Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4.

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.

Central nervous system control of food intake and body weight.

The capacity to adjust food intake in response to changing energy requirements is essential for survival. Recent progress has provided an insight into the molecular, cellular and behavioural mechanisms that link changes of body fat stores to adaptive adjustments of feeding behaviour. The physiological importance of this homeostatic control system is highlighted by the severe obesity that results from dysfunction of any of several of its key components. This new information provides a biological context within which to consider the global obesity epidemic and identifies numerous potential avenues for therapeutic intervention and future research.

Endocrine mechanisms mediating remission of diabetes after gastric bypass surgery.

Bariatric surgery is currently the most effective method to promote major, sustained weight loss. Roux-en-Y gastric bypass (RYGB), the most commonly performed bariatric operation, ameliorates virtually all obesity-related comorbid conditions, the most impressive being a dramatic resolution of type 2 diabetes mellitus (T2DM). After RYGB, 84% of patients with T2DM experience complete remission of this disease, and virtually all have improved glycemic control. Increasing evidence indicates that the impact of RYGB on T2DM cannot be explained by the effects of weight loss and reduced energy intake alone. Weight-independent antidiabetic actions of RYGB are apparent because of the very rapid resolution of T2DM (before weight loss occurs), the greater improvement of glucose homeostasis after RYGB than after an equivalent weight loss from other means, and the occasional development of very late-onset, pancreatic beta-cell hyperfunction. Several mechanisms probably mediate the direct antidiabetic impact of RYGB, including enhanced nutrient stimulation of L-cell peptides (for example, GLP-1) from the lower intestine, intriguing but still uncharacterized phenomena related to exclusion of the upper intestine from contact with ingested nutrients, compromised ghrelin secretion, and very probably other effects that have yet to be discovered. Research designed to prioritize these mechanisms and identify potential additional mechanisms promises to help optimize surgical design and might also reveal novel pharmaceutical targets for diabetes treatment.

Single-copy inverted repeats associated with regional genetic duplications in gamma fibrinogen and immunoglobulin genes.

We have found that a portion (150 base pairs) of the seventh exon of the human gamma fibrinogen gene is duplicated in the preceding intron. This duplicated sequence, termed a "pseudoexon," is flanked on each side by a single-copy inverted repeat sequence consisting of 102 base pairs. Frequencies of point substitutions indicate that both the pseudoexon and the inverted repeat sequence arose approximately 10 to 20 million years ago. The generality of this type of duplication is suggested by the occurrence of a similar duplication in the mouse immunoglobulin mu-delta region. As in the fibrinogen pseudoexon, the portion of the immunoglobulin mu-delta region containing the duplication and the inverted repeat was reported to be single-copy in the mouse genome. Since both of the first two single-copy inverted repeats to be sequenced are associated with regional duplications, it is likely that many of the single-copy inverted repeat sequences, which make up 1 to 2 percent of the genome, are also associated with regional duplications.

Overfeeding-induced weight gain suppresses plasma ghrelin levels in rats.

The elevation of plasma ghrelin associated with weight loss has been taken as evidence of a role for ghrelin in the adaptive response to body weight change. However, there has been no clear experimental evidence that circulating ghrelin is suppressed by weight gain. We investigate this issue using a model of involuntary (intra-gastric gavage) overfeeding-induced obesity. Rats were first maintained at normal body weight with 4 daily tube-feedings of liquid diet (2.11 kcal/ml), each delivered at a volume of 9 ml. Gavage volume was then increased to 13 ml/feeding for 2 weeks, during which rats gained 25% of their initial body weight. Fasting plasma ghrelin levels and the response to 9- and 13-ml intra-gastric load sizes were measured during the weight-stable and overfed conditions. We found that: 1) weight gain decreased circulating ghrelin levels; 2) this response could not be attributed to additional food in the gastrointestinal tract; 3) the ghrelin response to nutrient loads was diminished in the obese vs normal-weight conditions. Having discounted diet composition and differences in gastric contents at the time of blood sampling, the decrease in ghrelin levels with overfeeding can be unambiguously attributed to physiological correlates of weight gain.

Hormonal and metabolic defects in a prader-willi syndrome mouse model with neonatal failure to thrive.

Prader-Willi syndrome (PWS) has a biphasic clinical phenotype with failure to thrive in the neonatal period followed by hyperphagia and severe obesity commencing in childhood among other endocrinological and neurobehavioral abnormalities. The syndrome results from loss of function of several clustered, paternally expressed genes in chromosome 15q11-q13. PWS is assumed to result from a hypothalamic defect, but the pathophysiological basis of the disorder is unknown. We hypothesize that a fetal developmental abnormality in PWS leads to the neonatal phenotype, whereas the adult phenotype results from a failure in compensatory mechanisms. To address this hypothesis and better characterize the neonatal failure to thrive phenotype during postnatal life, we studied a transgenic deletion PWS (TgPWS) mouse model that shares similarities with the first stage of the human syndrome. TgPWS mice have fetal and neonatal growth retardation associated with profoundly reduced insulin and glucagon levels. Consistent with growth retardation, TgPWS mice have deregulated liver expression of IGF system components, as revealed by quantitative gene expression studies. Lethality in TgPWS mice appears to result from severe hypoglycemia after postnatal d 2 after depletion of liver glycogen stores. Consistent with hypoglycemia, TgPWS mice appear to have increased fat oxidation. Ghrelin levels increase in TgPWS reciprocally with the falling glucose levels, suggesting that the rise in ghrelin reported in PWS patients may be secondary to a perceived energy deficiency. Together, the data reveal defects in endocrine pancreatic function as well as glucose and hepatic energy metabolism that may underlie the neonatal phenotype of PWS.

Mutation of the RIIbeta subunit of protein kinase A prevents diet-induced insulin resistance and dyslipidemia in mice.

The mechanisms by which obesity contributes to diabetic phenotypes remain unclear. We evaluated the role of protein kinase A (PKA) signaling events in mediating diabetes associated with obesity. PKA comprises two regulatory subunits and two catalytic subunits and is activated by cAMP. The RIIbeta regulatory subunit is abundantly expressed in adipose tissue and brain. Knockout mice lacking this subunit are lean and display remarkable resistance to diet-induced obesity. We investigated whether these mice were also resistant to diet-induced diabetes and whether this effect was dependent on reduced adiposity. Mice were fed a high-fat, high-carbohydrate diet and weight gain and diabetes phenotypes were examined. RIIbeta(-/-) mice displayed decreased body weights, reduced insulin levels, improved insulin sensitivity, and improved total-body glucose disposal as compared with wild-type controls. Plasma levels of VLDL and LDL cholesterol were also reduced in high fat-fed RIIbeta(-/-) mice compared with wild-type mice. Taken together, these data demonstrate that loss of RIIbeta protects mice from diet-induced obesity, insulin resistance, and dyslipidemia.

A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans.

The recently discovered orexigenic peptide ghrelin is produced primarily by the stomach and circulates in blood at levels that increase during prolonged fasting in rats. When administered to rodents at supraphysiological doses, ghrelin activates hypothalamic neuropeptide Y/agouti gene-related protein neurons and increases food intake and body weight. These findings suggest that ghrelin may participate in meal initiation. As a first step to investigate this hypothesis, we sought to determine whether circulating ghrelin levels are elevated before the consumption of individual meals in humans. Ghrelin, insulin, and leptin were measured by radioimmunoassay in plasma samples drawn 38 times throughout a 24-h period in 10 healthy subjects provided meals on a fixed schedule. Plasma ghrelin levels increased nearly twofold immediately before each meal and fell to trough levels within 1 h after eating, a pattern reciprocal to that of insulin. Intermeal ghrelin levels displayed a diurnal rhythm that was exactly in phase with that of leptin, with both hormones rising throughout the day to a zenith at 0100, then falling overnight to a nadir at 0900. Ghrelin levels sampled during the troughs before and after breakfast correlated strongly with 24-h integrated area under the curve values (r = 0.873 and 0.954, respectively), suggesting that these convenient, single measurements might serve as surrogates for 24-h profiles to estimate overall ghrelin levels. Circulating ghrelin also correlated positively with age (r = 0.701). The clear preprandial rise and postprandial fall in plasma ghrelin levels support the hypothesis that ghrelin plays a physiological role in meal initiation in humans.

Elevated free fatty acids induce uncoupling protein 3 expression in muscle: a potential explanation for the effect of fasting.

The newly described uncoupling protein 3 (UCP3) may make an important contribution to thermogenesis in humans because of its high level of expression in skeletal muscle. Contrary to expectations, fasting, a condition that reduces resting energy expenditure, has been reported to increase UCP3 expression in muscle. We have confirmed that a 10-fold increase in UCP3 mRNA levels occurs in rat quadriceps muscle between 12 and 24 h of food removal. A less consistent twofold increase in muscle UCP2 mRNA levels was observed in animals fasted for up to 72 h. Administration of recombinant leptin to prevent a fall in circulating leptin levels did not eliminate the fasting-induced increase in quadriceps UCP3 expression. Administration of a high dose of glucocorticoid to fed animals to mimic the increase in corticosterone induced by fasting did not reproduce the increase in UCP3 expression observed in fasted animals. In contrast, elevation of circulating free fatty acid levels in fed animals by Intralipid plus heparin infusion caused significant increases in the UCP3/actin mRNA ratio compared with saline-infused fed controls in both extensor digitorum longus (2.01 +/- 0.34 vs. 0.68 +/- 0.11, P = 0.002) and soleus muscles (0.31 +/- 0.07 vs. 0.09 +/- 0.02, P = 0.014). We conclude that free fatty acids are a potential mediator of the increase in muscle UCP3 expression that occurs during fasting. This seemingly paradoxical induction of UCP3 may be linked to the use of free fatty acid as a fuel rather than an increased need of the organism to dissipate energy.

Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Reversal of cancer anorexia by blockade of central melanocortin receptors in rats.

Anorexia is a debilitating manifestation of many malignancies. The etiology of cancer anorexia is poorly understood, and effective treatment options are limited. To investigate the role of central melanocortin receptor signaling in the pathogenesis of cancer anorexia, we assessed the effects on food intake of the melanocortin receptor antagonist SHU9119 administered into the third cerebral ventricle of Lobund-Wistar rats that were anorexic from prostate cancer. In anorexic tumor-bearing rats, daily treatment with SHU9119 (0.35 nmol, intracerebroventricularly) increased food intake from 71 +/- 3% to 110 +/- 6% of preanorectic baseline and caused significant weight gain (13 +/- 5 vs. 5 +/- 1 g/3 d, SHU9119 vs. baseline in tumor-bearing rats). In control rats pair-fed to the intake of tumor-bearing animals, SHU9119 was ineffective at increasing food intake. The specificity of the SHU9119 feeding response was assessed using two other orexigenic peptides, NPY and the novel hormone ghrelin. Treatment of tumor-bearing rats with intracerebroventricular ghrelin (10 microg) increased food intake, but the effect was blunted relative to that in controls. Intracerebroventricular injections of NPY (1 microg) also failed to reverse anorexia in tumor-bearing rats. Because SHU9119 completely reverses cancer anorexia in this model, whereas ghrelin and NPY do not, increased central nervous system melanocortin signaling is implicated in the pathogenesis of this disorder. This suggests that new targets for the treatment of cancer anorexia may be found in the melanocortin pathways.

Prostate-sparing effects in primates of the potent androgen 7alpha-methyl-19-nortestosterone: a potential alternative to testosterone for androgen replacement and male contraception.

7alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that cannot be converted to dihydrotestosterone. In this study we determined the relative androgenic, antigonadotropic, and anabolic potencies of testosterone vs. MENT in the nonhuman primate M. fascicularis. In castrated monkeys, dose-response relationships were generated for the effects of testosterone and MENT on gonadotropin levels, prostate growth, body weight, and lipid metabolism. In a pilot study, four monkeys were castrated, and magnetic resonance imaging (MRI) was used to document a 50% loss of prostate volume within 8 weeks, verifying that MRI is a reliable means to measure prostate size in this species. Two additional groups of six monkeys each were then castrated and serially administered four graded dosages of testosterone or MENT via osmotic minipumps over 20 weeks. Complete suppression of LH was achieved with a minimum of 0.3 mg/day MENT, compared to 3.0 mg/day testosterone. MENT supported body weight 10 times more potently than did testosterone. Baseline prostate volumes were maintained with 0.1-0.2 mg/day MENT vs. 0.3 mg/day testosterone. Thus, in monkeys, MENT is 10 times more potent than testosterone with regard to the clinically desirable end points of gonadotropin suppression and anabolism, but only twice as potent at stimulating prostate growth. These results suggest that MENT may have a wider therapeutic index than testosterone for human androgen replacement and male contraception.

Prospects for new hormonal male contraceptives.

Overpopulation is a paramount global crisis, as it underlies virtually all major worldwide problems, including poverty, malnutrition, and warfare. It is imperative that proliferation be curbed by expediting reductions in fertility. This article discusses the demand for and acceptability of male contraceptives and the process of spermatogenesis. Endocrine control of spermatogenesis, and potential sites for disruption of male fertility, including steroid combinations and GnRH agonists and antagonists are reviewed also. Risks and benefits of these approaches are considered.

PIP: Despite a social trend in support of greater male involvement in fertility regulation, currently available male-oriented methods (e.g., condoms, coitus interruptus, and vasectomy) are flawed and lack widespread acceptability. Most experimental methods target the development, maturation, or ultimate function of sperm cells. This article focuses on efforts to develop an improved hormonal contraceptive for men that inhibits gonadotropin-releasing hormone (GnRH), gonadotropins, or both, using either exogenous steroids or GnRH analogs. Although GnRH antagonists suppress gonadotropins more rapidly and completely than any known agents and their pituitary effects are immediately reversible, these agents are currently unmarketable due to their high cost, insufficient potency, and impractical delivery systems. To ensure fertility, both follicle-stimulating hormone and intratesticular testosterone must be eliminated. Thus, any technique that suppresses gonadotropins must include testosterone replacement. The effects of prolonged testosterone administration on hypertrophy have not been determined. No candidate for reversible systemic male contraception is likely to achieve general marketability in the immediate future due to a combination of factors, including fundamental knowledge voids about the molecular control of spermatogenesis and a lack of financial support from pharmaceutical companies for research in this area.

Reduction of bacteria-containing spray produced during ultrasonic scaling.

Bacteria-containing spray (including both aerosols and splatter) has been shown to be a potential source of contagion in the dental environment. Bacterial air contamination increases during dental treatment; this is especially true during ultrasonic scaling procedures. This in vivo investigation evaluated the amount of bacteria-containing spray produced during ultrasonic scaling of 15 patients when using a suction-type aerosol reduction device (ARD) and/or a preoperative 0.12% chlorhexidine gluconate (CHX) antimicrobial rinse. When the study protocol was followed, the use of either an ARD or a CHX rinse produced significant bacterial reductions during ultrasonic scaling compared to the control. The use of an ARD produced the greatest bacterial reductions. Combining the ARD and the CHX rinse was no more effective than the use of an ARD alone.

Normal feeding and body weight in Fischer 344 rats lacking the cholecystokinin-1 receptor gene.

A large body of evidence has demonstrated that one mechanism by which cholecystokinin (CCK) inhibits food intake through activation of CCK1 receptors (CCK1R) on vagal afferent neurons that innervate the gastrointestinal tract and project to the hindbrain. OLETF rats, which carry a spontaneous null mutation of the CCK1R, are hyperphagic, obese, and predisposed to type 2 diabetes. Recently, by introgressing the OLETF-derived, CCK1R-null gene onto a Fischer 344 genetic background, we have been able to generate a CCK1R-deficient, congenic rat strain, F344.Cck1r(-/-), that in contrast to OLETF rats, possesses a lean and normoglycemic phenotype. In the present study, the behavioral and neurobiological phenotype of this rat strain was characterized more fully. As expected, intraperitoneal injections of CCK-8 inhibited intake of chow and Ensure Plus and induced Fos responses in the area postrema and the gelatinosus, commissural and medial subdivisions of the nucleus tractus solitarius of wild-type F344.Cck1r(+/+) rats, whereas CCK-8 was without effect on food intake or Fos induction in the F344.Cck1r(-/-) rats. F344.Cck1r(-/-) and F344.Cck1r(+/+) rats did not differ in body weight and showed comparable weight gain when maintained on Ensure Plus for 2 weeks. Also, no difference was found in 24-h food intake, and dark-phase meal frequency or meal size between F344.Cck1r(+/+) and F344.Cck1r(-/-) rats. As expected, blockade of endogenous CCK action at CCK1R increased food intake and blocked the effects of peripheral CCK-8 in wild-type F344.Cck1r(+/+) rats. These results confirm that in rats with a F344 background, CCK-1R mediates CCK-8-induced inhibition of food intake and Fos activation in the hindbrain and demonstrate that selective genetic ablation of CCK1R is not associated with altered meal patterns, hyperphagia, or excessive weight gain on a palatable diet.

Male contraception: ideas for the future.

PIP: Numerous global surveys have confirmed that men are willing to assume a more central role in family planning, especially if a wider range of contraceptive methods were available. At present, hormonal approaches to systemic male contraception represent the only hope for a marketable new technology within the next decade. Steroid regimens are reversible, inexpensive, free from major side effects, and can be delivered in convenient sustained-release formulations lasting several months. Gonadotropin-releasing hormone analogues are also promising. This article highlights recent advances in this important new area of research. All hormonal strategies require supplementation with androgens, whose long-term impact on lipid and prostate physiology needs further clarification. Unfortunately, research in the field of male contraception has been virtually abandoned by the pharmaceutical industry, which seems to underestimate men's interest in playing a more equitable role in family planning.^ieng

Age-related changes in growth hormone secretion: should the somatopause be treated?

Growth hormone (GH) secretion declines progressively with aging, and many age-related changes resemble those of the adult GH deficiency (GHD) syndrome, including a decrease in lean body mass; an increase in body fat, especially in the visceral/abdominal compartment; adverse changes in lipoproteins; and a reduction in aerobic capacity. The increase in central obesity can further inhibit GH secretion. GH replacement is effective in reversing many of these changes in adult GHD, and GH is now FDA approved for treatment of adults with documented GHD or hypopituitarism, although there is still only limited experience with its long-term benefits, side effects, and risks. This early experience with GHD has led to speculation that replacing GH or stimulating its secretion may also be beneficial in normal aging, and to widespread off-label use of GH in this context; however, there are still very few well controlled studies of the effects and side effects of GH or GH secretagogues in aging. All published studies are of 6 months or shorter treatment periods. From this limited experience there is a consensus that GH has effects on body composition, but reports disagree on effects on psychological or physical functional performance. Older adults are much more susceptible to the dose-related side effects of GH, including peripheral edema, carpal tunnel syndrome, and a variable decrease in insulin sensitivity; and it is not known whether chronic GH treatment affects the risk of malignancy or has other long-term risks. Thus while short-term results are somewhat encouraging, the evidence on risks and clinically pertinent benefits is still lacking to support the use of GH in normal aging outside of clinical studies. In evaluating patients with clinical features suggesting GHD, which can be quite nonspecific, it is important to assess the presence or absence of true GH deficiency by the context (pituitary disease or its treatment, childhood GHD) and by appropriate GH stimulation tests before considering GH replacement.

Phenotypic and functional properties of murine thymocytes. III. Kinetic analysis of the recovery of intrathymic cytolytic T lymphocyte precursors after in vivo administration of hydrocortisone acetate.

The phenotypic and functional properties of cells in the C57BL/6 mouse thymus regenerating after a single dose of 100 mg/kg hydrocortisone acetate (H/C) are described. Functionally, the frequency of anti-H-2d cytolytic T lymphocyte precursors (CTL-P) in thymuses from individual mice was determined by limit dilution analysis of mixed leukocyte microcultures. The initial increase in CTL-P frequency, seen 48 hr post-H/C, was followed at 6 to 8 days by a phase of rapid decrease. The CTL-P frequency returned to a normal level by 28 days post-H/C. Analysis of the results from individual mice suggested that changes in total thymic CTL-P content were independent of the kinetics of thymus regeneration. Phenotypically, whereas the thymus 48 hr after H/C was considerably depleted of Lyt-2+ cells, there followed a rapid increase in the proportion of such cells to normal levels by 14 days post-H/C. In addition, as measured by FLS, a subpopulation of larger, predominantly Lyt-2+ cells was found during the phase of rapid thymic regeneration. With the use of a monoclonal anti-Thy-1.2 antibody, the weakly Thy-1-staining subpopulation of cells was absent from the thymus at 14 days post-H/C. These changes in the phenotypic properties of the post-H/C regenerating thymus were correlated with changes in their functional properties.

Cloning of a mouse protein kinase A catalytic subunit pseudogene and chromosomal mapping of C subunit isoforms.

Two isoforms of the protein kinase A catalytic subunit, C alpha and C beta, have previously been described in the mouse. We now report the cloning and characterization of a novel C-related sequence, Cx, from a murine genomic library. Cx is 89.8% identical to part of the C alpha coding region, but lacks all of the introns present in this gene, suggesting that it arose via retroposition. The existence of several frameshift mutations, premature termination codons, and missense mutations at critical sites confirms that it is a pseudogene. Furthermore, we are unable to detect any expression. Homology with functional protein kinase genes commences exactly at the first intron splice junction in C alpha, downstream of the expected translational start codon. Cx is also truncated at its 3' end by the interposition of two distinct, contiguous LINE-1 elements. By fluorescence in situ hybridization, we demonstrate that Cx is located on the X Chromosome (Chr), at band F3. This is displaced from its functional homologs, C alpha and C beta, which we map to mouse Chrs 8 (band C3) and 3 (band H3), respectively.