Rat age-related benign prostate hyperplasia is concomitant with an increase in the secretion of low ramified α-glycosydic polysaccharides.

This work analysed the expression of prostate polysaccharides in rats with age-related benign prostatic hyperplasia (BPH) for a better understanding of the possible relationship between prostate polysaccharides secretion and BPH onset. For this, prostatic glands from 1 month-old, 3 months-old, 6 months-old and 12 months-old Sprague-Dawley rats were processed in order to identify their overall polysaccharide content. Additionally, serum testosterone was also determined. One-month old rats showed significantly (P < 0.05) lower testosterone levels (0.77 ng/mL±0.12 ng/mL) compared with the other groups, which showed no significant difference among them. PAS staining showed positive polysaccharides markings in both the prostatic lumen and inside of luminal prostatic cells in all groups. Semiquantitative analysis of intraluminal PAS showed that one month-old rats had significantly (P < 0.005) lower PAS intensity when compared with all other groups (100.0 ± 0.5, arbitrary units vs. 107.3 ± 0.6, arbitrary units in 3 months-old ones), whereas 12 months-old ones showed significantly (P < 0.005) higher values when compared with all other groups (133.6 ± 3.5, arbitrary units in 12 months-old rats vs. 108.6 ± 1.4, arbitrary units in 6 months-old ones). The PAS + content practically disappeared when tissues were pre-incubated with either α-amylase or amyloglucosidase, regardless of a previous incubation with proteinase K. Incubation of prostate extracts from 12 months-old rats for 2 h with α-amylase yielded a significantly higher amount of free glucose (1.47 nmol/mg protein±0.23 nmol/mg protein vs. 0.32 nmol/mg protein±0.01 nmol/mg protein in untreated extracts). Similar results were obtained when extracts were pre-incubated with amyloglucosidase. Contrarily, pre-incubation with N-glycosidase induced a significantly (P < 0.05), much lower increase of free glucose. Pre-treatment with proteinase K did not significantly modify these results, which indicate that BPH is related to an increase in the secretion of low ramified ductal α-glycosydic polysaccharides that were not protected against lysis by any type of protein protective core. These changes seem to not be related with concomitant variations in serum testosterone levels.

The onset of age-related benign prostatic hyperplasia is concomitant with increased serum and prostatic expression of VEGF in rats: Potential role of VEGF as a marker for early prostatic alterations.

The onset of age-related benign prostate hyperplasia (BPH) is linked with changes in the expression of specific prostatic chemokines. The aim of this work was to characterize those most relevant changes through the simultaneous analysis of 34 chemokines in both prostatic tissue and serum in rats at different ages with the aim to identify clinically workable parameters for the detection of early prostatic alterations. The study included 28 healthy Sprague-Dawley male rats that were distributed in four groups, 1 month-old (prepuberal; n = 7), 3 months-old (young; n = 7), 6 months-old (mature; n = 7) and 12 months-old (elder; n = 7). Chemokines were analyzed through a commercial mini-array system specially designed for rat tissues. Serum testosterone levels and prostatic histological status were also evaluated. Histological lesions indicative of BPH were detected in three mature rats and in all elder ones. Mini-arrays from prostatic tissue showed that young animals had an overall decreased expression of most of the analyzed chemokines when compared with prepuberal rats, with the exception of agrin, which showed a significant (P < 0.05) increase (100.0 ± 1.3, arbitrary units in prepuberal rats vs.148.2 ± 4.1, arbitrary units in young ones). Older animals showed further specific changes in 4 out 34 analyzed chemokines, namely agrin, platelet-derived growth factor (PDGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF). Additionally, elder rats showed the lowest intensity levels of agrin combined with the highest ones for PDGF, TIMP1 and VEGF when compared with all other groups. Finally, a significant increase of serum VEGF was detected in elder, BPH-affected rats when compared with young ones. Results indicated that the onset of both rat puberty and BPH would be related with specific changes in the prostatic expression of chemokines such as VEGF. Otherwise, the observed changes in serum VEGF levels could suggest the future possible utilization of serum VEGF levels to detect early pathological prostatic processes.

The achievement of boar sperm in vitro capacitation is related to an increase of disrupted disulphide bonds and intracellular reactive oxygen species levels.

The aim of this work was to determine the relationship of intracellular reactive oxygen species (ROS) and the disulphide bonds established between sperm proteins with the achievement of capacitation in boar spermatozoa. With this purpose, spermatozoa were incubated in a specifically designed in vitro capacitation medium (CM) in the presence or absence of reduced glutathione (GSH). Incubation of boar spermatozoa in CM for 4 h significantly (p < 0.05) increased free cysteine residues, which is a marker of disrupted disulphide bonds, and also intracellular ROS levels. The addition of GSH to the medium prevented most capacitation-like changes in sperm motility, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium levels and localization of tyrosine-phosphorylated proteins (pTyr), but not in tyrosine phosphorylation of P32. These effects were accompanied by the inhibition of the ability of sperm cells to trigger the acrosome exocytosis in response to progesterone. When GSH was added together with progesterone after 4 h of incubation, acrosome exocytosis was not altered, but the subsequent decrease in intracellular calcium observed in controls cells was inhibited. Furthermore, co-incubation of oocytes with spermatozoa previously incubated in CM in the presence of GSH for 4 h significantly (p < 0.05) increased the number of spermatozoa attached to the oocyte surface but decreased normal fertilization rates. Our results suggest that boar sperm capacitation is related to an increase in disrupted disulphide bonds and intracellular ROS levels and that both events are related to the regulation of hyperactivated motility, intracellular calcium dynamics, sperm binding ability to the oocyte and achievement of proper nuclear decondensation upon oocyte penetration.

Immunochemical Study of the Effect of F2Glc on Glycogen Synthase Translocation and Glycogen Synthesis in Isolated Rat Hepatocytes.

The compound 2-deoxy-2-fluoro-α-D-glucopyranosyl fluoride (F2Glc), which is a nonmetabolized superior glucose analogue, is a potent inhibitor of glycogen phosphorylase and pharmacological properties are reported. Glycogen phosphorylase (GP) and glycogen synthase (GS) are responsible of the degradation and synthesis, respectively, of glycogen which is a polymer of glucose units that provides a readily available source of energy in mammals. GP and GS are two key enzymes that modulate cellular glucose and glycogen levels; therefore, these proteins are suggested as potential targets for the treatment of diseases related to glycogen metabolism disorders. We studied by Western Blot technique that F2Glc decreased GP activity, and we also showed that F2Glc did not affect GS activity and its translocation from a uniform cytosolic distribution to the hepatocyte periphery, which is crucial for glycogen synthesis, using immunoblotting and immunofluorescence labeling techniques. F2Glc specifically inhibits glycogenolysis pathway and permits a greater deposition of glycogen. These observations open up the possibility of further develop drugs that act specifically on GP. The ability to selectively inhibit GP, which is a key enzyme for the release of glucose from the hepatic glycogen reserve, may represent a new approach for the treatment of hyperglycemia in type 2 diabetes.

Intracellular calcium movements of boar spermatozoa during 'in vitro' capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model.

This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

'In vitro' capacitation and further 'in vitro' progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa.

The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.

"In vitro" capacitation and subsequent acrosome reaction are related to changes in the expression and location of midpiece actin and mitofusin-2 in boar spermatozoa.

The induction of "in vitro" capacitation (IVC) and subsequent, progesterone-induced "in vitro" acrosome reaction (IVAR) was concomitant with an increase in actin polymerization, also showing an increase in actin presence at the apical area of the midpiece. The presence of mitofusin-2, a protein involved in the regulation of the coordinated mitochondrial function, expanded from midpiece to the principal piece after IVC and IVAR. All of these results indicate that the increase of boar sperm mitochondrial activity during IVC and the first minutes of IVAR is concomitant with changes in the expression and location of both actin and mitofusin-2. Our results suggest that both actin and mitofusin-2 play important roles in the modulation of boar sperm mitochondrial function, both by originating changes in the protein membrane environment and by changes in the mitochondrial structure itself.

Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm.

The main aim of this work is to gain insight into the mechanisms by which freezing-thawing alters the nucleoprotein structure of boar sperm. For this purpose, the freezing-thawing-related changes of structure and location of histones-DNA domains in the boar sperm head were analyzed through Western blot and immunocytochemistry. Afterwards, it was analyzed whether freezing-thawing induced changes in tyrosine phosphorylation levels of both protamine 1 and histone H1, through Western blot analyses in samples previously subjected to immunoprecipitation. This analysis was completed with the determination of the changes induced by freezing-thawing on the overall levels of sperm-head disulfide bonds through analysis of free-cysteine radicals levels. Freezing-thawing induced significant changes in the histones-DNA structures, which were manifested in the appearance of a freezing-thawing-linked histone H1-DNA aggregate of about a 35-kDa band and in the spreading of histone H1-positive markings from the caudal area of the sperm head to more cranial zones. Freezing-thawing did not have any significant effect on the tyrosine phosphorylation levels of either protamine 1 or histone H1. However, thawed samples showed a significant (P < 0.05) increase in the free cysteine radical content (from 3.1 ± 0.5 nmol/µg protein in fresh samples to 6.7 ± 0.8 nmol/µg protein). In summary, our results suggest that freezing-thawing causes significant alterations in the nucleoprotein structure of boar sperm head by mechanism/s linked with the rupture of disulfide bonds among the DNA. These mechanisms seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Furthermore, results suggest that the boar-sperm nuclear structure is heterogeneous suggesting the existence of a zonated pattern, differing in their total DNA density and the compactness of the precise nucleoprotein structures present in each zone.

Glucose and fructose as functional modulators of overall dog, but not boar sperm function.

The main aim of the present work was to test the effects of glucose and fructose on the phosphorylation levels of proteins linked to the control of overall sperm function in two species with very different metabolic characteristics, dog and boar. Incubation of dog spermatozoa with 10mM glucose increased serine phosphorylation of proteins related to cell cycle and signal transduction including cyclins B and E, Cdk2, Cdk6, Cdc6, PYK2, c-kit, Raf-1, TRK and several protein phosphatases. Incubation of dog spermatozoa with 10mM fructose decreased serine phosphorylation levels of cyclins B and D3, Cdk1/Cdc2, Cdk2, Cdk6, Akt, PI3 kinase, ERK-1 and protein kinase C. Incubation of boar spermatozoa with glucose or fructose did not modify any of the phosphorylation patterns studied. Given that one important difference between dog and boar spermatozoa is the presence of glucokinase (GK) in dog but not in boar, GK-transfected COS7 cells were incubated with either 10mM glucose or 10mM fructose. Incubation of GK-transfected cells with fructose decreased serine phosphorylation of cyclin A, ERK-2 and Hsp-70. In contrast, incubation of control COS7 cells with fructose increased serine phosphorylation of Cdk6, Cdk1/Cdc2, protein kinase C and Hsp-70. Incubation with glucose did not induce any significant effect. Our results indicate that monosaccharides act as signalling compounds in dog spermatozoa after ejaculation through changes in the phosphorylation levels of specific proteins. One of the factors that may be related to the action of sugars is the equilibrium of the total sperm hexokinase activity, in which the presence or absence of GK appears to be relevant.

'In vitro' capacitation and acrosome reaction are concomitant with specific changes in mitochondrial activity in boar sperm: evidence for a nucleated mitochondrial activation and for the existence of a capacitation-sensitive subpopulational structure.

The main scope of this manuscript is to analyse the dynamics of mitochondrial activity in boar sperm subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome reaction (IVAR). This was determined after analysis of the rhythm of O(2) consumption and concomitant changes in the mitochondria activity-specific JC-1 staining. Results showed that IVC, and especially IVAR, was concomitant with a peak in O(2) consumption (from 1.61 ± 0.08 nmol O(2)/min/10(7) viable sperm at 0 h of incubation to 2.62 ± 0.12 nmol O(2) /min/10(7) viable sperm after 5 min of IVAR induction). These results were accompanied by parallel changes in the mean intensity of JC-1 staining. Based on JC-1, mitochondrial activation followed a nucleated pattern, with specific, activation starting points at the midpiece from which mitochondrial activation was spread. Moreover, four separate sperm subpopulations were detected following the JC-1 orange-red/green ratio, and the observed changes in the mean JC-1 staining during IVC and IVAR were related to concomitant changes in both the orange-red/green JC-1 ratio and the percentage of sperm included in each subpopulation. All of these results indicate that IVC and the first minutes of IVAR are accompanied by a progressive increase in mitochondrial activity, which reached a peak coincidental with the achievement of IVAR. Moreover, results suggest the presence of separate sperm subpopulations, which show a different mitochondrial sensitivity to IVC and IVAR. Finally, mitochondrial activation, at least under JC-1 staining, seems to originate in concrete nucleation points at the midpiece, thus suggesting thus a well-coordinated pattern in boar-sperm mitochondrial activity modulation.

Cryopreservation-induced alterations in boar spermatozoa mitochondrial function are related to changes in the expression and location of midpiece mitofusin-2 and actin network.

The authors analyzed changes in mitochondrial activity of boar semen during a standard cryopreservation protocol. For this purpose, mitochondrial activity was evaluated simultaneously with the rhythm of mitochondrial formation of reactive oxygen species (mROS) through a double MitoTracker Red/proxylfluorescamine stain. Moreover, we analyzed changes in the expression and location of two key regulatory elements of mitochondrial function, namely mitofusin-2 (Mfn2) and actin, during the freezing-thawing protocol. Our results indicate that mitochondrial activity and mROS formation decreased during cyropreservation, with an initial decrease during the cooling phase of the protocol. This decrease was accompanied by an increase in the amount of solubilized Mfn2, which was concomitant with a progressive extension of Mfn2 location from the apical zone of the midpiece to the whole midpiece. Simultaneously, cryopreservation induced a decrease in solubilized actin, which was concurrent with significant changes in the midpiece actin location. The observed changes in the expression and location of both Mfn2 and actin were already present after the cooling phase of the cryopreservation protocol. Our results suggest that freezing-thawing impaired mitochondrial function. This impairment was concomitant with a decrease in the mitochondrial capacity to synthesize mROS. This impairment is attributed to changes in mitochondrial volume as a result of alterations in the expression and location of both Mfn-2 and the actin network. Finally, the alterations of mitochondrial function induced by the cryopreservation protocol were already apparent at the cooling phase. This observation indicates that the cooling phase is a crucial stage in which mitochondrial alterations occur during cryopreservation.

The degree of resistance to freezing-thawing is related to specific changes in the structures of motile sperm subpopulations and mitochondrial activity in boar spermatozoa.

The main aim of this work was to analyze the possible relationship between the structures of motile-sperm subpopulations and boar (Sus scrofa domesticus) sperm resistance to freezability. For this purpose, 45 boar ejaculates were subjected to a standard freezing-thawing protocol, and afterwards they were classified into three groups, in accordance with their resistance to freezing-thawing. Our analysis yielded four separate motile-sperm subpopulations in all of the studied ejaculates, both in fresh samples and after freezing-thawing. Furthermore, whereas curvilinear velocity (VCL), mean velocity (VAP), and dance (DNC) of sperm from Subpopulation 1 underwent significant increases after freezing-thawing in samples with a good response to freezing-thawing, the same parameters of Subpopulation 1 either did not undergo significant variations (VCL and DNC) or even showed a decrease (VAP) (from 20.4+/-0.4 microm/sec in fresh samples to 15.2+/-2.2 microm/sec after freezing-thawing) in samples with the poorest response. Similarly, the behavior of other motility parameters in each subpopulation was also very different in the worst samples when comparing them with those with a good or average response to cryopreservation. Additionally, the DNC of all four subpopulations was in all cases lower in samples with the poorest characteristics of freezability. This was not the only difference, and significant changes in parameters such as the VCL of Subpopulations 2 and 4, linearity coefficient (LIN) of Subpopulations 1, 2, and 3, and wobble coefficient (WOB) of Subpopulations 2 and 3 were also observed in samples with different response to freezing-thawing. Meanwhile, the determination of mitochondrial activity and mitochondrial-linked reactive oxygen species formation indicated that the samples with the poorest freezability characteristics were also those with the lowest mitochondrial activity. We conclude that boar ejaculate resistance to cryopreservation seems to be related to the specific, initial motile-sperm subpopulation structure. In turn, this structure would be closely related to the specific, overall mitochondrial activity, which would be a very important indicator of sperm function. Furthermore, and as a practical conclusion, an in-depth analysis of motile sperm subpopulation structure together with functional tests could improve the design of predictive strategies for the freezability of boar sperm.

Freeze-thawing induces alterations in the protamine-1/DNA overall structure in boar sperm.

The main aim of this work was to test the effects that freeze-thawing could have on the overall nuclear structure of boar sperm. This was done by analyzing both the DNA fragmentation and the protamine-1-DNA interaction of the boar-sperm nucleus. Our results indicate that freezing-thawing did not induce a significant degree of DNA fragmentation, as manifested through both the Sperm-Sus-Halomax stain and a random primed analysis prior to partial DNA digestion with enzymes BamHI-HinDIII. On the other hand, freeze-thawing induced significant changes in the protamine-1-DNA interaction, as revealed through both Western blot analysis and immunocytochemistry for protamine-1. These alterations caused, in turn, significant changes in the overall nuclear structure of boar sperm after thawing. Protamine-1-DNA alterations started to be apparent during the cooling phase of the freeze-thawing protocol. These results imply that one of the alterations that may be responsible for the loss of fertilizing ability of boar sperm after freeze-thawing may be an alteration in the correct formation of the overall nuclear structure, which, in turn, would induce alterations in the correct formation of the first nuclear structure after oocyte penetration.

Gluconeogenesis-linked glycogen metabolism is important in the achievement of in vitro capacitation of dog spermatozoa in a medium without glucose.

In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [(14)C]glycogen after the addition to l-CCM with [(14)C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

A biochemistry and molecular biology course for secondary school teachers*.

This article describes a course for reinforcing the knowledge of biochemistry in secondary school science teachers. The Department of Biochemistry and Molecular Biology of the University of Barcelona designed a course to bring these teachers up to date with this discipline. In addition to updating their knowledge of biochemistry and molecular biology, this course aims to provide teachers with a set of relevant laboratory practices that can be applied in their practical lessons.

Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa.

Incubation of dog spermatozoa with 10 mmol l(-1) glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of L-lactate and CO2. In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l(-1)). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l(-1) to 10 mmol l(-1), fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.

Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes.

Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8 h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.

Evidence for a functional glycogen metabolism in mature mammalian spermatozoa.

The glycogen content in fresh raw dog spermatozoa was 0.22+/-0.03 micromol/mg protein. This matched with the presence of a glycogen-like staining in the head and midpiece. Glycogen levels lowered to 0.05 micromol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 micromol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35+/-0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25+/-0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo.

Lithium's effects on rat liver glucose metabolism in vivo.

Oral administration of lithium carbonate to fed-healthy rats strongly decreased liver glycogen content, despite the simultaneous activation of glycogen synthase and the inactivation of glycogen phosphorylase. The effect seemed to be related to a decrease in glucose 6-phosphate concentration and to a decrease in glucokinase activity. Moreover, in these animals lithium markedly decreased liver fructose 2,6-bisphosphate, which could be a consequence of the fall in glucose 6-phosphate and of the inactivation of 6-phosphofructo-2-kinase. Liver pyruvate kinase activity and blood insulin also decreased after lithium administration. Lower doses of lithium carbonate had less intense effects. Lithium administration to starved-healthy and fed-streptozotocin-diabetic rats caused a slight increase in blood insulin, which was simultaneous with increases in liver glycogen, glucose 6-phosphate, and fructose 2, 6-phosphate. Glucokinase, 6-phosphofructo-2-kinase, and pyruvate kinase activities also increased after lithium administration in starved-healthy and fed-diabetic rats. Lithium treatment activated glycogen synthase and inactivated glycogen phosphorylase in a manner similar to that observed in fed-healthy rats. Glycemia was not modified in any group of animals. These results indicate that lithium acts on liver glycogen metabolism in vivo in at least two different ways: one related to changes in insulinemia, and the other related to the direct action of lithium on the activity of some key enzymes of liver glucose metabolism.

Intracellular distribution of hepatic glucokinase and glucokinase regulatory protein during the fasted to refed transition in rats.

We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1- and 2-h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo.