The effect of a low-energy food foam on appetite measures during a 1-day reduced-energy meal plan.

BACKGROUND/OBJECTIVES: We have previously shown that 500 ml of a foamed drink ('foam') significantly improved appetite versus a non-foamed control. The objectives of this research were to assess the effect of smaller volumes of foams on appetite, and the potential benefits of foam ingestion and its timing on appetite measures in a reduced-energy context. SUBJECTS/METHODS: Two randomized, parallel design studies (pre- and main study) were conducted using healthy adult subjects. Pre-study: 133 subjects (age 18-50 years, body mass index (BMI) 20-32 kg m(-2)) each consumed either 10, 25, 50, 100, 150 or 250 ml foamed meal replacer (~0.2 kcal ml(-1)), 150 min after a fixed breakfast. Main study: four groups of subjects (n=134; age 18-60 years, BMI 22.5-35.0 kg m(-2)) consumed 200 ml/22 kcal foam (based on pre-study results) immediately after main meals (M), after snacks (S), in-between snacks and main meals (I) or not at all (control, C) within 1 day of a reduced-energy meal plan consisting of three main meals and three snacks. Measurements included self-reported appetite (six scales, reported as area under the curve (AUC)) and (main study only) end-of-day appetite questionnaire. RESULTS: Pre-study: the strongest effect on appetite was produced by 250 ml (consistent across scales), whereas 150 ml showed more pronounced effects than 100 and 50 ml in most scales. Volumes 10 and 25 ml had no effects on any scale. Main study: 200 ml foam reduced appetite AUC substantially in all treatments, particularly M (for example, hunger AUC reduced by 35% (P <0.001), 28% (P <0.05) and 20% (P=0.11) for M, S and I, respectively versus C). A strong reduction in 'appetite for a snack' was seen for all timings (all P <0.05). The end-of-day appetite ratings confirmed these findings. CONCLUSIONS: Modest amounts of a low-energy foam can reduce appetite measures during a 1-day reduced-energy meal plan.

Blood coagulation in anhepatic pigs: effects of treatment with the AMC-bioartificial liver.

The function of a newly devised bioartificial liver (AMC-BAL) based on viable, freshly isolated porcine hepatocytes has been evaluated in anhepatic pigs. The aim of this study was to assess the contribution of BAL treatment on blood coagulation parameters. Pigs were anesthetized and a total hepatectomy was performed (n = 15). The infrahepatic caval vein and the portal vein were connected to the subdiaphragmatic caval vein using a three-way prosthesis. Animals received standard intensive care (control, n= 5), treatment with an empty BAL (device control, n= 5) or with a cell-loaded BAL (BAL-treatment, n= 5) for a period of 24 h starting 24 h after hepatectomy. Coagulation parameters studied concerned prothrombin time (PT), platelet count, the procoagulant system (factors (F)II, FV, FVII, FVIII and fibrinogen), anticoagulant system (AT III), fibrinolytic system (t-PA, PAI-1) as well as markers of coagulation factor activation (TAT complexes, prothrombin fragment F1 + 2). FII, FV, FVII, AT III and fibrinogen rapidly decreased after total hepatectomy in pigs in accordance with the anhepatic state of the animals. FVIII levels were not influenced by the hepatectomy. A mild drop in platelet count was seen in all groups. Treatment of anhepatic pigs with the cell-loaded BAL did not restore PT or clotting factor levels. TAT and F1 + 2 complexes, however, were significantly increased in this group. Levels of t-PA and PAI-1 were not influenced by cell-loaded BAL treatment. Treatment of anhepatic pigs with the AMC-BAL based on freshly isolated porcine hepatocytes does not result in an improved coagulation state due to extensive consumption of clotting factors. However, increased levels of TAT complexes and prothrombin fragments F1 + 2 during treatment of anhepatic pigs indicate synthesis and direct activation of coagulation factors, leading to thrombin generation. This demonstrates that this bioartificial liver is capable of synthesizing coagulation factors.

Treatment of acute liver failure in pigs reduces hepatocyte function in a bioartificial liver support system.

Several different types of bioartificial liver (BAL) support systems have been developed to bridge patients suffering from acute liver failure (ALF) to transplantation or liver regeneration. In this study we assessed the effects of ALF plasma on hepatocyte function in the BAL system that has been developed in our center. Pigs (40-60 kg) were anaesthetised and a total hepatectomy was performed. Cells were isolated from the resected livers and were transferred to the bioreactor of the BAL system. Twenty hours after cell isolation, hepatocytes in the BAL were tested for cell viability and functional activity by using a recirculating test medium in which assessment of LDH leakage, ammonia clearance, urea synthesis, 7-ethoxycoumarin O-deethylase (ECOD) activity and pseudocholine esterase production was performed. Subsequently, two groups were studied. In one group (I, n=5), the cell-loaded bioreactor was used to treat the donor pig, rendered anhepatic, for 24 hours. In the second group (II, n=5) the bioreactor was cultured for 24 h and served as a control. After 24 hours treatment or culturing, the cell viability count and functional activity tests were repeated. The results show that hepatocytes in the BAL remained viable after 24 h treatment of anhepatic pigs, as shown by the LDH release and pseudocholine esterase production. However, metabolic functions such as ammonia clearance, ECOD and urea synthesis were reduced after 24 h exposure of hepatocytes to autologous ALF plasma, whereas these functions were unaltered after 24 h culturing of the cells in the bioreactor.

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A.

The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux. Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion. Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells. The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux. Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport. Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates. However, reducing intracellular ATP contents completely blocked fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not. As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic anion transport system differs from MDRI and MRP2.

Neurotensin-and EGF-induced metabolic activation of colon carcinoma cells is diminished by dietary flavonoid cyanidin but not by its glycosides.

Dietary polyphenols, including anthocyanidins and their glycosides anthocyanins, are suggested to be involved in the protective effects of fruits and vegetables against cancer. Very few data are available concerning the effects of anthocyanidins/anthocyanins on cellular processes induced by growth factors such as neurotensin and epidermal growth factor (EGF), which are implicated in the pathophysiology of colon cancer. Here, we show that neurotensin and EGF caused an increase in the extracellular acidification rate, which could reflect the activity of cellular metabolism, in the human carcinoma cell line HT29 clone 19A. Neurotensin and EGF also caused a strong rise in the intracellular Ca2+ concentration, induced phosphorylation of extracellular signal-regulated kinases (ERK1 and ERK2), and stimulated growth of human carcinoma cells. Cyanidin (10 microM), but not its glycosides cyanin and idaein, was able to inhibit the neurotensin- and EGF-induced increased rate of extracellular acidification. In contrast to N-ethyl-N-isopropyl amiloride, an inhibitor of Na+/H+ exchange, cyanidin did not alter the rate of intracellular pH recovery of cells loaded by NH3/NH4+, indicating that cyanidin inhibits cellular metabolism, rather than directly altering Na+/H+ exchange. Cyanidin, but not cyanin and idaein, was able to inhibit an increase in intracellular Ca2+ concentration induced by neurotensin. Neurotensin- and EGF-induced phosphorylation of ERKs was not affected by cyanidin, cyanin, and idaein at < or = 100 microM. Only cyanidin (100 microM), but not cyanin and idaein, was able to inhibit cellular growth induced by EGF. Thus these findings suggest that a dietary polyphenol cyanidin, but not its glycosides, is a potent inhibitor of mitogen-induced metabolic activity, increase in free intracellular Ca2+, and cellular growth of cultured colon carcinoma cells.

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells.

INTRODUCTION: Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology. METHODS: We have measured H2O2-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H2O2 and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis ("Comet") Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H2O2-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage. RESULTS: A dose-response relationship was found for the H2O2-induced dye decomposition in NIH3T3 cells (7.8-125 microM H2O2) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62-1000 M H2O2). Fluorescence was significantly increased at 62microM H2O2 in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H2O2 than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells. CONCLUSIONS: Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H2O2-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H2O2.

Enhancement of ovalbumin-induced antibody production and mucosal mast cell response by mercury.

Food contaminants may contribute to the recent increased incidence of food allergies. We have investigated this hypothesis experimentally. It was our objective to determine whether toxicity to the intestinal tissue by orally applied mercury (Hg) could modulate the immune response to food allergens. Effective mechanisms were studied with functional immunological and toxicological parameters. Brown Norway rats were immunized intraperitoneally by ovalbumin (OVA). Before oral challenge with OVA, immunized and non-immunized animals were exposed to HgCl2. Immunological responses were measured by enzyme-linked immunosorbent assays [anti-OVA-IgE and-IgG, rat mast cell protease II (RMCPII), interferon-gamma, interleukin-4, lymphocyte proliferation] and by flow cytometry (lymphocyte subpopulations). Toxicity of Hg to the intestinal barrier was determined by measuring viability, DNA damage and induction of glutathione S-transferase in isolated intestinal epithelial cells and lymph node cells, and by measuring permeability, short-circuit current and tissue conductance of the intact intestinal epithelium. A single high oral dose of HgCl2 enhanced the serum concentrations of anti-OVA-IgE and IgG (P < 0.05) and of RMCPII (P < 0.05) in immunized rats. The treatment resulted in a higher number of CD4/CD25+ T cells in the lymph nodes (P < 0.05). The multiple application of low HgCl2 doses (5 x 0.2 mg/kg body weight) only resulted in an elevated RMCPII serum concentration (P < 0.05). Neither treatment schedules impaired proliferation and cytokine production of lymphocytes. In non-immunized rats only minor immunological changes were observed. Oral HgCl2 induced genotoxic damage in lymph node cells and in jejunal epithelial cells (P < 0.05). Moreover, HgCl2 increased the permeability of intestinal epithelial tissue and of Caco-2 monolayers and was genotoxic and cytotoxic to isolated intestinal epithelial cells in vitro. In conclusion, these studies indicate that the food contaminant Hg can stimulate the immune response to OVA in immunized rats. One possible mechanism could be the toxicity of Hg to the intestinal epithelial and the lymph node cells. Whether humans with allergies respond to high oral doses of Hg in a similar way needs to be investigated in further studies.

Analysis of DNA strand breaks, oxidized bases, and glutathione S-transferase P1 in human colon cells from biopsies.

The balance of genetic damage and deactivating enzymes is decisive for cancer risk. To assess these factors in normal human colon cells, we determined background levels of DNA breaks or oxidized bases and of glutathione S-transferases (GSTs) as potential biomarkers of risk and chemoprevention, respectively. Also, genotoxicity by compounds involved in lipid peroxidation was determined to elucidate possible sources of damage. Cells were isolated from sigmoid biopsies of 51 donors and processed with the comet assay to reveal genetic damage. GST proteins were analyzed immunologically. HT29 clone 19A colon tumor cells, resembling primary cells, were treated with 2-trans-hexenal (400 microM) or hydrogen peroxide (75 microM) and processed for damage. Fifteen percent of primary colon cells contained strand breaks; 22% contained additional oxidized bases, with distinct sex differences. Similar damage was found in HT29 clone cells and is induced by both test compounds. GST levels were similar in both cell types. The comet assay is sufficiently sensitive to detect oxidative genetic damage in small amounts of cells from small amounts of biopsies. Lipid peroxidation is a possible risk factor. Together with GST as a potential biomarker of chemoprevention, the technique may serve as a valuable biomarker to assess exposure to risk factors.

Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells.

Short chain fatty acids (SCFA) are considered to be beneficial fermentation products in the gut by exerting trophic effects in non-transformed colon cells and by slowing proliferation and enhancing differentiation in colonic tumour cells. We have studied the further effects of SCFA on cellular events of early carcinogenesis, genotoxicity and cytotoxicity in rat distal colon cells. Cytotoxicity was assessed by measuring trypan blue exclusion and by determining the H2O2-induced changes in intracellular calcium concentration ([Ca2+]i) using a fluorospectrophotometer and the calcium-sensitive fluorescent dye Fura-2. The microgel electrophoresis technique (COMET assay) was used to assess oxidative DNA damage. Individual SCFA and physiological SCFA mixtures were investigated for their potential to prevent DNA and cell damage induced by H2O2. For this, freshly isolated colon cells were treated with H2O2 (100-500 microM) and 6.25 mM SCFA. We have found 100-500 microM H2O2 to cause a fast initial increase in [Ca2+]i, whereafter the levels gradually further increased. Addition of SCFA did not affect [Ca2+]i nor did it reduce the H2O2-induced increase in [Ca2+]i. Butyrate and acetate were able to reduce the induction of DNA damage by 100, 200 and 500 microM H2O2, respectively. In contrast, i-butyrate and propionate were ineffective. The degree of reduction of DNA damage for the two protective SCFA was similar. Physiological mixtures containing acetate, propionate and butyrate in ratios of 41:21:38 or 75:15:10 that are expected to arise in the colon after fermentation of resistant starches and pectin, respectively, did not show significant antigenotoxic effects. The major difference between butyrate and acetate, on one hand, and i-butyrate and propionate, on the other hand, is that the former compounds are utilized best as energy sources by the colon cells. Therefore, our results on antigenotoxicity coupled with the findings on [Ca2+]i homeostasis indicate that molecular effects on the energy system render these non-transformed, freshly isolated colon cells to be less susceptible to H2O2.

Apical membrane isolation of surface and crypt cells from rabbit distal colon.

Surface and crypt cells of rabbit distal colon were separately isolated, and amiloride-sensitive 22Na+ uptake could only be demonstrated in a crude membrane fraction derived from surface cells. For purification of apical membranes of surface and crypt cells (H(+)-K+)-ATPase and alkaline phosphatase were used as putative apical membrane markers. Apical membranes of surface cells were isolated after mild homogenization, low speed centrifugation, and subsequent fractionation on a Percoll density gradient. Apical membranes of crypt cells were collected after more vigorous homogenization, followed by high speed centrifugation, and fractionation on a Percoll gradient. In surface and crypt cells, (H(+)-K+)-ATPase and alkaline phosphatase activity accumulated in a low and a high density Percoll band. Further fractionation of the low density Percoll band from crypt cells on a discontinuous sucrose gradient yielded a vesicle fraction with 7- to 10-fold enrichment in (H(+)-K+)-ATPase activities. To demonstrate the usefulness of the isolated fractions in studying transport mechanisms, vesicle volume was determined and planar lipid bilayer studies were performed. In the latter studies, a 83-pS 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive Cl(-)-channel, resembling the outward rectifying intermediate conductance (ORIC) Cl(-)-channel of secretory epithelia, was encountered most frequently. This channel was present in fractions of surface and crypt cells.

Cellular acidification occurs during anoxia in cultured, but not in freshly isolated, rabbit proximal tubular cells.

In a variety of cells it has been shown that acidosis is protective against anoxic injury. We have demonstrated previously that proximal tubule (PT) cells in primary culture were more resistant to anoxia-induced cell injury than were freshly isolated cells. Therefore, we asked the question of whether a difference in cellular acidification during anoxia could explain this difference in susceptibility to anoxia. To answer this question, intracellular pH (pHi) was measured during anoxic incubation of PT cells in culture and those that were freshly isolated. PT cells were incubated in an anoxic chamber at 37 degrees C after loading with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) or fura-2 acetoxymethyl ester (fura-2-AM). pHi and cytosolic free Ca2+ ([Ca2+]i) were measured by digital imaging fluorescence microscopy. During anoxia, pHi in cultured PT cells decreased from 7.3 +/- 0.1 to 6.8 +/- 0.1, whereas pHi in freshly isolated cells did not decrease significantly. In addition, the intrinsic buffering capacities (beta i) in cultured and freshly isolated PT cells were determined and turned out to be the same at a pHi greater than or equal to 7.3. Below pHi 7.3, beta i increased several fold in freshly isolated PT cells, and rose to significantly higher levels than in cultured PT cells. During 1 h of anoxia, cell viability of freshly isolated PT cells decreased significantly to 54% +/- 2% (P < 0.05), while no loss in viability was observed in cultured PT cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of intracellular pH in crypt cells from rabbit distal colon.

H+ secretory mechanisms and intrinsic intracellular buffering capacity were studied in crypt cells from rabbit distal colon. To this end crypts of Lieberkühn were isolated by microdissection, and intracellular pH (pHi) was measured using digital imaging fluorescence microscopy and the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein. In the absence of HCO(3-)-CO2 and presence of Na+, resting pHi was 7.51 +/- 0.04 (n = 237/23, cells/crypts). However, 6 min after superfusion with a solution containing zero Na+, 1 x 10(5) M Sch-28080 and 5 x 10(-8) M bafilomycin A1, pHi in cells at the bottom of the crypts was significantly reduced, whereas pHi in cells at the top of the crypts remained unchanged. The intrinsic buffering capacity of cells from the middle to the top portion of crypts was significantly higher in the pHi range 7.2-7.6 than of cells at the bottom of the crypt. H+ secretion after an NH(4+)-NH3 pulse amounted to 245 +/- 53 microM/s (n = 73/7) at pHi 7.1 and was largely Na+ dependent and ethylisopropylamiloride sensitive. The Na(+)-independent recovery of pHi after an acid load was insensitive to Sch-28080 and bafilomycin A1. In conclusion, pHi in colonic crypt cells is regulated through Na+/H+ exchange activity in the absence of HCO3-. In addition, intracellular buffering capacity varied with the position along the crypt axis, whereas Na+/H+ exchange activity and pHi did not.

Effects of pH on apical calcium entry and active calcium transport in rabbit cortical collecting system.

Rabbit connecting tubules and cortical collecting ducts were isolated by immunodissection and cultured on permeable supports. The monolayers actively transported Ca2+ with a net transcellular rate of 92 +/- 3 nmol.h-1.cm-2. Methoxyverapamil, felodipine, diltiazem, omega-conotoxin GVIA, and omega-agatoxin IVA when added to the apical side had no effect on Ca2+ absorption. Neither hyperpolarization nor depolarization of the apical membrane affected Ca2+ transport rates significantly. Stepwise lowering of the apical pH (pHa) from 8.0 to 5.6 gradually inhibited Ca2+ transport from 88 +/- 5 to 7 +/- 2 nmol.h-1.cm-2. Measuring the intracellular pH (pHi) with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein revealed that lowering the pHa from 8.0 to 5.6 decreased pHi from 7.8 to 6.7. To determine whether inhibition of Ca2+ absorption results from intracellular acidification, pHi was lowered using an NH4Cl pulse while extracellular pH was kept constant. Intracellular acidification from 7.4 +/- 0.2 to 6.9 +/- 0.1 reduced Ca2+ absorption by 26 +/- 6% only. In addition, lowering of the basolateral pH to 6.2 resulted in a pHi of 6.8 +/- 0.1, without affecting Ca2+ absorption rates. In conclusion, the basal Ca2+ influx mechanism in the apical membrane is most likely a voltage-independent Ca2+ transporter, insensitive to Ca2+ channel blockers, but strongly inhibited by apical acidification.

The colon carcinoma cell line Caco-2 contains an H+/K(+)-ATPase that contributes to intracellular pH regulation.

The presence of an H+/K(+)-ATPase and its contribution to the regulation of intracellular pH (pHi) was investigated in Caco-2 cells. The H+/K(+)-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H+/K(+)-ATPase. Cell pH was determined using the pH-sensitive dye 2',7'-bis(carboxyethyl)-carboxyfluorescein. Control pHi, measured in HCO(3-)-free medium, was 7.62 +/- 0.03 (n = 27) when cells were cultured for 14 days and decreased to 7.40 +/- 0.03 (n = 18) after 35 days in culture. Recovery of pHi following a NH+4/NH3 pulse could be reduced by either 100 microM SCH 28080 or 1 mM amiloride, or by removing extracellular Na+. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H+/K(+)-ATPase and a Na+/H+ exchanger in regulating pHi. Recovery rates at pHi 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na+/H+ exchange to the recovery. Recovery of pHi was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethyl-maleimide inhibited a vacuolar type of H+-ATPase, since bafilomycin A1 had no effect on pHi recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pHi, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na+/H+ exchanger to pHi regulation is decreased.

Circadian Rhythmicity of the Crustacean Hyperglycemic Hormone (CHH) in the Hemolymph of the Crayfish.

The crustacean hyperglycemic hormone (CHH) is involved in the regulation of endogenous blood glucose metabolism. In this paper we describe the daily rhythmicity in the blood glucose and the blood CHH content of the crayfish Orconectes limosus. Both blood CHH and blood glucose levels increase during the first hours after the beginning of darkness. The bioactivity of released CHH is far higher than that of CHH stored in the sinus gland. Moreover, the released hyperglycemic material shows an affinity for high molecular weight proteins in the hemolymph. Preliminary results suggest that subunits of hemocyanin may act as potential carrier-proteins for bioactive CHH.