Supplementation of conjugated linoleic acid in dairy cows reduces endogenous glucose production during early lactation.

Trans-10,cis-12 conjugated linoleic acid (CLA) supplementation causes milk fat depression in dairy cows, but CLA effects on glucose metabolism are not clear. The objective of the study was to investigate glucose metabolism, especially endogenous glucose production (eGP) and glucose oxidation (GOx), as well as hepatic genes involved in endogenous glucose production in Holstein cows supplemented either with 50 g of rumen-protected CLA (9% trans-10,cis-12 and 10% cis-9,trans-11; CLA; n=10) or 50 g of control fat (24% C18:2; Ctrl; n=10) from wk 2 before parturition to wk 9 of lactation. Animal performance data were recorded and blood metabolites and hormones were taken weekly from 2 wk before to 12 wk after parturition. During wk 3 and 9 after parturition, glucose tolerance tests were performed and eGP and GOx were measured by [U-(13)C] glucose infusion. Liver biopsies were taken at the same time to measure total fat and glycogen concentrations and gene expression of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and carnitine palmitoyl-transferase 1. Conjugated linoleic acid feeding reduced milk fat, but increased milk lactose output; milk yield was higher starting 5 wk after parturition in CLA-fed cows than in Ctrl-fed cows. Energy balance was more negative during CLA supplementation, and plasma concentrations of glucose were higher immediately after calving in CLA-fed cows. Conjugated linoleic acid supplementation did not affect insulin release during glucose tolerance tests, but reduced eGP in wk 3, and eGP and GOx increased with time after parturition. Hepatic gene expression of cytosolic phosphoenolpyruvate carboxykinase tended to be lower in CLA-fed cows than in Ctrl-fed cows. In spite of lower eGP in CLA-fed cows, lactose output and plasma glucose concentrations were greater in CLA-fed cows than in Ctrl-fed cows. This suggests a CLA-related glucose sparing effect most likely due to lower glucose utilization for milk fat synthesis and probably because of a more efficient whole-body energy utilization in CLA-fed cows.

Pancreatic β cells synthesize and secrete nerve growth factor

Differentiation and function of pancreatic β cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic β cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in β cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic β-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

MgATP activates the β cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (KATP) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.

Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits

The ATP-sensitive potassium channel (K-ATP channel) plays a key role in insulin secretion from pancreatic β-cells. It is closed by glucose metabolism, which stimulates secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and MgADP concentration, which inhibit and potentiate channel activity, respectively. The β-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. The site at which ATP mediates channel inhibition lies on Kir6.2, while the potentiatory action of MgADP involves the nucleotide-binding domains of SUR1. K-ATP channels are also activated by MgGTP and MgGDP. Furthermore, both nucleotides support the stimulatory actions of diazoxide. It is not known, however, whether guanine nucleotides mediate their effects by direct interaction with one or more of the K-ATP channel subunits or indirectly via a GTP-binding protein. We used a truncated form of Kir6.2, which expresses independently of SUR1, to show that GTP blocks K-ATP currents by interaction with Kir6.2 and that the potentiatory effects of GTP are endowed by SUR1. We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. This argues that the stimulatory effects of guanine nucleotides require the presence of both Walker A lysines.

Phentolamine block of KATP channels is mediated by Kir6.2

The ATP-sensitive K+-channel (KATP channel) plays a key role in insulin secretion from pancreatic β cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca2+ entry and insulin release. The β cell KATP channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K+-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit KATP channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. KATP channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac KATP channel.

A common muscarinic pathway for diapause recovery in the distantly related nematode species Caenorhabditis elegans and Ancylostoma caninum

Converging TGF-β and insulin-like neuroendocrine signaling pathways regulate whether Caenorhabditis elegans develops reproductively or arrests at the dauer larval stage. We examined whether neurotransmitters act in the dauer entry or recovery pathways. Muscarinic agonists promote recovery from dauer arrest induced by pheromone as well as by mutations in the TGF-β pathway. Dauer recovery in these animals is inhibited by the muscarinic antagonist atropine. Muscarinic agonists do not induce dauer recovery of either daf-2 or age-1 mutant animals, which have defects in the insulin-like signaling pathway. These data suggest that a metabotropic acetylcholine signaling pathway activates an insulin-like signal during C. elegans dauer recovery. Analogous and perhaps homologous cholinergic regulation of mammalian insulin release by the autonomic nervous system has been noted. In the parasitic nematode Ancylostoma caninum, the dauer larval stage is the infective stage, and recovery to the reproductive stage normally is induced by host factors. Muscarinic agonists also induce and atropine potently inhibits in vitro recovery of A. caninum dauer arrest. We suggest that host or parasite insulin-like signals may regulate recovery of A. caninum and could be potential targets for antihelminthic drugs.

The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.

Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

Control of the endocrine pancreas by gastrointestinal peptides

This thesis concerns the mechanism through which enteral delivery of glucose results in a larger insulin response than an equivalent parenteral glucose load. Preliminary studies in which mice received a glucose solution either intragastrically or intraperitoneally confirmed this phenomenon. An important regulatory system in this respect is the entero-insular axis, through which insulin secretion is influenced by neural and endocrine communication between the gastrointestinal tract and the pancreatic islets of Langerhans. Using an in vitro system involving static incubation of isolated (by collagenase digestion) islets of Langerhans, the effect of a variety of gastrointestinal peptides on the secretion of the four main islet hormones, namely insulin, glucagon, somatostatin and pancreatic polypeptide, was studied. The gastrointestinal peptides investigated in this study were the secretin family, comprising secretin, glucagon, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and growth hormone releasing factor (GRF). Gastrin releasing peptide (GRP) was also studied. The results showed that insulin release was stimulated by all peptides studied except PHI, glucagon release was stimulated by all peptides tested, except GRF which suppressed glucagon release, somatostatin release was stimulated by GIP and GRF but suppressed by VIP, PHI, glucagon and secretin, and PP release was stimulated by GIP and GRF, but suppressed by PHI. The insulinotropic effect of GRP was investigated further. A perifusion system was used to examine the time-course of insulin release from isolated islets after stimulation with GRP. GRP was shown to be insulinotropic only in the presence of physiologically elevated glucose concentrations and both first and second phases of insulin release were augmented. There was no effect at substimulatory or very high glucose concentrations. Studies using a cultured insulin-secreting islet cell line, the RINm5F cell line, were undertaken to elucidate the intracellular mechanism of action of GRP. This peptide did not enhance insulin release via an augmentation of glucose metabolism, or via the adenylate cyclase/cyclic AMP secondary messenger system. The pattern of changes of cytosolic free calcium in response to GRP, which involved both mobilization of intracellular stores and an influx of extracellular calcium, suggested the involvement of phosphatidylinositol bisphosphate breakdown as a mediator of the effect of GRP on insulin secretion.

Microencapsulation strategies for islet transplantation

A variety of islet microencapsulation techniques have been investigated to establish which method provides the least occlusive barrier to net insulin release in vitro, and optimum biocompatibility for islet implantation in vivo. NMRI mouse islets have been microencapsulated with Na+ -alginate-poly-L-lysine (PLL)/poly-L-ornithine (PLO)-alginate, Ba2+ -alginate and agarose gels. Both free and microencapsulated islets responded to glucose challenge in static incubation and perifusion by significantly increasing their rate of insulin release and theophylline significantly potentiated the insulin response to glucose. While little insulin was released from microencapsulated islets after short term (2 hours) static incubation, significantly greater amounts were released in response to glucose challenge after extended (8-24 hours) incubation. However, insulin release from all types of microencapsulated islets was significantly reduced compared with free islets. Na+ -alginate-PLO-alginate microencapsulated islets were significantly more responsive to elevated glucose than Na+ -alginate-PLL-alginate microencapsulated islets, due to the enhanced porosity of PLO membranes. The outer alginate layer created a significant barrier to glucose/insulin exchange and reduced the insulin responsiveness of microencapsulated islets to glucose. Ba2+ -alginate membrane coated islets, generated by the density gradient method, were the most responsive to glucose challenge. Low concentrations of NG-monomethyl L-arginine (L-NMMA) had no significant effect on glucose stimulated insulin release from either free or microencapsulated islets. However, 1.0 mmol/1 L-NMMA significantly inhibited the insulin response of both free and microencapsulated islets to glucose challenge. In vivo work designed to evaluate the extent of pericapsular fibrosis after 28 days ip. and sc. implantation of microencapsulated islets into STZ-diabetic recipients, revealed that the inclusion of islets within microcapsules increased their immunogenicity and markedly increased the extent of pericapsular fibrosis. When the outer alginate layer was omitted from microcapsules, little or no pericapsular mononuclear cell deposition was observed. The subcutaneous site was not suitable for microencapsulated islet transplantation in NMRI recipient mice. Systemic immunosuppression using cyclosporin A was effective in preventing pericapsular mononuclear cell deposition, while L-NMMA loading into microcapsules had no significant effect on pericapsular fibrosis, although it did maintain the integrity of microencapsulated islets.

Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets

Elevated islet uncoupling protein-2 (UCP-2) impairs β-cell function and UCP-2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP-2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5–22 mmol/l)±leptin (0–10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose-dependently increased UCP-2 expression in all islet batches, maximally by three-fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP-2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP-2 expression in human islets and provides novel evidence of its role in the loss of β-cell function in diabetes.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala8-substituted analogues of GLP-1, (Abu8)GLP-1 and (Val8)GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu8)GLP-1 and (Val8)GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu8)GLP-1 and (Val8)GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val8)GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu8 )GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val8)GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala8 in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val8)GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Glycemic index role on visceral obesity, subclinical inflammation and associated chronic diseases

Background: It is believed that the glycemic index (GI) may be used as a strategy to prevent and control noncommunicable diseases (NCD). Obesity is a multifactorial condition, a risk factor for development of other NCDs. Among the different types, abdominal obesity is highlighted, which is essential for the diagnosis of metabolic syndrome, and it is related to insulin resistance, dyslipi-demia, hypertension and changes in levels of inflammatory markers. Such indicators are closely related to the development of Type 2 Diabetes and cardiovascular disease. Objectives: Discuss the role of GI as a strategy for the prevention and/or treatment of visceral obesity, subclinical inflammation and chronic diseases. Results and discussion: The intake of low GI diets is associated with glycemic decreases, and lower and more consistent postprandial insulin release, avoiding the occurrence of hypoglycemia. Moreover, consumption of a low GI diet has been indicated as beneficial for reducing body weight, total body fat and visceral fat, levels of proinflammatory markers and the occurrence of dyslipidemia and hypertension. The intake of low GI foods should be encouraged in order to prevent and control non-communicable diseases.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.