Identification of the Alzheimer's disease amyloid precursor protein (APP) and its homologue APLP2 as essential modulators of glucose and insulin homeostasis and growth.

The amyloid precursor protein (APP), the source of the neurotoxic amyloid beta (A beta) peptide involved in Alzheimer's disease (AD), belongs to a conserved family of related proteins. In mammals, the APP family contains amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2). Whilst a number of activities have been attributed to the APP family, an overall function has not been definitively established. While ablating either the APP or APLP2 gene in mice produces minimal phenotypic change, the combined knockout of these genes in mice causes postnatal mortality. Postnatal survival therefore requires a shared but unknown function of APP and APLP2. To investigate the biochemical basis for the postnatal lethality, plasma was analysed from double knockout mice (APP-/- APLP2-/-) 2 days before birth, at gestational day E17, and from mice at 12-16 h after birth. The postnatal double knockouts had 66% lower plasma glucose levels than their wild-type controls and 50% lower than their single knockout counterparts. Interestingly, the postnatal double knockouts displayed hyperinsulinaemia, as shown by inappropriate plasma insulin levels, given their degree of hypoglycaemia. The single knockout mice also showed hyperinsulinaemia and had 31% lower plasma glucose than the wild-types. While the double knockouts did not survive more than 24 h after birth, the single knockouts reached adulthood and their hypoglycaemia continued. Therefore, APP and APLP2 expression modulates plasma insulin and glucose concentrations. Plasma calcium, magnesium and phosphate were also significantly reduced in the double knockouts compared to the wild-types, and they showed distinctive growth restriction, suggesting the involvement of a metabolic impairment. These results link the expression of the APP and APLP2 genes with glucose homeostasis and growth and therefore identify a novel function for the APP family.

Modulation of central leptin sensitivity and energy balance in a rat model of diet-induced obesity.

AIM: The aim of this study was to further explore the time-dependent changes in leptin sensitivity using a rat model of dietary fat-induced obesity and to investigate the potential mechanisms governing these changes. METHODS: We used male, adult Sprague-Dawley rats that were fed either a standard laboratory chow diet (3% fat) or a high-saturated fat (HF) diet (60% fat) for 2 or 5 weeks. Energy balance (body weight, energy intake and energy expenditure); sensitivity to central leptin and central alpha-melanin stimulating hormone (alpha-MSH) administration and expression levels of hypothalamic ObRb, signal transducers and activators of transcription factor (STAT)-3 phosphorylation, suppressor of cytokine signalling-3 (SOCS-3), proopiomelanocortin (POMC) processing hormones (prohormone convertase-1 and prohormone convertase-2) and neuropeptide Y (NPY) were measured. RESULTS: After 2 weeks of feeding HF diet, there was an increase in total energy intake (TEI) but a reduction in food intake as measured by the mass of food ingested. Body weight at this time was not significantly different between the two diet groups; however, white adipose tissue (WAT) weight was significantly greater in the HF-fed rats than in the chow-fed rats. In addition, spontaneous physical activity levels were increased, but no changes were observed in resting energy expenditure. Furthermore, chow-fed lean rats responded to central leptin administration by reducing the energy intake by approximately 67 kJ compared with saline treatment (p < 0.05), while the HF-fed diet-induced obese (DIO) rats responded by reducing their energy intake by approximately 197 kJ compared with saline treatment (p < 0.05). After 5 weeks of feeding HF diet, TEI remained significantly higher, body weight was significantly increased by 5% in the HF-fed rats and WAT weight was significantly heavier in HF-fed rats than in the chow-fed lean rats. After leptin treatment, the chow-fed lean rats reduced their energy intake by approximately 97 kJ (p < 0.05); yet, leptin had no significant effect in the HF-fed DIO rats. ObRb protein expression, STAT-3 phosphorylation levels, content and messenger RNA (mRNA) expression of NPY, SOCS-3 mRNA and protein expression and energy intake response to central alpha-MSH administration were not altered after HF diet feeding. CONCLUSION: These results suggest that early in the course of HF diet-induced weight gain, there was a period of central leptin hypersensitivity, and as the obesity progresses, central leptin insensitivity develops. This insensitivity does not appear to be explained by a downregulation of ObRb protein levels, reduced leptin signalling, an increase in either SOCS-3 or NPY expression or reduced function of the melanocortin system. The effect of an HF diet on other actions of leptin such as its effect on the endocannabinoid system should be investigated.

Increased nicotinamide nucleotide transhydrogenase levels predispose to insulin hypersecretion in a mouse strain susceptible to diabetes.

AIMS/HYPOTHESIS: Insulin hypersecretion may be an independent predictor of progression to type 2 diabetes. Identifying genes affecting insulin hypersecretion are important in understanding disease progression. We have previously shown that diabetes-susceptible DBA/2 mice congenitally display high insulin secretion. We studied this model to map and identify the gene(s) responsible for this trait. METHODS: Intravenous glucose tolerance tests followed by a genome-wide scan were performed on 171 (C57BL/6 x DBA/2) x C57BL/6 backcross mice. RESULTS: A quantitative trait locus, designated hyperinsulin production-1 (Hip1), was mapped with a logarithm of odds score of 7.7 to a region on chromosome 13. Production of congenic mice confirmed that Hip1 influenced the insulin hypersecretion trait. By studying appropriate recombinant inbred mouse strains, the Hip1 locus was further localised to a 2 Mb interval, which contained only nine genes. Expression analysis showed that the only gene differentially expressed in islets isolated from the parental strains was Nnt, which encodes the mitochondrial proton pump, nicotinamide nucleotide transhydrogenase (NNT). We also found in five mouse strains a positive correlation (r2 = 0.90, p < 0.01) between NNT activity and first-phase insulin secretion, emphasising the importance of this enzyme in beta cell function. Furthermore, of these five strains, only those with high NNT activity are known to exhibit severe diabetes after becoming obese. CONCLUSIONS/INTERPRETATION: Insulin hypersecretion is associated with increased Nnt expression. We suggest that NNT must play an important role in beta cell function and that its effect on the high insulin secretory capacity of the DBA/2 mouse may predispose beta cells of these mice to failure.

Increase of fat oxidation and weight loss in obese mice caused by chronic treatment with human growth hormone or a modified C-terminal fragment.

OBJECTIVE: To observe the chronic effects of human growth hormone (hGH) and AOD9604 (a C-terminal fragment of hGH) on body weight, energy balance, and substrate oxidation rates in obese (ob/ob) and lean C57BL/6Jmice. In vitro assays were used to confirm whether the effects of AOD9604 are mediated through the hGH receptor, and if this peptide is capable of cell proliferation via the hGH receptor. METHOD: Obese and lean mice were treated with hGH, AOD or saline for 14 days using mini-osmotic pumps. Body weight, caloric intake, resting energy expenditure, fat oxidation, glucose oxidation, and plasma glucose, insulin and glycerol were measured before and after treatment. BaF-BO3 cells transfected with the hGH receptor were used to measure in vitro 125I-hGH receptor binding and cell proliferation. RESULTS: Both hGH and AOD significantly reduced body weight gain in obese mice. This was associated with increased in vivo fat oxidation and increased plasma glycerol levels (an index of lipolysis). Unlike hGH, however, AOD9604 did not induce hyperglycaemia or reduce insulin secretion. AOD9604 does not compete for the hGH receptor and nor does it induce cell proliferation, unlike hGH. CONCLUSIONS: Both hGH and its C-terminal fragment reduce body weight gain, increase fat oxidation, and stimulate lipolysis in obese mice, yet AOD9604 does not interact with the hGH receptor. Thus, the concept of hGH behaving as a pro-hormone is further confirmed. This data shows that fragments of hGH can act in a manner novel to traditional hGH-stimulated pathways.

Novel anti-obesity drugs.

There is increasing evidence that body weight is homeostatically regulated and that in obesity this regulation maintains weight at a high level. Weight loss activates mechanisms that are designed to return individuals to their pre-existing weight. This explains the universally poor results of current strategies to maintain weight loss. On this basis, life-long drug therapy may be justified for those with significant obesity. Currently available drugs include selective serotonin re-uptake inhibitors (e.g., fluoxetine), noradrenergic re-uptake inhibitors (e.g., phentermine), a serotonin and noradrenergic re-uptake inhibitor (sibutramine) and an intestinal lipase inhibitor (orlistat). An active research program is underway to develop new agents based on the rapidly expanding knowledge of the complex mechanisms regulating body weight. Leptin, a hormone produced by adipocytes that inhibits food intake, has undergone clinical trials and analogues are currently being developed. Other agents include amylin, melanocortin-4 receptor agonists, neuropeptide Y antagonists, beta(3) adrenergic agonists and glucagon-like peptide-1 agonists. As some redundancy exists in the central regulatory system controlling body weight, some agents might need to be used in combination to be effective.

Short-term, high-fat diets lower circulating leptin concentrations in rats.

BACKGROUND: Leptin is produced in proportion to body fat mass and can act on the brain to induce satiety and regulate adipose tissue mass; factors other than adipose tissue mass may influence circulating leptin concentrations. OBJECTIVE: We explored the possibility that short-term, moderately high-fat diets induce weight gain by producing inappropriately low circulating leptin concentrations. DESIGN: Female Hooded Wistar rats were fed either a moderately high-fat diet or control diet. Body weight, energy intake, body composition, and fasting plasma leptin were compared after 4 and 14 wk of dietary treatment. RESULTS: After 4 wk, abdominal fat mass was 38% greater in rats fed the high-fat diet than in those fed the control diet (P < 0.01). However, plasma leptin concentrations were 24% lower in animals fed the high-fat diet (P < 0.05), resulting in significantly lower plasma leptin concentrations per unit abdominal fat mass than in control animals (P < 0.005). From 4 to 14 wk, animals fed the high-fat diet gained twice as much weight and consumed 32 kJ/d more than controls (both P < 0.05). At 14 wk, plasma leptin concentrations per unit abdominal fat mass were 27% lower in rats fed the high-fat diet (P = 0.058) and there was a significant negative association between leptin concentrations per unit abdominal fat mass and body weight (r = 0.44, P < 0.05). CONCLUSIONS: In the short term, a moderately high-fat diet is associated with lower than expected circulating leptin concentrations, which correlate with a higher body weight. A high-fat diet may therefore contribute to weight gain by reducing leptin secretion in adipose tissue.

Leptin in the pathophysiology of human obesity and the clinical potential of leptin-based therapy.

Leptin is a circulating hormone that is secreted in proportion to fat mass. It can reduce bodyweight by activating signalling molecules in the brain. Leptin appears to affect bodyweight primarily by decreasing food intake; there is no direct evidence that it significantly influences energy expenditure in humans. Its discovery in 1994 raised the possibility that it may be a useful, satiety-inducing, anti-obesity drug. However, treating obese patients with leptin alone does not induce substantial bodyweight loss because most obese patients are insensitive to leptin and are not leptin deficient. In combination with diet therapy, however, leptin treatment has the potential to eliminate the dramatic fall in circulating leptin levels (and the subsequent increase in hunger) caused by calorie restriction. Used in this manner, leptin may play a very useful role in the maintenance of bodyweight loss. In the future, leptin analogues and the development of compounds that increase leptin sensitivity may also prove to be valuable therapeutic approaches for obesity.

Protein crystals and their evil twins.

Different types of crystal twinning are reviewed with an emphasis on how to detect the phenomenon from protein diffraction data. The recent literature and a database survey both serve as reminders to perform routine checks whenever twinning is a possibility.

Breast calcifications: analysis of imaging properties.

The imaging characteristics of microcalcifications in both benign and malignant breast conditions were analyzed in 48 digitized film mammograms. Each case included in this analysis had findings considered suggestive of malignancy by the radiologist, with the underlying histologic structure determined by excisional biopsy. Imaging properties of each microcalcification--such as pixel intensity, relative location, distribution, size, and local neighborhood intensities--were recorded. This information was statistically analyzed at the population level according to such selection criteria as histologic type, size of calcification, and cluster size. Distribution ranges were determined for these criteria. Statistical differences between data from benign and malignant cases show the average distance between calcifications in malignant conditions was greater than in benign conditions, and tissue region averages surrounding calcifications associated with malignant conditions were consistently higher than those for benign conditions.

Algorithm for the detection of fine clustered calcifications on film mammograms.

An algorithmic process for the detection and marking of clustered calcifications in digitized film-screen mammograms has been applied to mammograms from 50 clinical cases sampled at two digitization levels, in both the craniocaudal and mediolateral views. In all but one case the detector accurately located suggestive clusters found by radiologists in normal screening. In five cases additional clusters were also found by the detector. The detector has a negligible false-positive rate for the detection of clustered calcifications, although it is sensitive to clusters of emulsion defects displayed as artifactual calcification densities in the original film. The detector is flexible in structure and is easily adapted to various calcification/cluster criteria. The detector shows considerable promise when applied to clinical examples but will require refinement before formal testing.

Experience in vibratory and electro-ejaculation techniques in spinal cord injury patients: a preliminary report.

More than 90 per cent of complete spinal cord injury patients have major fertility problems, depending upon the site and type of injury. During the last 5 years 34 patients were treated by vibratory and/or electrostimulation at our center, and semen was produced in all but 5. In 8 patients ejaculation was attempted by vibratory stimulation alone and in 22 electrostimulation also was used. Vibratory stimulation is the easier and less cumbersome of the 2 methods. No major side effects were noted with either technique. Stimulation was performed by a rectal electrode incorporated in a silicone finger glove with a current of 0.1 msec. in duration, a frequency of 30 Hz. and an average of 60 volts. Vibratory stimulation was applied to the frenulum and/or glans penis with a specially constructed vibrator at a frequency of 80 Hz. and a peak-to-peak oscillation of 1.6 to 2.4 mm. Semen obtained during the first 6 months after injury was not of a quality consistent with successful fertilization owing to poor motility. However, semen quality and motility were better in patients who had been injured for more than 6 months. Repeated electro-ejaculation did not improve the quality of semen. The effects of bladder outlet surgery and autonomic blockers were noted in 5 patients.

5-microtransducer catheter in evaluation of neurogenic bladder function.

Recent use of the multiple microtransducer catheter in the evaluation of neurogenic bladder due to spinal-cord injuries leads us to believe that the use of the inferior edge of the symphysis pubis as the zero point for resting bladder pressure is more accurate than its superior edge, changes in resting bladder pressure at various volumes are influenced more by body position than by intravesical position of the sensor, back-to-back microtransducers indicate significant pressure difference at the external sphincter zone, and detrusor bladder neck dyssynergia during autonomic dysreflexia in patients with spinal cord injury is more likely of skeletal than of smooth muscle origin.

Management of neurogenic bladder in female spinal cord injury patients.

Twenty-two female spinal cord injury patients were admitted to the Spinal Cord Injury Service at the West Roxbury VAMC during a period of 17 years (1965-1982). Bladder status and means of drainage were evaluated. Twelve patients (55%) required no means of drainage, nine of them were dry all the time, while the other three needed pamper support to counteract occasional wetness. Seven were on constant indwelling catheters, two were on self-catheterization, while one had an intestinal loop diversion. It appears that female spinal cord injury patients depend more on constant indwelling catheters than their male counterparts. In some instances, female paraplegics do well on self-catheterization. Catheter complications in female spinal cord injury patients appear to be less than in males.

Indication and results of semirigid penile prostheses in spinal cord injury patients: long-term followup.

During the last 7 years semirigid intracorporeal penile prostheses were inserted in 36 spinal cord injury patients between 21 and 58 years old (average age 38.5 years). An operation was done 1 to 32 years after the initial injury (average 10 years). Surgical intervention was intended to provide an adequate body to the penile shaft so as to hold an external urinary drainage device in 11 patients, for treatment of sexual dysfunction only in 17 and for an external urinary drainage device plus treatment of sexual inadequacy in 8. Although a number of complications causing extrusion or removal of the prosthesis occurred in 6 patients (16.5 per cent), as well as an aborted operation in 1 (19.5 per cent), there were no permanent sequelae. Because of loss of sensation and vasomotor control, and pressure produced by the penile prosthesis spinal cord injury patients represent a higher operative risk than other patients without neurological or vascular impairments. In addition, urinary tract infection should not be overlooked as another major risk factor. Penile prostheses were most successful in maintenance of external urinary appliances in patients with a short or retractile penis. Whenever the prosthesis was intended for sexual intercourse an important prerequisite to a successful surgical outcome was the retention of some reflexogenic or psychogenic erection over and above the rods. Careful individual preoperative assessment is advised if a satisfactory result is to be achieved.

Detrusor urethral sphincter dyssynergia: micturitional vesicourethral pressure profile patterns.

Vesicourethral static pressure recordings were attempted in patients with detrusor urethral sphincter dyssynergia. The technique consisted of recording vesicourethral pressures at successive points, commencing in the lower segment of the bladder during micturition. A small catheter with side holes was used for this purpose and static (lateral) pressure profile recordings were attempted during voiding. Successful static pressure recordings were obtained only in those patients who could expel urine as uninterrupted stream. Patients who could not void or those who could void only with interrupted stream have demonstrated profile patterns that required careful interpretation. The accuracy of urodynamic interpretation also depended upon careful clinical evaluation and awareness of the built-in artifacts of the technique.

Role of striated and smooth muscle components in the urethral pressure profile in traumatic neurogenic bladders: a neuropharmacological and urodynamic study. Preliminary report.

Urodynamic investigations with urethral pressure profile, and vesical, intrarectal and anal pressure recordings were performed in 37 patients with spinal cord lesions. The recordings were done before and after phentolamine injections and/or pudendal nerve blocks to evaluate the respective contribution of sympathetic and somatic innervation to the maximum urethral closure pressure in the mid and distal portions of the membranous urethra. A pressure gradient was demonstrated in the membranous urethra with higher values in the distal than in the mid portion. These results emphasize that the interrupted withdrawal technique is superior to the continuous technique in patients with upper motor neuron bladders. Mid urethral striated and smooth muscle components were shown to represent approximately 60 and 30 per cent of the maximum urethral closure pressure, respectively. In the distal urethra striated and smooth components are more abundant than in the mid portion and contribute in equal proportion to the maximum urethral closure pressure. No substantial role was found for the vascular bed in the maximum urethral closure pressure. The greatest pressure decrease in the mid and distal urethra of patients with lower motor neuron bladders was believed to be an effect of denervation supersensitivity. The results of pudendal blocks showed sphincter dyssynergia to be mediated through pudendal nerves via spinal reflex arcs. Phentolamine effects on bladder activity suggest that blockade of alpha-adrenergic receptors inhibits primarily the transmission in vesical and/or pelvic parasympathetic ganglia and acts secondarily through direct depression of the vesical smooth muscle. Our neuropharmacological results raise strong doubts as to the existence of a sympathetic innervation of the striated urethral muscle in humans.