Effect of feeding fat or intrajugular infusion of glucagon-like peptide-1 and cholecystokinin on dry matter intake, digestibility, and digesta rate of passage in growing wethers

A cause and effect relationship between glucagon-like peptide 1 (7, 36) amide (GLP-1) and cholecystokinin (CCK) and DMI regulation has not been established in ruminants. Three randomized complete block experiments were conducted to determine the effect of feeding fat or infusing GLP-1 or CCK intravenously on DMI, nutrient digestibility, and Cr rate of passage (using Cr(2)O(3) as a marker) in wethers. A total of 18 Targhee × Hampshire wethers (36.5 ± 2.5 kg of BW) were used, and each experiment consisted of four 21-d periods (14 d for adaptation and 7 d for infusion and sampling). Wethers allotted to the control treatments served as the controls for all 3 experiments; experiments were performed simultaneously. The basal diet was 60% concentrate and 40% forage. In Exp. 1, treatments were the control (0% added fat) and addition of 4 or 6% Ca salts of palm oil fatty acids (DM basis). Treatments in Exp. 2 and 3 were the control and 3 jugular vein infusion dosages of GLP-1 (0.052, 0.103, or 0.155 µg•kg of BW(-1)•d(-1)) or CCK (0.069, 0.138, or 0.207 µg•kg of BW(-1)•d(-1)), respectively. Increases in plasma GLP-1 and CCK concentrations during hormone infusions were comparable with increases observed when increasing amounts of fat were fed. Feeding fat and infusion of GLP-1 tended (linear, P = 0.12; quadratic, P = 0.13) to decrease DMI. Infusion of CCK did not affect (P > 0.21) DMI. Retention time of Cr in the total gastrointestinal tract decreased (linear, P < 0.01) when fat was fed, but was not affected by GLP-1 or CCK infusion. In conclusion, jugular vein infusion produced similar plasma CCK and GLP-1 concentrations as observed when fat was fed. The effects of feeding fat on DMI may be partially regulated by plasma concentration of GLP-1, but are not likely due solely to changes in a single hormone concentration.

Insulin, glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide and insulin-like growth factor I as putative mediators of the hypolipidemic effect of oligofructose in rats

The addition of oligofructose as a dietary fiber decreases the serum concentration and the hepatic release of VLDL-triglycerides in rats. Because glucose, insulin, insulin-like growth factor I (IGF-I) and gut peptides [i.e., glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1)]) are factors involved in the metabolic response to nutrients, this paper analyzes their putative role in the hypolipidemic effect of oligofructose. Male Wistar rats were fed a nonpurified diet with or without 10% oligofructose for 30 d. Glucose, insulin, IGF-I and GIP concentrations were measured in the serum of rats after eating. GIP and GLP-1 contents were also assayed in small intestine and cecal extracts, respectively. A glucose tolerance test was performed in food-deprived rats. Serum insulin level was significantly lower in oligofructose-fed rats both after eating and in the glucose tolerance test, whereas glycemia was lower only in the postprandial state. IGF-I serum level did not differ between groups. GIP concentration was significantly higher in the serum of oligofructose-fed rats. The GLP-1 cecal pool was also significantly higher. In this study, we have shown that cecal proliferation induced by oligofructose leads to an increase in GLP-1 concentration. This latter incretin could be involved in the maintenance of glycemia despite a lower insulinemia in the glucose tolerance test in oligofructose-fed rats. We discuss also the role of hormonal changes in the antilipogenic effect of oligofructose.

Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice. Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signalling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein

In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.

Feeding rumen-inert fats differing in their degree of saturation decreases intake and increases plasma concentrations of gut peptides in lactating dairy cows

Our objective was to determine the effect of feeding rumen-inert fats differing in their degree of saturation on dry matter intake (DMI), milk production, and plasma concentrations of insulin, glucagon-like peptide 1 (7-36) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin (CCK) in lactating dairy cows. Four midlactation, primiparous Holstein cows were used in a 4 x 4 Latin square experiment with 2-wk periods. Cows were fed a control mixed ration ad libitum, and treatments were the dietary addition (3.5% of ration dry matter) of 3 rumen-inert fats as sources of mostly saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), or polyunsaturated fatty acids (PUFA). Daily DMI, milk yield, and composition were measured on the last 4 d of each period. Jugular vein blood was collected every 30 min over a 7-h period on d 12 and 14 of each period for analysis of plasma concentrations of hormones, glucose, and nonesterified fatty acids. Feeding fat decreased DMI, and the decrease tended to be greater for MUFA and PUFA compared with SFA. Plasma concentration of GLP-1 increased when fat was fed and was greater for MUFA and PUFA. Feeding fat increased plasma glucose-dependent insulinotropic polypeptide and CCK concentrations and decreased plasma insulin concentration. Plasma CCK concentration was greater for MUFA and PUFA than for SFA and was greater for MUFA than PUFA. Decreases in DMI in cows fed fat were associated with increased plasma concentrations of GLP-1 and CCK and a decreased insulin concentration. The role of these peptides in regulating DMI in cattle fed fat requires further investigation.

Plasma concentrations of gut peptides in dairy cattle increase after calving

Effects of transition from late gestation to early lactation on plasma concentrations of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1-(7-36) amide (GLP-1), and cholecystokinin (CCK) have not been reported in cattle. The objective of the present study was to measure plasma concentrations of GLP-1, GIP, CCK, insulin, glucose, and nonesterified fatty acids in blood plasma obtained from the coccygeal vein of 32 Holstein cows at an average of 11 d before, and 5, 12, and 19 d after calving. Feed dry matter intake (DMI) averaged 14.4, 17.7, and 19.9 kg/d on d 5, 12, and 19 of lactation, respectively, as milk yield increased (30.6, 36.6, and 39.7 kg/d, respectively). Plasma concentrations of insulin and glucose were lower postpartum than prepartum, but did not differ among samples collected after calving. In contrast, plasma concentration of gut peptides increased linearly after calving, perhaps as a consequence of increased feed intake and nutrient absorption; however, the increases in plasma concentrations of GIP and GLP-1 as lactation progressed were not associated with increased DMI per se, and likely reflect the endocrine and metabolic adaptations of lactogenesis. In contrast, increased concentration of CCK was related both to increasing days in milk and DMI. By 19 d postpartum, concentrations of GLP-1, GIP, and CCK increased by 2.3-, 1.8-, and 2.8-fold, respectively, compared with values at 11 d before calving. Although these peptides have direct and indirect effects that reduce appetite and DMI in other species (including increased insulin secretion), these may be glucose- or insulin-dependent functions, and insulin and glucose concentrations were reduced in early lactation.

Abomasal infusion of casein, starch and soybean oil differentially affect plasma concentrations of gut peptides and feed intake in lactating dairy cows

The effects of specific nutrients on secretion and plasma concentrations of gut peptides (glucagon-like peptide-1((7-36)) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin-8 (CCK)) differ across species, but are not reported for cattle. Our objective was to determine acute (hours) and chronic (1 week) effects of increased abomasal supply of protein, carbohydrate, or fat to the small intestine on dry matter intake (DMI) and plasma concentrations of GLP-1, GIP, CCK, and insulin. Four mid-lactation Holstein cows were used in a 4 x 4 Latin square design experiment. Treatments were 7-day abomasal infusions of water, soybean oil (500 g/d), corn starch (1100 g/d), or casein (800 g/d). Jugular vein plasma was obtained over 7 h at the end of the first and last day of infusions. Oil infusion decreased DMI on day 7, but total metabolizable energy (ME) supply (diet plus infusate) did not differ from water infusion. Casein and starch infusion had no effect on feed DMI; thus, ME supply increased. Decreased DMI on day 7 of oil infusion was accompanied by increased plasma GLP-1 concentration, but decreased plasma CCK concentration. Increased plasma GIP concentration was associated with increased ME supply on day 7 of casein and starch infusion. Casein infusion tended to increase plasma CCK concentration on both days of sampling, and increased plasma GLP-1 and insulin concentration on day 1 of infusion. The present data indicate a sustained elevation of plasma concentration of GLP-1, but not CCK, may contribute to the reduced DMI observed in dairy cows provided supplemental fat. (C) 2008 Elsevier Inc. All rights reserved.

Supramolecular peptide helix from a novel double turn forming peptide containing a beta-amino acid

A single-crystal X-ray diffraction study of the terminally protected tetrapeptide Boc-beta-Ala-Aib-Leu-Aib-OMe 1 (Aib: alpha-aminoisobutyric acid; beta-Ala: beta-Alanine) reveals that it adopts a new type of double turn structure which self-associates to form a unique supramolecular helix through intermolecular hydrogen bonds. Scanning electron microscopic studies show that peptide 1 exhibits amyloid-like fibrillar morphology in the solid state. (C) 2003 Elsevier Science Ltd. All rights reserved.

Can a consecutive double turn conformation be considered as a peptide based molecular scaffold for supramolecular helix in the solid state?

Helices and sheets are ubiquitous in nature. However, there are also some examples of self-assembling molecules forming supramolecular helices and sheets in unnatural systems. Unlike supramolecular sheets there are a very few examples of peptide sub-units that can be used to construct supramolecular helical architectures using the backbone hydrogen bonding functionalities of peptides. In this report we describe the design and synthesis of two single turn/bend forming peptides (Boc-Phe-Aib-Ile-OMe 1 and Boc-Ala-Leu-Aib-OMe 2) (Aib: alpha-aminoisobutyric acid) and a series of double-turn forming peptides (Boc-Phe-Aib-IIe-Aib-OMe 3, Boc-Leu-Aib-Gly-Aib-OMe 4 and Boc-gamma-Abu-Aib-Leu-Aib-OMe 5) (gamma-Abu: gamma-aminobutyric acid). It has been found that, in crystals, on self-assembly, single turn/bend forming peptides form either a supramolecular sheet (peptide 1) or a supramolecular helix (peptide 2). unlike self-associating double turn forming peptides, which have only the option of forming supramolecular helical assemblages. (c) 2005 Elsevier Ltd. All rights reserved.

Effect of feed restriction and supplemental dietary fat on gut peptide and hypothalamic neuropeptide mRNA concentrations in growing wethers

The objectives of the present study were 1) to evaluate the effects of supplemental fat and ME intake on plasma concentrations of glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide, ghrelin, and oxyntomodulin; and 2) to determine the association of these peptides with DMI and the hypothalamic concentration of mRNA for the following neuropeptides: neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC). In a completely randomized block design with a 2 x 2 factorial arrangement of treatments, 32 pens with 2 wethers each were restricted-fed (2.45 Mcal/lamb per day) or offered diets ad libitum (n = 16) with or without 6% supplemental fat (n = 16) for a period of 30 d. Dry matter intake was measured daily. On d 8, 15, 22, and 29, BW was measured before feeding, and 6 h after feeding, blood samples were collected for plasma measurement of insulin, GLP-1, CCK, ghrelin, glucose-dependent insulinotropic polypeptide, oxyntomodulin, glucose, and NEFA concentrations. On d 29, blood was collected 30 min before feeding for the same hormone and metabolite analyses. At the end of the experiment, wethers were slaughtered and the hypothalami were collected to measure concentrations of NPY, AgRP, and POMC mRNA. Offering feed ad libitum (resulting in greater ME intake) increased plasma insulin and NEFA concentrations (P = 0.02 and 0.02, respectively) and decreased hypothalamic mRNA expression of NPY and AgRP (P = 0.07 and 0.02, respectively) compared with the restricted-fed wethers. There was a trend for the addition of dietary fat to decrease DMI (P = 0.12). Addition of dietary fat decreased insulin and glucose concentrations (P < 0.05 and 0.01, respectively) and tended to increase hypothalamic mRNA concentrations for NPY and AgRP (P = 0.07 and 0.11, respectively). Plasma GLP-1 and CCK concentrations increased in wethers offered feed ad libitum compared with restricted-fed wethers, but the response was greater when wethers were offered feed ad libitum and had supplemental fat in the diet (fat x intake interaction, P = 0.04). The prefeeding plasma ghrelin concentration was greater in restricted-fed wethers compared with those offered feed ad libitum, but the concentrations were similar 6 h after feeding (intake x time interaction, P < 0.01). Supplemental dietary fat did not affect (P = 0.22) plasma ghrelin concentration. We conclude that insulin, ghrelin, CCK, and GLP-1 may regulate DMI in sheep by regulating the hypothalamic gene expression of NPY, AgRP, and POMC.

Synthesis, crystal structure, and magnetic properties of a very rare double μ-1,1-azido- and a μ-1,1-(OMe)-bridged FeIII dimer containing a N,N,O-donor tridentate Schiff base ligand

Two new Fe-III complexes, [Fe2L2(mu-OMe)(2)(NCS)(2)] (1) and [Fe2L2(mu-N-3)(2)(N-3)(2)] (2), have been synthesized using a N,N,O-donor tridentate Schiff base ligand HL {2-[(2-dimethylaminoethylimino)methyl]phenol}, the condensation product of salicylaldehyde and N,N-dimethyl-1,2-diaminoethane. The complexes were characterized by X-ray structural analyses and variable-temperature magnetic susceptibility measurements. Both crystal structures are centrosymmetric dimers containing two Fe-III atoms, which are bridged in compound 1 by two methoxy anions and in compound 2 by two mu-1,1-azides. The chelating tridentate Schiff base and a terminal thiocyanato (for 1) or azido (for 2) group complete the hexacoordination of the distorted octahedral environment of each iron center. The magnetic properties of compound 1 show the presence of antiferromagnetic exchange interactions mediated by double methoxy bridges (J = -29.45 cm(-1)). Compound 2 shows the presence of very weak ferromagnetic exchange interactions mediated by double mu-1,1-N-3 bridges (J = 1.08 cm(-1)).

Dry matter intake is decreased more by abomasal infusion of unsaturated free fatty acids than by unsaturated Triglycerides

Previous experiments from our group have demonstrated that abomasal infusion of unsaturated free fatty acids (FFA) markedly decreases dry matter intake (DMI) in dairy cows. In contrast, experiments from other groups have noted smaller decreases in DMI when unsaturated triglycerides (TG) were infused postruminally. Our hypothesis was that unsaturated FFA would be more potent inhibitors of DMI than an equivalent amount of unsaturated TG. Four Holstein cows in late lactation were used in a single reversal design. Cows were fed a total mixed ration containing (DM basis) 23% alfalfa silage, 23% corn silage, 40.3% ground shelled corn, and 10.5% soybean meal. Two cows received soy FFA (UFA; 0, 200, 400, 600 g/d) and 2 received soy oil (TG) in the same amounts; cows then were switched to the other lipid source. Cows were abomasally infused with each amount for 5-d periods. The daily amount of lipid was pulse-dosed in 4 equal portions at 0600, 1000, 1700, and 2200 h; no emulsifiers were used and there was no sign of digestive disturbance. Both lipid sources linearly decreased DMI, with a significant interaction between lipid source and amount. Slope-ratio analysis indicated that UFA were about 2 times more potent in decreasing DMI than were TG. Decreased DMI led to decreased milk production. Milk fat content was increased linearly by lipid infusion. Milk fat yield decreased markedly for UFA infusion but was relatively unaffected by infusion of TG. Contents of short- and medium-chain fatty acids in milk fat decreased as the amount of either infusate increased. Contents of C-18:2 and C18: 3 in milk fat were increased linearly by abomasal infusion of either fat source; cis-9 C-18:1 was unaffected. Transfer of infused C18: 2 to milk fat was 35.6, 42.5, and 27.8% for 200, 400, and 600 g/d of UFA, and 34.3, 39.6, and 34.0% for respective amounts of TG. Glucagon-like peptide-1 (7-36) amide (GLP-1) concentration in plasma significantly increased as DMI decreased with increasing infusion amount of UFA or TG. Plasma concentration of cholecystokinin-octapeptide (CCK-8) was unaffected by lipid infusion. These results indicate that unsaturated FFA reaching the duodenum are more potent inhibitors of DMI than are unsaturated TG; the effect may be at least partially mediated by GLP-1.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

A suite of parallel vectors for baculovirus expression

The expression of proteins using recombinant baculoviruses is a mature and widely used technology. However, some aspects of the technology continue to detract from high throughput use and the basis of the final observed expression level is poorly understood. Here, we describe the design and use of a set of vectors developed around a unified cloning strategy that allow parallel expression of target proteins in the baculovirus system as N-terminal or C-terminal fusions. Using several protein kinases as tests we found that amino-terminal fusion to maltose binding protein rescued expression of the poorly expressed human kinase Cot but had only a marginal effect on expression of a well-expressed kinase IKK-2. In addition, MBP fusion proteins were found to be secreted from the expressing cell. Use of a carboxyl-terminal GFP tagging vector showed that fluorescence measurement paralleled expression level and was a convenient readout in the context of insect cell expression, an observation that was further supported with additional non-kinase targets. The expression of the target proteins using the same vectors in vitro showed that differences in expression level were wholly dependent on the environment of the expressing cell and an investigation of the time course of expression showed it could affect substantially the observed expression level for poorly but not well-expressed proteins. Our vector suite approach shows that rapid expression survey can be achieved within the baculovirus system and in addition, goes some way to identifying the underlying basis of the expression level obtained. (c) 2006 Elsevier Inc. All rights reserved.

The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat ( RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT ( HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 ( AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract - but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.