-1 secretion in the model, suggesting a crucial role for SGLT1 in triggering GLP-1 release in agreement with experimental studies. We suggest a secondary, but participating, role of GLUT2 and glucose metabolism for GLP-1 secretion via an amplifying pathway that increases the secretion rate at a given Ca²⁺ level.     

Glucose-mediated control of ghrelin release from primary cultures of gastric mucosal cells

The peptide hormone ghrelin is released from a distinct group of gastrointestinal cells in response to caloric restriction, whereas its levels fall after eating. The mechanisms by which ghrelin secretion is regulated remain largely unknown. Here, we have used primary cultures of mouse gastric mucosal cells to investigate ghrelin secretion, with an emphasis on the role of glucose. Ghrelin secretion from these cells upon exposure to different d-glucose concentrations, the glucose antimetabolite 2-deoxy-d-glucose, and other potential secretagogues was assessed. The expression profile of proteins involved in glucose transport, metabolism, and utilization within highly enriched pools of mouse ghrelin cells and within cultured ghrelinoma cells was also determined. Ghrelin release negatively correlated with d-glucose concentration. Insulin blocked ghrelin release, but only in a low d-glucose environment. 2-Deoxy-d-glucose prevented the inhibitory effect of high d-glucose exposure on ghrelin release. mRNAs encoding several facilitative glucose transporters, hexokinases, the ATP-sensitive potassium channel subunit Kir6.2, and sulfonylurea type 1 receptor were expressed highly within ghrelin cells, although neither tolbutamide nor diazoxide exerted direct effects on ghrelin secretion. These findings suggest that direct exposure of ghrelin cells to low ambient d-glucose stimulates ghrelin release, whereas high d-glucose and glucose metabolism within ghrelin cells block ghrelin release. Also, low d-glucose sensitizes ghrelin cells to insulin. Various glucose transporters, channels, and enzymes that mediate glucose responsiveness in other cell types may contribute to the ghrelin cell machinery involved in regulating ghrelin secretion under these different glucose environments, although their exact roles in ghrelin release remain uncertain.     

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.     

ATP-sensitive K+ channels in human isolated pancreatic B-cells

Glucose-stimulated insulin release from rodent pancreatic B-cells is thought to be initiated by the closing of ATP-sensitive K+ channels in the plasma membrane as a consequence of glucose metabolism. We have identified an ATP-sensitive K+ channel in membrane patches excised from human B-cells which is similar to that found in rodent B-cells in conductance, kinetics, ATP sensitivity and its inhibition by sulphonylureas. In man, the ATP-sensitive K+ channel may also have a central role in glucose-stimulated insulin secretion and may be (linked to) the receptor for the hypoglycemic sulphonylureas.     

A novel mechanism for the suppression of a voltage-gated potassium channel by glucose-dependent insulinotropic polypeptide: protein kinase A-dependent endocytosis

The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive (K(ATP)) and calcium-activated (K(CA)) potassium channels have been extensively studied, but less is known about the role of voltage-gated (K(V)) potassium channels in pancreatic beta-cells. The incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) stimulates insulin secretion by potentiating events underlying membrane depolarization and exerting direct effects on exocytosis. In the present study, we identified a novel role for GIP in regulating K(V)1.4 channel endocytosis. In GIP receptor-expressing HEK293 cells, GIP reduced A-type peak ionic current amplitude of K(V)1.4 via activation of protein kinase A (PKA). Using mutant forms of K(V)1.4 with Ala-Ser/Thr substitutions in a potential PKA phosphorylation site, C-terminal phosphorylation was shown to be linked to GIP-mediated current amplitude decreases. Proteinase K digestion and immunocytochemical studies on mutant K(V)1.4 localization following GIP stimulation demonstrated phosphorylation-dependent rapid endocytosis of K(V)1.4. Expression of K(V)1.4 protein was also demonstrated in human beta-cells; GIP treatment resulting in similar decreases in A-type potassium current peak amplitude to those in HEK293 cells. Transient overexpression in INS-1 beta-cells (clone 832/13) of wild-type (WT) K(V)1.4, or a T601A mutant form resistant to PKA phosphorylation, resulted in reduced glucose-stimulated insulin secretion; WT K(V)1.4 overexpression potentiated GIP-induced insulin secretion, whereas this response was absent in T601A cells. These results strongly support an important novel role for GIP in regulating K(V)1.4 cell surface expression and modulation of A-type potassium currents, which is likely to be critically important for its insulinotropic action.     

Fasting-induced suppression of LH secretion does not require activation of ATP-sensitive potassium channels

Reproductive hormone secretions are inhibited by fasting and restored by feeding. Metabolic signals mediating these effects include fluctuations in serum glucose, insulin, and leptin. Because ATP-sensitive potassium (K(ATP)) channels mediate glucose sensing and many actions of insulin and leptin in neurons, we assessed their role in suppressing LH secretion during food restriction. Vehicle or a K(ATP) channel blocker, tolbutamide, was infused into the lateral cerebroventricle in ovariectomized mice that were either fed or fasted for 48 h. Tolbutamide infusion resulted in a twofold increase in LH concentrations in both fed and fasted mice compared with both fed and fasted vehicle-treated mice. However, tolbutamide did not reverse the suppression of LH in the majority of fasted animals. In sulfonylurea (SUR)1-null mutant (SUR1(-/-)) mice, which are deficient in K(ATP) channels, and their wild-type (WT) littermates, a 48-h fast was found to reduce serum LH concentrations in both WT and SUR(-/-) mice. The present study demonstrates that 1) blockade of K(ATP) channels elevates LH secretion regardless of energy balance and 2) acute fasting suppresses LH secretion in both SUR1(-/-) and WT mice. These findings support the hypothesis that K(ATP) channels are linked to the regulation of gonadotropin-releasing hormone (GnRH) release but are not obligatory for mediating the effects of fasting on GnRH/LH secretion. Thus it is unlikely that the modulation of K(ATP) channels either as part of the classical glucose-sensing mechanism or as a component of insulin or leptin signaling plays a major role in the suppression of GnRH and LH secretion during food restriction.     

Discovery of a cholecystokinin-releasing peptide: biochemical characterization and physiological implications

Recent studies indicate that the secretion of CCK is mediated by a trypsin sensitive peptide secreted by the proximal small intestine that has been designated "CCK-releasing factor" (CCK-RF). This CCK-RF was found to be identical to the porcine diazepam binding inhibitor by peptide sequencing and mass spectrometry analysis. This peptide is present in abundance in the epithelial cells in the duodenal mucosa. Its release into the lumen is mediated by intestinal submucosal cholinergic neurons. Functionally, this peptide appears to mediate feedback regulation of pancreatic secretion and CCK release in response to peptone and lipid stimulation. It fulfills all the criteria as a physiological regulator of CCK secretion. This represents the first chemical characterization of a luminally secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release.     

Syntaxin 1A regulates surface expression of beta-cell ATP-sensitive potassium channels

The pancreatic ATP-sensitive potassium (K(ATP)) channel consisting of four inwardly rectifying potassium channel 6.2 (Kir6.2) and four sulfonylurea receptor SUR1 subunits plays a key role in insulin secretion by linking glucose metabolism to membrane excitability. Syntaxin 1A (Syn-1A) is a plasma membrane protein important for membrane fusion during exocytosis of insulin granules. Here, we show that Syn-1A and K(ATP) channels endogenously expressed in the insulin-secreting cell INS-1 interact. Upregulation of Syn-1A by overexpression in INS-1 leads to a decrease, whereas downregulation of Syn-1A by small interfering RNA (siRNA) leads to an increase, in surface expression of K(ATP) channels. Using COSm6 cells as a heterologous expression system for mechanistic investigation, we found that Syn-1A interacts with SUR1 but not Kir6.2. Furthermore, Syn-1A decreases surface expression of K(ATP) channels via two mechanisms. One mechanism involves accelerated endocytosis of surface channels. The other involves decreased biogenesis and processing of channels in the early secretory pathway. This regulation is K(ATP) channel specific as Syn-1A has no effect on another inward rectifier potassium channel Kir3.1/3.4. Our results demonstrate that in addition to a previously documented role in modulating K(ATP) channel gating, Syn-1A also regulates K(ATP) channel expression in β-cells. We propose that physiological or pathological changes in Syn-1A expression may modulate insulin secretion by altering glucose-secretion coupling via changes in K(ATP) channel expression.     

Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic β-Cells

In pancreatic β-cells, voltage-gated potassium 2.1 (Kv2.1) channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β-cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase (AMPK). Activation of AMPK mimics whereas inhibition of AMPK occludes the effect of leptin. Inhibition of Ca²⁺/calmodulin-dependent protein kinase kinase β, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase (PKA) is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, whereas stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization, and pharmacologically induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (K_ATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface K_ATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. This study supports a model in which leptin exerts concerted trafficking regulation of K_ATP and Kv2.1 channels to coordinately inhibit insulin secretion.     

Regulation of K(ATP) channel by 17β-estradiol in pancreatic β-cells

ATP-sensitive potassium channels (K_ATP) regulate electrical activity and insulin secretion in pancreatic β-cells. When glucose concentration increases, the [ATP]/[ADP] ratio rises closing K_ATP channels, and the membrane potential depolarizes, triggering insulin secretion. This pivotal role of K_ATP channels is used not only by glucose but also by neurotransmitters, hormones and other physiological agents to modulate electrical and secretory β-cell response.In recent years, it has been demonstrated that estrogens and estrogen receptors are involved in glucose homeostasis, and that they can modulate the electrical activity and insulin secretion of pancreatic β-cells. The hormone 17β-estradiol (E2), at physiological levels, is implicated in maintaining normal insulin sensitivity for β-cell function. Long term exposure to E2 increases insulin content, insulin gene expression and insulin release via the estrogen receptor α (ERα), while rapid responses to E2 can regulate K_ATP channels increasing cGMP levels through the estrogen receptor β (ERβ) and type A guanylate cyclase receptor (GC-A). This review summarizes the main actions of 17β-estradiol on K_ATP channels and the subsequent insulin release in pancreatic β-cells.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Studies with GIP/Ins cells indicate secretion by gut K cells is KATP channel independent

K cells are a subpopulation of enteroendocrine cells that secrete glucose-dependent insulinotropic polypeptide (GIP), a hormone that promotes glucose homeostasis and obesity. Therefore, it is important to understand how GIP secretion is regulated. GIP-producing (GIP/Ins) cell lines secreted hormones in response to many GIP secretagogues except glucose. In contrast, glyceraldehyde and methyl pyruvate stimulated hormone release. Measurements of intracellular glucose 6-phosphate, fructose 1,6-bisphosphate, and pyruvate levels, as well as glycolytic flux, in glucose-stimulated GIP/Ins cells indicated that glycolysis was not impaired. Analogous results were obtained using glucose-responsive MIN6 insulinoma cells. Citrate levels increased similarly in glucose-treated MIN6 and GIP/Ins cells. Thus pyruvate entered the tricarboxylic acid cycle. Glucose and methyl pyruvate stimulated 1.4- and 1.6-fold increases, respectively, in the ATP-to-ADP ratio in GIP/Ins cells. Glyceraldehyde profoundly reduced, rather than increased, ATP/ADP. Thus nutrient-regulated secretion is independent of the ATP-dependent potassium (K(ATP)) channel. Antibody staining of mouse intestine demonstrated that enteroendocrine cells producing GIP, glucagon-like peptide-1, CCK, or somatostatin do not express detectable levels of inwardly rectifying potassium (Kir) 6.1 or Kir 6.2, indicating that release of these hormones in vivo may also be K(ATP) channel independent. Conversely, nearly all cells expressing chromogranin A or substance P and approximately 50% of the cells expressing secretin or serotonin exhibited Kir 6.2 staining. Compounds that activate calcium mobilization were potent secretagogues for GIP/Ins cells. Secretion was only partially inhibited by verapamil, suggesting that calcium mobilization from intracellular and extracellular sources, independent from K(ATP) channels, regulates secretion from some, but not all, subpopulations of enteroendocrine cells.     

Tolbutamide and diazoxide modulate phospholipase C-linked Ca(2+) signaling and insulin secretion in beta-cells

Arginine vasopressin (AVP), bombesin, and ACh increase cytosolic free Ca(2+) and potentiate glucose-induced insulin release by activating receptors linked to phospholipase C (PLC). We examined whether tolbutamide and diazoxide, which close or open ATP-sensitive K(+) channels (K(ATP) channels), respectively, interact with PLC-linked Ca(2+) signals in HIT-T15 and mouse beta-cells and with PLC-linked insulin secretion from HIT-T15 cells. In the presence of glucose, the PLC-linked Ca(2+) signals were enhanced by tolbutamide (3-300 microM) and inhibited by diazoxide (10-100 microM). The effects of tolbutamide and diazoxide on PLC-linked Ca(2+) signaling were mimicked by BAY K 8644 and nifedipine, an activator and inhibitor of L-type voltage-sensitive Ca(2+) channels, respectively. Neither tolbutamide nor diazoxide affected PLC-linked mobilization of internal Ca(2+) or store-operated Ca(2+) influx through non-L-type Ca(2+) channels. In the absence of glucose, PLC-linked Ca(2+) signals were diminished or abolished; this effect could be partly antagonized by tolbutamide. In the presence of glucose, tolbutamide potentiated and diazoxide inhibited AVP- or bombesin-induced insulin secretion from HIT-T15 cells. Nifedipine (10 microM) blocked both the potentiating and inhibitory actions of tolbutamide and diazoxide on AVP-induced insulin release, respectively. In glucose-free medium, AVP-induced insulin release was reduced but was again potentiated by tolbutamide, whereas diazoxide caused no further inhibition. Thus tolbutamide and diazoxide regulate both PLC-linked Ca(2+) signaling and insulin secretion from pancreatic beta-cells by modulating K(ATP) channels, thereby determining voltage-sensitive Ca(2+) influx.     

Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels

ATP-sensitive potassium (KATP) channels couple the metabolic status of a cell to its membrane potential-a property that endows pancreatic beta-cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that KATP channels undergo rapid internalisation from the plasma membrane by clathrin-mediated endocytosis. We present several lines of evidence to demonstrate that endocytosis is mediated by a tyrosine based signal (330YSKF333) located in the carboxy-terminus of Kir6.2 and that SUR1 has no direct role. We show that genetic mutations, Y330C and F333I, which cause permanent neonatal diabetes mellitus, disrupt this motif and abrogate endocytosis of reconstituted mutant channels. The resultant increase in the surface density of KATP channels would predispose beta-cells to hyperpolarise and may account for reduced insulin secretion in these patients. The data imply that endocytosis of KATP channels plays a crucial role in the (patho)-physiology of insulin secretion.     

Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density

Pancreatic ATP-sensitive potassium (K_ATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed K_ATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that K_ATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases K_ATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native K_ATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated K_ATP channel trafficking may play a role in the regulation of insulin secretion.     

Glucosensing by GnRH neurons: inhibition by androgens and involvement of AMP-activated protein kinase

GnRH neurons integrate steroidal and metabolic cues to regulate fertility centrally. Central glucoprivation reduces LH secretion, which is governed by GnRH release, suggesting GnRH neuron activity is modulated by glucose availability. Here we tested whether GnRH neurons can sense changes in extracellular glucose, and whether glucosensing is altered by the steroids dihydrotestosterone (DHT) and/or estradiol (E). Extracellular recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice ± DHT and/or E implants. Firing rate was reduced by a switch from 4.5 to 0.2 mm glucose in cells from OVX, OVX+E, and OVX+DHT+E mice, but not OVX+DHT mice. This suggests that androgens reduce the sensitivity of GnRH neurons to changes in extracellular glucose, but E mitigates this effect. Next we investigated potential mechanisms. In the presence of the ATP-sensitive potassium channel antagonist tolbutamide, glucosensing persisted. In contrast, glucosensing was attenuated in the presence of compound C, an antagonist of AMP-activated protein kinase (AMPK), suggesting a role for AMPK in glucosensing. The AMPK activator N1-(b-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR) mimicked the effect of low glucose and was less effective in cells from DHT-treated mice. The effect of DHT to diminish responses to low glucose and AICAR was abolished by blockade of fast synaptic transmission. Both AICAR and low glucose activated a current with a reversal potential near -50 mV, suggesting a nonspecific cation current. These studies indicate that glucosensing is one mechanism by which GnRH neurons sense fuel availability and point to a novel role for AMPK in the central regulation of fertility.     

Brain glucose metabolism controls the hepatic secretion of triglyceride-rich lipoproteins

Increased production of very low-density lipoprotein (VLDL) is a critical feature of the metabolic syndrome. Here we report that a selective increase in brain glucose lowered circulating triglycerides (TG) through the inhibition of TG-VLDL secretion by the liver. We found that the effect of glucose required its conversion to lactate, leading to activation of ATP-sensitive potassium channels and to decreased hepatic activity of stearoyl-CoA desaturase-1 (SCD1). SCD1 catalyzed the synthesis of oleyl-CoA from stearoyl-CoA. Curtailing the liver activity of SCD1 was sufficient to lower the hepatic levels of oleyl-CoA and to recapitulate the effects of central glucose administration on VLDL secretion. Notably, portal infusion of oleic acid restored hepatic oleyl-CoA to control levels and negated the effects of both central glucose and SCD1 deficiency on TG-VLDL secretion. These central effects of glucose (but not those of lactate) were rapidly lost in diet-induced obesity. These findings indicate that a defect in brain glucose sensing could play a critical role in the etiology of the metabolic syndrome.     

Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels.     

Guanidinated casein hydrolysate stimulation of cholecystokinin release via pancreatic enzyme- and cholinergic-independent mechanisms in rats.

We had demonstrated that a peptic hydrolysate of guanidinated casein that is made from casein by the conversion of lysine to homoarginine stimulated pancreatic exocrine secretion in rats with chronic bile-pancreatic juice (BPJ) diversion from the proximal small intestine. This modified protein also stimulated cholecystokinin (CCK) release from dispersed rat intestinal cells. In this study, we found that guanidinated casein hydrolysate stimulates CCK release in chronic BPJ-diverted rats with cholinergic control blocked by atropine. Intraduodenal guanidinated casein hydrolysate increased portal plasma CCK concentration and pancreatic secretion in atropine-treated BPJ-diverted rats. In contrast, the portal plasma CCK concentration was not increased by intact casein hydrolysate. We conclude that guanidinated casein hydrolysate directly stimulates CCK release from the intestine via some cholinergic-independent mechanism, and an increase of the pancreatic exocrine secretion is regulated by CCK released by guanidinated casein hydrolysate. A guanidyl residue is likely to be involved in this control.     

Effect of glucose and K+ depolarization on cytosolic free Ca2+ in cultured neonatal islets.

In cultured neonatal islet cells, glucose (16.7 mM) and K+ (50 mM) increased cytosolic free Ca2+ ([Ca2+]i). The increments in [Ca2+]i induced by either glucose or K+ were similar to those obtained in cultured adult islet cells but only half of that recorded in freshly isolated adult islet cells. These data indicate that, in neonatal islet cells, the reduced insulin release in response to glucose is associated with a diminished increase in [Ca2+]i. This reduced insulin response may not solely be due to an impaired regulation of the ATP-sensitive K+ channels as previously suggested. It may also result from some alteration in the process of Ca2+ inflow through voltage-sensitive Ca2+ channels.