Longitudinal evaluation of intramyocellular lipids (IMCLs) in tibialis anterior muscle of ob/ob and ob/+ control mice using a cryogenic surface coil at 9.4 T

Insulin resistance is a central feature of type II diabetes and is associated with alterations in skeletal muscle lipid metabolism, which manifest themselves, in part, in increased intramyocellular lipid (IMCL) accumulation. The objective of this study was to assess noninvasively the levels of IMCL longitudinally in the tibialis anterior muscle of Lep(ob) /Lep(ob) (ob/ob) mice, a genetic model of obesity and mild diabetes, and Lep(ob) /+ (ob/+) heterozygous control animals, using (1) H MRS at 9.4 T. The use of a cryogenic surface coil transceiver leads to significant increases in sensitivity. Method implementation included the assessment of the reproducibility and spatial heterogeneity of the IMCL signal and the determination of T(2) relaxation times, as IMCL levels were expressed relative to the total creatine signal, and therefore the signal ratios had to be corrected for differences in T(2) relaxation. IMCL levels were found to be significantly higher in ob/ob mice relative to ob/+ heterozygous control mice that do not develop disease. An increase in IMCL levels was observed for ob/ob mice until weeks 16/17; after this time point, IMCL levels decreased again, reaching final levels that were slightly higher than the initial values. These noninvasively detected alterations in skeletal muscle lipid metabolism in ob/ob mice were accompanied by a transient increase in plasma insulin concentrations. This study indicates that IMCL may be reliably assessed in mouse tibialis anterior muscle using a cryogenic surface coil, implying that (1) H MRS at 9.4 T represents a useful technology for the noninvasive measurement of changes in lipid metabolism in the skeletal muscle that accompany obesity.     

Lipid accumulation in non-adipose tissue and lipotoxicity

Obesity is a well-known risk factor for the development of type 2 diabetes mellitus and cardiovascular disease. Importantly, obesity is not only associated with lipid accumulation in adipose tissue, but also in non-adipose tissues. The latter is also known as ectopic lipid accumulation and may be a possible link between obesity and its comorbidities such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease.

In skeletal muscle and liver, lipid accumulation has been associated with the development of insulin resistance, an early hallmark of developing type 2 diabetes mellitus. More specifically, accumulation of intermediates of lipid metabolism, such as diacylglycerol (DAG) and Acyl-CoA have been shown to interfere with insulin signaling in these tissues. Initially, muscular and hepatic insulin resistance can be overcome by an increased insulin production by the pancreas, resulting in hyperinsulinemia. However, during the progression towards overt type 2 diabetes, pancreatic failure occurs resulting in reduced insulin production. Interestingly, also in the pancreas lipid accumulation has been shown to precede dysfunction.

Finally, accumulation of fat in the heart has been associated with cardiac dysfunction and heart failure, which may be an explanation for diabetic cardiomyopathy.

Taken together, we conclude that evidence for deleterious effects of lipid accumulation in non-adipose tissue (lipotoxicity) is strong. However, while ample human data is available for skeletal muscle and the liver, future research should focus on lipid accumulation in the pancreas and the heart.     

Regulation of myoplasmic Ca(2+) in genetically obese (ob/ob) mouse single skeletal muscle fibres

The present study examined whether calcium handling in skeletal muscle fibres from ob/ob mice was abnormal compared to normal mice. Simultaneous measurements of free myoplasmic calcium and force were made in mouse single intact muscle fibres at rest, during repetitive stimulation and for 30 min afterwards. Fibres were subjected to two bouts of intermittent tetanic contractions 1 h apart. The first bout consisted of 50 tetani only, while during the second bout stimulation was continued until force fell to 40% of control. During a bout of 50 repeated contractions, muscle fibres from ob/ob mice were unable to maintain basal calcium and tetanic calcium transients. During a second series of contractions, muscle fibres from ob/ob mice showed a marked improvement in calcium handling compared to the first series but still fatigued more rapidly than control fibres. It is concluded that calcium handling in skeletal muscle fibres from ob/ob mice is abnormal compared to fibres from normal mice and this contributes to premature fatigue.     

Lipotoxicity, overnutrition and energy metabolism in aging

The safest place to store lipids is the white adipose tissue, but its storage capacity may become saturated resulting in excess of fat "overspilled" to non-adipose tissues. This overspill of fat occurs in apparently opposite pathological states such as lipodistrophy or obesity. When the excess of energy is redirected towards peripheral organs, their initial response is to facilitate the storage of the surplus in the form of triacylglycerol, but the limited triacylglycerol buffer capacity becomes saturated soon. Under these conditions excess of lipids enter alternative non-oxidative pathways that result in production of toxic reactive lipid species that induce organ-specific toxic responses leading to apoptosis. Reactive lipids can accumulate in non-adipose tissues of metabolically relevant organs such as pancreatic beta-cells, liver, heart and skeletal muscle leading to lipotoxicity, a process that contributes substantially to the pathophysiology of insulin resistance, type 2 diabetes, steatotic liver disease and heart failure. The effects of this lipotoxic insult can be minimised by several strategies: (a) decreased incorporation of energy, (b) a less orthodox approach such as increased adipose tissue expandability and/or (c) increased oxidation of fat in peripheral organs. Aging should be considered as physiological degenerative process potentially accelerated by concomitant lipotoxic insults. Conversely, the process of aging can sensitise cells to effects of lipid toxicity.     

Lipogenic regulators are elevated with age and chronic overload in rat skeletal muscle

Aim: Both muscle mass and strength decline with ageing, but the loss of strength far surpasses what is projected based on the decline in mass. Interestingly, the accumulation of fat mass has been shown to be a strong predictor of functional loss and disability. Furthermore, there is a known attenuated hypertrophic response to skeletal muscle overload with ageing. The purpose of this study was to determine the effect of 28 days of overload on the storage of intramuscular triglycerides (IMTG) and metabolic regulators of lipid synthesis in young and old skeletal muscle. Methods: The phosphorylation and expression of essential lipogenic regulators were determined in the plantaris of young (YNG; 6‐month‐old) and aged (OLD; 30‐month‐old) rats subjected to bilateral synergist ablation (SA) of two‐thirds of the gastrocnemius muscle or sham surgery. Results: We demonstrate that age‐induced increases in IMTG are associated with enhancements in the expression of lipogenic regulators in muscle. We also show that the phosphorylation and concentration of the 5′AMP‐activated protein kinase (AMPK) isoforms are altered in OLD. We observed increases in the expression of lipogenic regulators and AMPK signalling after SA in YNG, despite no increase in IMTG. Markers of oxidative capacity were increased in YNG after SA. These overload‐induced effects were blunted in OLD. Conclusion: These data suggest that lipid metabolism may be altered in ageing skeletal muscle and is unaffected by mechanical overload via SA. By determining the role of increased lipid storage on skeletal muscle mass during ageing, possible gene targets for the treatment of sarcopenia may be identified.     

Longitudinal evaluation of hepatic lipid deposition and composition in ob/ob and ob/+ control mice

Obesity is associated with insulin resistance (IR) and hepatosteatosis. Understanding the link between IR and hepatosteatosis could be relevant to chronic clinical outcomes. The objective of this study was to quantitatively assess lipid deposition (fractional lipid mass, fLM) and composition (fraction of polyunsaturated lipids, fPUL and mean chain length, MCL) in livers of ob/ob mice, a genetic model of obesity and mild diabetes, and ob/+ heterozygous control animals in a noninvasive manner using ¹H‐MRS at 9.4T. For accurate quantification, intensity values were corrected for differences in T₂ values while T₁ effects were considered minimal due to the long T_R values used. Values of fLM, fPUL and MCL were derived from T₂‐corrected signal intensities of lipids and water resonance. Hepatic lipid signals were compared with fasted plasma insulin, glucose and lipid levels. Statistically significant correlations between fPUL and fasting plasma insulin/glucose levels were found in adolescent ob/ob mice. A similar correlation was found between fLM and fasting plasma insulin levels; however, the correlation between fLM and fasting plasma glucose levels was less obvious in adolescent ob/ob mice. These correlations were lost in adult ob/ob mice. The study showed that in adolescent ob/ob mice, there was an obvious link between lipid deposition/composition in the liver and plasma insulin/glucose levels. This correlation was lost in adult animals, probably due to the limited lipid storage capacity of the liver. Copyright © 2013 John Wiley & Sons, Ltd.     

Upregulated Expression of AQP 7 in the Skeletal Muscles of Obese ob/ob Mice

Aquaporin (AQP) is suggested to be regulated by leptin through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway. AQP7 and AQP9 are membrane proteins with water and glycerol channels, the latter of which is essential for triglyceride synthesis. We conjectured that the expression of AQP7 and AQP9 would be altered in the skeletal myofibers in obese leptin deficient ob/ob mice as compared with that of wild mice. RNA and protein levels were studied in the quadriceps femoris muscles of ob/ob and wild mice. Real time quantitative RT-PCR analysis showed that mouse AQP7 mRNA levels in skeletal muscles were significantly higher in ob/ob mice than in wild mice (P<0.01), whereas mouse AQP9 mRNA level was not different between the two groups (P>0.05). Histologically the type 1 myofibers of ob/ob mice contained numerous lipid droplets in oil red O stain samples. Immunohistochemical staining of ob/ob mouse muscles revealed enhanced expression of AQP7 at myofiber surface membranes, while AQP9 expression appeared to be similar to that of wild mice. The findings suggest that the upregulated expression of AQP7 in ob/ob mouse muscles facilitates the secretion of glycerol from myocytes.     

Na+,K+-ATPase enzyme units in lean and obese (ob/ob) thyroxine-injected mice.

The possible involvement of Na+,K+-ATPase in the etiology of obesity in the obese (ob/ob) mouse was explored. The number of Na+,K+-ATPase enzyme units in skeletal muscle, liver, and kidneys from 4- and 8-wk-old obese and lean mice was estimated from saturable [3H]ouabain binding to particulate fractions. Neither phenotype nor age altered the Kd value for ouabain binding in these three tissue preparations. The total number of [3H]ouabain binding sites in hindlimb muscles was 35--55% lower in 4- and 8-wk-old obese mice than in their lean counterparts. However, the total number of [3H]ouabain binding sites in liver and kidneys of obese mice was similar to values observed in their lean counterparts. Because it has been suggested that ob/ob mice are hypothyroid, we investigated the response of Na+,K+-ATPase in these mice to thyroid hormone treatment (approximately 5 microgram thyroxine/day for 2 wk). The number of [3H]ouabain binding sites in the three tissues increased in both obese and lean mice injected with this relatively large dose of thyroxine, but the obese mice were 2--3 times more responsive than lean mice.     

硫化氢在ob/ob小鼠皮肤创面愈合中的作用与调控机制

目的:观察硫化氢(H2S)对ob/ob小鼠皮肤创面愈合的影响并探讨其作用机制。方法:将ob/ob小鼠随机分为生理盐水组、胰岛素组和NaHS(H2S供体)组,C57BL/6小鼠作为对照组,构建小鼠背部皮肤创面模型。干预后检测各组H2S释放量;用Western blot检测胱硫醚γ-裂解酶(CSE)及基质金属蛋白酶-9(MMP-9)蛋白的表达差异;用RT-qPCR检测CSE的mRNA表达变化;使用免疫组织化学法检测中性粒细胞及单核/巨噬细胞的浸润数量;使用ELISA检测肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6的水平;用Masson染色检测胶原沉积情况。结果:ob/ob小鼠皮肤创面肉芽组织中H2S释放及CSE蛋白、mRNA的表达水平以及胶原沉积显著低于C57BL/6小鼠(P0.05)。外源性H2S可加速ob/ob小鼠皮肤创面愈合(P0.05),增加胶原沉积。ob/ob小鼠创面中性粒细胞及单核/巨噬细胞浸润数量,TNF-α、IL-6的水平及MMP-9蛋白表达水平显著增加(P0.05),NaHS组显著降低。结论:H2S可显著改善糖尿病难愈性溃疡的愈合,作用机制可能与其抗炎作用有关。     

Autonomic dysregulation in ob/ob mice is improved by inhibition of angiotensin-converting enzyme

The leptin-deficient ob/ob mice are insulin resistant and obese. However, the control of blood pressure in this model is not well defined. The goal of this study was to evaluate the role of leptin and of the renin-angiotensin system in the cardiovascular abnormalities observed in obesity using a model lacking leptin. To this purpose, we measured blood pressure in ob/ob and control animals by radiotelemetry combined with fast Fourier transformation before and after both leptin and enalapril treatment. Autonomic function was assessed pharmacologically. Blood pressure during daytime was slightly higher in the ob/ob compared to control mice, while no difference in heart rate was observed. Blood pressure response to trimetaphane and heart rate response to metoprolol were greater in ob/ob mice than in control littermates indicating an activated sympathetic nervous system. Heart rate response to atropine was attenuated. Baroreflex sensitivity and heart rate variability were blunted in ob/ob mice, while low frequency of systolic blood pressure variability was found increased. Chronic leptin replacement reduced blood pressure and reversed the impaired autonomic function observed in ob/ob mice. Inhibition of angiotensin-converting enzyme by enalapril treatment had similar effects, prior to the loss of weight. These findings suggest that the renin-angiotensin system is involved in the autonomic dysfunction caused by the lack of leptin in ob/ob mice and support a role of this interplay in the pathogenesis of obesity, hypertension, and metabolic syndrome.     

Decrease in particle-induced osteolysis in obese (ob/ob) mice

There may be variability in the susceptibility of different individuals to osteolysis from wear debris, and it is not clear whether some individuals may have a genetic predisposition for a more marked osteolytic response. The purpose of this study in mice was to determine whether genetically determined obesity can alter the response to particulate debris. Polyethylene particles were implanted onto the calvaria of seven wild-type mice and seven obese mice (ob/ob). Calvaria from unimplanted wild-type and obese mice served as controls. Calvaria were harvested after 7 days, stained with toluidine blue and for tartrate-specific alkaline phosphatase, and analyzed by histomorphometry. The osteoclast number per mm total bone perimeter was 8.000+/-3.464 in wild-type animals with particles and 2.857+/-1.676 in ob/ob animals with particles (p=0.002; Fisher's PLSD). Bone resorption was 1.895+/-0.713 mm/mm(2) in wild-type animals with particles and 1.265+/-0.494 mm/mm(2) in ob/ob animals with particles (p=0.0438; Fisher's PLSD). Particles induced a diminished osteolytic response in genetically determined obese mice, suggesting that obesity may have a protective role against particle-induced bone resorption-similar to obesity and osteoporosis. These important new findings may help to stimulate clinical studies which may define criteria to better identify patients at risk to develop particle-induced osteolysis.     

The Qγ component of intra-membrane charge movement is present in mammalian muscle fibres, but suppressed in the absence of S100A1

S100A1 is a Ca²⁺ binding protein that modulates excitation–contraction (EC) coupling in skeletal and cardiac muscle. S100A1 competes with calmodulin for binding to the skeletal muscle SR Ca²⁺ release channel (the ryanodine receptor type 1, RyR1) at a site that also interacts with the C-terminal tail of the voltage sensor of EC coupling, the dihydropyridine receptor. Ablation of S100A1 leads to delayed and decreased action potential evoked Ca²⁺ transients, possibly linked to altered voltage sensor activation. Here we investigate the effects of S100A1 on voltage sensor activation in skeletal muscle utilizing whole-cell patch clamp electrophysiology to record intra-membrane charge movement currents in isolated flexor digitorum brevis (FDB) muscle fibres from wild-type and S100A1 knock-out (KO) mice. In contrast to recent reports, we found that FDB fibres exhibit two distinct components of intra-membrane charge movement, an initial rapid component ( Q _β), and a delayed, steeply voltage dependent 'hump' component ( Q _γ) previously recorded primarily in amphibian but not mammalian fibres. Surprisingly, we found that Q _γ was selectively suppressed in S100A1 KO fibres, while the Q _β component of charge movement was unaffected. This result was specific to S100A1 and not a compensatory result of genetic manipulation, as transient intracellular application of S100A1 restored Q _γ. Furthermore, we found that exposure to the RyR1 inhibitor dantrolene suppressed a similar component of charge movement in FDB fibres. These results shed light on voltage sensor activation in mammalian muscle, and support S100A1 as a positive regulator of the voltage sensor and Ca²⁺ release channel in skeletal muscle EC coupling.     

Muscle denervation promotes opening of the permeability transition pore and increases the expression of cyclophilin D

Loss of neural input to skeletal muscle fibres induces atrophy and degeneration with evidence of mitochondria-mediated cell death. However, the effect of denervation on the permeability transition pore (PTP), a mitochondrial protein complex implicated in cell death, is uncertain. In the present study, the impact of 21 days of denervation on the sensitivity of the PTP to Ca²⁺-induced opening was studied in isolated muscle mitochondria. Muscle denervation increased the sensitivity to Ca²⁺-induced opening of the PTP, as indicated by a significant decrease in calcium retention capacity (CRC: 111 ± 12 versus 475 ± 33 nmol (mg protein)⁻¹ for denervated and sham, respectively). This phenomenon was partly attributable to in vivo mitochondrial and whole muscle Ca²⁺ overload. Cyclosporin A, which inhibits PTP opening by binding to cyclophilin D (CypD), was significantly more potent in mitochondria from denervated muscle and restored CRC to the level observed in mitochondria from sham-operated muscles. In contrast, the CypD independent inhibitor trifluoperazine was equally effective at inhibiting PTP opening in sham and denervated animals and did not correct the difference in CRC between groups. This phenomenon was associated with a significant increase in the content of the PTP regulating protein CypD relative to several mitochondrial marker proteins. Together, these results indicate that Ca²⁺ overload in vivo and an altered expression of CypD could predispose mitochondria to permeability transition in denervated muscles.     

Pathology of the liver in obese and diabetic ob/ob and db/db mice fed a standard or high-calorie diet

Non‐alcoholic fatty liver disease (NAFLD) is one of the commonest liver diseases in Western countries. Although leptin deficient ob/ob and db/db mice are frequently used as murine models of NAFLD, an exhaustive characterization of their hepatic lesions has not been reported to date, particularly under calorie overconsumption. Thus, liver lesions were characterized in 78 ob/ob and db/db mice fed either a standard or high‐calorie (HC) diet, for one or three months. Steatosis, necroinflammation, apoptosis and fibrosis were assessed and the NAFLD activity score (NAS) was calculated. Steatosis was milder in db/db mice compared to ob/ob mice and was more frequently microvesicular. Although necroinflammation was usually mild in both genotypes, it was aggravated in db/db mice after one month of calorie overconsumption. Apoptosis was observed in db/db mice whereas it was only detected in ob/ob mice after HC feeding. Increased apoptosis was frequently associated with microvesicular steatosis. In db/db mice fed the HC diet for three months, fibrosis was aggravated while steatosis, necroinflammation and apoptosis tended to alleviate. This was associated with increased plasma β‐hydroxybutyrate suggesting an adaptive stimulation of hepatic mitochondrial fatty acid oxidation (FAO). Nevertheless, one‐third of these db/db mice had steatohepatitis (NAS ≥ 5), whereas none of the ob/ob mice developed non‐alcoholic steatohepatitis under the same conditions. Steatosis, necroinflammation, apoptosis and fibrosis are modulated by calorie overconsumption in the context of leptin deficiency. Association between apoptosis and microvesicular steatosis in obese mice suggests common mitochondrial abnormalities. Enhanced hepatic FAO in db/db mice is associated with fibrosis aggravation.     

Catecholamine-induced lipolysis in adipose tissue and skeletal muscle in obesity

Increased fat storage in adipose and non-adipose tissue (e.g. skeletal muscle) characterizes the obese insulin resistant state. Disturbances in pathways of lipolysis may play a role in the development and maintenance of these increased fat stores. A reduced catecholamine-induced lipolysis may contribute to the development and maintenance of increased adipose tissue stores. To data, a reduced hormone-sensitive lipase (HSL) expression is the best characterized defect contributing to this catecholamine resistance. The recently discovered adipose triglyceride lipase (ATGL) seems not to be involved in the catecholamine resistance of lipolysis observed in abdominal subcutaneous adipose tissue of obese subjects, which contrasts with findings in mice studies. So far, little is known on the regulation of skeletal muscle lipolysis. There is evidence of both HSL and ATGL activity and/or expression in skeletal muscle. A blunted fasting and/or catecholamine-induced lipolysis has been reported in skeletal muscle, but data require confirmation. It is tempting to speculate that an imbalance between ATGL and HSL expression results in incomplete lipolysis and increased accumulation of lipid intermediates in skeletal muscle of obese insulin resistant subjects. The latter may inhibit insulin signalling and play a role in the development of type 2 diabetes. This review summarizes the current knowledge on (intracellular) adipose tissue and skeletal muscle lipolysis in obesity, discusses the underlying mechanisms of these disturbances and will finally address the question whether disturbances in the lipolytic pathways may be primary factors in the etiology of obesity or adaptational responses to the obese insulin resistant state.     

Y4 receptor knockout rescues fertility in ob/ob mice

Hypothalamic neuropeptide Y (NPY) has been implicated in the regulation of energy balance and reproduction, and chronically elevated NPY levels in the hypothalamus are associated with obesity and reduced reproductive function. However, it is not known which one of the five cloned Y receptors mediates these effects. Here we show that crossing the Y4 receptor knockout mouse (Y4(-/-)) onto the ob/ob background restores the reduced plasma testosterone levels of ob/ob mice as well as the reduced testis and seminal vesicle size and morphology to control values. Fertility in the sterile ob/ob mice was greatly improved by Y4 receptor deletion, with 100% of male and 50% of female Y4(-/-),ob/ob double knockout mice producing live offspring. Development of the mammary ducts and lobuloalveoli was significantly enhanced in pregnant Y4(-/-) and Y4(-/-),ob/ob females. Consistent with the improved fertility and enhanced mammary gland development, gonadotropin releasing hormone (GnRH) expression was significantly increased in Y4(-/-) and Y4(-/-),ob/ob animals. Y4(-/-) mice displayed lower body weight and reduced white adipose tissue mass accompanied by increased plasma levels of pancreatic polypeptide (PP). However, Y4 deficiency had no beneficial effects to reduce body weight or excessive adiposity of ob/ob mice. These data suggest that central Y4 receptor signaling specifically inhibits reproductive function under conditions of elevated central NPY-ergic tonus.     

Norepinephrine turnover in obese (ob/ob) mice: effects of age, fasting, and acute cold

The possibility that low sympathetic nervous system (SNS) activity in brown adipose tissue (BAT) of 8-wk-old obese (ob/ob) mice results from their gross obesity at that age was investigated. Norepinephrine (NE) turnover, an estimator of SNS activity, was measured in BAT and other organs of 2-wk-old preobese ob/ob mice, and at 4 and 8 wk of age. Rates of NE turnover were 36% slower in BAT of preobese ob/ob mice than in lean littermates and remained slow in their BAT at 4 (-66%) and 8 (-56%) wk of age. In heart, rates of NE turnover were 48% slower in preobese ob/ob mice than in lean littermates, but the difference diminished at 4 (-21%) and 8 (-16%) wk of age. Rates of NE turnover in white adipose tissue, liver, and pancreas of obese mice were generally comparable with rates in these organs of lean mice. Effects of fasting (24 h) and acute cold exposure (14 degrees C for 8 h) were also examined. In general, fasting lowered and cold exposure elevated NE turnover equally in obese and lean mice. Ob/ob mice housed at 23-25 degrees C exhibit low SNS activity in their BAT prior to the onset of gross obesity, even though SNS activity in their BAT responds normally to an acute cold stress. This low SNS activity probably contributes to their subsequent high efficiency of energy retention.     

Reduced cholesterol accumulation by leptin deficient (ob/ob) mouse macrophages

Objective Although presenting many aspects of the metabolic syndrome, leptin deficient (ob/ob) mice do not spontaneously develop atherosclerosis. To examine the role of leptin in foam cell formation we analyzed ob/ob leukocyte inflammation markers and macrophage cholesterol accumulation. Methods Resident and thioglycollate (TG) elicited peritoneal cells of ob/ob and wildtype mice were studied. Activation markers, scavenger receptors (SR) and cholesterol accumulation were analyzed using flow cytometry and Taqman analysis. Cytokines, haptoglobin, adiponectin and amyloid A levels were analyzed with ELISA. Results Macrophages of ob/ob mice had reduced expression of MHC class II, CD11b, CD40, SR-A and CD36 and reduced cholesterol accumulation in vitro. Plasma haptoglobin was increased and T-cell IFNγ was reduced in ob/ob mice. Peritoneal TG instillation induced an unexpectedly weak inflammatory response in ob/ob mice. Conclusions The ob/ob mice had a reduced inflammatory response and reduced macrophage cholesterol accumulation in vitro. The data suggest decreased foam cell formation and atherosclerosis development in ob/ob mice. Received 27 January 2006; returned for revision 27 February 2006; accepted by A. Falus 27 March 2006     

A differential role for nitric oxide in two forms of physiological angiogenesis in mouse

NO plays a role in a variety of in vitro models of angiogenesis, although confounding effects of NO on non-endothelial tissues make its role during in vivo angiogenesis unclear. We therefore examined the effects of NO on two physiological models of angiogenesis in mouse skeletal muscle: (1) administration of prazosin (50 mg l⁻¹) thereby increasing blood flow; and (2) muscle overload from surgical ablation of a functional synergist. These models induce angiogenesis via longitudinal splitting and capillary sprouting, respectively. Administration of N ^G-nitro- l -arginine ( l -NNA) abolished the increase in capillary to fibre ratio (C:F) in response to prazosin administration, along with the increases in luminal filopodia and large endothelial vacuoles. l -NNA prevented luminal filopodia and vacuolisation in response to extirpation, but had no effect on abluminal sprouting, and little effect on C:F. Comparison of mice lacking endothelial (eNOS^−/−) and neuronal NO synthase (nNOS^−/−) showed that longitudinal splitting is eNOS-dependent, and Western blotting demonstrated an increase in eNOS but not inducible NOS (iNOS) expression. These data show that there are two pathways of physiological angiogenesis in skeletal muscle characterised by longitudinal splitting and capillary sprouting, respectively. NO generated by eNOS plays an essential role in splitting but not in sprouting angiogenesis, which has important implications for angiogenic therapies that target NO.     

Autophagy and Cell Death of Purkinje Cells Overexpressing Doppel in Ngsk Prnp-deficient Mice

In Ngsk prion protein (PrP)-deficient mice ( NP^0/0 ), ectopic expression of PrP-like protein Doppel (Dpl) in central neurons induces significant Purkinje cell (PC) death resulting in late-onset ataxia. NP^0/0 PC death is partly prevented by either knocking-out the apoptotic factor BAX or overexpressing the anti-apoptotic factor BCL-2 suggesting that apoptosis is involved in Dpl-induced death. In this study, Western blotting and immunohistofluorescence show that both before and during significant PC loss, the scrapie-responsive gene 1 ( Scrg1 )—potentially associated with autophagy—and the autophagic markers LC3B and p62 increased in the NP^0/0 PCs whereas RT-PCR shows stable mRNA expression, suggesting that the degradation of autophagic products is impaired in NP^0/0 PCs. At the ultrastructural level, autophagic-like profiles accumulated in somatodendritic and axonal compartments of NP^0/0 , but not wild-type PCs. The most robust autophagy was observed in NP^0/0 PC axon compartments in the deep cerebellar nuclei suggesting that it is initiated in these axons. Our previous and present data indicate that Dpl triggers autophagy and apoptosis in NP^0/0 PCs. As observed in amyloid neurodegenerative diseases, upregulation of autophagic markers as well as extensive accumulation of autophagosomes in NP^0/0 PCs are likely to reflect a progressive dysfunction of autophagy that could trigger apoptotic cascades.