Functional human to mouse adipose tissue xenotransplantation.

White adipose tissue (WAT) produces a number of metabolically important factors and, therefore, some inborn errors of metabolism may potentially be corrected by transplantation of normal allogeneic WAT. To explore the ability of human WAT (HuWAT) to compensate for a missing factor and to induce allogeneic immune response, we created leptin-deficient, immunodeficient mice and transplanted them with either 2·5 or 5 ml HuWAT. Recipient mice showed stable levels of human leptin in circulation, reduced body mass gain, and amelioration of hepatic steatosis. Food consumption and plasma insulin levels were reduced only in recipients of 5 ml WAT. Transfer of 2×10(7) human mononuclear cells to reject WAT as an allograft was ineffective and resulted only in some reduction of circulating leptin and a limited damage to the WAT grafts followed by the loss of human leukocytes.

Mapping Loci for fox domestication: deconstruction/reconstruction of a behavioral phenotype.

During the second part of the twentieth century, Belyaev selected tame and aggressive foxes (Vulpes vulpes), in an effort known as the "farm-fox experiment", to recapitulate the process of animal domestication. Using these tame and aggressive foxes as founders of segregant backcross and intercross populations we have employed interval mapping to identify a locus for tame behavior on fox chromosome VVU12. This locus is orthologous to, and therefore validates, a genomic region recently implicated in canine domestication. The tame versus aggressive behavioral phenotype was characterized as the first principal component (PC) of a PC matrix made up of many distinct behavioral traits (e.g. wags tail; comes to the front of the cage; allows head to be touched; holds observer's hand with its mouth; etc.). Mean values of this PC for F1, backcross and intercross populations defined a linear gradient of heritable behavior ranging from tame to aggressive. The second PC did not follow such a gradient, but also mapped to VVU12, and distinguished between active and passive behaviors. These data suggest that (1) there are at least two VVU12 loci associated with behavior; (2) expression of these loci is dependent on interactions with other parts of the genome (the genome context) and therefore varies from one crossbred population to another depending on the individual parents that participated in the cross.

Lipopolysaccharide inhibition of glucose production through the Toll-like receptor-4, myeloid differentiation factor 88, and nuclear factor kappa b pathway.

UNLABELLED: Acute exposure to lipopolysaccharide (LPS) can cause hypoglycemia and insulin resistance; the underlying mechanisms, however, are unclear. We set out to determine whether insulin resistance is linked to hypoglycemia through Toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappaB (NFkappaB), a cell signaling pathway that mediates LPS induction of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha). LPS induction of hypoglycemia was blocked in TLR4(-/-) and MyD88(-/-) mice but not in TNFalpha(-/-) mice. Both glucose production and glucose utilization were decreased during hypoglycemia. Hypoglycemia was associated with the activation of NFkappaB in the liver. LPS inhibition of glucose production was blocked in hepatocytes isolated from TLR4(-/-) and MyD88(-/-) mice and hepatoma cells expressing an inhibitor of NFkappaB (IkappaB) mutant that interferes with NFkappaB activation. Thus, LPS-induced hypoglycemia was mediated by the inhibition of glucose production from the liver through the TLR4, MyD88, and NFkappaB pathway, independent of LPS-induced TNFalpha. LPS suppression of glucose production was not blocked by pharmacologic inhibition of the insulin signaling intermediate phosphatidylinositol 3-kinase in hepatoma cells. Insulin injection caused a similar reduction of circulating glucose in TLR4(-/-) and TLR4(+/+) mice. These two results suggest that LPS and insulin inhibit glucose production by separate pathways. Recovery from LPS-induced hypoglycemia was linked to glucose intolerance and hyperinsulinemia in TLR4(+/+) mice, but not in TLR4(-/-) mice. CONCLUSION: Insulin resistance is linked to the inhibition of glucose production by the TLR4, MyD88, and NFkappaB pathway.

Neuroendocrine inhibition of glucose production and resistance to cancer in dwarf mice.

Pit1 null (Snell dwarf) and Proph1 null (Ames dwarf) mutant mice lack GH, PRL and TSH. Snell and Ames dwarf mice also exhibit reduced IGF-I, resistance to cancer and a longer lifespan than control mice. Endogenous glucose production during fasting is reduced in Snell dwarf mice compared to fasting control mice. In view of cancer cell dependence on glucose for energy, low endogenous glucose production may provide Snell dwarf mice with resistance to cancer. We investigated whether endogenous glucose production is lower in Snell dwarf mice during feeding. Inhibition of endogenous glucose production by glucose injection was enhanced in 12 to 14 month-old female Snell dwarf mice. Thus, we hypothesize that lower endogenous glucose production during feeding and fasting reduces cancer cell glucose utilization providing Snell dwarf mice with resistance to cancer. The elevation of circulating adiponectin, a hormone produced by adipose tissue, may contribute to the suppression of endogenous glucose production in 12 to 14 month-old Snell dwarf mice. We compared the incidence of cancer at time of death between old Snell dwarf and control mice. Only 18% of old Snell dwarf mice had malignant lesions at the time of death compared to 82% of control mice. The median ages at death for old Snell dwarf and control mice were 33 and 26 months, respectively. By contrast, previous studies showed a high incidence of cancer in old Ames dwarf mice at the time of death. Hence, resistance to cancer in old Snell dwarf mice may be mediated by neuroendocrine factors that reduce glucose utilization besides elevated adiponectin, reduced IGF-I and a lack of GH, PRL and TSH, seen in both Snell and Ames dwarf mice. Proteomics analysis of pituitary secretions from Snell dwarf mice confirmed the absence of GH and PRL, the secretion of ACTH and elevated secretion of Chromogranin B and Secretogranin II. Radioimmune assays confirmed that circulating Chromogranin B and Secretogranin II were elevated in 12 to 14 month-old Snell dwarf mice. In summary, our results in Snell dwarf mice suggest that the pituitary gland and adipose tissue are part of a neuroendocrine loop that lowers the risk of cancer during aging by reducing the availability of glucose.

PohnB6F1: a cross of wild and domestic mice that is a new model of extended female reproductive life span.

In the search for novel genetic diversity that affects the timing of life history traits, we investigated a wild-derived stock of mice (Pohn). Early generations showed extended reproductive life span; however, this phenotype diminished with propagation of the stock. Out-crossing latter generation Pohn mice to C57BL/6J (B6) mice produced PohnB6F1 hybrids with remarkably extended reproductive life spans-mean age at last litter of 647 +/- 32 days-longer than for the parental strains (70% longer than Pohn, 88% longer than B6) and longer than for highly heterogeneous crosses of laboratory mice. Litter size among young PohnB6F1 adults was similar to parental stocks, but their age-related decline in litter size was delayed by 150-200 days, resembling the earlier Pohn generations. Possibly, out-crossing Pohn mice unmasked cryptic alleles that promote extended female reproduction. This work establishes the PohnB6F1 hybrid as a new model for delayed reproductive aging.

Low utilization of circulating glucose after food withdrawal in Snell dwarf mice.

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.

Acute rejection of white adipose tissue allograft.

White adipose tissue (WAT) transplantation, although widely used in humans, has been done for cosmetic and reconstructive purposes only. Accumulating evidence indicates, however, that WAT is an important endocrine organ and, therefore, WAT transplantation may become valuable as a replacement therapy for a number of hereditary human diseases. Because the most readily available source for such transplantations would be allogeneic tissue, the mechanisms involved in the rejection of WAT allograft should be explored. We have established a model in which leptin-producing allogeneic WAT is transplanted into leptin-deficient ob/ob mice. Because ob/ob mice are obese, hyperphagic, and hypothermic, WAT allograft function is monitored as the reversal of this leptin-deficient phenotype. Here we report that allografted WAT is primarily nonfunctional. However, when WAT is transplanted into immunodeficient (Rag1-/-) ob/ob mice, or into ob/ob mice depleted of T cells by anti-CD3 antibody, a long-term graft survival is achieved as indicated by the reversal of hyperphagia, weight loss, and normalization of body temperature. The symptoms of leptin deficiency rapidly recur when normal spleen cells of the recipient type are injected, or when the antibody treatment is terminated. In contrast, selective depletion of either CD4+ or CD8+ cells alone does not prevent WAT allograft rejection. Similarly, WAT allografts that do not express MHC class I or class II molecules are rapidly rejected, suggesting that both CD4+ and CD8+ T cells may independently mediate WAT allograft rejection.

Reduced adiposity in ob/ob mice following total body irradiation and bone marrow transplantation.

OBJECTIVE: The objective of this study was to assess long-term metabolic consequences of total body irradiation (TBI) and bone marrow transplantation. Severe obesity develops due to both hypertrophy and hyperplasia of adipocytes. We hypothesized that TBI would arrest adipose tissue growth and would affect insulin resistance (IR). RESEARCH METHODS AND PROCEDURES: We exposed 2-month-old female ob/ob mice to 8 Grays of TBI followed by bone marrow transplantation and tested the animals for body weight (BW) gain, body composition, blood glucose, and insulin sensitivity. RESULTS: Two months after TBI, irradiated mice stopped gaining BW, whereas non-treated mice continued to grow. At the age of 9.5 months, body mass of irradiated mice was 60.6 +/- 1.4 grams, which was only 61% of that in non-treated ob/ob controls (99.4 +/- 1.6 grams). Body composition measurements by DXA showed that decreased BW was primarily due to an impaired fat accumulation. This could not result from the production of leptin by bone marrow-derived adipocyte progenitors because inhibition of the obese phenotype was identical in recipients of both B6 and ob/ob bone marrow. Inability of the irradiated mice to accumulate fat was associated with hepatomegaly, lower levels of monocyte chemoattractant protein-1 expression in adipose tissue, and increased IR. DISCUSSION: Our data argue in favor of the hypothesis that inability of adipose tissue to expand may increase IR. This mouse model may be valuable for studies of late-onset radiation-induced IR in humans.

Can short-term dietary restriction and fasting have a long-term anticarcinogenic effect?

Long-term dietary restriction (DR) robustly inhibits various types of carcinogenesis in rodents. Because malignancies are a major cause of death in humans, reducing the incidence or, at least, delaying the time of onset of neoplasia may significantly increase longevity of a large proportion of the human population. Long-term DR may not however be practical in humans and, judging from religious practices, several days of fasting to several weeks of DR is what a large segment of the human population can adhere to. In contrast to long-term DR, a single episode of fasting or several fasting-refeeding cycles did not have any long-lasting beneficial and usually had even a deleterious effect on carcinogenesis in rodent models. On the other hand, DR of a relatively short (1-3 months) duration often significantly increased latency and reduced the incidence of cancer over the entire life span. These results suggest that the immediate anticarcinogenic action of DR is to slow down the expansion of initiated clones, but that several months of DR may be sufficient for the elimination of a significant portion of initiated precancerous clones through apoptosis. The development of optimized DR regimens for humans will be contingent on further advances in our understanding of the mechanisms of cancer suppression by DR.

Susceptibility to induced and spontaneous carcinogenesis is increased in fatless A-ZIP/F-1 but not in obese ob/ob mice.

Obesity is typically associated with increased tumor susceptibility, whereas caloric restriction, a regimen resulting in leanness, inhibits carcinogenesis. The link between adiposity and malignancies suggests that adipose tissue may influence carcinogenesis. An adipose tissue hormone, leptin, could be procarcinogenic because it stimulates proliferation in various tissues and tumor cell lines. Leptin may contribute to the correlation between adiposity and malignancies as its levels are usually increased in obese subjects and reduced by caloric restriction. We hypothesized that leptin deficiency, despite obesity, would inhibit carcinogenesis in leptin-null ob/ob mice and tested this hypothesis in two models: (a) two-stage skin carcinogenesis initiated by 7,12-dimethylbenz(a)anthracene and promoted by phorbol 12-myristate 13-acetate (PMA) and (b) p53 deficiency. Contrary to a typical association between obesity and enhanced carcinogenesis, obese ob/ob mice developed induced skin papillomas and spontaneous p53-deficient malignancies, mostly lymphomas, similarly to their lean littermates. Surprisingly, lipodystrophic (ZIP) mice that had very little both adipose tissue and leptin were highly susceptible to carcinogenesis. Hyperphagia, hyperinsulinemia, and hyperglycemia are unlikely to have contributed significantly to the enhancement of carcinogenesis in ZIP mice because similarly hyperphagic, hyperinsulinemic, and hyperglycemic ob/ob mice had normal susceptibility to carcinogenesis. Our data suggest that, in contrast to a well-known correlation between obesity and cancer, the direct effect of adipose tissue may rather be protective.

Adipose tissue transplantation protects ob/ob mice from obesity, normalizes insulin sensitivity and restores fertility.

Adipose tissue affects metabolism by secreting various adipokines. Lipodystropic mice benefit both from leptin replacement therapy and from transplantation of normal fat. Leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice can also be treated with leptin. Surprisingly, there have been no reports of successful treatment of obese ob/ob mice by transplantation of normal white adipose tissue (WAT). If WAT transplantation is ineffective in treating insulin resistance and diabetes in obese individuals, its applicability may be limited in humans as such abnormalities are usually associated with obesity. In the current study, we tested whether WAT transplantation might prevent, and even reverse, abnormalities characteristic of ob/ob mice. To assess the preventive potential, 6-week-old ob/ob mice were transplanted, subcutaneously, with gonadal fat pads from normal mice. Profound effects on multiple physiological phenotypes were achieved despite leptin levels below 25% of those in control mice. WAT from one donor reduced body weight gain, and WAT from 4 or 8 donors prevented obesity in ob/ob mice. Nonfasting insulin levels and insulin tolerance test were normalized. Corticosterone elevation was also prevented. Finally, WAT from 4 donors restored fertility in ob/ob females. The effects of WAT transplantation were long-lasting, with body weight gain suppressed for at least 40 weeks. To assess the therapeutic potential, obese 13-month-old ob/ob mice with a long history of leptin deficiency were used. Their body weight decreased by approximately 50% when transplanted with WAT from 8 donors. As in young recipients, transplantation greatly reduced nonfasting insulin, suggesting normalized insulin sensitivity. Thus, WAT transplantation was effective for both prevention and therapy. In the future, WAT transplantation may become a useful alternative to hormone replacement in treating not only lipodystropy, but also certain types of obesity.

Sexual differentiation, pregnancy, calorie restriction, and aging affect the adipocyte-specific secretory protein adiponectin.

Adiponectin or adipocyte complement-related protein of 30 kDa (Acrp30) is a circulating protein produced exclusively in adipocytes. Circulating Acrp30 levels have been associated with insulin sensitivity in adult mice and humans, yet the Acrp30 profile over the lifespan and its hormonal regulation in vivo have not been previously described. Hence, we set forth to determine whether hormonal and metabolic changes associated with sexual maturation, reproduction, aging, and calorie restriction affect Acrp30. In mice, Acrp30 levels increase during sexual maturation by 4-fold in males and 10-fold in females. Neonatal castration (CX) allows Acrp30 of adults to reach female levels. CX in adults does not lead to female Acrp30 levels unless glucocorticoid exposure is elevated simultaneously by implant. Ovariectomy of infant mice does not interfere with the pubertal rise of Acrp30. However, ovariectomy in adults increases Acrp30. Estrogen suppressed Acrp30 in mice and 3T3-L1 adipocytes. In parallel to changes in estrogen action, Acrp30 decreased in late gestation but increased in both calorie-restricted and old (anovulatory) mice. The reduction of Acrp30 in lactating dams is consistent with a suppressive effect of prolactin and a stimulating effect of bromocriptine. In summary, Acrp30 levels in serum are under complex hormonal control and may play a key role in determining systemic insulin sensitivity under the respective conditions.

Assessing effects of long-term food restriction on myocardial energetics in the isolated heart preparation.

Food restriction (FR) may increase longevity by increasing the efficiency of energy utilization by some organs. We tested whether any effect of FR on the energy efficiency of isolated, isovolumically beating hearts could be observed, by studying four groups of rats: (1). AL fed (AL) 10-13-month-old rats, (2). age matched, FR at 60% of AL rats, (3). young AL, heart weight matched to FR rats and (4). 10-13-month-old AL rats, short-term FR for the last 3 weeks of life. The oxygen cost of tension development was not different among the groups. With contractility changed by calcium, the oxygen cost of contractility was higher in the young AL than in the adult rats either AL or short-term FR. With isoproterenol, it was higher in FR than in AL groups. The basal metabolic rate of hearts was higher in the adult AL than in the short-term, but not long-term, FR rats. In the long run, FR did not significantly change the pattern of cardiac energy utilization of isolated, isovolumically beating hearts. Our observations do not lend support to the hypothesis that the anti-aging action of FR is mediated by changes in cardiac efficiency.