Increased density of inhibitory noradrenergic parenchymal nerve fibers in hypertrophic islets of Langerhans of obese mice.

BACKGROUND AND AIM: We sought to identify mechanisms of beta cell failure in genetically obese mice. Little is known about the role of pancreatic innervation in the progression of beta cell failure. In this work we studied adrenergic innervation, in view of its potent inhibitory effect on insulin secretion. We analyzed genetically obese ob/ob and db/db mice at different ages (6- and 15-week-old), corresponding to different compensatory stages in the course of beta cell dysfunction. 15 week-old HFD mice were also studied. METHODS AND RESULTS: All mice were characterized by measures of plasma glucose, insulin, and HOMA. After perfusion, pancreata were dissected and studied by light microscopy, electron microscopy, and morphometry. Insulin, Tyrosine Hydroxylase-positive fibers and cells and Neuropeptide Y-positive cells were scored by immunohistochemistry. Islets of obese mice showed increased noradrenergic fiber innervation, with significant increases of synaptoid structures contacting beta cells compared to controls. Noradrenergic innervation of the endocrine area in obese db/db mice tended to increase with age, as diabetes progressed. In ob/ob mice, we also detected an age-dependent trend toward increased noradrenergic innervation that, unlike in db/db mice, was unrelated to glucose levels. We also observed a progressive increase in Neuropeptide Y-immunoreactive elements localized to the islet core. CONCLUSIONS: Our data show increased numbers of sympathetic nerve fibers with a potential to convey inhibitory signals on insulin secretion in pancreatic islets of genetically obese animals, regardless of their diabetic state. The findings suggest an alternative interpretation of the pathogenesis of beta cell failure, as well as novel strategies to reverse abnormalities in insulin secretion.

Time course of histomorphological changes in adipose tissue upon acute lipoatrophy.

BACKGROUND AND AIMS: Crown-like structures (CLS) are characteristic histopathology features of inflamed adipose tissues in obese mice and humans. In previous work, we suggested that these cells derived from macrophages primarily involved in the reabsorption of dead adipocytes. Here, we used a well-characterized transgenic mouse model in which the death of adipocytes in adult mice is inducible and highly synchronized. In this "FAT ATTAC" model, apoptosis is induced through forced dimerization of a caspase-8 fusion protein. METHODS AND RESULTS: 0, 0.5, 1, 2, 3 and 10 days post induction of adipocyte cell death, we analyzed mesenteric and epididymal adipose depots by histology, immunohistochemistry and electron microscopy. Upon induction of caspase-8 dimerization, numerous adipocytes lost immunoreactivity for perilipin, a marker for live adipocytes. In the same areas, we found adipocytes with hypertrophic mitochondria and signs of organelle degeneration. Neutrophils and lymphocytes were the main inflammatory cells present in the tissue, and the macrophages were predominantly Mac-2 negative. Over the course of ablation, Mac-2 positive macrophages substituted for Mac-2 negative macrophages, followed by CLS formation. All perilipin negative, dead adipocytes were surrounded by CLS structures. The time course of histopathology was similar in both fat pads studied, but occurred at earlier stages and was more gradual in mesenteric fat. CONCLUSION: Our data demonstrate that CLS formation results as a direct consequence of adipocyte death, and that infiltrating macrophages actively uptake remnant lipids of dead adipocytes. Upon induction of adipocyte apoptosis, inflammatory cells infiltrate adipose tissue initially consisting of neutrophils followed by macrophages that are involved in CLS formation.

The adipose organ of obesity-prone C57BL/6J mice is composed of mixed white and brown adipocytes.

White and brown adipocytes are believed to occupy different sites in the body. We studied the anatomical features and quantitative histology of the fat depots in obesity and type 2 diabetes-prone C57BL/6J mice acclimated to warm or cold temperatures. Most of the fat tissue was contained in depots with discrete anatomical features, and most depots contained both white and brown adipocytes. Quantitative analysis showed that cold acclimation induced an increase in brown adipocytes and an almost equal reduction in white adipocytes; however, there were no significant differences in total adipocyte count or any signs of apoptosis or mitosis, in line with the hypothesis of the direct transformation of white into brown adipocytes. The brown adipocyte increase was accompanied by enhanced density of noradrenergic parenchymal nerve fibers, with a significant correlation between the density of these fibers and the number of brown adipocytes. Comparison with data from obesity-resistant Sv129 mice disclosed a significantly different brown adipocyte content in C57BL/6J mice, suggesting that this feature could underpin the propensity of the latter strain to develop obesity. However, the greater C57BL/6J browning capacity can hopefully be harnessed to curb obesity and type 2 diabetes in patients with constitutively low amounts of brown adipose tissue.

The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.

The origin of brown adipocytes arising in white adipose tissue (WAT) after cold acclimatization is unclear. Here, we demonstrate that several UCP1-immunoreactive brown adipocytes occurring in WAT after cold acclimatization have a mixed morphology (paucilocular adipocytes). These cells also had a mixed mitochondrioma with classic "brown" and "white" mitochondria, suggesting intermediate steps in the process of direct transformation of white into brown adipocytes (transdifferentiation). Quantitative electron microscopy disclosed that cold exposure (6 degrees C for 10 days) did not induce an increase in WAT preadipocytes. beta(3)-adrenoceptor-knockout mice had a blunted brown adipocyte occurrence upon cold acclimatization. Administration of the beta(3)-adrenoceptor agonist CL316,243 induced the occurrence of brown adipocytes, with the typical morphological features found after cold acclimatization. In contrast, administration of the beta(1)-adrenoceptor agonist xamoterol increased only the number of preadipocytes. These findings indicate that transdifferentiation depends on beta(3)-adrenoceptor activation, whereas preadipocyte recruitment is mediated by beta(1)-adrenoceptor. RT-qPCR experiments disclosed that cold exposure induced enhanced expression of the thermogenic genes and of genes expressed selectively in brown adipose tissue (iBAT) and in both interscapular BAT and WAT. beta(3)-adrenoceptor suppression blunted their expression only in WAT. Furthermore, cold acclimatization induced an increased WAT expression of the gene coding for C/EBPalpha (an antimitotic protein), whereas Ccna1 expression (related to cell proliferation) was unchanged. Overall, our data strongly suggest that the cold-induced emergence of brown adipocytes in WAT predominantly reflects beta(3)-adrenoceptor-mediated transdifferentiation.

Morphological and immunohistochemical features of brown adipocytes and preadipocytes in a case of human hibernoma.

BACKGROUND AND AIM: The role of brown adipose tissue physiology and pathology in humans is debated. A greater knowledge of its developmental aspects could play a pivotal role in devising treatments for obesity and diabetes. METHODS AND RESULTS: Tissue from a rare case of hibernoma, removed from a 17-year-old boy, was examined by light and electron microscopy, morphometry and immunohistochemistry. The tumour was well vascularised and innervated and contained mature adipocytes with the characteristics of both brown and white adipocytes. Numerous, poorly differentiated cells resembling brown adipocyte precursors were seen in a pericytic position in close association with the capillary wall. On immunohistochemistry mature brown adipocytes were seen to express the marker protein UCP1. On morphometry the intensity of uncoupling protein 1 (UCP1) immunostaining varied in relation to the morphological features of adipocytes: the "whiter" their appearance, the weaker their UCP1 immunoreactivity. CONCLUSIONS: Our data suggest that in humans, as in rodents, brown adipocyte precursors arise in close association with vessel walls and that intermediate forms between white and brown adipocytes can also be documented in human adults.

Noradrenergic parenchymal nerve fiber branching after cold acclimatisation correlates with brown adipocyte density in mouse adipose organ.

The mammalian adipose organ is composed of subcutaneous and visceral depots containing white and brown adipocytes. Cold acclimatisation induces an increase in the brown component without affecting the overall number of adipocytes; this form of plasticity is associated to obesity and diabetes resistance in experimental models. Cold activates the drive of the sympathetic nervous system to the adipose organ, where the vast majority of nerve fibers are in fact noradrenergic. However, it is unclear whether and how such fibers are involved in the plastic changes of the adipose organ. We thus conducted a systematic study of the distribution and number of sympathetic noradrenergic nerve fibers in the adipose organ of mice kept at different environmental temperatures. Adult Sv129 female mice were kept at 28 degrees C or 6 degrees C for 10 days. The density of tyrosine hydroxylase (noradrenergic)-positive nerve fibers (no. of fibers per 100 adipocytes) was calculated in the subcutaneous and visceral depots of the adipose organ, and a correlation was sought between fiber density and proportion of brown adipocytes. Tyrosine hydroxylase-positive parenchymal fibers were detected in all subcutaneous and visceral depots among white as well as brown adipocytes, the mediastinal depot displaying the densest innervation. Cold acclimatisation induced a threefold increase in the total number of TH fibers in the whole organ. The proportion of brown adipocytes positively correlated with noradrenergic fiber density in the organ. Taken together, these data suggest that cold acclimatisation induces noradrenergic fiber branching in the adipose organ of adult mice, and that such changes may be a precondition for its plastic transformation into a brown phenotype.

Tumor necrosis factor in the cerebrospinal fluid of children with central nervous system leukemia.

To clarify the role of cytokines in cerebrospinal fluid (CSF) in the pathogenesis of central nervous system (CNS) leukemia, three cytokine activities, interleukin 1 (IL-1)-beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, and their correlations with other laboratory studies of the CSF were analysed in 23 children with acute leukemia. These patients were classified into three groups: group A (n = 8)--patients with overt CNS leukemia, group B (n = 5)--patients with CNS leukemia in remission, group C (n = 10)--patients without CNS disease. IFN-gamma in the CSF was undetectable in these 23 patients. There was no difference in IL-1-beta levels among the three groups. However, TNF-alpha levels were significantly higher in group A than in group B, and higher in group B than in group C. By Kendall's rank sum test, high TNF levels in CSF correlated with high CSF leukemic cell counts and low sugar levels. In two patients with overt CNS leukemia, the TNF level in the CSF decreased gradually with intrathecal chemotherapy. These results indicate that TNF released from stimulated cells in the cerebrospinal space may induce CNS leukemia-related symptoms or alter laboratory parameters measured in the CSF. TNF levels in CSF may also prove useful in diagnosing early CNS involvement in children with acute leukemia.

Cardio-facio-cutaneous (CFC) syndrome: report of two patients without hyperkeratotic skin lesions.

We report on two boys with the cardio-faciocutaneous (CFC) syndrome, but without hyperkeratotic skin involvement. They showed most of the manifestations of the CFC syndrome: growth and developmental retardation, relative macrocephaly, distinct facial appearance, sparse hair, and heart defects. Their skin was not hyperkeratotic, but patient 1 had mild atopic dermatitis and keloid-like depigmented spots.

Pigmentary dysplasias and chromosomal mosaicism: report of 9 cases.

Chromosomes were studied in 9 individuals with pigmentary dysplasias of the skin and other abnormalities. Of the 9 individuals, 5 were chromosomal mosaics in both blood lymphocytes and skin fibroblasts (46,XY/47,XY, + 13;46,XX/47,XX, + 14;46,XY/47,XY, + 18;46,XX/47,XX, + 18;46, XX/47,XX, + mar), while the other 4 individuals were chromosomally normal in both tissues studied. The pigmentary dysplasias involved hypo- or hyperpigmented patches/flecks or lines/whorls. The latter ran along Blachko lines on the back, abdomen and the limbs. These patterns varied not only between individuals but also between different regions of an individual. The possibility of chimerism was studied but ruled out (1/32 to 1/256) in 7 individuals, using chromosomal heteromorphisms in the patients and their parents as markers.

Premature aging and immunodeficiency: Mulvihill-Smith syndrome?

We report on a 30-year-old woman with premature aging, immunodeficiency, and other abnormalities. She had many manifestations of the Mulvihill-Smith syndrome, a disorder that has been described in 4 sporadic individuals, ranging in age from 4 to 17 years. The common manifestations include short stature, microcephaly, a senile face with an underdeveloped lower half, diminished facial subcutaneous fat, multiple pigmented nevi, sensorineural hearing loss, and a low IgG level. Our patient also had severe mental retardation, brachydactyly, severe T cell dysfunction, and suffered from severe verruca vulgaris and a chronic, active Epstein-Barr virus infection. The fact that her parents were first cousins suggests autosomal recessive inheritance of her disorder. Two alternative possibilities were considered: the disorder in the patient represents the Mulvihill-Smith syndrome with immune deficiency as a sign of its advanced stage, or a hitherto undescribed syndrome.

Giant hepatic granuloma caused by Bartonella henselae.

We report a 10-year-old girl with a 3.0- by 3.5-cm giant hepatic granuloma caused by Bartonella henselae. Such a solitary and large granuloma associated with B. henselae infection has not been previously reported. We believe that B. henselae infection is a consideration in the differential diagnosis of a large hepatic mass.

[Two patients with Bartonella henselae infection from a dog].

Two patients were reported as having been infected with Bartonella henselae after having contact with a dog. Both of the patients owned a dog, but had no contact with cats. One patient was a 10-year-old boy who had experienced a fever of 38-39 degrees C for 11 days, as well as having bilateral cervical lymphadenopathy. The boy's serum IgM antibodies to B. henselae were negative on the 6th and 16th day of his illness, whereas his IgG value, using indirect fluorescence antibody (IFA) method, was found to be elevated from 1:256 to 1:1,024. B. henselae DNA was detected, by PCR method, in swabs from the gingiva and buccal membrane of the dog with which the boy had been in contact. The boy was first treated with cefdinir (300 mg daily) for 6 days without beneficial effect. He responded, however, to minocycline (100 mg daily) with symptom resolution in four days. The other patient was a 64-year-old man who had experienced a fever of 38-39 degrees C for 27 days, as well as having right inguinal lymphadenopathy. The man's serum IgM antibody to B. henselae was negative, although his IgG value, determined by IFA, was 1:1,024. In addition, B. henselae DNA was detected, by PCR method, in parafin-embedded tissue obtained from the biopsied inguinal lymph nodes. The man was treated with cefazolin (2 g daily). His fever resolved, but his lymph nodes remained swollen. After a regimen of erythromycin (1,200 mg daily), the swelling in his inguinal lymphnodes gradually disappeared. Careful review of suspected CSD victims' history of contact with animals is important in making a prompt diagnosis of B. henselae infection.

[Three children with systemic cat scratch disease].

Three girls with systemic cat scratch disease, aged 10, 13 and 9 years, were reported. They presented a prolonged fever and back pain in the early stage of the disease, and had no regional lymphadenopathy. Two of them had hepatosplenic granulomas, one with multiple 5 mm hypoechoic lesions in the liver and spleen, and the other with a single 2.5 cm hypodense lesion in the left hepatic lobe. The latter patient underwent a partial left hepatic lobectomy. All patients had elevated titers of antibodies to Bartonella henselae. Polymerase chain reaction detected B. henselae DNA in tissue specimens of the patient who underwent a hepatic lobectomy. Cat scratch disease should be recognized as a cause of fever of unknown origin because the prevalence of B henselae infection might be higher in Japan.

Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation.

The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel role for Mdm2 in the initiation of adipocyte differentiation that is independent of its ability to regulate p53. We show that Mdm2 is required for cAMP-mediated induction of CCAAT/enhancer-binding protein δ (C/EBPδ) expression by facilitating recruitment of the cAMP regulatory element-binding protein (CREB) coactivator, CREB-regulated transcription coactivator (Crtc2)/TORC2, to the c/ebpδ promoter. Our findings reveal an unexpected role for Mdm2 in the regulation of CREB-dependent transactivation during the initiation of adipogenesis. As Mdm2 is able to promote adipogenesis in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination.

Lipoatrophy induced by subcutaneous insulin infusion: ultrastructural analysis and gene expression profiling.

CONTEXT AND OBJECTIVE: Subcutaneous adipose tissue (SAT) lipoatrophy (LA) is a rare complication of insulin therapy. We aimed to analyze the ultrastructural and molecular aspects of LA lesions. SETTING AND PATIENTS: Macroscopic and microscopic morphology of SAT beneath the LA areas from patients with type 1 diabetes treated with Lispro insulin by continuous sc insulin infusion was studied using magnetic resonance imaging, immunohistochemistry, electron microscopy, and quantitative PCR for adipose tissue-specific genes. RESULTS: SAT was present in LA lesions characterized by: 1) smaller, unilocular perilipin-positive adipocytes, with lipofuscin granules; 2) some "slimmed cells" losing lipid droplets as those we observed during starvation; and 3) numerous perivascular preadipocytes. We did not identify inflammatory cells. SAT in LA areas displayed a strong leptin down-regulation and an increase of AEBP1, a preadipocyte marker. CONCLUSIONS: Our results clearly indicate that the remarkable reduction in fat cell lipid droplets and adipocyte size justifies the decrease of SAT without a reduction in adipocyte number because of necrosis or apoptosis. Thus, immune cells and any other toxic damaging fat cells were not involved in the generation of LA. We speculate that adipocytes chronically exposed to high local insulin concentrations could become severely insulin resistant, dramatically increasing lipolysis and giving rise to "slimmed cells." Clinical LA regression could be explained by the active recruitment of preadipocytes, even if they were unable to differentiate and regenerate adipose tissue unless the insulin injection was removed.

Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice.

Accumulation of visceral fat is a key phenomenon in the onset of obesity-associated metabolic disorders. Macrophage infiltration induces chronic mild inflammation widely considered as a causative factor for insulin resistance and eventually diabetes. We previously showed that >90% of macrophages infiltrating the adipose tissue of obese animals and humans are arranged around dead adipocytes, forming characteristic crown-like structures (CLS). In this study we quantified CLS in visceral and subcutaneous depots from two strains of genetically obese mice, db/db and ob/ob. In both strains, CLS were prevalent in visceral compared with subcutaneous fat. Adipocyte size and CLS density exhibited a positive correlation both in visceral and in subcutaneous depots; however, the finding that adipocyte size was smallest and CLS density highest in visceral fat suggests a different susceptibility of visceral and subcutaneous adipocytes to death. Visceral fat CLS density was 3.4-fold greater in db/db than in ob/ob animals, which at the age at which our experimental strain was used are more prone to glucose metabolic disorders.

Fibroblast-specific common fragile sites induced by aphidicolin.

The distribution and frequency of aphidicolin-induced common fragile sites were studied in chromosomes of cultured skin fibroblasts and PHA-stimulated lymphocytes from five normal individuals; 0.2 microM aphidicolin was added for the last 26 h of culture. Skin fibroblasts from five fra(X)-positive patients were also studied in the same manner. Fragile sites most frequently found in fibroblasts from normal individuals were 3q26.2, 7q11.23, 16q23, 1p31, 10q11.2, 12q23 and 7q31, whereas those in lymphocytes from the same individuals were 3p14, 16q23, Xp22, 7q32 and 14q24. The distribution of fragile sites in fibroblasts from fra(X)-positive patients was essentially identical with that in normal individuals. The average number of gaps and breaks in 100 metaphases was 36.8 in fibroblasts from normal individuals, 113.8 in those from fra(X)-positive patients, and 279 in lymphocytes from normal individuals. Their rates of chromosome-type breaks and gaps were 7.9%, 29.7% and 54.5%, respectively. Thus, the distribution and frequency of aphidicolin-induced fragile sites were different between skin fibroblasts and lymphocytes, possibly reflecting differences in their DNA replication sequence or gene activity.

Cell type-dependent difference in the distribution and frequency of aphidicolin-induced fragile sites: T and B lymphocytes and bone marrow cells.

The distribution and frequency of aphidicolin-induced common fragile sites were studied in Epstein-Barr virus-transformed B lymphocytes from eight normal individuals, and in bone marrow cells from six children in remission from malignant blood diseases. PHA-stimulated helper T lymphocytes from the same individuals were also studied. These cells were cultured in MEM, and treated with 0.2 microM aphidicolin for 26 h. The results, together with those of our previous study on cultured skin fibroblasts, indicated that the distribution and frequency of aphidicolin-induced fragile sites are different among different types of cells.

Cell type-dependent difference in the distribution and frequency of excess thymidine-induced common fragile sites: T lymphocytes and skin fibroblasts.

Common fragile sites were induced by excess thymidine in phytohemagglutinin-stimulated T lymphocytes from 4 normal individuals, and skin fibroblasts from 4 normal and 5 fra(X) positive individuals. The results indicate that the frequency and distribution of excess thymidine-induced fragile sites are different between these two types of cells. The sites at 1p13 and 2p11.2, induced in both types of cells, have not previously been described, and are thus considered to be excess thymidine-specific fragile sites. These findings extend and support our previous studies on cell type-dependent difference in aphidicolin-induced common fragile sites.

Prenatal diagnosis of infantile hypophosphatasia.

Prenatal diagnosis was attempted in a pregnant Japanese woman whose son had died of infantile hypophosphatasia, using chorionic villi sampled at 10 weeks of gestation. Southern blot analysis of restriction fragment length polymorphism was used as a guide, with cDNA for the human liver-type alkaline phosphatase as a probe, and BclI as a restriction enzyme. The fetus was found to be a heterozygote; the pregnancy was allowed to continue; and the baby born was phenotypically normal.