Valerie B. Holcomb

Oestrogen alters adipocyte biology and protects female mice from adipocyte inflammation and insulin resistance

Aims: Obesity is associated with insulin resistance, liver steatosis and low‐grade inflammation. The role of oestrogen in sex differences in the above co‐morbidities is not fully understood. Our aim was to assess the role oestrogen has in modulating adipocyte size, adipose tissue oxidative stress, inflammation, insulin resistance and liver steatosis.

Deletion of Ku70, Ku80, or Both Causes Early Aging without Substantially Increased Cancer

ABSTRACT Ku70 forms a heterodimer with Ku80, called Ku, that is critical for repairing DNA double-stand breaks by nonhomologous end joining and for maintaining telomeres. Mice with either gene mutated exhibit similar phenotypes that include increased sensitivity to ionizing radiation and severe combined immunodeficiency. However, there are also differences in the reported phenotypes. For example, only Ku70 mutants are reported to exhibit a high incidence of thymic lymphomas while only Ku80 mutants are reported to exhibit early aging with very low cancer levels. There are two explanations for these differences. First, either Ku70 or Ku80 functions outside the Ku heterodimer such that deletion of one is not identical to deletion of the other. Second, divergent genetic backgrounds or environments influence the phenotype. To distinguish between these possibilities, the Ku70 and Ku80 mutations were crossed together to generate Ku70, Ku80, and double-mutant mice in the same genetic background raised in the same environment. We show that these three cohorts have similar phenotypes that most resemble the previous report for Ku80 mutant mice, i.e., early aging without substantially increased cancer levels. Thus, our observations suggest that the Ku heterodimer is important for longevity assurance in mice since divergent genetic backgrounds and/or environments likely account for these previously reported differences.

Adaptive stress response in segmental progeria resembles long-lived dwarfism and calorie restriction in mice.

How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPDG602D/R722W/XPA−/−) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80 −/− mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage.

Ku80 and p53 suppress medulloblastoma that arise independent of Rag-1-induced DSBs.

Ku80 maintains the genome by repairing DNA double-strand breaks (DSBs) through nonhomologous end joining (NHEJ), a pathway that repairs nonspecific DSBs and Rag-1 Rag-2 (Rag)-specific DSBs. As a result, Ku80 deletion results in phenotypes characteristic of defective repair for both nonspecific DSBs (γ-radiation hypersensitivity and genomic instability) and Rag-specific DSBs (immunodeficiency). ku80−/− mice also exhibit neuronal apoptosis, but we do not know the type of DSBs responsible for this response. In spite of genomic instability and immunodeficiency, cancer incidence is not increased in ku80−/− mice. However, deletion of the tumor suppressor, p53 greatly increases pro-B-cell lymphoma in ku80−/− mice due to IgH/c-Myc translocations suggesting that responses to Rag-specific DNA DSBs suppress cancer. Like suppression of pro-B-cell lymphoma, neuronal apoptosis requires p53 presenting the intriguing possibility that Rag-specific DSBs mediate neuronal development as they do lymphocyte development. Here we delete Rag-1 from ku80−/−p53−/− mice to differentiate the impact nonspecific vs Rag-specific DSBs have on ku80−/− mice. We find that deleting Rag-1 prevents pro-B cell lymphoma confirming Rag-induced DSBs induce this form of cancer. Both the triple mutant mice and the p53−/−rag-1−/− mice exhibit T-cell lymphoma and medulloblastoma; incidence of T-cell lymphoma is the same for both cohorts whereas incidence of medulloblastoma is higher for the triple-mutant cohort. Thus, p53-mediated neuronal apoptosis likely suppresses medulloblastoma in Ku80-deleted mice and Ku80 likely suppresses medulloblastoma by repairing nonspecific DNA DSBs instead of Rag-specific DSBs. Our observations are the first to show that Ku80 suppresses cancer caused by nonspecific DNA damage and we present a novel mouse model for medulloblastoma.

Alcohol Promotes Mammary Tumor Development via the Estrogen Pathway in Estrogen Receptor Alpha-Negative HER2/neu Mice

BACKGROUND Alcohol consumption is an established risk factor for breast cancer. Yet, the mechanism by which alcohol affects breast cancer development remains unresolved. The transition from the premenopausal to the postmenopausal phase is associated with a drastic reduction in systemic estrogen levels. It is not clear whether the risk of breast cancer attributable to alcohol consumption is modified by the different levels of estrogen found in pre- and postmenopausal women. The objective of this study is to determine whether the effects of alcohol on mammary tumor development are dependent on the presence of ovarian estrogen. METHODS As a model of breast cancer, we used mouse mammary tumor virus (MMTV)-neu transgenic mice that overexpress the human epidermal growth factor receptor 2 (HER2/neu) in the mammary epithelium, resulting in the development of estrogen receptor alpha (ERα)-negative mammary tumors. The mammary tumorigenesis process in these mice is similar to that of patients with HER2 breast cancer. Nonovariectomized (NOVX) and ovariectomized (OVX) MMTV-neu mice were exposed to 0, 5, and 20% ethanol in the drinking water. Breast cancer development and progression were determined alongside the effects of alcohol on estrogen availability and signaling. RESULTS Our data show that 20% alcohol consumption promoted tumor development in MMTV-neu mice only in the presence of ovarian hormones. Tumor promotion was associated with increased systemic estrogen levels, increased expression of aromatase (the rate-limiting enzyme in estrogen synthesis), and increased expression of ERα in the tumors of 20% alcohol-consuming MMTV-neu mice. Additionally, we show that ovariectomy (removal of the ovaries and ovarian hormone production) blocked the effects of 20% alcohol on tumor development. CONCLUSIONS Our results support the notion that alcohol consumption promotes HER2 breast cancer development via the estrogen signaling pathway. Additionally, they suggest that the effects of alcohol on breast cancer may be prevented by blocking estrogen signaling.

Effects of body weight and alcohol consumption on insulin sensitivity

BackgroundObesity is a risk factor for the development of insulin resistance, which can eventually lead to type-2 diabetes. Alcohol consumption is a protective factor against insulin resistance, and thus protects against the development of type-2 diabetes. The mechanism by which alcohol protects against the development of type-2 diabetes is not well known. To determine the mechanism by which alcohol improves insulin sensitivity, we fed water or alcohol to lean, control, and obese mice. The aim of this study was to determine whether alcohol consumption and body weights affect overlapping metabolic pathways and to identify specific target genes that are regulated in these pathways.MethodAdipose tissue dysfunction has been associated with the development of type-2 diabetes. We assessed possible gene expression alterations in epididymal white adipose tissue (WAT). We obtained WAT from mice fed a calorie restricted (CR), low fat (LF Control) or high fat (HF) diets and either water or 20% ethanol in the drinking water. We screened the expression of genes related to the regulation of energy homeostasis and insulin regulation using a gene array composed of 384 genes.ResultsObesity induced insulin resistance and calorie restriction and alcohol improved insulin sensitivity. The insulin resistance in obese mice was associated with the increased expression of inflammatory markers Cd68, Il-6 and Il-1α; in contrast, most of these genes were down-regulated in CR mice. Anti-inflammatory factors such as Il-10 and adrenergic beta receptor kinase 1 (Adrbk1) were decreased in obese mice and increased by CR and alcohol. Also, we report a direct correlation between body weight and the expression of the following genes: Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Lpin2 (lipin2), and Dusp9 (dual-specificity MAP kinase phosphatase 9).ConclusionWe show that alcohol consumption increased insulin sensitivity. Additionally, alterations in insulin sensitivity related with obesity were coupled with alterations in inflammatory genes. We provide evidence that alcohol may improve insulin sensitivity by up-regulating anti-inflammatory genes. Moreover, we have indentified potential gene targets in energy metabolic pathways and signal transducers that may contribute to obesity-related insulin resistance as well as calorie restriction and alcohol-induced insulin sensitivity.

Ku80 Deletion Suppresses Spontaneous Tumors and Induces a p53-Mediated DNA Damage Response

Ku80 facilitates DNA repair and therefore should suppress cancer. However, ku80(-/-) mice exhibit reduced cancer, although they age prematurely and have a shortened life span. We tested the hypothesis that Ku80 deletion suppresses cancer by enhancing cellular tumor-suppressive responses to inefficiently repaired DNA damage. In support of this hypothesis, Ku80 deletion ameliorated tumor burden in APC(MIN) mice and increased a p53-mediated DNA damage response, DNA lesions, and chromosomal rearrangements. Thus, contrary to its assumed role as a caretaker tumor suppressor, Ku80 facilitates tumor growth most likely by dampening baseline cellular DNA damage responses.

Deletion of Ku80 causes early aging independent of chronic inflammation and Rag-1-induced DSBs

Animal models of premature aging are often defective for DNA repair. Ku80-mutant mice are disabled for nonhomologous end joining; a pathway that repairs both spontaneous DNA double-strand breaks (DSBs) and induced DNA DSBs generated by the action of a complex composed of Rag-1 and Rag-2 (Rag). Rag is essential for inducing DSBs important for assembling V(D)J segments of antigen receptor genes that are required for lymphocyte development. Thus, deletion of either Rag-1 or Ku80 causes severe combined immunodeficiency (scid) leading to chronic inflammation. In addition, Rag-1 induces breaks at non-B DNA structures. Previously we reported Ku80-mutant mice undergo premature aging, yet we do not know the root cause of this phenotype. Early aging may be caused by either defective repair of spontaneous DNA damage, defective repair of Rag-1-induced breaks or chronic inflammation caused by scid. To address this issue, we analyzed aging in control and Ku80-mutant mice deleted for Rag-1 such that both cohorts are scid and suffer from chronic inflammation. We make two observations: (1) chronic inflammation does not cause premature aging in these mice and (2) Ku80-mutant mice exhibit early aging independent of Rag-1. Therefore, this study supports defective repair of spontaneous DNA damage as the root cause of early aging in Ku80-mutant mice.

Alcohol consumption promotes mammary tumor growth and insulin sensitivity

Epidemiological data show that in women, alcohol has a beneficial effect by increasing insulin sensitivity but also a deleterious effect by increasing breast cancer risk. These effects have not been shown concurrently in an animal model of breast cancer. Our objective is to identify a mouse model of breast cancer whereby alcohol increases insulin sensitivity and promotes mammary tumorigenesis. Our results from the glucose tolerance test and the homeostasis model assessment show that alcohol consumption improved insulin sensitivity. However, alcohol-consuming mice developed larger mammary tumors and developed them earlier than water-consuming mice. In vitro results showed that alcohol exposure increased the invasiveness of breast cancer cells in a dose-dependent manner. Thus, this animal model, an in vitro model of breast cancer, may be used to elucidate the mechanism(s) by which alcohol affects breast cancer.

Estrogen inhibits the effects of obesity and alcohol on mammary tumors and fatty liver.

The risk of developing breast cancer and fatty liver is increased by alcohol consumption. The objective of the present study was to determine if obesity and exogenous estrogen supplementation alter the effects of alcohol on mammary tumorigenesis and fatty liver. Ovariectomized female mice were (1) fed diets to induce overweight and obese phenotypes, (2) provided water or 20% alcohol, (3) implanted with placebo, low- or high-dose estrogen pellets and (4) injected with Met-1 mouse mammary cancer cells. Alcohol-consuming mice were more insulin sensitive and developed larger tumors than water consuming mice. Obese mice developed slightly larger tumors than control mice. Alcohol consumption and obesity increased growth factors, hepatic steatosis, activation of Akt, and inhibited the caspase-3 cascade. Estrogen treatment triggered the loss of body fat, induced insulin sensitivity, suppressed tumor growth, reduced growth factors and improved hepatic steatosis. Results show that the effects of alcohol on mammary tumor and fatty liver are modified by obesity and estrogen supplementation.