Rhonda L. Maple

Amyloid precursor protein expression is upregulated in adipocytes in obesity.

The aim of this study was to determine whether amyloid precursor protein (APP) is expressed in human adipose tissue, dysregulated in obesity, and related to insulin resistance and inflammation. APP expression was examined by microarray expression profiling of subcutaneous abdominal adipocytes (SAC) and cultured preadipocytes from obese and nonobese subjects. Quantitative real‐time PCR (QPCR) was performed to confirm differences in APP expression in SAC and to compare APP expression levels in adipose tissue, adipocytes, and stromal vascular cells (SVCs) from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) specimens. Adipose tissue samples were also examined by western blot and immunofluorescence confocal microscopy. Microarray studies demonstrated that APP mRNA expression levels were higher in SAC (∼2.5‐fold) and preadipocytes (∼1.4) from obese subjects. Real‐time PCR confirmed increased APP expression in SAC in a separate group of obese compared with nonobese subjects (P = 0.02). APP expression correlated to in vivo indices of insulin resistance independently of BMI and with the expression of proinflammatory genes, such as monocyte chemoattractant protein‐1 (MCP‐1) (R = 0.62, P = 0.004), macrophage inflammatory protein‐1α (MIP‐1α) (R = 0.60, P = 0.005), and interleukin‐6 (IL‐6) (R = 0.71, P = 0.0005). Full‐length APP protein was detected in adipocytes by western blotting and APP and its cleavage peptides, Aβ40 and Aβ42, were observed in SAT and VAT by immunofluorescence confocal microscopy. In summary, APP is highly expressed in adipose tissue, upregulated in obesity, and expression levels correlate with insulin resistance and adipocyte cytokine expression levels. These data suggest a possible role for APP and/or Aβ in the development of obesity‐related insulin resistance and adipose tissue inflammation.

Plasma Amyloid-β Peptide Levels Correlate with Adipocyte Amyloid Precursor Protein Gene Expression in Obese Individuals

Background/Aims: Several studies have demonstrated that midlife obesity increases the risk for dementia and Alzheimer’s disease. Moreover, plasma 42-amino-acid amyloid-β (Aβ42) levels appear to correlate with BMI. We recently demonstrated that adipocyte amyloid precursor protein (APP) expression is upregulated in obesity and correlates with insulin resistance and adipose tissue inflammation. In this study, we aimed to investigate the relation between adipocyte APP expression and plasma Aβ peptide levels. Methods: We conducted a pilot study in which we measured adipocyte APP gene expression and the circulating plasma levels of Aβ40 in 10 obese individuals before and after a 6-month behaviorally based weight loss intervention. Subjects had an oral glucose tolerance test with measurement of insulin levels, Aβ40 levels measured by ELISA and transcript levels of APP in subcutaneous abdominal adipocytes measured by quantitative real-time PCR. Results: At baseline, adipocyte APP expression correlated significantly with plasma Aβ40 levels and with 2-hour insulin concentrations. Following the 6-month weight loss intervention, body weight and BMI decreased significantly. Fasting plasma concentrations of glucose and insulin were improved. Adipocyte APP expression was significantly decreased (p < 0.001) after weight loss. Changes in adipocyte APP expression correlated with changes in plasma Aβ40 levels (R = 0.74, p = 0.01) and changes in 2-hour insulin (R = 0.75, p = 0.01). Conclusion: The results of this pilot study suggest that increased circulating plasma levels of Aβ peptides in obesity may be due to increased adipocyte APP gene expression. While these results suggest a possible mechanism linking midlife obesity with the later development of Alzheimer’s disease, further research is necessary to elucidate the regulation and functional significance of APP in adipocytes.

The cannabinoid CB1 receptor is expressed in pancreatic δ-cells

Antagonists of cannabinoid CB1 receptor (CB1, CNR1) promote weight loss and decrease hyperglycemia in patients with type 2 diabetes. While the endocannabinoid system may modulate islet hormone secretion, the cell-type expressing CB1 receptor in islets has not been fully resolved. In this study, we verified receptor gene expression in rodent islets and cell lines and examined the distribution of CB1 receptor in mouse, rat, and human islets by confocal immunofluorescence (IF) microscopy. IF demonstrated CB1 receptor was present in beta-cell lines, but co-localized solely with somatostatin in the islet delta-cells of Zucker rats, C57BL/6 mice, and humans; no CB1 receptor expression was observed in alpha-, beta-, or pp-cells. Similarly, a rat somatostatinoma cell line, MSL-G2-Tu6, was found to express CB1 receptor. We also found monoacylglycerol lipase (MAGL) to be expressed in delta-cells and fatty acid amide hydrolase (FAAH) to be expressed in alpha-cells. The specific expression of CB1 in delta-cells suggests that the ECS may play a role in modulating islet hormone secretion. As there are some differences between our findings and previous reports, further studies, including detailed physiological studies of the effects of the ECS on islet function, are warranted.

Progesterone stimulates DNA synthesis and lobulo-alveolar development in mammary glands in ovariectomized mice

The objective of this study was to determine whether sustained progesterone (P) use in the absence of estrogen could influence mammary development in mice. Three‐week‐old intact or ovariectomized mice were primed with subcutaneous (s.c.) cholesterol (C), estrogen (E), P, or estrogen and progesterone (E/P) together. Nine days after priming, mammary glands were removed and incubated as a whole organ in media supplemented with various combinations of lactogenic hormones. After 5 days in whole organ culture, glands were removed and end buds, alveolar buds and lobulo‐alveoli were quantified. Glands from mice primed with C or E developed significantly less lobulo‐alveoli than glands from mice primed with P or E/P. While the development was greater in animals treated with E/P compared to those treated with P, it was clear that P in the absence of E could still induce lobulo‐alveolar development. We have shown in this paper that P, in the absence of E, can stimulate cell proliferation during priming. Subsequently, the P primed glands can differentiate in response to lactogenic hormones. J. Cell. Physiol. 180:298–304, 1999.

Hypergravity disruption of homeorhetic adaptations to lactation in rat dams include changes in circadian clocks.

Summary Altered gravity load induced by spaceflight (microgravity) and centrifugation (hypergravity) is associated with changes in circadian, metabolic, and reproductive systems. Exposure to 2-g hypergravity (HG) during pregnancy and lactation decreased rate of mammary metabolic activity and increased pup mortality. We hypothesize HG disrupted maternal homeorhetic responses to pregnancy and lactation are due to changes in maternal metabolism, hormone concentrations, and maternal behavior related to gravity induced alterations in circadian clocks. Effect of HG exposure on mammary, liver and adipose tissue metabolism, plasma hormones and maternal behavior were analyzed in rat dams from mid-pregnancy (Gestational day [G]11) through early lactation (Postnatal day [P]3); comparisons were made across five time-points: G20, G21, P0 (labor and delivery), P1 and P3. Blood, mammary, liver, and adipose tissue were collected for analyzing plasma hormones, glucose oxidation to CO2 and incorporation into lipids, or gene expression. Maternal behavioral phenotyping was conducted using time-lapse videographic analyses. Dam and fetal-pup body mass were significantly reduced in HG in all age groups. HG did not affect labor and delivery; however, HG pups experienced a greater rate of mortality. PRL, corticosterone, and insulin levels and receptor genes were altered by HG. Mammary, liver and adipose tissue metabolism and expression of genes that regulate lipid metabolism were altered by HG exposure. Exposure to HG significantly changed expression of core clock genes in mammary and liver and circadian rhythms of maternal behavior. Gravity load alterations in dams circadian system may have impacted homeorhetic adaptations needed for a successful lactation.

Lipogenesis impaired in periparturient rats exposed to altered gravity is independent of prolactin and glucocorticoid secretion

Perturbed prolactin (PRL) secretion and concomitant downregulation of PRL receptor (PRLR) in periparturient dams exposed to altered gravity are linked to aberrant lipogenesis and reduced neonatal survival. PRL and glucocorticoids (GC) are known to modulate PRLR expression. We hypothesized that improving levels of PRLR would mitigate the increased gravity [hypergravity (HG)]-induced effects of impaired mammary lipogenesis and increase neonatal survival. The objective of this study was to determine if prepartum PRL or GC supplementation would override the HG-induced repression of PRLR along with lipogenic genes and increase tissue fatty acid synthesis. Pregnant rats were exposed to either 2g (HG) or kept at 1g (control) from day 11 of gestation (G11) through Postnatal day 1 (P1). HG exposed rats were supplemented with either PRL or corticosterone or a placebo from G13 to P1. On P1, mammary, liver and adipose tissues were collected to measure glucose incorporation into lipids and mRNA abundance of PRL long and short form receptors (Prlr-l, Prlr-s), glucocorticoid receptor (Nr3c1), Acetyl CoA carboxylase-α (Acaca), fatty acid synthase (Fasn), lipoprotein lipase (Lpl), Sterol Regulatory Element Binding Protein-1 (Srebp1) and protein kinase B (Akt1) genes by quantitative polymerase chain reaction (qPCR). PRL and GC supplementation had a limited effect on lipogenesis in the three tissues of HG group likely due to their inability to increase abundance of key down-regulated genes, including Prlr-l and Nr3c1. There was no difference in the abundance of genes coding for milk proteins or those associated with milk fat globule formation and secretion. These data suggest that reduced lipogenesis in HG exposed dams is independent of PRL and GC secretion but may be associated with dysregulation of multiple metabolic regulators at the level of mRNA expression.

Contents Vol. 90, 2009

93 Abstracts of the 6th Annual ENETS Conference Granada, Spain, March 5–7, 2009 147 ENETS Newsletter

Validation of Transforming Growth Factor ß-1 Binding Assay for Bovine Mammary Tissue

Active transforming growth factor-beta-1 (TGF-s1) implanted in the mammary gland prior to puberty inhibits DNA synthesis in the end buds (Daniel et al., 1989). However, implantation of the same pellet during pregnancy does not influence lobulo-alveolar development (Daniel et al., 1989). This inability of the mammary gland to respond to active TGF-sl may be regulated at the level of the receptor for TGF-s1.

Effect of Plane of Nutrition on Mammary Gland Development in Prepubertal Goats

A high plane of nutrition fed prior to puberty has been shown to negatively affect mammary gland development in heifers by decreasing secretory tissue and increasing adipose tissue (Sejrsen et al.,1982; Stelwagen and Grieve, 1990). Heifers reared on a high plane of nutrition have been shown to have depressed milk yields throughout their productive lives (Peri et al., 1992). Presently, it is not known why a high plane of nutrition inhibits mammary parenchymal development. Hormones and local growth factors play an important role in mammary gland development (Plaut, 1993). It is possible that a high plane of nutrition alters levels of hormones or growth factors that are needed for parenchymal development. Because heifers take up to 10 months to reach puberty, research in this area has been limited. Goats reach puberty 3–5 months sooner than heifers, therefore a similar study of caprine mammary development could be carried out in almost half the time. This study was conducted to test the usefulness of goats for such studies, and to test if a high plane of nutrition affects prepubertal mammary development.