PAT proteins (perilipin, adipophilin, and TIP47) are hypothesized to be critical regulators of lipid accumulation in eukaryotic cells. We investigated the developmental relationships between the expression of these proteins and cytoplasmic lipid droplet (CLD) accumulation in differentiating secretory epithelial cells in mouse mammary glands. Adipophilin (ADPH) specifically localized to CLD in differentiating and lactating mammary glands and was found exclusively in the secreted lipid droplet fraction of mouse milk. ADPH transcripts were selectively detected in secretory epithelial cells, and steady-state levels of both ADPH mRNA and protein increased during secretory differentiation in patterns consistent with functional linkage to CLD accumulation. TIP47 also was detected in secretory epithelial cells; however, it had a diffuse punctate appearance, and its mRNA and protein expression patterns did not correlate with CLD accumulation. Perilipin-positive adipose cells and steady-state levels of perilipin mRNA and protein decreased during mammary gland differentiation, suggesting a progressive loss of adipose lipid storage during this process.Collectively, these data demonstrate that increased ADPH expression is a specialized property of differentiated secretory epithelial cells and provide developmental evidence specifically linking increased ADPH expression to increased CLD accumulation. In addition, evidence is presented that the epithelial and adipose compartments of the mammary gland undergo concerted, developmentally regulated shifts in lipid metabolism that increase the availability of fatty acids necessary for lipid synthesis by milk-secreting cells.-

Xanthine oxidoreductase (XOR), a key enzyme of purine metabolism, has been implicated in the secretion of the milk fat droplet in lactating mammary epithelial cells, possibly through structural interactions with other milk fat globule proteins including butyrophilin (Btn) and adipophilin (ADPH). To help determine the mechanism by which XOR is regulated, we examined the expression and localization of XOR in the non‐secretory states of late pregnancy and induced involution compared with the state of active secretion. XOR mRNA levels started to increase at mid‐pregnancy, turned sharply upwards at the onset of lactation and decreased rapidly with forced involution, indicating transcriptional control of the enzyme level by differentiation and secretory function. During pregnancy and involution the enzyme was diffusely distributed in the cytoplasm, but moved rapidly to the apical membrane of the cells when secretion was activated, where it colocalized with both Btn and ADPH, similar to the situation in the milk fat globule itself. Size‐exclusion chromatography of solubilized milk fat globule membrane proteins showed that XOR formed a sulphydryl‐bond‐dependent complex with Btn and ADPH in the milk fat globule membrane. XOR returned to a diffuse cytoplasmic localization shortly after induced involution, while Btn remained localized to the apical membrane, suggesting that localization of XOR is not dependent on the presence of Btn in the apical membrane. Our findings indicate that the expression and membrane association of XOR in the mammary gland are tightly regulated by secretory activity, and suggest that the apical membrane association of XOR regulates the coupling of lipid droplets to the apical plasma membrane during milk lipid secretion.

Nutrition education and counseling for the purposes of reducing caries in children is aimed at teaching parents the importance of reducing high frequency exposures to obvious and hidden sugars. Guidelines include: avoiding frequent consumption of juice or other sugar-containing drinks in the bottle or sippy cup, discouraging the behavior of a child sleeping with a bottle, promoting noncariogenic foods for snacks, fostering eating patterns consistent with the Food Guide Pyramid, limiting cariogenic foods to mealtimes, rapidly clearing cariogenic foods from the child's oral cavity either by toothbrushing or by consumption of protective foods, and restricting sugar-containing snacks that are slowly eaten (e.g., candy, cough drops, lollipops, suckers). Along with nutritional factors, a comprehensive approach to preventing dental caries in preschool children must include improved general dietary habits, good oral hygiene, appropriate use of fluorides, and access to preventive and restorative dental care.

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CCAAT/enhancer-binding protein ␤ (C/EBP␤) plays a key role in initiation of adipogenesis in adipose tissue and gluconeogenesis in liver; however, the role of C/EBP␤ in hepatic lipogenesis remains undefined. Here we show that C/EBP␤ inactivation in Lepr db/db mice attenuates obesity, fatty liver, and diabetes. In addition to impaired adipogenesis, livers from C/EBP␤ ؊/؊ x Lepr db/db mice had dramatically decreased triglyceride content and reduced lipogenic enzyme activity. C/EBP␤ deletion in Lepr db/db mice down-regulated peroxisome proliferator-activated receptor ␥2 (PPAR␥2) and stearoyl-CoA desaturase-1 and up-regulated PPAR␣ independent of SREBP1c. Conversely, C/EBP␤ overexpression in wild-type mice increased PPAR␥2 and stearoyl-CoA desaturase-1 mRNA and hepatic triglyceride content. In FAO cells, overexpression of the liver inhibiting form of C/EBP␤ or C/EBP␤ RNA interference attenuated palmitate-induced triglyceride accumulation and reduced PPAR␥2 and triglyceride levels in the liver in vivo. Leptin and the anti-diabetic drug metformin acutely down-regulated C/EBP␤ expression in hepatocytes, whereas fatty acids up-regulate C/EBP␤ expression. These data provide novel evidence linking C/EBP␤ expression to lipogenesis and energy balance with important implications for the treatment of obesity and fatty liver disease.Obesity is the most common nutritional disorder in Western societies. Today in the United States, more than 60% of people are either overweight (body mass index (BMI) Ͼ 25) or obese (BMI Ͼ 30) (1). Obesity is frequently associated with type II diabetes, hypertension, and hyperlipidemia, all known risk factors for cardiovascular disease (2). Obesity is also a major risk factor for non-alcoholic fatty liver disease, one of the most common emerging liver diseases in Western countries coinciding with the worldwide obesity epidemic (3, 4). The underlying transcriptional events that contribute to obesity and its associated disorders are not well understood. Some of the genes that regulate body weight have been identified as well as additional neuropeptides, hormones, and nutritional factors that play a role in body weight regulation, particularly through the ␤-adrenergic system (5, 6). Discovery of the hormone leptin and its receptors, which suppress appetite and reduce fat mass, has dramatically increased our understanding of the regulation of energy balance (7,8). More recently, the study of specific transcription factor genes and their metabolism has provided powerful new tools for understanding the integrated mechanisms underlying obesity and diabetes (9 -11). This is most elegantly illustrated using tissue-specific gene knockouts and overexpression models to elucidate the mechanism of action of the PPAR 5 family of nuclear hormone receptors (12). The CCAAT/enhancer-binding protein (C/EBP) family includes five nuclear transcription factors, C/EBP ␣, ␤, ␥, ␦, and ⑀, encoded by separate genes located on different chromosomes (13,14). Collectively, C/EBPs are expressed across a variety of cell types, and...

Adipophilin (ADPH), a member of the perilipin family of lipid droplet-associated proteins, is hypothesized to mediate milk lipid formation and secretion. Unexpectedly, the fat content of milk from ADPH-null mice was only modestly lower than that of wild-type controls, and neither TIP47 nor perilipin appeared to fully compensate for ADPH loss. This prompted us to investigate the possibility that the mutated ADPH gene was not a genuine null mutation. ADPH transcripts were detected in ADPH-null mammary tissue by quantitative real-time PCR, and C-terminal-specific, but not N-terminal-specific, ADPH antibodies detected a single lower molecular weight product and immunostained cytoplasmic lipid droplets (CLDs) and secreted milk fat globules in ADPH-null mammary tissue. Furthermore, stable cell lines expressing cDNA constructs corresponding to the ADPH-null mutation produced a product comparable in size to the one detected in ADPH-null mammary glands and localized to CLDs. Based on these data, we conclude that ADPH-null mice express an N-terminally truncated form of ADPH that retains the ability to promote the formation and secretion of milk lipids.-Russell, T.

During pregnancy the mammary epithelium and its supporting vasculature rapidly expand to prepare for lactation, resulting in dramatic changes in the microenvironment. In order to investigate the role of oxygenation and metabolism in these processes, the oxygen-responsive component of the hypoxia-inducible factor (HIF) 1 complex, HIF1α, was deleted in the murine mammary gland. Although vascular density was unchanged in the HIF1α null mammary gland, loss of HIF1α impaired mammary differentiation and lipid secretion, culminating in lactation failure and striking changes in milk composition. Transplantation experiments confirmed that these developmental defects were mammary epithelial cell autonomous. These data make clear that HIF1α plays a critical role in the differentiation and function of the mammary epithelium.

SummaryMilk lipids originate by secretion of triglyceride-rich cytoplasmic lipid droplets (CLDs) from mammary epithelial cells. Adipophilin (ADPH)/Plin2, a member of the perilipin family of CLD binding proteins, is hypothesized to regulate CLD production in these cells during differentiation of the mammary gland into a secretory organ. We tested this hypothesis by comparing CLD accumulation in differentiating mammary glands of wild-type and ADPH-deficient mice. ADPH deficiency did not prevent CLD formation; however, it disrupted the increase in CLD size that normally occurs in differentiating mammary epithelial cells. Failure to form large CLDs in ADPH-deficient mice correlated with localization of adipose triglyceride lipase (ATGL) to the CLD surface, suggesting that ADPH promotes CLD growth by inhibiting lipolytic activity. Significantly, mammary alveoli also failed to mature in ADPH-deficient mice, and pups born to these mice failed to survive. The possibility that CLD accumulation and alveolar maturation defects in ADPH-deficient mice are functionally related was tested by in vivo rescue experiments. Transduction of mammary glands of pregnant ADPH-deficient mice with adenovirus encoding ADPH as an N-terminal GFP fusion protein prevented ATGL from localizing to CLDs and rescued CLD size and alveolar maturation defects. Collectively, these data provide direct in vivo evidence that ADPH inhibition of ATGL-dependent lipolysis is required for normal CLD accumulation and alveolar maturation during mammary gland differentiation. We speculate that impairing CLD accumulation interferes with alveolar maturation and lactation by disrupting triglyceride homeostasis in mammary epithelial cells.