Amelioration of lipid-induced insulin resistance in rat skeletal muscle by overexpression of Pgc-1ß involves reductions in long-chain acyl-CoA levels and oxidative stress.

AIMS/HYPOTHESIS: To determine if acute overexpression of peroxisome proliferator-activated receptor, gamma, coactivator 1 beta (Pgc-1ß [also known as Ppargc1b]) in skeletal muscle improves insulin action in a rodent model of diet-induced insulin resistance. METHODS: Rats were fed either a low-fat or high-fat diet (HFD) for 4 weeks. In vivo electroporation was used to overexpress Pgc-1ß in the tibialis cranialis (TC) and extensor digitorum longus (EDL) muscles. Downstream effects of Pgc-1ß on markers of mitochondrial oxidative capacity, oxidative stress and muscle lipid levels were characterised. Insulin action was examined ex vivo using intact muscle strips and in vivo via a hyperinsulinaemic-euglycaemic clamp. RESULTS: Pgc-1ß gene expression was increased >100% over basal levels. The levels of proteins involved in mitochondrial function, lipid metabolism and antioxidant defences, the activity of oxidative enzymes, and substrate oxidative capacity were all increased in muscles overexpressing Pgc-1ß. In rats fed a HFD, increasing the levels of Pgc-1ß partially ameliorated muscle insulin resistance, in association with decreased levels of long-chain acyl-CoAs (LCACoAs) and increased antioxidant defences. CONCLUSIONS: Our data show that an increase in Pgc-1ß expression in vivo activates a coordinated subset of genes that increase mitochondrial substrate oxidation, defend against oxidative stress and improve lipid-induced insulin resistance in skeletal muscle.

Muscle ceramide content in man is higher in type I than type II fibers and not influenced by glycogen content.

Human muscle is studied during glycogen depletion and repletion to understand the influence of exercise and muscle glycogen on total ceramide content. In addition, fiber-type-specific ceramide storage is investigated. Ten healthy males (26.4 +/- 0.9 years, BMI 24.4 +/- 0.7 kg m(-2) and VO2max 57 +/- 2 mL O2 min(-1) kg(-1)) participated in the study. On the first day, one leg was glycogen-depleted (DL) by exhaustive intermittent exercise followed by low carbohydrate diet. Next day, in the overnight fasted condition, muscle biopsies were excised from vastus lateralis before and after exhaustive exercise from both DL and control leg (CL). Muscle glycogen was analyzed biochemically and total muscle ceramide content by 2D quantitative lipidomic approach. Furthermore, fiber-type ceramide content was determined by fluorescence immunohistochemistry. Basal muscle glycogen was decreased (P < 0.05) with 50 +/- 6% in DL versus CL. After exhaustive exercise, muscle glycogen was similar in CL and DL 139 +/- 38 and 110 +/- 31 mmol kg(-1), respectively. Total muscle ceramide 58 +/- 1 pmol mg(-1) was not influenced by glycogen or exercise. Ceramide content was consistently higher (P < 0.001) in type I than in type II muscle fibers. In conclusion, human skeletal muscle, ceramide content is higher in type I than in type II. Despite rather large changes in muscle glycogen induced by prior depletion, exercise to exhaustion and repletion, total muscle ceramide concentration remained unchanged.

Aberrant extrathymic T cell receptor gene rearrangement in the small intestinal mucosa: a risk factor for coeliac disease?

BACKGROUND: Coeliac disease is a small intestine enteropathy caused by permanent intolerance to wheat gluten. Gluten intake by patients with coeliac disease provokes a strong reaction by intestinal intraepithelial lymphocytes (IELs), which normalises on a gluten-free diet. AIM: To investigate whether impaired extrathymic T cell maturation and/or secondary T cell receptor (TCR) gene recombination in IELs are features of coeliac disease which could contribute to the failure of establishing tolerance to gluten. METHODS: Expression levels of the four splice-forms of recombination activating gene-1 (RAG1) mRNA and preT alpha-chain (preTalpha) mRNA were determined in IEL-subsets of children with coeliac disease and controls. Frequencies of RAG1 expressing IELs were determined by immunomorphometry. RESULTS: In controls, the RAG1-1A/2 splice-form selectively expressed outside the thymus, was dominant and expressed in both mature (TCR(+)) and immature (CD2(+)CD7(+)TCR(-)) IELs ( approximately 8 mRNA copies/18S rRNA U). PreTalpha was expressed almost exclusively in CD2(+)CD7(+)TCR(-) IELs ( approximately 40 mRNA copies/18S rRNA U). By contrast, RAG1 and preTalpha mRNA levels were low in patients with coeliac disease compared to controls, both with active disease and with inactive, symptom-free disease on a gluten-free diet (p values <0.01 for mature and <0.05 for immature IELs). Similarly, the frequencies of RAG1+ IELs were significantly lower in patients with coeliac disease compared to controls (p<0.001). CONCLUSIONS: Patients with coeliac disease appear to have an impaired capacity for extrathymic TCR gene rearrangement. This is an inherent feature, which probably plays a pivotal role in the failure to efficiently downregulate the T cell response to gluten.

The crystal structure of staphylococcal enterotoxin H: implications for binding properties to MHC class II and TcR molecules.

The X-ray structure of the superantigen staphylococcal enterotoxin H (SEH) has been determined at 1.69 A resolution. In this paper we present two structures of zinc-free SEH (apoSEH) and one zinc-loaded form of SEH (ZnSEH). SEH exhibits the conventional superantigen (SAg) fold with two characteristic domains. In ZnSEH one zinc ion per SEH molecule is bound to the C-terminal beta-sheet in the region implicated for major histocompatibility complex class II (MHC class II) binding in SEA, SED and SEE. Surprisingly, the zinc ion has only two ligating amino acid residues His206 and Asp208. The other ligands to the zinc ion are two water molecules. An extensive packing interaction between two symmetry-related molecules in the crystal, 834 A(2)/molecule, forms a cavity that buries the zinc ions of the molecules. This dimer-like interaction is found in two crystal forms. Nevertheless, zinc-dependent dimerisation is not observed in solution, as seen in the case of SED. A unique feature of SEH as compared to other staphylococcal enterotoxins is a large negatively charged surface close to the Zn(2+) site. The interaction of SEH with MHC class II is the strongest known among the staphylococcal enterotoxins. However, SEH seems to lack a SEB-like MHC class II binding site, since the side-chain properties of structurally equivalent amino acid residues in SEH and those in SEB-binding MHC class II differ dramatically. There is also a structural flexibility between the domains of SEH. The domains of two apoSEH structures are related by a 5 degrees rotation leading to at most 3 A difference in C(alpha) positions. Since the T-cell receptor probably interacts with both domains, SEH by this rotation may modulate its binding to different TcR Vbeta-chains.

Fusion proteins in biotechnology.

Gene fusion techniques allow the production of recombinant proteins featuring the combined characteristics of the parental products. Originally, these techniques were used to probe transcriptional and translational activity, to translocate proteins across cell membranes, and to facilitate the recovery of proteins. Recently, new applications have emerged in areas such as protein refolding, immunology, drug targeting and protein display. A slightly modified version of this review is also published in Current Opinion in Structural Biology 1992, 2:569-575.

Naloxone stimulates sexual behaviour in lactating rats.

During lactation the display of sexual receptivity in response to treatment with oestradiol benzoate (OB; 2 or 10 micrograms) and progesterone (0.5 mg) was inhibited, but the behaviour could be activated by i.p. (5 mg) or intracerebroventricular (i.c.v.; 100 micrograms) but not intrathecal (i.t.; 100 or 500 micrograms) injections of the opioid peptide receptor antagonist naloxone. The behaviour was also inhibited in ovariectomized rats in which serum progesterone and prolactin levels had been raised by treatment with progesterone implants and the dopamine receptor antagonist domperidone, and the uterine cervix had been stimulated. Intraperitoneal injections of naloxone (1 mg) reactivated the behaviour of cervically stimulated rats. The concentration of beta-endorphin-like immunoreactivity in the serum of lactating rats (42.8 +/- 9.2 pmol/l) was not raised above that of ovariectomized rats (35.8 +/- 8.4 pmol/l) nor was the concentration of beta-endorphin-like immunoreactivity altered in the pituitary gland (22.5 +/- 2.5 pmol/l), midbrain central grey (6.3 +/- 2.2 pmol/l) or hypothalamus (5.6 +/- 2.6 pmol/l) of lactating rats in comparison with ovariectomized rats (24.8 +/- 4.4, 4.0 +/- 2.0 and 4.7 +/- 1.4 pmol/l respectively). Adrenalectomy facilitated the display of sexual behaviour in lactating rats treated with OB plus progesterone and caused a slight increase in serum beta-endorphin-like immunoreactivity (30.5 +/- 2.7 pmol/l) compared with that in non-adrenalectomized lactating rats (26.1 +/- 2.1 pmol/l). It is suggested that an opioid peptide, but probably not beta-endorphin, inhibits sexual behaviour during lactation and after cervical stimulation.

Role of the uterine cervix in inhibition of sexual behaviour in lactating rats.

Treatment of ovariectomized rats with progesterone-filled constant-release implants, which increased serum progesterone concentrations to 99.4 +/- 5.0 nmol/l, facilitated the induction of lordosis behaviour by subsequent treatment with oestradiol benzoate (OB, 10 micrograms). Concurrent treatment with the dopamine receptor antagonist domperidone (two daily injections of 2.5 mg/rat), which increased serum prolactin concentrations, did not inhibit the behavioural response of ovariectomized progesterone-treated rats to OB. If the treatment was combined with stimulation of the uterine cervix it inhibited lordosis to a level which was comparable with that of progesterone-domperidone-treated rats, which had been ovariectomized and from which the pups had been removed on the day of parturition. The cervical stimulation did not increase the amount of prolactin secreted by the pituitary gland in response to an injection of domperidone. The behavioural effect of cervical stimulation was blocked by injecting an anaesthetic paste (0.1 ml lidocain-prilocain) intravaginally against the cervix. The effect of cervical stimulation, or of parturition, lasted only for a few days and sexual behaviour was inhibited during a prolonged period of lactation. Sucking by the pups on the nipples of the mother may be required for preventing sexual behaviour during the entire period of lactation.

Naloxone reverses post-ejaculatory inhibition of sexual behaviour in female rats.

Sexual receptivity was inhibited in ovariectomized rats treated with oestradiol benzoate (OB: two injections of 2 micrograms) and progesterone (0.5 mg) immediately after ejaculation by the male and restored after the end of the post-ejaculatory refractory period in the male. The post-ejaculatory inhibition of sexual receptivity was reversed by i.p. (5 mg), intracerebroventricular (50 micrograms) or intrathecal (50 micrograms) injection of the opioid peptide receptor antagonist naloxone. The concentration of serum beta-endorphin-like immunoreactivity in ovariectomized rats treated with OB plus progesterone was unaltered by sexual interactions with males (18.3 +/- 6.0 (S.E.M.), 26.4 +/- 2.1 and 21.8 +/- 6.1 pmol/l before sexual activity, after ejaculation and after the end of the post-ejaculatory interval) but reduced to non-detectable by hypophysectomy. Subcutaneous injection of 10 micrograms beta-endorphin raised serum concentrations of beta-endorphin-like immunoreactivity but did not affect the display of sexual behaviour. The behaviour was also unaffected by intracerebroventricular injection of 0.1, 0.2 or 1.0 microgram beta-endorphin or by injections of 0.25 microgram beta-endorphin in the periaqueductal central grey of the mesencephalon. The results show that ejaculation by male rats causes a transient inhibition of sexual receptivity in the female which may be dependent upon opioid peptide receptor mechanisms in the brain and spinal cord. It is unlikely that the peptide is beta-endorphin.

Effects of restricted maternal contact in neonatal rats on sexual behaviour in the adult.

Rats, deprived of maternal contact and nutrition every alternate day starting on day 5 of life, attained a body weight at 45 days of age which was 50% of that of rats which had free access to maternal contact and nutrition. After 55 days of unrestricted food availability the body weight of the neonatally deprived rats was approximately 15% lower than that of the controls. Malnourished female rats showed normal behavioural oestrous cycles and became pregnant and lactated normally as young adults. After ovariectomy they showed higher lordosis quotients in response to treatment with oestradiol benzoate and progesterone than controls but lost less body weight in response to treatment with oestradiol-filled constant-release implants. Malnourished male rats ejaculated less frequently than controls in tests with sexually receptive female rats but this difference disappeared with repeated testing. The malnourished males showed longer ejaculation latencies and had somewhat higher serum concentrations of LH than controls after castration and treatment with testosterone-filled constant-release implants which reduced serum androgen concentrations to about 30% of the intact level. The results show that rats are capable of sustaining a rather severe neonatal nutritional deprivation without losing the capacity for essentially normal mating behaviour in adulthood.

Plasma concentrations of cholecystokinin octapeptide and food intake in male rats treated with cholecystokinin octapeptide.

Intraperitoneal injection of 5 micrograms cholecystokinin octapeptide (CCK-8) into male rats deprived of food for 48 h produced a transient (less than 15 min) increase in plasma levels of CCK-8 but suppressed food intake for an extended period (45 min). Plasma concentrations of CCK-8 after i.p. injection of CCK-8 were raised to levels which were fairly comparable to those after feeding. Intracerebroventricular (i.c.v.) injection of the CCK antagonist proglumide (100 micrograms) reversed the effect of CCK-8 on food intake, while i.p. injection of proglumide (100 micrograms) did not have this effect. Feeding increased the plasma concentrations of somatostatin and gastrin but not of oxytocin, and somatostatin and oxytocin but not gastrin were released in response to i.p. injection of CCK-8. However, neither somatostatin nor oxytocin affected food intake, and their release in response to CCK-8 was unaffected by i.c.v. injection of proglumide. These results support the suggestion that CCK-8 is a physiological 'satiety' peptide, which can affect food intake in rats by mechanisms involving both peripheral and central CCK receptors.

Sexual activity increases plasma concentrations of cholecystokinin octapeptide and offsets hunger in male rats.

Male rats deprived of food for 48 h ignored food pellets immediately after ejaculating with a sexually receptive female rat as did males tested 5 min after i.p. injection of 5 micrograms cholecystokinin octapeptide (CCK-8) or males which had consumed food pellets for 1 h. The concentration of CCK-8 in plasma was increased to comparable levels (10.1 +/- 3.8 (S.E.M.); 11.3 +/- 3.7 and 7.2 +/- 0.5 pmol/l respectively, compared with 1.2 +/- 0.3 pmol/l for NaCl-injected controls) under each of these conditions, whereas the concentration of gastrin increased only after food consumption (18.4 +/- 3.8; 17.7 +/- 5.4 and 54.3 +/- 7.0 pmol/l respectively, compared with 21.5 +/- 2.1 pmol/l for NaCl-injected controls). The effect of ejaculation on the latency to starting to eat was partially reversed by i.p. injection of 50 mg of the CCK-8 antagonist proglumide, but neither treatment with proglumide or CCK-8 nor consumption of food affected the display of sexual behaviour. The results support the suggestion that CCK-8 is a 'satiety peptide' in the rat.

Treatment with tumor-reactive Fab-IL-2 and Fab-staphylococcal enterotoxin A fusion proteins leads to sustained T cell activation, and long-term survival of mice with established tumors.

C215Fab-IL-2 fusion protein, with full IL-2 and antigen binding activity, was produced in E. coli at high level (>50 mg/l). When co-administered with Fab-superantigen fusion protein (C215Fab-SEA) in mice strong and sustained T cell activation was observed. Combination treatment of mice carrying B16 melanoma transfected with C215 antigen was also more efficient than using C215Fab-SEA (p<0.01) or C215Fab-IL-2 alone (p<0.001). In a long-term survival experiment 5/12 mice having received combination treatment 5 days after i.v. inoculation of B16 cells survived >85 days. Improved therapeutic efficacy correlated with increased tumor infiltration by activated CD25+ T cells, indicating a T cell mediated mechanism.

Determination of Enkephalins in Rat Cerebrospinal Fluid with High-Performance Liquid Chromatography-Electrochemical Detection: Increased Concentrations During Lactation are Controlled by Sucking.

Abstract Methionine- and leucine-enkephalin were measured in the cerebrospinal fluid of lactating rats by high-performance liquid chromatography and electrochemical detection. The concentration of both peptides was high while the rats were nursing their litter. The concentration of methionine-enkephalin decreased rapidly when the mother left her litter and increased equally rapidly after mother-young reunion, provided the pups were allowed direct contact with the nipples of the mother. The level of leucine-enkephalin did not change during the period of time the lactating rat normally stayed away from its litter but decreased after prolonged (12 h) mother-pup separation. These results show that the concentration of methionine-, but not leucine-enkephalin in the cerebrospinal fluid fluctuates as the lactating rat interacts with its litter and is directly dependent upon the suckling stimulus. Although methionine-enkephalin may contribute to the inhibition of sexual behaviour which occurs during lactation, the role of the enkephalins in the other behavioural and endocrine adaptations of lactation is unknown.

Involvement of dopamine in inhibition of food intake by cholecystokinin octapeptide in male rats.

Abstract Deprivation of food reduced the level of dopamine in the cerebrospinal fluid of male rats and subsequent ingestion of food or intraperitoneal injection of Cholecystokinin octapeptide restored the level. Injection of a dopamine receptor agonist (apomorphine) or Cholecystokinin octapeptide inhibited food intake and these effects were reversed by pretreatment with a dopamine receptor antagonist (cis-flupentixol). Blockade of cholecystokinin-A receptors, by treatment with L-364,718, but not cholecystokinin-B receptors, by treatment with L-365,260, blocked the inhibitory effect of Cholecystokinin octapeptide on food intake but did not affect the inhibitory effect of apomorphine. It is suggested that Cholecystokinin interacts with dopamine in the control of food intake.

Involvement of Cholecystokinin in Food Intake: III. Oestradiol Potentiates the Inhibitory Effect of Cholecystokinin Octapeptide on Food Intake in Ovariectomized Rats.

Abstract The role of Cholecystokinin in a model of hypophagia, oestradiol-treated Ovariectomized rats, was investigated. Implantation of oestradiol-filled constant-release implants in rats made obese by ovariectomy potentiated the inhibitory effect of intraperitoneal injection of Cholecystokinin octapeptide on food intake after 24 h of food deprivation. The alterations in the concentration of Cholecystokinin in pjasma and of cholecystokinin-like immunoreactivity in cerebrospinal fluid produced by deprivation of food for 24 h and subsequent food intake for 1 h were unaffected by the oestradiol treatment as was the amount of food consumed during 1 h. Oestradiol-treated rats deprived of food for 6 h, however, consumed less food during a 15-min test than controls. Treatment with oestradiol blunted the decrease in the concentration of cholecystokinin-like immunoreactivity in the cerebrospinal fluid in response to 6 h of food deprivation. No alterations in the concentration of Cholecystokinin in plasma occurred after this period of food deprivation and subsequent feeding during 15 min in either oestradiol-treated or control rats. Thus, treatment with oestradiol enhances responsivity to exogenous Cholecystokinin octapeptide and changes the response of endogenous levels of cholecystokinin-like immunoreactivity in the cerebrospinal fluid to a short period of food deprivation. It is suggested that these effects are caused by an action of oestradiol on Cholecystokinin pathways in the brain. The results support the suggestion that hunger in the rat is inversely related to the decrease in the concentration of cholecystokinin-like immunoreactivity in the cerebrospinal fluid.

Involvement of Cholecystokinin in Food Intake: II. Lactational Hyperphagia in the Rat.

Abstract The role of Cholecystokinin in the hyperphagia of lactation was studied by measuring the concentration of this hormone in plasma and cerebrospinal fluid in relation to food intake in lactating rats. Cholecystokinin was measured by high-performance liquid chromatography and radioimmunoassay in plasma and by radioimmunoassay in cerebrospinal fluid. Plasma concentrations of Cholecystokinin were increased in freely-fed lactating rats compared with non-lactating, regularly cycling rats. However, after 24 h of food deprivation the concentration of plasma Cholecystokinin was markedly decreased in the lactating rats to levels which were lower than those of non-lactating animals. Furthermore, plasma levels of Cholecystokinin did not increase in response to 1 h of feeding in lactating rats, whereas in non-lactating rats they did. In contrast, the concentration of cholecystokinin-like immunoreactivity in the cerebrospinal fluid was the same in freely-fed lactating and non-lactating rats. As in plasma, food deprivation markedly decreased the levels of cholecystokinin-like immunoreactivity in the cerebrospinal fluid of lactating rats but unlike in plasma, the levels were restored by feeding. The levels of cholecystokinin-like immunoreactivity were not changed under these conditions in the non-lactating rats. These results show that there is no correlation between the concentration of Cholecystokinin in plasma and cerebrospinal fluid, which supports the suggestion that the cholecystokinin-like immunoreactivity in the cerebrospinal fluid is derived from the brain. Removal of the litter from lactating rats deprived of food for 24 h reduced food intake and increased the concentration of cholecystokinin-like immunoreactivity in the cerebrospinal fluid, but not in plasma. The inhibition of food intake caused by an intraperitoneal injection of Cholecystokinin octapeptide increased after litter removal. It is suggested that hunger in the lactating rat is reflected by a decrease in the levels of cholecystokinin-like immunoreactivity in the cerebrospinal fluid and satiety by the restoration of these levels.

Increased cerebrospinal fluid concentration of nitrite in Parkinson's disease.

The concentration of nitrite, a metabolite of nitric oxide (NO), was increased in the cerebrospinal fluid (CSF) of untreated patients with Parkinson's disease and in patients treated with L-DOPA in comparison with a group of patients without dopaminergic dysfunction. There was no difference in the concentration of L-arginine (ARG), a precursor of NO, between the groups. There was a highly significant, linear relationship between the concentration of nitrite and ARG in the CSF suggesting that the production of NO is dependent on the availability of ARG. The results support the possibility that production of NO is increased in the brain in Parkinson's disease.

Inhibition of sexual behavior in female rats by intracerebral injections of Met-enkephalin in combination with an inhibitor of enkephalin degrading enzymes.

Injection of Met-enkephalin, either subcutaneously (10 or 100 microgram, s.c.), intracerebroventricularly (100 ng or 1 microgram, i.c.v.) or into the mesencephalic central gray (100 ng), had no inhibitory effect on sexual behavior in female rats. If combined with [(R)-3-(N-hydroxy)-carboxamido-2-benzylpropanoyl]-L-alanine (kelatorphan, 5 micrograms i.c.v.; 1.5 micrograms in the central gray; 200 micrograms s.c.), an inhibitor of enkephalin degrading enzymes, however, i.c.v. or central gray (100 ng), but not s.c. (100 micrograms), injection of Met-enkephalin suppressed the behavior. Injection of Leu-enkephalin, s.c. or i.c.v. alone or in combination with kelatorphan, had no inhibitory effect. Peptidases may rapidly inactivate Met-enkephalin after intracerebral injection and prevent the behavioral effect. Inhibition of sexual behavior by Met-enkephalin may occur during lactation, a physiological state when the behavior is suppressed by an opioid peptide.

The effect of naloxone on sexual behavior in female rats depends on the site of injection.

Intraperitoneal injections of the opiate receptor antagonist naloxone (0.1, 1, 5 or 10 mg) had no effects on sexual behavior in ovariectomized female rats made either partially or fully receptive by injections of ovarian hormones. Intracerebroventricular (i.c.v.) injections of naloxone (10, 50 or 100 micrograms) had no effects on the behavior of partially receptive rats and in fully receptive rats an i.c.v. injection of a high dose of naloxone (100 micrograms) inhibited the behavior. Intrathecal (i.t.) injections of naloxone (50 or 100 micrograms) facilitated sexual behavior in partially receptive rats while i.t. injections of even very high doses of naloxone (500 micrograms) had no behavioral effects in fully receptive rats. Thus, the effects of naloxone on female sexual behavior depend on the site of injection.

beta-Endorphin acts on the reproductive tract of female rats to suppress sexual receptivity.

Sexual receptivity was suppressed in female rats immediately after the male had ejaculated. The inhibition was prevented by intravaginal injection of naloxone (100 micrograms) before testing and intravaginal injection of beta-endorphin (1.0 micrograms) inhibited sexual behavior in female rats in a manner comparable to that of ejaculation by the male. beta-Endorphin was present in ejaculatory plugs collected from the vagina of female rats (8.2 +/- 0.6 pM) and in seminal fluid collected from male rats (7.7 +/- 0.2 pM). The results suggest that beta-endorphin in the ejaculate of the male rat can act on the reproductive tract of the female rat to suppress her sexual behavior.