Changes in daily leptin, ghrelin and adiponectin profiles following a diet with carbohydrates eaten at dinner in obese subjects.

BACKGROUND AND AIMS: Our recently published randomised clinical trial evaluated the effect of a low-calorie diet with carbohydrates eaten at dinner. This dietary pattern led to lower hunger scores, and better anthropometric, biochemical and inflammatory outcomes compared to a standard low-calorie diet. In the same study, changes in diurnal secretion patterns of leptin, ghrelin and adiponectin were investigated. METHODS AND RESULTS: Seventy-eight police officers (body mass index (BMI) > 30) were randomly allocated to experimental (carbohydrates at dinner) or control weight loss diets for 6 months. Sixty-three subjects finished the programme. On days 0, 7, 90 and 180 blood samples and hunger scores were collected every 4 h from 8:00 to 20:00. Hormonal profiles were available for 39. The dietary manipulation led to changes in daylight hormonal profiles in the experimental group. Leptin's secretion curve became convex, with a nadir later in the day (significant difference compared to baseline at morning and evening, p = 0.023, p = 0.021, respectively). Ghrelin's secretion curve became concave, peaking only in the evening hours. Adiponectin's curve was elevated only after the experimental diet (significant difference compared to baseline at afternoon, p = 0.044). CONCLUSIONS: We propose that a low-calorie diet with carbohydrates eaten at dinner can modulate daytime hormonal profiles. Taken together with our earlier results, we believe this diet regime may prevent mid-day hunger, better support weight loss and improve metabolic outcomes compared to conventional weight loss diets. The trial is registered at controlled-trials.com, ISRCTN37829376, December 2009.

Highly selective hydrolysis of kinins by recombinant prolylcarboxypeptidase.

We have previously cloned a cDNA encoding human prolylcarboxypeptidase (PRCP) and expressed the cDNA in the Schneider 2 (S2) drosophila cell line. Here, we further characterized this recombinant enzyme. Investigations were performed to determine whether recombinant PRCP (rPRCP) metabolizes kinins (BK 1-9 and BK 1-8). The metabolites of these kinins were identified by LC/MS. rPRCP metabolized BK 1-8 to BK 1-7, whereas rPRCP was ineffective in metabolizing BK 1-9. The hydrolysis of BK 1-8 by rPRCP was dose- and time-dependent. A homology model of PRCP was developed based upon the sequence of dipeptidyl-peptidase 7 (DPP7, PDB ID: 3JYH), and providentially, the structure of PRCP (PDB ID: 3N2Z) was characterized during the course of our investigation. Docking studies of bradykinin oligopeptides were performed both from the homology model, and from the crystal structure of PRCP. These docking studies may provide a better understanding of the contribution of specific residues involved in substrate selectivity of human PRCP.

Identification of lipopolysaccharide binding site on high molecular weight kininogen.

Plasma kallikrein kinin system (KKS) activation along with its cellular receptors expression are increased after injury and in patients with septic shock, hypotensive bacteremia and rhesus monkey infected with Salmonella typhimurium. KKS signaling cascade is activated by activated factor XII (FXIIa, Hageman factor)- and prolylcarboxypeptidase (PRCP)-dependent pathways on endothelial cells. Among the many entities that comprise the KKS, high molecular weight kininogen (HK), a bradykinin precursor, is critical in the assembly and activation of this system. HK is primarily expressed in the liver and secreted into the bloodstream. The activation of the KKS influences the permeability of the endothelium by liberating bradykinin (BK) from HK. BK is a potent inflammatory peptide which stimulates constitutive bradykinin B2 and inducible B1 receptors to release nitric oxide and prostacyclin. Regardless of the triggers, PK can only be activated on HK bound to the artificial negatively charged or to cell membrane surfaces. Since LPS has a negatively charged moiety and the ability to induce inflammatory responses in human, we determined the interaction between LPS and HK. HKH19 (HK cell binding site) and heparin inhibited LPS binding to HK with IC(50)s of 15nM and 20 microg/ml, respectively. C1-inhibitor and N-acetylglucosamine glycan inhibited LPS binding to HK with IC(50)s of about 10 microg/ml and 10mM, respectively. This novel study underscores the implication of HK in infection. We propose that HKH19, heparin, and C1-inhibitor present therapeutic potential for the treatment of sepsis and hypotensive bacteremia.

The functional importance of the N-terminal region of human prolylcarboxypeptidase.

The renin-angiotensin-system cascade pathway generates the vasopressor and prothrombotic hormones, angiotensin II (Ang II) and angiotensin III (Ang III) from angiotensinogen. One of the key enzymes for the generation of angiotensin 1-7 (Ang 1-7) and angiotensin 2-7 (Ang 2-7) from Ang II and III, respectively, is prolylcarboxypeptidase (PRCP). To understand the contribution of the N-terminal region to catalysis, an N-terminal truncated form, lacking 179 N-terminal residues of PRCP (rPRCP(40)) was constructed. The circular dichroism (CD) spectrum of rPRCP(40) illustrated that it was structured with significant helical content as indicated by local minima at approximately 220 and 208nm. The main products of Ang III metabolized by rPRCP(40) were Ang 2-7 plus phenylalanine as determined by LC-MS. Angiotensin I (Ang I) blocked the metabolism of Ang III by rPRCP(40). These investigations showed that the C-terminal region of the rPRCP(40) contributes to PRCP's catalytic function, and provided additional experimental evidence for this suggestion.

Kininogen-cytokeratin 1 interactions in endothelial cell biology.

This review brings available data together to put in perspective the binding properties of high molecular weight kininogen (HK) to cytokeratin 1 (CK1) to direct future investigations. We summarize our knowledge of the structure and function of HK and discuss the influence of HK on the biologic activities of the plasma kallikrein/kinin system. To better understand the role of HK in regulating the activation of the kallikrein/kinin system, we discuss the interaction of HK with endothelial cell CK1 and its influence on prekallikrein activation. Last, we address the question on how a cytoskeletal protein could be a membrane-binding site and its possible role in disease states.

Estrogen stimulation of 3-o-methyl-D-glucose uptake in isolated rat hepatocytes.

The effect of in vivo and in vitro estrogen treatment on 3-O-methyl-D-glucose (30MDG) uptake in isolated rat hepatocytes was examined. A 1-h preincubation of isolated hepatocytes with 17 beta-estradiol (E2) or 17 alpha-ethynyl estradiol stimulated uptake of 30MDG in a dose-dependent fashion. The response appeared to be sterospecific, in that 17 alpha-estradiol was approximately 100-fold less effective than its 17 beta isomer. By itself, the triarylethylene antiestrogen, nafoxidine, slightly increased hepatocyte 30MDG uptake, while in combination with estrogen, nafoxidine completely blocked the effects of both E2 and 17 alpha-ethynyl estradiol. The protein synthesis inhibitor, cycloheximide, inhibited basal 30MDG uptake and abolished the stimulatory effect of estrogen. Kinetic analysis indicated that the estrogen effect resulted from an increase in the Vmax of the 30MDG transport system, with no measurable change in its Km. In vivo estrogen treatment, using either estrogen injections or continuous release Silastic E2 capsules, produced an increase in hepatocyte 30MDG uptake of 52-86%. A similar (66%) increase was seen in pregnant animals between the 14th and 19th days of gestation. These findings demonstrate that estrogens exert a rapid stimulatory effect on hepatocyte hexose transport. This effect is qualitatively similar to the response to estrogen of the hexose transport systems in other estrogen target tissues, such as the uterus. In addition, the results provide further support for the concept that the effects of the triarylethylene antiestrogens are both tissue and end-point dependent. Although previous studies have shown that nafoxidine exerts estrogen-like effects on hepatic renin substrate production, the present data indicate that, with respect to hepatocyte 30MDG, nafoxidine is almost a pure estrogen antagonist.

Cellular mechanisms of insulin resistance in polycystic ovarian syndrome.

Insulin resistance is a predominant feature in women with polycystic ovarian syndrome (PCO). The cellular mechanisms for this insulin resistance have not been defined. In this study, major steps in the insulin action cascade, receptor binding, kinase activity, and glucose transport activity were evaluated in isolated adipocytes prepared from PCO subjects (n = 8) without acanthosis nigricans and in a group of age and weight-matched controls [normal cycling (NC) n = 8]. The PCO group was hyperinsulinemic and displayed elevated insulin responses to an iv glucose load. The binding of 125I-insulin to adipocytes was similar in cells from PCO and NC subjects. In PCO, autophosphorylation of the insulin receptor-subunit in the absence of insulin was normal but a significant decrease (30% below control) in maximal insulin stimulated autophosphorylation was observed. However, receptor kinase activity measured against the exogenous substrate poly glu:tyr (4:1) was normal. Cells from PCO subjects transported glucose at the same rate, in both the absence and presence of a maximal insulin concentration, as those from NC subjects. Strikingly, there was a large rightward shift in the insulin dose-response curve for transport stimulation in PCO cells (EC50 = 87 +/- 14 pmol in NC vs. 757 +/- 138 in PCO, P less than 0.0005); 8-fold greater insulin concentrations were required to attain comparable glucose transport rates in cells from PCO against NC. In conclusion, our results suggest that insulin resistance in PCO, as assessed in the adipocyte, is accompanied by normal function of insulin receptors, but involves a novel postreceptor defect in the insulin signal transduction chain between the receptor kinase and glucose transport.

Effect of brown rice and soybean dietary fiber on the control of glucose and lipid metabolism in diabetic rats.

We have studied the effect of brown rice and soybean dietary fiber on the oral glucose tolerance test, plasma-insulin, -glucagon, -triglycerides, and cholesterol levels in control and streptozotocin-induced diabetic rats. Plasma glucose level after glucose loading in diabetic rats fed soybean fiber was considerably lower (60 and 120 min) than the corresponding group not receiving the fiber. Soybean fiber given to control rats decreased the plasma glucose at 60 min after the oral glucose test. Rice fiber decreased the plasma glucose of diabetic rats only at 60 min and the control rats fed with or without fiber elicited a comparable glycemic response. While soybean fiber significantly lowered the glucagon and triglyceride levels in both control and diabetic rats, rice fiber had no effect on these blood parameters. In conclusion, this study demonstrated the potential benefit of soybean dietary fiber over rice fiber in diabetes treatment with additional advantages resulting from its ease in usage either in a mixture of water or milk products and cooking. As well, being devoid of a disagreeable taste so characteristic of other fibers, patient acceptance is more forthcoming.

Metabolic consequences of the alpha-glucosidase inhibitor BAY-M-1099 given to nondiabetic and diabetic rats fed a high-carbohydrate diet.

The effect of a new alpha-glucosidase inhibitor BAY-M-1099 on postprandial glucose levels in nondiabetic and diabetic rats after sucrose loading was studied. Evaluation was also made of the metabolic consequences of the addition of BAY-M-1099 to a high-carbohydrate diet consisting of equal quantities of wheat starch and sucrose (Diet A). This drug significantly reduced (p less than 0.05) postprandial glucose levels in nondiabetic and diabetic rats after sucrose loading. BAY-M-1099 led to a significant reduction in urinary glucose loss (177.8 +/- 54.2 vs 98.9 +/- 35.6 mmol/L) and in postprandial plasma glucose levels in diabetic rats fed diet A. Addition of BAY-M-1099 to the diet of nondiabetic rats significantly (p less than 0.05) decreased the postprandial plasma glucose level at 45, 90, 180, and 225 min after a meal test. Addition of BAY-M-1099 to a diet containing starch plus sucrose led to reduced glycosuria and serum glucose levels and may have potential benefit in the management of diabetes mellitus.

Effect of the alpha-glucosidase inhibitor Bay-O-1248 on the metabolic response of nondiabetic and diabetic rats to a high-carbohydrate diet.

The metabolic consequences of the addition of a new alpha-glucosidase inhibitor (BAY-O-1248) to a high carbohydrate diet (67% by calories) in which the carbohydrate comprised equal quantities (50% wt/wt) of wheat starch and sucrose (Diet A) or 100% glucose (Diet B) was studied in diabetic and nondiabetic rats. BAY-O-1248 led to a significant reduction in daily food intake and weight gain in rats fed Diet A but not Diet B. In diabetic rats fed Diet A with BAY-O-1248, daily urinary glucose was significantly diminished (6820 +/- 402 vs 3796 +/- 210 mg), while the postprandial plasma glucose excursions were similar. In nondiabetic rats, the addition of BAY-O-1248 decreased the postprandial plasma glucose level with no change in urine glucose. In summary, addition of an alpha-glucosidase inhibitor to a starch plus sucrose containing diet led to reductions in glycosuria (diabetic rats) and serum glucose levels (normal rats).

Glycemic and insulinemic responses after ingestion of ethnic foods by NIDDM and healthy subjects.

In an attempt to apply the concept of glycemic index (GI) and insulinemic index (II) to local eating habits, we examined the plasma glucose and insulin responses in subjects with non-insulin-dependent diabetes mellitus (NIDDM) and healthy subjects to five mixed meals of different ethnic origins. All meals contained 50 g carbohydrate and were compared with a 50-g glucose load. The GI was highest for the Polish dish and lowest for the Syrian dish (66 +/- 5.5 vs 24 +/- 5.1). However, the II was the highest for the standard meal and lowest again for the Syrian dish (174 +/- 27 vs 66 +/- 25). A high correlation was found between the area under the glucose curve and the predicted GI in both NIDDM and healthy subjects. The GI concept is valid and potentially useful in diet planning and legume foods should be incorporated as a carbohydrate source when diets are being planned for NIDDM subjects or individuals with impaired glucose tolerance.

Factor XI assembly and activation on human umbilical vein endothelial cells in culture.

Biotin-FXI optimally bound to HUVEC in the presence of 40 nM high molecular weight kininogen (HK) and > or =7 microM Zn2+. There was little specific FXI binding in the absence of added HK and at concentrations of Zn2+ <15 microM. FXI and prekallikrein, but not prothrombin, blocked biotin-FXI binding to HUVEC in the presence of HK with an IC50 of 18 nM and 180 nM, respectively. Monoclonal antibody HKL16 and peptide SDD31 also inhibited biotin-XI binding in the presence of HK with an IC50 of 4.7 nM and 50 microM, respectively. Alternatively, peptide T249-F260 of FXI's apple domain 3 and heparin monosulfate were weak inhibitors of FXI binding to HUVEC. FXI bound to HUVEC with an apparent Kd of 6.9 +/- 3.0 nM and Bmax of 13 +/- 2.6 x 10(6) sites/cell. FXI bound to HK on HUVEC, but not prothrombin, became converted to FXIa. FXI activation on HUVEC resulted from tissue culture media bovine factor XIIa. HUVEC grown in human factor XI-deficient serum did not support FXI activation. FXI binding to HUVEC in culture was mostly mediated by HK and FXI activation on HUVEC is dependent on cell-associated factor XIIa.

Assembly of high molecular weight kininogen and activation of prekallikrein on cell matrix.

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires > or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.

Binding sites of human growth hormone and ovine and bovine prolactins in the mammary gland and the liver of lactating dairy cow.

Membrane preparations and Triton X-100 solubilized fractions from the mammary gland and liver of the lactating dairy cow were capable of specific binding of [125I]hGH and [125I]oPRL. The specific binding of the latter was significantly lower and could not be increased by higher receptor levels. Displacement studies of [125I]hGh by hGH, bPRL and oPRL revealed that the two latter hormones have a 20-40-fold lower affinity for the receptor than hGH, although strong indications exist that they all bind or the same sites. This feature is unique for cows and does not exist or is much less pronounced in rodents.

Partial purification and characterization of bovine mammary gland prolactin receptor.

Prolactin (PRL) receptors from the mammary gland of the lactating cow were solubilized with 3-[(3-cholamidopropyl)dimethylamonio]-1-propane sulfonate (CHAPS). Affinite chromatography on human growth hormone (hGH) coupled to Affi-Gel 10 resulted in over 500-fold purification, as compared to microsomal fractions. Scatchard analysis of the binding of hGH indicated an increase in the affinity constant of 2.5-fold after solubilization and of further 2-fold after the affinity purification. The specific binding activity of the affinity-purified fraction was 9000 fmol hGH/mg protein. Complexes of Triton X-100-solubilized receptors with [125I]hGH were analyzed by gel filtration on Sephadex G-150, in the presence of Triton X-100. A minor fraction of the complexes eluted as high molecular weight (Mr) aggregates, whereas a major fraction eluted as a 150 kDa peak. Assuming a contribution of approximately 30% to the Mr by the bound detergent and a hormone: receptor ratio of 1:1 in the complex, a Mr of 80-85 kDa can be calculated for the receptor molecule. Affinity labelling of the receptor with [125I]hGH revealed a Mr of 37 +/- 0.5 kDa (n = 7) for the binding subunit. Specific high Mr aggregates were also observed after crosslinking; however, the size of the labelled species was not affected by reducing agents. Homologous and heterologous competitive binding studies with ovine PRL (oPRL) or hGH revealed a considerably higher affinity for hGH as compared to oPRL. The competitive displacement patterns obtained with oPRL or hGH as tracers were similar, indicating that both hormones bound to the same receptor sites with different affinities. A similar difference in affinity was retained by the affinity-purified receptors.

Short-term regulation of prolactin receptors in the liver, mammary gland and kidney of pregnant and lactating rats infused with ovine prolactin or human growth hormone.

Short-term regulation of prolactin (PRL) receptors was studied in ketamine-anaesthetized 18-day pregnant or 7-day lactating female rats, by infusing them with various doses of oPRL or human growth hormone (hGH) for 0-3 h and measuring the binding of [125I]oPRL of [125I]hGH to the microsomal fractions prepared from the liver, mammary gland and kidneys of animals sacrificed at various states of infusion. Our main findings are: In pregnant rats, only 30% of liver receptors are unoccupied and infusion with 25 micrograms/h for 3 h of either oPRL of hGH decreased both free and total receptors by 22-30% while infusion with 250 micrograms/ml caused an additional decrease only in the free receptors. In the mammary gland and the kidney of pregnant rats, all receptors seem to be unoccupied; infusion with 25 micrograms/ml had none or a slight elevating effect on the number of both free and total receptors in the mammary gland but caused a significant 3-fold increase in the kidney; infusion with 250 micrograms/ml, however, resulted in a slight decrease in the mammary gland and a significant decrease in the kidney in both total and free receptors. In the liver of the lactating rats, there was no significant difference between the number of free and total receptors, but in mammary gland, specific binding to the total receptor was higher than to the free ones indicating partial occupancy; infusion with 25 micrograms/ml caused a significant decrease in free and total liver receptors without a remarkable change in the mammary gland and some decrease (by infusion with hGH only) in the kidney. In all cases, the changes in the specific binding resulted from the increase or decrease in receptor number and not from the change in receptor-hormone affinity. In almost all cases, infusion with oPRL or hGH yielded similar results. Infusion with both hormones did not affect the level of the endogenous rat prolactin. In conclusion, our results indicate the short-term regulation of PRL receptors by exogenous hormones is a complicated process which is affected by the level of the infused hormone, physiological state of the animal and may yield, simultaneously, different or even opposite changes in receptor number in various organs.

The role of dietary factors in prevention of chemically-induced cancers (review).

The role of dietary factors in prevention of chemically-induced cancer was reviewed on two models: i) the role of high fiber diets in prevention of colon cancer and ii) the role of high fat diets in prevention of mammary gland cancer, i) Experiments in colon cancer showed that 20% cellulose content decreased tumor incidence caused by 1,2-dimethylhydrazine (DMH) to 33% compared with 92% of tumors developed in animals fed a fiber-free diet. The tumor-preventive effect of a cellulose diet was accompanied by increased enzyme concentrations, such as ornithine decarboxylase, thymidine kinase and beta-glucuronidase. Corncob fiber (15%), treated with the fungus Pleurotus os., had a significant protective effect against DMH-induced rat colon cancer. This effect was accompanied by activation of some cellular mechanisms, i.e. apoptosis, proliferating cell nuclear antigen (PCNA) and p53 protein synthesis. A high positive correlation was found between tumor grade and p53 protein in the serum (r=0.97) or in the cell cytoplasm (r=0.77), and between tumor grade and PCNA (r=0.81). An inverse relationship was found between tumor grade and apoptosis (r=-0.63). ii) Experiments in mammary gland cancer showed that a 15% olive-oil diet reduced tumor incidence caused by 9,10-dimethyl-1,2-benzanthracene to 30%, compared with 55% in the control group. The antitumor effect of the olive oil diet was connected to its content of monounsaturated fatty acids, such as oleic and palmitic acids. The promotive tumorigenic effects of other high-fat diets (avocado, soybeans) were associated with high content of some polyunsaturated fatty acids (linoleic and alpha-linolenic). We concluded that different diets have different targets. The effect of the same diet depends on its content of anti-tumor substances.

Glycemic indices of various foods given to pregnant diabetic subjects.

The applicability of the glycemic index concept in pregnancy has not been established. The postprandial glucose curves were measured for nine foods (glucose, bread, raisins, dates, sweet corn, bananas, oranges, spaghetti, and green peas) in 28 gestational diabetic subjects. Uniform glycemic indices were observed for each food, similar to those reported by others in nonpregnant subjects. Postprandial glucose levels reached their peak later after glucose and bread ingestion than after the remaining seven foods. These results demonstrate that despite known changes in gastrointestinal function in pregnancy, glycemic indices are uniform after the ingestion of foods. Pregnancy does not appear to alter the glycemic indices of the foods tested.

Composition and histological observations of enlarged pancreases of chicks adapted to raw soybean meal diets.

Chicks fed raw soybean meal (RSBM) as a sole protein source developed a significantly enlarged pancreas as compared to chicks fed heated soybean meal. No difference in DNA, RNA and protein content was observed, but the number of acinar cells was significantly higher in pancreases of chicks fed RSBM, after 14 and 28 days of feeding. Histological and ultrastructural analyses revealed that the enlargement of the pancreas resulted both from the hyperplasmic increase in the number of acinar cells and from the dilution of the acini by intercellular connective tissue and the appearance of cytopathogenetic areas.

The comparative effect of prolonged feeding with raw and heated soybean mean on the growth response, pancreatic enlargement and pancreatic enzymes of chicks.

Prolonged feeding of chicks with raw soybean meal as the main protein source in the diet resulted in growth inhibition, pancreas enlargement, and increased content of pancreatic proteolytic enzymes. The effect of feeding with raw soybean meal as compared to heated soybean meal upon total intestinal enzymatic activities in chicks was tested. The tendency of chicks to overcome the effects of raw soybean meal is expressed mainly by the increase of biosynthesis and secretion of proteolytic enzymes in the pancreas.