Nutrient responsive nesfatin-1 regulates energy balance and induces glucose-stimulated insulin secretion in rats.

Nesfatin-1 is a recently discovered anorexigen, and we first reported nesfatin-like immunoreactivity in the pancreatic ß-cells. The aim of this study was to characterize the effects of nesfatin-1 on whole-body energy homeostasis, insulin secretion, and glycemia. The in vivo effects of continuous peripheral delivery of nesfatin-1 using osmotic minipumps on food intake and substrate partitioning were examined in ad libitum-fed male Fischer 344 rats. The effects of nesfatin-1 on glucose-stimulated insulin secretion (GSIS) were examined in isolated pancreatic islets. L6 skeletal muscle cells and isolated rat adipocytes were used to assess the effects of nesfatin-1 on basal and insulin-mediated glucose uptake as well as on major steps of insulin signaling in these cells. Nesfatin-1 reduced cumulative food intake and increased spontaneous physical activity, whole-body fat oxidation, and carnitine palmitoyltransferase I mRNA expression in brown adipose tissue but did not affect uncoupling protein 1 mRNA in the brown adipose tissue. Nesfatin-1 significantly enhanced GSIS in vivo during an oral glucose tolerance test and improved insulin sensitivity. Although insulin-stimulated glucose uptake in L6 muscle cells was inhibited by nesfatin-1 pretreatment, basal and insulin-induced glucose uptake in adipocytes from nesfatin-1-treated rats was significantly increased. In agreement with our in vivo results, nesfatin-1 enhanced GSIS from isolated pancreatic islets at both normal (5.6 mM) and high (16.7 mM), but not at low (2 mM), glucose concentrations. Furthermore, nesfatin-1/nucleobindin 2 release from rat pancreatic islets was stimulated by glucose. Collectively, our data indicate that glucose-responsive nesfatin-1 regulates insulin secretion, glucose homeostasis, and whole-body energy balance in rats.

High-fat diet increases thyrotropin and oxygen consumption without altering circulating 3,5,3'-triiodothyronine (T3) and thyroxine in rats: the role of iodothyronine deiodinases, reverse T3 production, and whole-body fat oxidation.

This study investigated the effects of obesity induced by high-fat (HF) diet on thyroid function and whole-body energy balance. To accomplish that, we assessed the effects of 8 wk of HF diet on several parameters of hypothalamus-pituitary-thyroid axis function. Serum total T(4) and T(3), rT(3), and TSH, the activity of type 1 and type 2 deiodinases in central and peripheral tissues were determined. Also, we measured in vivo energy balance, substrate partitioning, and markers of leptin resistance. Here we provide novel evidence that prolonged positive energy balance acquired by feeding a HF diet induced hyperactivation of the hypothalamus-pituitary-thyroid axis, which was characterized by 2.24-, 1.6-, and 3.7-fold elevations in hypothalamic TRH expression, thyroid iodide uptake, and serum TSH, respectively. Serum T(4) and T(3) were normal together with augmented deiodinase type 1 activity in liver (1.3-fold) and kidney (1.2-fold) and increased (1.5-fold) serum rT3 in HF rats. Despite no increase in circulating levels of T(3) and T(4), whole-body oxygen consumption was increased, and substrate metabolism was shifted toward fat oxidation in HF rats. These in vivo metabolic adjustments were mainly driven by the fat content of the diet. Furthermore, spontaneous dark cycle physical activity was reduced by 30% in rats fed a HF diet, which limited energy expenditure and favored the development of obesity. Our findings provide new insight into the endocrine and physiological mechanisms that underlie the alterations in thyroid hormone availability, energy balance, and metabolic partitioning in HF diet-induced obesity.

A robust xenotransplantation model for acute myeloid leukemia.

Xenotransplantation of human acute myeloid leukemia (AML) in immunocompromised animals has been critical for defining leukemic stem cells. However, existing immunodeficient strains of mice have short life spans and low levels of AML cell engraftment, hindering long-term evaluation of primary human AML biology. A recent study suggested that NOD/LtSz-scid IL2Rgammac null (NSG) mice have enhanced AML cell engraftment, but this relied on technically challenging neonatal injections. Here, we performed extensive analysis of AML engraftment in adult NSG mice using tail vein injection. Of the 35 AML samples analyzed, 66% showed bone marrow engraftment over 0.1%. Further, 37% showed high levels of engraftment (>10%), with some as high as 95%. A 2-44-fold expansion of AML cells was often seen. Secondary and tertiary recipients showed consistent engraftment, with most showing further AML cell expansion. Engraftment did not correlate with French-American-British subtype or cytogenetic abnormalities. However, samples with FLT3 mutations showed a higher probability of engraftment than FLT3 wild type. Importantly, animals developed organomegaly and a wasting illness consistent with advanced leukemia. We conclude that the NSG xenotransplantation model is a robust model for human AML cell engraftment, which will allow better characterization of AML biology and testing of new therapies.

Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1alpha represses MEF2-dependent gene expression.

The myocyte enhancer factor 2 (MEF2) transcription factors play important roles in neuronal, cardiac, and skeletal muscle tissues. MEF2 serves as a nuclear sensor, integrating signals from several signaling cascades through protein-protein interactions with kinases, chromatin remodeling factors, and other transcriptional regulators. Here, we report a novel interaction between the catalytic subunit of protein phosphatase 1alpha (PP1alpha) and MEF2. Interaction occurs within the nucleus, and binding of PP1alpha to MEF2 potently represses MEF2-dependent transcription. The interaction utilizes uncharacterized domains in both PP1alpha and MEF2, and PP1alpha phosphatase activity is not obligatory for MEF2 repression. Moreover, a MEF2-PP1alpha regulatory complex leads to nuclear retention and recruitment of histone deacetylase 4 to MEF2 transcription complexes. PP1alpha-mediated repression of MEF2 overrides the positive influence of calcineurin signaling, suggesting PP1alpha exerts a dominant level of control over MEF2 function. Indeed, PP1alpha-mediated repression of MEF2 function interferes with the prosurvival effect of MEF2 in primary hippocampal neurons. The PP1alpha-MEF2 interaction constitutes a potent locus of control for MEF2-dependent gene expression, having potentially important implications for neuronal cell survival, cardiac remodeling in disease, and terminal differentiation of vascular, cardiac, and skeletal muscle.

Absence of Mycobacterium bovis infection in dogs and cats residing on infected cattle farms: Michigan, 2002.

A cross-sectional field study was performed to evaluate infection in dogs and cats living on farms with Mycobacterium bovis-infected cattle. The purpose was to determine pet infection status and assess their risk to farm families and/or tuberculosis-free livestock. Data and specimens were collected from 18 cats and five dogs from nine participating farms. ELISA testing for M. bovis and M. avium was conducted. Fifty-one biological samples were cultured; all were negative for M. bovis, although other Mycobacterium species were recovered. No radiographic, serological or skin test evidence of mycobacterial infection was found. These negative results may be due to the low level of M. bovis infection in the cattle and the limited duration of exposure of pets to infected cattle residing on the same farm. No evidence was found to indicate that pets residing on M. bovis-infected Michigan cattle farms pose a risk to humans or M. bovis-free livestock; however, precautionary advice for farm owners was provided.

Explanation for the enhanced dissolution of silica column packing in high pH phosphate and carbonate buffers.

It has been reported that at high pH, the rate of bonded phase packing degradation in methanol/water mobile phases is greater for carbonate and phosphate buffers than for amine buffers. This conclusion was based on buffer pH determined in the aqueous buffer before dilution with methanol. Changes in buffer species pKa, and therefore buffer pH, upon methanol dilution are consistent with the observed degradation results. Measurements of pH in the methanol/water solutions confirm that the carbonate and phosphate buffers were considerably more basic than the amine buffer, even though all the buffers were pH 10 before dilution with methanol. These results demonstrate that it can be misleading to extrapolate aqueous pH data to partially aqueous solutions. Measurements of pH in the mixed solvent provide more reliable predictions of column and sample stability.

Activated MEK1 binds the nuclear MyoD transcriptional complex to repress transactivation.

To elucidate the mechanism through which MAPK signaling regulates the MyoD family of transcription factors, we investigated the role of the signaling intermediate MEK1 in myogenesis. Transfection of activated MEK1 strongly repressed gene activation and myogenic conversion by the MyoD family. This repression was not mediated by direct phosphorylation of MyoD or by changes in MyoD stability or subcellular distribution. Deletion mapping revealed that MEK1-mediated repression required the MyoD amino-terminal transactivation domain. Moreover, activated MEK1 was nuclearly localized and bound a complex containing MyoD in a manner that is dependent on the presence of the MyoD amino terminus. Together, these data demonstrate that MEK1 signaling has a strong negative effect on MyoD activity via a novel mechanism involving binding of MEK1 to the nuclear MyoD transcriptional complex.

Repellent signaling by Slit requires the leucine-rich repeats.

Slit is a repellent axon guidance cue produced by the midline glia in Drosophila that is required to regulate the formation of contralateral projections and the lateral position of longitudinal tracts. Four sequence motifs comprise the structure of Slit: a leucine-rich repeat (LRR), epidermal growth factor-like (EGF) repeats, a laminin-like globular (G)-domain, and a cysteine domain. Here we demonstrate that the LRR is required for repellent signaling and in vitro binding to Robo. Repellent signaling by slit is reduced by point mutations that encode single amino acid changes in the LRR domain. By contrast to the EGF or G-domains, the LRR domain is required in transgenes to affect axon guidance. Finally, we show that the midline repellent receptor, Robo, binds Slit proteins with internal deletions that also retain repellent activity. However, Robo does not bind Slit protein missing the LRR. Taken together, our data demonstrate that Robo binding and repellent signaling by Slit require the LRR region.

Effect of tibial plateau leveling on cranial and caudal tibial thrusts in canine cranial cruciate-deficient stifles: an in vitro experimental study.

OBJECTIVES: To investigate the effect of tibial plateau leveling (TPL) on tibial subluxation and tibial axial rotation; to determine the minimal tibial plateau rotation (MinTPR) angle that provides stifle stability; and to evaluate caudal cruciate ligament (CaCL) strain following tibial plateau rotation in cranial cruciate ligament (CrCL)-deficient stifles. ANIMALS: Fifteen canine cadaver hind limbs. METHODS: Tibial subluxation was measured from lateral radiographs in intact, loaded stifles and after sequential CrCL transection, MinTPR, TPL, and CaCL transection. The MinTPR angle was determined using a custom-made hinge plate and compared with the TPL angle. Tibial axial rotation was evaluated in CrCL-deficient stifles before and after TPL. Finally, CaCL strain was recorded in intact, loaded stifles, and following MinTPR, TPL, and tibial plateau over-rotation (MaxTPR) using a force probe. RESULTS: Cranial tibial subluxation in CrCL-deficient stifles was eliminated with TPL. Tibial plateau rotation, however, induced caudal tibial subluxation, which significantly increased from MinTPR to TPL before and after CaCL transection. The MinTPR angle was 6.5 degrees +/- 0.9 degrees less than the TPL angle (P <.05). Tibial internal rotation decreased significantly after TPL in CrCL-deficient stifles. Finally, CaCL strain increased with increasing tibial plateau rotation. CONCLUSIONS: This study suggests that, during stance phase, TPL transforms cranial tibial thrust into caudal tibial thrust, thereby stabilizing the stifle in the cranio-caudal plane via the constraint of the CaCL. The increase in CaCL stress, which results from tibial plateau rotation, could predispose the CaCL to fatigue failure and therefore would caution against tibial plateau over-rotation.

Molecular mechanisms regulating myogenic determination and differentiation.

The myogenic regulatory factors are necessary for the determination and terminal differentiation of skeletal muscle. Gene targeting experiments have demonstrated that MyoD and Myf5 are important for myogenic determination whereas myogenin and MRF4 are important for terminal differentiation and lineage maintenance. During development, all trunk skeletal muscle is derived from the somite. Two spatially distinct sources of myogenic progenitors are defined by the expression of MyoD or Myf5 and these give rise to hypaxial and epaxial musculature. Both in vivo and in vitro analyses have provided a detailed picture regarding the molecular events controlling lineage determination, cell migration, terminal differentiation and tissue repair. Signal transduction pathways regulating cell cycle, protein-protein interactions and myogenic factor gene activation are implicated in the regulation of myogenesis. Recent experiments examining the origin and stem-cell capacity of satellite cells suggest that these cells may originate from the vascular system, are multipotential and may be useful for the treatment of several degenerative diseases.

Determination of aliphatic anhydrides and acids by reversed-phase liquid chromatography.

Few chromatography methods have been reported for the determination of anhydrides in mixtures or as mixed anhydrides. The potential reactivity of anhydrides with water and other common eluent components complicates possible schemes for separation and analysis. By optimizing variables that affect hydrolysis, including the stationary phase, conditions can be found to successfully analyze anhydrides as reactive as acetic anhydride. The corresponding acids can be determined at the same time. The effect of the stationary phase on anhydride hydrolysis rates may prove to be a sensitive means of probing stationary phase chemistry.

Regression of hypertrophic osteopathy in a cat after surgical excision of an adrenocortical carcinoma.

A 12-year-old, spayed, female domestic shorthair cat was diagnosed with severe and extensive hypertrophic osteopathy of the appendicular skeleton. Diagnostic ultrasound detected a mass lesion in the right adrenal gland. A right adrenalectomy was performed, and histopathological examination confirmed an adrenocortical carcinoma. No radiographic evidence of pulmonary metastasis was found on initial presentation or recheck thoracic radiographs taken 15 weeks later. Almost complete regression of periosteal new bone formation occurred 15 weeks following the successful surgical removal of the adrenal tumor.

Severe cardiomyopathy in mice lacking dystrophin and MyoD.

The mdx mouse, a mouse model of Duchenne muscular dystrophy, carries a loss-of-function mutation in dystrophin, a component of the membrane-associated dystrophin-glycoprotein complex. Unlike humans, mdx mice rarely display cardiac abnormalities and exhibit dystrophic changes only in a small number of heavily used skeletal muscle groups. By contrast, mdx:MyoD-/- mice lacking dystrophin and the skeletal muscle-specific bHLH transcription factor MyoD display a severe skeletal myopathy leading to widespread dystrophic changes in skeletal muscle and premature death around 1 year of age. The severely increased phenotype of mdx:MyoD-/- muscle is a consequence of impaired muscle regeneration caused by enhanced satellite cell self-renewal. Here we report that mdx:MyoD-/- mice developed a severe cardiac myopathy with areas of necrosis associated with hypertrophied myocytes. Moreover, heart tissue from mdx:MyoD-/- mice exhibited constitutive activation of stress-activated signaling components, similar to in vitro models of cardiac myocyte adaptation. Taken together, these results support the hypothesis that the progression of skeletal muscle damage is a significant contributing factor leading to development of cardiomyopathy.

The effect of triple pelvic osteotomy on the articular contact area of the hip joint in dysplastic dogs: an in vitro experimental study.

OBJECTIVE: To investigate the effect of triple pelvic osteotomy (TPO) on articular contact area and acetabular coverage of dysplastic hip joints in dogs. STUDY DESIGN: Articular contact area and femoral head coverage by the acetabulum were computed in vitro in normal and dysplastic canine hips. The effect of TPO on articular contact and coverage was then analyzed in the dysplastic hips. Sample Population-Five normal and six dysplastic canine cadaver specimens. METHODS: Contact area and coverage of loaded hips were computed using serial computed tomography scan images before and after TPO. Three angles of acetabular ventroversion (AVV) were studied (20 degrees, 30 degrees, and 40 degrees). Using a custom-designed hinge plate, angles of spontaneous hip reduction in dysplastic hips were compared with previously recorded angles of reduction determined by the Ortolani test. RESULTS: Contact area significantly increased from 0 degrees to 30 degrees of AVV, then remained virtually unchanged. Coverage significantly increased from 0 degrees to 20 degrees of AVV. Both contact and coverage of normal hips were similar, yet significantly smaller than those of dysplastic hips once reduction had occurred. The experimental angles of reduction were significantly smaller and poorly correlated with the angles of reduction determined by the Ortolani test. Although coverage continued to increase with AVV, the actual joint contact area did not significantly vary after relocation of the femoral head. CONCLUSIONS: This study suggests that increasing AVV beyond 20 degrees does not significantly improve the beneficial effects of TPO and therefore should be carefully weighed against increased risks of postoperative complications associated with large angles of AVV.

Elevating intracellular calcium levels in human sperm using an internal calcium ATPase inhibitor, 2,5-di(tert-butyl) hydroquinone (TBQ), initiates capacitation and the acrosome reaction but only in the presence of extracellular calcium.

The aim of this study was to investigate the effect of an internal calcium ATPase inhibitor, TBQ, on human sperm capacitation and the acrosome reaction during incubation in a calcium-depleted media. Sperm were isolated into and incubated for up to 6 hr in media depleted of Ca2+ and two Ca(2+)-containing media controls. At set time intervals, sperm in each media group were treated with 100 microM TBQ for 5 min. Afterwards, sperm were induced to acrosome react using the divalent cation ionophore, A23187, as a measure of sperm fertilizing potential. It was established, using the Chlortetracycline assay, that incubation of sperm in a Ca(2+)-depleted media inhibited or delayed sperm capacitation resulting in fewer spontaneous or A23187-induced acrosome reacted sperm. However, incubation of sperm in a Ca(2+)-depleted media did not appear to inhibit sperm motility. The treatment of sperm with TBQ during their incubation in Ca(2+)-depleted media was found to have very little effect resulting in low numbers of capacitated and acrosome reacted sperm. The results from this study suggest that human sperm have an obligatory requirement for extracellular calcium during capacitation and the acrosome reaction, but may require either very little extracellular Ca2+ to maintain motility or possess internal Ca2+ stores sufficient for their requirements. In addition, TBQ did not increase the number of capacitated and acrosome reacted sperm during incubation in a Ca(2+)-depleted media suggesting that the TBQ-effect of accelerating sperm capacitation is dependent on presence of extracellular Ca2+.

Response of human spermatozoa to an internal calcium ATPase inhibitor, 2,5-di(tert-butyl) hydroquinone.

This study has investigated the effect of elevating intracellular calcium levels, using an internal calcium ATPase inhibitor, 2,5-di(tert-butyl) hydroquinone (TBQ), on human sperm function. Isolated sperm samples from five fertile donors were incubated in a capacitating media for up to 6 hr. After 0, 3, and 6 hr incubation, sperm were exposed to a range of TBQ concentrations; 100 microM, 10 microM, and 1 microM, for a fixed incubation period of 5 min. Controls were run for each experiment where sperm were incubated for 5 min in the absence of TBQ. Sperm capacitation and the acrosome reaction were monitored prior to and after exposure to TBQ, using the Chlortetracycline assay. In addition, sperm motility was assessed at each time point and after sperm had been exposed to TBQ. The treatment of sperm with TBQ caused a significant increase in the number of capacitated sperm with an optimum response being achieved in the presence of 100 microM TBQ. However, sperm motility was found not to be effected by the addition of TBQ. The results from the present study suggest that elevating intracellular calcium levels in human sperm by short exposure to a high concentration of TBQ can rapidly accelerate the capacitation process. Furthermore, the observation that TBQ did not elicit a change in sperm motility suggests that TBQ may be highly specific in its mode of action by acting within the head region of human sperm.

Pregnancy in early adolescence: are there obstetric risks?

The purpose of this study was to determine if early adolescence imparts a significant obstetric risk in young primiparas relative to adult primiparas. The records of 239 young primiparas (< 16 years) and 148 older primiparas (18-29 years) were reviewed for demographic information, antepartum complications, mode of delivery, length of labor, episiotomy, lacerations, birthweight, and length of gestation. The young adolescents were shorter, had an earlier age at menarche, a lower pregravid body mass index, and a higher gestational weight gain. The young teens were less likely to smoke cigarettes but were more likely to be Medicaid recipients. The incidence of most antenatal complications (chronic hypertension, pregnancy-induced hypertension, placental abruption, placenta previa, premature rupture of the membranes, urinary tract infections, and anemia) were similar between the two groups. Preterm labor and contracted pelvis were more common among the young adolescent, while gestational diabetes was less common. The young primiparas were significantly (P < .05) less likely to have a Cesarean delivery and to lacerate with vaginal delivery. The length of labor and its stages were similar, as were overall birthweight and length of gestation. Thus, obstetric concerns regarding pregnancy in early adolescence may be unfounded. With the exception of an increased risk for preterm labor, it appears that pregnancy, labor, and delivery do not pose inordinate obstetric and medical risk to the very young adolescent primipara.

Comparative study of the effect of human cervical mucus and a cervical mucus substitute, Healonid, on capacitation and the acrosome reaction of human spermatozoa in vitro.

The present study was designed to investigate the effect of human cervical mucus on capacitation and the acrosome reaction of human spermatozoa and compare its effect to that of a cervical mucus substitute, sodium hyaluronate (Healonid). Spermatozoa from donors of proven fertility were isolated from semen using cervical mucus, Healonid or a direct swim-up (acting as the control). Sperm capacitation and the acrosome reaction were monitored by the chlortetracycline assay. In the mucus-treated group, there was a significantly higher percentage of capacitated spermatozoa, but a low incidence of spontaneous and A23187-induced acrosome reactions compared to the control. The use of Healonid during sperm isolation mimicked the effect of mucus relatively successfully. Since mucus and Healonid show very little chemical similarity, this finding would imply that cervical mucus exerts a physical effect during its interaction with spermatozoa, although a chemical effect cannot be completely dismissed. In conclusion, this study confirms early reports describing the ability of cervical mucus to capacitate spermatozoa but at the same time conserve sperm function. The finding that Healonid exerts an almost identical effect on spermatozoa would lend support to its use as a cervical mucus substitute during in-vitro fertility assessments and research studies.