Changes in calmodulin levels during development of the silkworm,Bombyx mori

Calmodulin levels were measured in various tissues during the larval-adult development of the silkworm, Bombyx mori. In the larval period, calmodulin levels in fat body, midgut and testis were in a range of 0.3–1.7 μg/mg protein and remained almost constant during larval growth. The silk gland contained a relatively high (0.2 μg/mg protein) level of calmodulin early in the fifth instar which gradually decreased during maturation of the larva. At pupation, testis calmodulin dropped from 1.5 to 1.7 μg/mg protein to about 1 μg/mg, and remained constant thereafter. The most striking change occurred in fat body calmodulin which fell from 0.5 to 0.6 μg/mg in the larval stage to 0.01–0.03 μg/mg during pupal-adult metamorphosis. Midgut calmodulin levels were unchanged at pupation and remained constant during pupal-adult development.When expressed on per g wet weight basis, calmodulin levels in silkworm tissues were comparable to mammalian tissue levels. However, only 2–4% of the total calmodulin in silkworm tissues was in a membrane-bound form compared to 20–60% for membrane-bound calmodulin in mammals.     

The 20-kD human growth hormone reduces body fat by increasing lipolysis and decreasing lipoprotein lipase activity

AIM: The aim of this study was to estimate the lipolytic activity of the human growth hormone variant, 20-kD human growth hormone (20K-hGH). METHODS: Obese KV-A(y) mice were given daily subcutaneous injections of 20K-hGH (0.25, 0.5, 1.0 mg/kg), 22K-hGH (0.25 mg/kg) or saline as a control for 2 weeks. Body composition (fat, water and protein), lipolysis and lipoprotein lipase (LPL) activity were measured 24 h after the final injection. RESULTS: Both growth hormone isoforms significantly reduced relative fat pad and whole body lipids. In addition, 20K-hGH produced an inhibition of LPL activity in adipose tissue and stimulated lipolysis in adipocytes. CONCLUSION: These data strongly suggest that inhibition of LPL activity in adipose tissue and stimulation of lipolysis in adipocytes by 20K-hGH treatment reduce adipose tissue mass, resulting in body fat reduction.     

Stimulation of trehalose efflux from cockroach (Periplaneta americana) fat body by hypertrehalosemic hormone is dependent on protein kinase C and calmodulin

Protein kinase C and calmodulin play key roles in cockroach fat body during activation of phosphorylase and trehalose efflux by HTH-II. The data support the view that an increase in cytosolic Ca2+ is prerequisite for enhanced activity of protein kinase C and calmodulin. Chelation of Ca2+ (i) with BAPTA blocks HTH-II-induced trehalose efflux from the fat body whereas thapsigargin, which raises [Ca2+]i to the same level as HTH-II, produces only a small, yet significant increase in trehalose efflux. Sphingosine, an inhibitor of protein kinase C, inhibits HTH-II-induced trehalose efflux in a concentration-dependent manner. Trehalose efflux is not activated by the protein kinase C activators OAG or PMA alone but in the presence of thapsigargin both agents increase trehalose efflux to a level comparable to that obtained with HTH-II. Thapsigargin has only a moderate activating effect on phosphorylase but in combination with OAG produces an activation indistinguishable from that provoked by HTH-II. Each of the structurally different calmodulin inhibitors, trifluoperazine, W-7, and calmidazolium, blocks completely the action of HTH-II on trehalose efflux, thus confirming the importance of calmodulin in HTH-II initiated trehalose efflux.     

Changes in the biochemical composition of fat body stores during adult development of female crickets, Gryllus bimaculatus

Age-dependent changes in the fat body composition and aspects of lipogenesis in the free abdominal fat body of female crickets, Gryllus bimaculatus, were studied. Lipid, protein, glycogen, and free carbohydrate content of the fat body, and fat body wet weight increased simultaneously and sharply from day 0 onwards and were doubled/almost doubled by day 2 after adult emergence. Lipogenic activity of the fat body, fat body weight, and the energy stores in the fat body peaked on day 2, except for free carbohydrate, which peaked on day 3. On day 2, the fat body was mainly comprised of lipid (53.8%) and protein (6.6%), while glycogen and free carbohydrate together contributed less than 1% of the fat body wet weight. After peaking, both lipogenesis and energy stores decreased in a synchronous manner. The depletion of the fat body energy stores and the consequent decrease in the fat body weight were concomitant with a fast and massive gain in ovary weight (day 2: 19.5 +/- 1.5 mg; day 4: 332.8 +/- 31.5 mg) due to the vitellogenic oocyte growth that started on day 2. Our data clearly underline the importance of the free abdominal fat body as a source of energy for reproduction in the cricket. Fat body fatty acid synthase activity coincided with lipogenic activity. Adipokinetic hormone inhibits lipid synthesis in the fat body, but treatment of the fat body with adipokinetic hormone in vitro showed no consistent effect on fatty acid synthase activity.     

Involvement of altered cytoskeletal protein phosphorylation in aluminum-induced CNS dysfunction

In the present investigation, we have studied the effects of aluminum (10 mg/kg of body weight per day, i.p.) and desferrioxamine (6 mg/kg of body weight per day, i.p.) alone and in combination for 4 weeks on the regulation of phosphorylation of neuronal proteins. A marked decrease in the biological activity of calmodulin was observed after aluminum treatment; however, in combination with desferrioxamine, a reversal in the levels of calmodulin, in terms of nmol cAMP hydrolyzed/min/mg protein, was observed. Exogenous addition of calmodulin had an inhibitory effect on calmodulin-mediated synaptosomal protein phosphorylation in aluminum-exposed animals. An almost complete reversal of this inhibition was observed following coexposure to aluminum and desferrioxamine. Cyclic AMP-dependent synaptosomal protein phosphorylation was, however, stimulated following aluminum exposure. Coadministration of desferrioxamine along with aluminum was found to mitigate the neurotoxic effect of aluminum. © 1997 John Wiley & Sons, Inc.     

Ovarian development in autogenous and anautogenous Lucilia cuprina in relation to protein storage in the larval fat body

Females of Lucilia cuprina from field populations and from normally maintained laboratory cultures are anautogenous. Normal anautogenous females were compared with females from a laboratory selected autogenous strain especially as regards protein storage and ovarian protein content. The following differences were found: (1) autogenous females at emergence from the puparium contain about 1 mg (20%) more protein than anautogenous females of equal fresh weight, (2) about 70% of this additional protein is accounted for by increased protein content of the abdominal larval fat body, (3) the larval fat body of autogenous females has greater volume and greater protein content per unit volume than that of anautogenous females, (4) the greater volume of larval fat body in autogenous females is the result of increased cell size rather than cell number, (5) the protein content of the ovaries of mature non-protein-fed autogenous females is about 1 mg. That of non-protein-fed anautogenous females of similar age is negligible, (6) the larval fat body of non-protein-fed anautogenous females disappears more rapidly after emergence than does that of autogenous females.     

Estimates of individual factors of the tryptophan requirement based on protein and tryptophan accretion responses to increasing tryptophan supply in broiler chickens 8-21 days of age

Ten diets (196 g CP and 13.0 MJ ME(N)/kg) with graded levels of tryptophan (Trp) between 0.9 and 2.7 g/kg were offered ad libitum to Ross broilers from day 8-21 post-hatch. Each diet was allocated to three pens of ten birds each. In addition to growth and feed conversion, the accretions of protein, Trp, fat, and energy were determined by comparative whole body analyses. A sigmoidal function was fitted to the data. Responses to Trp supply were nonlinear and attained plateau values (y(max)) within the studied range of Trp supply. The Y(max) values estimated for BW gain, whole body protein gain, and Trp gain during the 14 days under study were 615, 104, and 0.87 g/bird, respectively. Based on the response in whole body protein gain, the estimated requirement was 1.9 g Trp/kg of diet or 0.15 g Trp/MJ of MEN. Estimates made from the other response criteria were similar to this value. While the protein concentration in gained BW was unaffected by Trp intake (169 g of protein per kg of gained BW), fat and energy concentrations of gained BW increased with increasing Trp supply, approaching plateau values of 146 g fat and 9.8 MJ energy per kg of gained BW. This is supposed to result from feed intake increasing with Trp supplementation. The Trp concentration in gained whole body protein (0.84 g Trp/ 16 g of gained N) was not significantly affected by Trp intake. As long as Trp intake was the limiting factor, 59% of supplemented Trp was transferred to gained whole body protein. During the 14 days of the study, broilers inevitably lost 87 mg Trp. This is equivalent to a daily loss of 30 mg/kg BW or a maintenance requirement of 52 mg/kg BW per day. Data determined for the individual factors may be used for future modelling of broilers' Trp requirement.     

Development and Regional Distribution of Methionine Synthetase in Rabbit Brain

The development and regional distribution of methionine synthetase (EC 2.1.1.13) in rabbit brain was determined. In adult rabbits, the specific activity (units per milligram protein) of methionine synthetase in cortex, cerebellum, brain stem, and corpus striatum was comparable to the specific activity in whole brain (0.5 units/mg). In the first few weeks of life, the specific activity of methionine synthetase in whole rabbit brain declined from a value of 1.1 units/mg at 1 day of age to 0.5 units/mg at 6–10 weeks. Two-year-old rabbits had 0.6 units/mg in whole brain. These results show that: (a) methionine synthetase is distributed widely in mammalian brain and (b) methionine synthetase activity in brain declines relatively little with development.     

The relationships between corpora allata and fat body and haemolymph lipids in the adult female desert locust

Allatectomy of 1-day-old female desert locusts resulted in an accumulation of lipid in the fat body. This accumulation of lipid was due to the continuation of lipid deposition in the fat body after the period of somatic growth. Somatic growth and feeding activity were unaffected by allatectomy, and so could not be indirect causes of fat body lipid accumulation. Lipid accumulation in allatectomized locusts is more likely to be related directly to a lack of juvenile hormone. Implantation of active corpora allata into 1-day-old adult female locusts resulted in a premature development of oöcytes and a decrease in fat body lipid accumulation; somatic growth was not inhibited. Implantation of active corpora allata into old allatectomized locusts resulted in a decrease in the fat body lipid content and the onset of oöcyte development. The lipid synthetic activity of the fat body, measured by the incorporation of ¹⁴C-actate into total fat body lipid, was greatly increased in allatectomized locusts after the period of somatic growth. The protein synthetic activity of the fat body, measured by the incorporation of ³H-leucine into total fat body protein, remained low after the period of somatic growth in allatectomized insects. Juvenile hormone might thus have a dual effect on fat body metabolism, that is suppressing lipid synthesis and stimulating vitellogenic protein synthesis. Increased synthesis of lipid by the fat body would then account for the accumulation of lipid in the fat body after allatectomy. Inhibition of release of lipid from the fat body is unlikely to play a part in the accumulation as allatectomy had no effect on haemolymph lipid concentrations.     

Difructose anhydride III does not contribute to body energy accumulation in rats

We evaluated the body energy accumulation as fat and protein from ingestion of difructose anhydride III (DFAIII). Male Wistar rats were fed 0, 0.25, 0.5, 1.0, or 1.5 g per d of sucrose or DFAIII added to a 7 g of basal diet for 20 d. Supplements of DFAIII did not increase whole body or peripheral fat or total body energy, whereas sucrose increased them in a dose-dependent manner. Dose-dependent increases in body water were observed in both groups. The body protein was influenced by the dose of sugars. The estimated available energy value of DFAIII was 0.263 kcal per gram; this value is one-fifteenth that of sucrose. Ingestion of DFAIII dose-dependently increased the cecal SCFA pool. DFAIII was not detected in feces, showing complete degradation of DFAIII in the intestine. These results indicate that DFAIII is a fermentable saccharide with quite low available energy for fat accumulation.     

Effects of chronic spinal cord injury on body weight and body composition in rats fed a standard chow diet

The inability to maintain body weight within prescribed ranges occurs in a significant portion of the human spinal cord injury (SCI) population. Using a rodent model of long-term high thoracic (spinal level T3) spinal cord transection (TX), we aimed to identify derangements in body weight, body composition, plasma insulin, glucose tolerance, and metabolic function, as measured by uncoupling protein 1 (UCP1) expression in interscapular brown adipose tissue (IBAT). Sixteen weeks after SCI, body weights of injured female rats stabilized and were significantly lower than surgical control animals. At the same time point, SCI rats had a significantly lower whole body fat:lean tissue mass ratio than controls, as measured indirectly by NMR. Despite lower body weight and fat mass, the cumulative consumption of standard laboratory chow (4.0 kcal/g) and mean energy intake (kcal.day(-1).100 g body wt(-1)) of chronic SCI rats was significantly more than controls. Glucose tolerance tests indicated a significant enhancement in glucose handling in 16-wk SCI rats, which were coupled with lower serum insulin levels. The post mortem weight of gonadal and retroperitoneal fat pads was significantly reduced after SCI and IBAT displayed significantly lower real-time PCR expression of UCP1 mRNA. The reduced fat mass and IBAT UCP1 mRNA expression are contraindicative of the cumulative caloric intake by the SCI rats. The prolonged postinjury loss of body weight, including fat mass, is not due to hypophagia but possibly to permanent changes in gastrointestinal transit and absorption, as well as whole body homeostatic mechanisms.     

Inhibition of the formation of protein storage granules by glutaurine in the larval fat body cells of Mamestra brassicae (Insecta, Lepidoptera)

Correlative changes in the protein contents of haemolymph and fat body and the accumulation of protein storage granules in the fat body cells of Mamestra brassicae were investigated during the last larval stage in normally developing larvae and following administration of glutaurine (1 X 10(-4) mg/g body weight). The protein content of the haemolymph of untreated larvae increased up to the 4th day of the stage, declined during days 5 and 6, and increased again before pupation. In the glutaurine-treated larvae the amount of proteins in the haemolymph was as high as in the controls during the first four days but continued to rise up to the end of the stage. The protein content of the fat body started to increase from the 3rd day and heavy accumulation of protein storage granules in the cells of fat body was observed on the 5th and following days. The protein content of the fat body of glutaurine-treated larvae remained at a low level and the protein storage granules were absent in the cells. The inhibition of the selective uptake of haemolymphatic storage proteins by fat body following glutaurine treatment is suggested.     

Estimates of individual factors of the tryptophan requirement based on protein and tryptophan accretion responses to increasing tryptophan supply in broiler chickens 8 – 21 days of age

Ten diets (196?g CP and 13.0 MJ ME_N/kg) with graded levels of tryptophan (Trp) between 0.9 and 2.7?g/kg were offered ad libitum to Ross broilers from day 8?–?21 post-hatch. Each diet was allocated to three pens of ten birds each. In addition to growth and feed conversion, the accretions of protein, Trp, fat, and energy were determined by comparative whole body analyses. A sigmoidal function was fitted to the data. Responses to Trp supply were nonlinear and attained plateau values (y_max) within the studied range of Trp supply. The y_max values estimated for BW gain, whole body protein gain, and Trp gain during the 14 days under study were 615, 104, and 0.87?g/bird, respectively. Based on the response in whole body protein gain, the estimated requirement was 1.9?g Trp/kg of diet or 0.15?g Trp/MJ of ME_N. Estimates made from the other response criteria were similar to this value. While the protein concentration in gained BW was unaffected by Trp intake (169?g of protein per kg of gained BW), fat and energy concentrations of gained BW increased with increasing Trp supply, approaching plateau values of 146?g fat and 9.8 MJ energy per kg of gained BW. This is supposed to result from feed intake increasing with Trp supplementation. The Trp concentration in gained whole body protein (0.84?g Trp/16?g of gained N) was not significantly affected by Trp intake. As long as Trp intake was the limiting factor, 59% of supplemented Trp was transferred to gained whole body protein. During the 14 days of the study, broilers inevitably lost 87?mg Trp. This is equivalent to a daily loss of 30?mg/kg BW or a maintenance requirement of 52?mg/kg BW per day. Data determined for the individual factors may be used for future modelling of broilers' Trp requirement.     

Exogenous human growth hormone reduces body fat in obese women

The effects of biosynthetic methionyl human growth hormone (met-hGH) on body composition and endogenous secretion of insulin-like growth factor I (IGF-I) were studied in obese women ranging between 138 and 226% of ideal body weight. Following double-blind procedures, 12 subjects were assigned at random to either treatment with met-hGH (n = 6, 0.08 mg/kg desirable body weight) or placebo (n = 6, bacteriostatic water diluent). Treatments were delivered intramuscularly three times per week for a period of 27-28 days. Subjects were instructed to follow a weight-maintaining diet and their pre- and posttreatment kilocaloric intake was monitored for verification. The baseline peak serum GH response to L-dopa/arginine stimulation for the study population as a whole, was in the hyposecretory range (9.6 +/- 1.9 ng/ml), accompanied by a low level of circulating IGF-I (0.56 +/- 0.09 U/ml). Hydrodensitometry revealed that the met-hGH-treated subjects had a significant reduction in body fat, while an observed mean increase in fat-free mass (FFM) approached significance. The percent change in body fat was unrelated to pretreatment levels of body fat, total body weight, or initial endogenous GH status. Changes in circulating IGF-I were similar to those for FFM, with increases approaching significance. There were no significant changes in body composition or IGF-I in the placebo-treated subjects. No significant differences were observed in the self-reported dietary intake of kilocalories during the experimental period between the two groups. We conclude that exogenous GH reduces body fat in obese women in the apparent absence of significant kilocaloric restriction. The effect appears to be unrelated to endogenous GH secretion or body composition.     

Effect of dietary fat on whole body fatty acid synthesis in weanling rats

The effect of dietary fat on body composition, whole body lipogenesis, and enzyme activity was measured in rats over the first 16 weeks post-weaning. Rats were fed either a low fat (5% w/w fat) or high fat (20% w/w fat) diet for the first 4 weeks. After this time all rats were fed the low fat diet. The results showed no significant effect of diet on the rate of fat synthesis over the first 8 weeks of the experiment. However, the activities of the enzymes of fatty acid synthesis [glucose 6-phosphate dehydrogenase, malic enzyme, adenosine triphosphate-citrate lyase, acetyl-coenzyme A carboxylase (ACCX), fatty acid synthetase] were dependent on the age and dietary status of the animals. The exact pattern depended on the specific enzyme and the tissue source. No significant differences in pyruvate dehydrogenase (PDH) activity were observed. Mathematical analysis of the enzyme activities suggested that ACCX and PDH were the most likely sites of fat synthesis regulation. In addition, an examination of body composition and overall weight retention showed that the "weight increasing" effect of a high fat diet could be completely reversed by subsequent feeding of a low fat diet. However, the reversal required an additional 12 weeks. Interestingly, at this time the rats switched from a high fat to a low fat diet had a lower body weight and lower body fat content than rats fed a low fat diet throughout the course of the experiment.     

Adipokinetic hormone is dependent on extracellular Ca2+ for its stimulatory action on the glycogenolytic pathway in locust fat body in vitro

Inclusion of glucose or trehalose in the medium during the incubation of locust fat body in vitro leads to a reduction of the relative amount of active (AMP-independent) glycogen phosphorylase. The presence of adipokinetic hormone (AKH I) results in a rapid activation of phosphorylase, reaching a maximum within 5 min. This AKH effect is highly dependent on added Ca²⁺, and requires ⩾ 1 mM Ca²⁺ for maximal enzyme activation. Ca²⁺ alone has no effect on phosphorylase activity, but it does activate the enzyme when the ionophore A23187 is also included in the medium. In a cell-free system from locust fat body the activation of endogenous phosphorylase by phosphorylase kinase is stimulated by Ca²⁺. Activity of the latter enzyme can be increased further by high doses of calmodulin. Both in the presence and in the absence of external calmodulin, the calmodulin antagonist trifluoperazine has an inhibitory effect on phosphorylase kinase. Results are discussed in relation to the possible mechanisms underlying hormonal control of glycogenolysis.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Calmodulin and calmodulin-binding proteins in liver cell nuclei

Three nuclear subfractions were prepared from isolated hepatocytes nuclei. The calmodulin content in whole nuclei was 79 ng/mg of protein. The soluble fraction obtained after digestion of the nuclei with DNase I and RNase A (S1 fraction) contained 252 ng of calmodulin/mg of protein. The pellet obtained after the digestion with nucleases was treated with 1.6 M NaCl, and the soluble fraction and the residual structures obtained after the treatment were called S2 fraction and nuclear matrix, respectively. The calmodulin contents of the S2 fraction and of the nuclear matrix were 68 and 190 ng/mg of protein, respectively. If nuclei were digested only with DNase I, the calmodulin content in the soluble fraction increased to 703 ng/mg of protein, indicating that part of the nuclear calmodulin is associated with active DNA. Five nuclear calmodulin-binding proteins were identified. Two, having apparent molecular masses of 240 and 150 kDa were only found in the nuclear matrix, whereas the other three, having molecular masses of 120, 65, and 40 kDa were found in different proportions in all nuclear subfractions. A calmodulin-dependent inhibition of protein phosphorylation in the S1 fraction was discovered. Purification attempts on the calmodulin-binding proteins of the S1 subfraction by calmodulin affinity chromatography yielded four major polypeptides with apparent molecular masses of about 41, 46, and 120 (two products) kDa. These polypeptides retained the ability to inhibit protein phosphorylation but not the sensitivity to calmodulin.     

Calmodulin-dependent regulation of Ca,Mg-ATPase activity in plasma membranes of the swine myometrium

Highly purified plasma membrane (PM) preparations of pig myometrium were found to contain 0.91 +/- 0.22 microgram calmodulin per mg of PM protein. Treatment of membranes with 1 mM EGTA in the presence of 0.2 M NaCl causes the diminution of the calmodulin content down to 3% of the original level. The activity of Ca, Mg-ATPase is thereby decreased by 40%. Exogenous calmodulin restores the enzyme activity up to 1.94 +/- +/- 0.30 mumol Pi/mg protein/hour. The maximal activation of Ca, Mg-ATPase is observed with 10(-7) M calmodulin. Calmodulin increases the total ATPase activity of myometrium PM without affecting the Mg-ATPase activity. Trifluoroperazine (20 microM) diminishes the activating effect of exogenous calmodulin on Ca, Mg-ATPase. Calmodulin stimulates Ca, Mg-ATPase at low concentrations of Ca2+(10(-8)-10(-6) M) by decreasing Km for Ca2+ from 0.4.10(-6) M to 2.10(-8) M as well as by increasing Vmax--from 0,8 to 1.42 mumol Pl/mg protein/hour. It is supposed that the activating effect of calmodulin on Ca, Mg-ATPase is based on electrostatic interactions of Ca2+-free calmodulin with the enzyme.