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Regulation of diurnal and circadian rhythms and cell proliferation are coupled in all mammals, including humans. However, the molecular mechanisms by which diurnal and circadian rhythms regulate cell proliferation are relatively poorly understood. In this study, we report that tumor growth in nude rats bearing human steroid receptor-negative MCF-7 breast tumors can be significantly accelerated by exposing the rats to light at night (LAN). Under normal conditions of an alternating light/dark cycle, proliferating cell nuclear antigen (PCNA) levels in tumors were maximal in the early light phase but remained at very low levels throughout the daily 24-hour cycle period monitored. Surprisingly, PCNA was expressed in tumors continually at a high level throughout the entire 24-hour period in LAN-exposed nude rats. Daily fluctuations of Akt and mitogen activated protein kinase activation in tumors were also disrupted by LAN. These fluctuations did not track with PCNA changes, but we found that activation of the Akt stimulatory kinase phosphoinositide-dependent protein kinase 1 (PDK1) directly correlated with PCNA levels. Expression of insulin-like growth factor 1 receptor (IGF-1R), an upstream signaling molecule for PDK1, also correlated with fluctuations of PDK1/PCNA in the LAN group. In addition, circulating IGF-1 concentrations were elevated in LAN-exposed tumor-bearing nude rats. Finally, RNAi-mediated knockdown of PDK1 led to a reduction in PCNA expression and cell proliferation in vitro and tumor growth in vivo, indicating that PDK1 regulates breast cancer growth in a manner correlated with PCNA expression. Taken together, our findings demonstrate that LAN exposure can accelerate tumor growth in vivo, in part through continuous activation of IGF-1R/PDK1 signaling. Cancer Res; 71(7); 2622–31. ©2011 AACR.

Supplementary Figure 1 from Light at Night Activates IGF-1R/PDK1 Signaling and Accelerates Tumor Growth in Human Breast Cancer Xenografts

nonalcoholic fatty liver disease (NAFLD), an obesity and insulin resistance associated clinical condition - ranges from simple steatosis to nonalcoholic steatohepatitis. To model the human condition, a high-fat Western diet that includes liquid sugar consumption has been used in mice. Even though liver pathophysiology has been well characterized in the model, little is known about the metabolic phenotype (e.g., energy expenditure, activity, or food intake). Furthermore, whether the consumption of liquid sugar exacerbates the development of glucose intolerance, insulin resistance, and adipose tissue dysfunction in the model is currently in question. In our study, a high-fat Western diet (HFWD) with liquid sugar [fructose and sucrose (F/S)] induced acute hyperphagia above that observed in HFWD-fed mice, yet without changes in energy expenditure. Liquid sugar (F/S) exacerbated HFWD-induced glucose intolerance and insulin resistance and impaired the storage capacity of epididymal white adipose tissue (eWAT). Hepatic TG, plasma alanine aminotransferase, and normalized liver weight were significantly increased only in HFWD+F/S-fed mice. HFWD+F/S also resulted in increased hepatic fibrosis and elevated collagen 1a2, collagen 3a1, and TGFβ gene expression. Furthermore, HWFD+F/S-fed mice developed more profound eWAT inflammation characterized by adipocyte hypertrophy, macrophage infiltration, a dramatic increase in crown-like structures, and upregulated proinflammatory gene expression. An early hypoxia response in the eWAT led to reduced vascularization and increased fibrosis gene expression in the HFWD+F/S-fed mice. Our results demonstrate that sugary water consumption induces acute hyperphagia, limits adipose tissue expansion, and exacerbates glucose intolerance and insulin resistance, which are associated with NAFLD progression.

Highlights • Rural families are at risk for diet-related diseases because of food disparities. • An online produce market increases redemption of produce prescriptions. • Photovoice illuminates the benefits and limits of produce prescription programs. • Produce prescriptions, online produce market, and cooking lessons affect behavior. • Fruit and vegetable consumption increases for families when they cook together., Despite being surrounded by farms, rural families are often at risk for diet-related diseases because of food disparities. Barriers such as distance and transportation to a full service grocery store, lack of cooking knowledge and skills, and the cost of fresh produce, force families to shop at convenience stores that carry predominantly unhealthy, processed foods. We combined a Fruit and Vegetable Prescription Program (F&VRx) with family cooking/nutrition classes and a pilot online produce shopping to promote lifestyle dietary changes in rural families. In 2 rural upstate New York communities school health staff referred low income families with one or more children at risk for chronic disease related to obesity for this pilot program. Each family was given a weekly online produce credit for 5 months, September/October 2017 through January/February 2018. Two monthly nutrition/cooking lessons were provided for the entire family. Evaluation was done using pre and post program surveys and Photovoice. Families took photos in response to the question “How has the F&VRx program affected my family?”. Redemption of online produce credit was 94% and class attendance was 80%. Fruit and vegetable consumption rose for children. Confidence, culinary skills, and food literacy increased slightly. Three months after program completion, 60% of the families continued weekly online produce shopping without the F&VRx. Healthy behaviors for shopping, preparing, and consuming fruits and vegetables can be increased using a F&VRx, online produce ordering, and family cooking classes.

The association between a Western Diet and colon cancer suggests that dietary factors and/or obesity may contribute to cancer progression. Our objective was to develop a new animal model of obesity and the associated pathophysiology to investigate human cancer independent of dietary components that induce obesity. A novel congenic rat strain was established by introducing the fa allele from the Zucker rat into the Rowett Nude rat to generate a "fatty nude rat". The obese phenotype was first characterized in the new model. To then examine the utility of this model, lean and obese rats were implanted with HT-29 human colon cancer xenografts and tumor growth monitored. Fatty nude rats were visibly obese and did not develop fasting hyperglycemia. Compared to lean rats, fatty nude rats developed fasting hyperinsulinemia, glucose intolerance, and insulin resistance. Colon cancer tumor growth rate and final weight were increased (P0.05) in fatty nude compared to lean rats. Final tumor weight was associated with p38 kinase phosphorylation (P0.01) in fatty nude rats. We have established a novel model of obesity and pre-type 2 diabetes that can be used to investigate human cancer and therapeutics in the context of obesity and its associated pathophysiology.

Strong epidemiologic evidence links colon cancer to obesity. The increasing worldwide incidence of colon cancer has been linked to the spread of the Western lifestyle, and in particular consumption of a high-fat Western diet. In this study, our objectives were to establish mouse models to examine the effects of high-fat Western diet-induced obesity on the growth of human colon cancer tumor xenografts, and to examine potential mechanisms driving obesity-linked human colon cancer tumor growth. We hypothesize that mice rendered insulin resistant due to consumption of a high-fat Western diet will show increased and accelerated tumor growth. Homozygous Rag1tm1Mom mice were fed either a low-fat Western diet or a high-fat Western diet (HFWD), then human colon cancer xenografts were implanted subcutaneously or orthotopically. Tumors were analyzed to detect changes in receptor tyrosine kinase-mediated signaling and expression of inflammatory-associated genes in epididymal white adipose tissue. In both models, mice fed an HFWD weighed more and had increased intra-abdominal fat, and tumor weight was greater compared with in the low-fat Western diet-fed mice. They also displayed significantly higher levels of leptin; however, there was a negative correlation between leptin levels and tumor size. In the orthotopic model, tumors and adipose tissue from the HFWD group displayed significant increases in both c-Jun N-terminal kinase activation and monocyte chemoattractant protein 1 expression, respectively. In conclusion, this study suggests that human colon cancer growth is accelerated in animals that are obese and insulin resistant due to the consumption of an HFWD.

Regulation of diurnal and circadian rhythms and cell proliferation are coupled in all mammals, including humans. However, the molecular mechanisms by which diurnal and circadian rhythms regulate cell proliferation are relatively poorly understood. In this study, we report that tumor growth in nude rats bearing human steroid receptor-negative MCF-7 breast tumors can be significantly accelerated by exposing the rats to light at night (LAN). Under normal conditions of an alternating light/dark cycle, proliferating cell nuclear antigen (PCNA) levels in tumors were maximal in the early light phase but remained at very low levels throughout the daily 24-hour cycle period monitored. Surprisingly, PCNA was expressed in tumors continually at a high level throughout the entire 24-hour period in LAN-exposed nude rats. Daily fluctuations of Akt and mitogen activated protein kinase activation in tumors were also disrupted by LAN. These fluctuations did not track with PCNA changes, but we found that activation of the Akt stimulatory kinase phosphoinositide-dependent protein kinase 1 (PDK1) directly correlated with PCNA levels. Expression of insulin-like growth factor 1 receptor (IGF-1R), an upstream signaling molecule for PDK1, also correlated with fluctuations of PDK1/PCNA in the LAN group. In addition, circulating IGF-1 concentrations were elevated in LAN-exposed tumor-bearing nude rats. Finally, RNAi-mediated knockdown of PDK1 led to a reduction in PCNA expression and cell proliferation in vitro and tumor growth in vivo, indicating that PDK1 regulates breast cancer growth in a manner correlated with PCNA expression. Taken together, our findings demonstrate that LAN exposure can accelerate tumor growth in vivo, in part through continuous activation of IGF-1R/PDK1 signaling. Cancer Res; 71(7); 2622–31. ©2011 AACR.

Hepatic steatosis can progress to the clinical condition of non-alcoholic steatohepatitis (NASH), which is a precursor of more serious liver diseases. The novel PKC isoforms δ and ε are activated by lipid metabolites and have been implicated in lipid-induced hepatic disease. Using a methionine- and choline-deficient (MCD) dietary model of NASH, we addressed the question of whether hepatic PKCδ and PKCε are activated. With progression from steatosis to steatohepatitis, there was activation and increased PKCδ protein content coincident with hepatic endoplasmic reticulum (ER) stress parameters. To examine whether similar changes could be induced in vitro, McA-RH 7777 (McA) hepatoma cells were used. We observed that McA cells stored triglyceride and released alanine aminotransferase (ALT) when treated with MCD medium in the presence of fatty acids. Further, MCD medium with palmitic acid, but not oleic or linoleic acids, maximally activated PKCδ and stimulated ER stress. In PKCδ knockdown McA cells, MCD/fatty acid medium-induced ALT release and ER stress induction were completely blocked, but triglyceride storage was not. In addition, a reduction in the uptake of propidium iodide and the number of apoptotic nuclei and a significant increase in cell viability and DNA content were observed in PKCδ knockdown McA cells incubated in MCD medium with palmitic acid. Our studies show that PKCδ activation and protein levels are elevated in an animal model of steatohepatitis, which was recapitulated in a cell model, supporting the conclusion that PKCδ plays a role in ALT release, the ER stress signal, and cell death.

TNFalpha plays key roles in the regulation of inflammation, cell death, and proliferation and its signaling cascade cross-talks with the insulin signaling cascade. PKCdelta, a novel PKC isoform, is known to participate in proximal TNFalpha signaling events. However, it has remained unclear whether PKCdelta plays a role in distal TNFalpha signaling events. Here we demonstrate that PKCdelta is activated by TNFalpha in a delayed fashion that is temporally associated with JNK activation. To investigate the signaling pathways activating PKCdelta and JNK, we used pharmacological and genetic inhibitors of NFkappaB. We found that inhibition of NFkappaB attenuated PKCdelta and JNK activations. Further analysis revealed that ER stress contributes to TNFalpha-stimulated PKCdelta and JNK activations. To investigate the role of PKCdelta in TNFalpha action, we used 29-mer shRNAs to silence PKCdelta expression. A reduction of ~90% in PKCdelta protein levels reduced TNFalpha-stimulated stress kinase activation, including JNK. Further, PKCdelta was necessary for thapsigargin-stimulated JNK activation. Because thapsigargin is a potent inducer of ER stress, we determined whether PKCdelta was necessary for induction of the UPR. Indeed, a reduction in PKCdelta protein levels reduced thapsigargin-stimulated CHOP induction, a hallmark of the UPR, but not BiP/GRP78 induction, suggesting that PKCdelta does not globally regulate the UPR. Next, the role of PKCdelta in TNFalpha mediated cross-talk with the insulin signaling pathway was investigated in cells expressing human IRS-1 and a 29-mer shRNA to silence PKCdelta expression. We found that a reduction in PKCdelta protein levels reversed the TNFalpha-mediated reduction in insulin-stimulated IRS-1 Tyr phosphorylation, Akt activation, and glycogen synthesis. In addition, TNFalpha-stimulated IRS protein Ser/Thr phosphorylation and degradation were blocked. Our results indicate that: 1) NFkappaB and ER stress contribute in part to PKCdelta activation; 2) PKCdelta plays a key role in the propagation of the TNFalpha signal; and 3) PKCdelta contributes to TNFalpha-induced inhibition of insulin signaling events.

Previous reports have demonstrated that normal serum can increase the rate of adipocyte lipolysis in vitro. However, the nature of the lipolytic activity has remained obscure. We have investigated the lipolytic activity of human serum using isolated rat adipocytes. Human serum resulted in a dose-dependent stimulation of lipolysis (glycerol release) in adipocytes, with a half-maximal effective dose of 0.05% serum and with maximal stimulation with 1% serum. The effect of serum on glycerol release was rapid (within 30 min), and the effect was reversible. Partial purification of this lipolytic activity using gel filtration and ion-exchange chromatography demonstrates that a protein of approximately 80 kDa contributes to the lipolytic activity. Human transferrin mimicked the activity of partially purified serum, resulting in a maximal 50% increase in basal lipolysis. In addition, ferrous sulfate heptahydrate induced a biphasic increase in the rate of lipolysis, with a maximal increase of 50% at approximately 0.6 microg/ml iron. Inhibitors of protein kinase A (H89) and mitogen-activated protein kinase (PD98059) did not block the effect of serum on lipolysis, whereas the free radical scavenger N-acetyl-l-cysteine completely inhibited the effect. These findings suggest that the stimulatory effect of serum on lipolysis is in part mediated by iron, probably through a prooxidant mechanism.

PKCδ has been linked to key pathophysiological features of non-alcoholic fatty liver disease (NAFLD). Yet, our knowledge of PKCδ's role in NAFLD development and progression in obese models is limited. PKCδ(-/-)/Lepr(db)(/)(db) mice were generated to evaluate key pathophysiological features of NAFLD in mice. Hepatic histology, oxidative stress, apoptosis, gene expression, insulin signaling, and serum parameters were analyzed in Lepr(db)(/)(db) and PKCδ(-/-)/Lepr(db)(/)(db) mice. The absence of PKCδ did not abrogate the development of obesity in Lepr(db)(/)(db) mice. In contrast, serum triglyceride levels and epididymal white adipose tissue weight normalized to body weight were reduced in PKCδ(-/-)/Lepr(db)(/)(db) mice compared Lepr(db)(/)(db) mice. Analysis of insulin signaling in mice revealed that hepatic Akt and GSK3β phosphorylation were strongly stimulated by insulin in PKCδ(-/-)/Lepr(db)(/)(db) compared Lepr(db)(/)(db) mice. PKCδ may be involved in the development of obesity-associated NAFLD by regulating hepatic lipid metabolism and insulin signaling.

Insulin resistance can arise when pathological levels of free fatty acids (FFAs) and proinflammatory cytokines disrupt insulin signaling. Protein kinase C delta (PKCδ) is a FFA- and a proinflammatory cytokine-regulated protein kinase that is associated with inhibition of insulin signaling and action. To gain insight into the role of PKCδ in insulin resistance, PKCδ activation was studied in a genetic model of obesity-linked insulin resistance. PKCδ was found to be activated in the liver of obese insulin-resistant Zucker rats and in isolated cultured hepatocytes. PKCδ was further studied in PKCδ-null mice and their wild-type littermates fed a high-fat or control diet for 10 weeks. PKCδ-null mice on a high-fat diet had improved insulin sensitivity and hepatic insulin signaling compared to wild-type littermates. Additionally, the deleterious effect of a high-fat diet on glucose tolerance in wild-type mice was completely blocked in PKCδ-null mice. To directly test the role of PKCδ in cellular insulin resistance, primary hepatocytes from the high-fat diet mice were isolated and stimulated with insulin. Primary hepatocytes from PKCδ-null mice had improved insulin-stimulated Akt and FOXO phosphorylation compared to hepatocytes from wild-type littermates. Consistent with this result, tumor necrosis factor alpha-mediated inhibition of insulin signaling was blocked in PKCδ knockdown primary hepatocytes. These results indicate that PKCδ plays a role in insulin resistance and is consistent with the hypothesis that PKCδ is a negative regulator of insulin signaling and thus may be a therapeutic target for the treatment of type 2 diabetes.

TNFalpha plays key roles in the regulation of inflammation, cell death, and proliferation and its signaling cascade cross-talks with the insulin signaling cascade. PKCdelta, a novel PKC isoform, is known to participate in proximal TNFalpha signaling events. However, it has remained unclear whether PKCdelta plays a role in distal TNFalpha signaling events. Here we demonstrate that PKCdelta is activated by TNFalpha in a delayed fashion that is temporally associated with JNK activation. To investigate the signaling pathways activating PKCdelta and JNK, we used pharmacological and genetic inhibitors of NFkappaB. We found that inhibition of NFkappaB attenuated PKCdelta and JNK activations. Further analysis revealed that ER stress contributes to TNFalpha-stimulated PKCdelta and JNK activations. To investigate the role of PKCdelta in TNFalpha action, we used 29-mer shRNAs to silence PKCdelta expression. A reduction of ~90% in PKCdelta protein levels reduced TNFalpha-stimulated stress kinase activation, including JNK. Further, PKCdelta was necessary for thapsigargin-stimulated JNK activation. Because thapsigargin is a potent inducer of ER stress, we determined whether PKCdelta was necessary for induction of the UPR. Indeed, a reduction in PKCdelta protein levels reduced thapsigargin-stimulated CHOP induction, a hallmark of the UPR, but not BiP/GRP78 induction, suggesting that PKCdelta does not globally regulate the UPR. Next, the role of PKCdelta in TNFalpha mediated cross-talk with the insulin signaling pathway was investigated in cells expressing human IRS-1 and a 29-mer shRNA to silence PKCdelta expression. We found that a reduction in PKCdelta protein levels reversed the TNFalpha-mediated reduction in insulin-stimulated IRS-1 Tyr phosphorylation, Akt activation, and glycogen synthesis. In addition, TNFalpha-stimulated IRS protein Ser/Thr phosphorylation and degradation were blocked. Our results indicate that: 1) NFkappaB and ER stress contribute in part to PKCdelta activation; 2) PKCdelta plays a key role in the propagation of the TNFalpha signal; and 3) PKCdelta contributes to TNFalpha-induced inhibition of insulin signaling events.

Low-carbohydrate (LC) weight-reducing diets are popular choices for self-dieters. Eighteen adults (BMI/= 25 kg/m(2)) were enrolled in this short-term longitudinal study to evaluate dietary intake and weight on their "usual" diets and LC diet. Subjects were instructed to follow the first two phases of the diet described in Dr. Atkins' New Diet Revolution (2 weeks each). Total daily intake of calories and nutrients were calculated from 3-day food diaries. Body weight was measured at the end of each 2-week diet session. All enrolled subjects completed the study (age = 39.8 +/- 8.1 years, BMI = 36.6 +/- 6.6 kg/m(2)). Mean caloric intakes were 1400 +/- 472 kcal/day (Induction diet) and 1558 +/- 490 kcal/day (Ongoing Weight Loss diet) both p/= 0.001 compared to "usual" (Baseline diet) 2481 +/- 723 kcal/day. Body weights were 107.4 +/- 24.2 kg, 103.6 +/- 23.0 kg and 102.1 +/- 22.6 kg at the conclusion of the Baseline, Induction, and Ongoing Weight Loss diets, respectively (both p/= 0.001). Decreases in daily caloric intake correlated with weight loss. Pearson correlation coefficients were, r = 0.64 (p/= 0.001) and r = 0.40 (p/= 0.001) for Induction and Ongoing Weight Loss diets versus "usual" diet. Caloric intake is decreased when otherwise healthy overweight and obese adults self-implement Atkins' Induction and Ongoing Weight Loss diets and significantly altered their dietary micronutrient intake. Weight loss can be explained by the self-selected lower caloric intake on The Atkins' Diet.

Steatosis, oxidative stress, and apoptosis underlie the development of nonalcoholic steatohepatitis (NASH). Protein kinase C delta (PKCδ) has been implicated in fatty liver disease and is activated in the methionine and choline-deficient (MCD) diet model of NASH, yet its pathophysiological importance towards steatohepatitis progression is uncertain. We therefore addressed the role of PKCδ in the development of steatosis, inflammation, oxidative stress, apoptosis, and fibrosis in an animal model of NASH. We fed PKCδ(-/-) mice and wildtype littermates a control or MCD diet. PKCδ(-/-) primary hepatocytes were used to evaluate the direct effects of fatty acids on hepatocyte lipid metabolism gene expression. A reduction in hepatic steatosis and triglyceride levels were observed between wildtype and PKCδ(-/-) mice fed the MCD diet. The hepatic expression of key regulators of β-oxidation and plasma triglyceride metabolism was significantly reduced in PKCδ(-/-) mice and changes in serum triglyceride were blocked in PKCδ(-/-) mice. MCD diet-induced hepatic oxidative stress and hepatocyte apoptosis were reduced in PKCδ(-/-) mice. MCD diet-induced NADPH oxidase activity and p47(phox) membrane translocation were blunted and blocked, respectively, in PKCδ(-/-) mice. Expression of pro-apoptotic genes and caspase 3 and 9 cleavage in the liver of MCD diet fed PKCδ(-/-) mice were blunted and blocked, respectively. Surprisingly, no differences in MCD diet-induced fibrosis or pro-fibrotic gene expression were observed in 8 week MCD diet fed PKCδ(-/-) mice. Our results suggest that PKCδ plays a role in key pathological features of fatty liver disease but not ultimately in fibrosis in the MCD diet model of NASH.

e14632 Background: Obesity has reached epidemic proportions globally. Colon cancer is one of the leading causes of cancer related deaths in United States and obesity is associated with one-third of incidence of colon cancer. Obesity is also associated with a worse prognosis of colon cancer. However, the mechanistic details are unknown and animal models to investigate human colon cancer in the setting of obesity are lacking. Therefore, we developed a diet-induced animal model of obesity to determine whether obesity alters the efficacy of a standard colon cancer therapeutic, 5-Fluorouracil, in mice with human colon cancer xenografts. Methods: Male Rag1 mice were placed on a High Fat Western Diet (HFWD) or a Low Fat Western Diet (LFWD) for up to 12 weeks. Body weight, biochemical parameters, and glucose and insulin tolerance were measured. To determine the efficacy of 5-Fluorouracil on human colon cancer xenograft growth, Rag1 mice were fed a LFWD or HFWD for 8 weeks and then implanted with tissue-isolated HT-29 human colon cancer tumors. After 3 weeks of tumor growth, the mice were randomly assigned to a drug (5-Flurouracil 40 mg/kg) or control (normal saline) treatment group. Tumor growth was monitored for 14 days. Results: Significant differences in body weight, and glucose and insulin tolerance were observed in the four week HFWD fed mice. Obesity, glucose intolerance, and insulin resistance were more pronounced in 8 and 12 week fed HFWD mice. Estimated HT-29 human colon cancer tumor growth was significantly greater in obese HFWD fed mice compared to lean LFWD fed mice. Treatment with 5-Fluorouracil significantly reduced HT-29 human colon cancer tumor growth in lean LFWD fed mice but was without effect in obese HFWD fed mice. Conclusions: In conclusion, our findings demonstrate that diet induced obesity impairs the efficacy of 5-Fluorouracil in an obese animal model of human colon cancer. Given that obesity is growing exponentially worldwide, more studies to understand the mechanistic links between obesity and various cancers are required.

To determine whether equilibrium binding between albumin and hepatocytes involves a cell surface receptor for albumin, we incubated freshly isolated rat hepatocytes with 125I-albumin and determined the amount of albumin associated with the cells as a function of the total albumin concentration. The resulting two-phase binding curve showed the rat albumin-hepatocyte interaction to consist of a saturable binding interaction with a dissociation constant of 1.1 microM and 2 X 10(6) sites/cell in addition to a weak, nonsaturable binding interaction. However, the saturable binding of albumin to hepatocytes did not appear to result from the presence of an albumin receptor on the cell surface; the interaction was the same for different species of albumin, for chemically modified albumins, and for fragments of albumin representing mutually exclusive domains of the molecule. The saturable binding was, instead, found to involve a subpopulation of albumin with an enhanced affinity for the cell surface. We show that this subpopulation of albumin is generated upon contact with either solid surfaces or cell surfaces and can be transferred from one surface to another. We propose that the two-phase Scatchard binding curve and the "albumin receptor effect" reflect two populations of albumin that bind to the cell surface with different affinities rather than one population of albumin that binds to two classes of binding sites.