Downregulation of plasma insulin levels and hepatic PPARγ expression during the first week of caloric restriction in mice

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Abstract

Calorie restriction extends lifespan by decreasing the rate of tumor formation, an effect occurring within 8 weeks of initiating a restricted diet. Our goal was to define how the first weeks of a calorie restricted diet (60% of ad libitum calories) affects putative mediators of the calorie restriction phenotype, focusing on regulators of fatty acid biosynthesis. In C57Bl/6 mice, insulin decreased over 50% (p < 0.05) during the first week of calorie restriction whereas IGF-1 was unaffected. In the liver, PPARγ mRNA fell to 13% of baseline after 1 week of calorie restriction (p < 0.05), whereas hepatic SREBP-1c and SIRT1 mRNA levels were unaffected. No changes in abdominal or subcutaneous adipose tissue were observed until after 4 weeks of caloric restriction. We conclude that calorie restriction-induced decreases in insulin and hepatic PPARγ are rapid enough to support a role for these molecules in triggering the initial phase of the calorie restriction phenotype.

Introduction

In all species studied to date, restricting caloric intake by 20–50% while still providing adequate micronutrients significantly extends mean and maximal lifespan, largely by retarding age-associated diseases, most importantly cancer (Weindruch, 1996, Weindruch et al., 1986). Not surprisingly there is intense interest in elucidating the molecular basis for the anti-aging and anti-cancer effects of calorie restriction (Ingram et al., 2006, Wolf, 2006). We have reported that 16 weeks of calorie restriction decreases protein levels in the liver of acetyl CoA carboxylase (ACC), the rate-limiting enzyme in fatty acid biosynthesis, to approximately 25% of baseline (Gonzalez et al., 2004). This observation is significant in that fatty acid biosynthesis appears to be closely linked to the timing and extent of tumor development, most notably in a subset of aggressive malignancies requiring a high rate of lipogenesis for growth (De Schrijver et al., 2003, Kuhajda, 2000, Menendez et al., 2005, Pizer et al., 1998). In fact, specific inhibition of fatty acid synthase (FAS) and lipogenesis have been used successfully to inhibit growth of malignancies (De Schrijver et al., 2003, Pizer et al., 1998). Thus calorie restriction-induced downregulation of ACC protein in the liver could be part of a general inhibition of fatty acid biosynthesis contributing to the observed decrease in spontaneous and induced tumor development in calorie restricted animals (Fu et al., 1994, Yoshida et al., 1999). Interestingly, the transcription factors PPARγ and SREBP-1c which regulate ACC and FAS expression have additional effects on processes central to aging such as cell cycling, apoptosis and inflammation (De Schrijver et al., 2003, Ettinger et al., 2004, Gonzalez et al., 2004, Kirkland et al., 2002, Picard and Guarente, 2005, Rossi et al., 2003, Semple et al., 2006). Thus any calorie restriction-induced downregulation of PPARγ, SREBP-1c, or other factors that influence fatty acid biosynthesis (i.e. insulin) could have collateral effects contributing to the anti-aging and anti-tumor phenotype of these animals.

Accordingly, our goal was to define how a calorie restricted diet affects the following regulators of fatty acid biosynthesis gene expression: PPARγ, SREBP-1c, SIRT1 and plasma insulin levels. Importantly, the caloric restriction phenotype of retarded aging and tumor development is evident within 8 weeks after initiating calorie restriction, with half of the eventual changes in hepatic gene expression already detectible at 2 weeks (Dhahbi et al., 2004). Thus the critical molecular events triggering this phenotype must occur within the first several weeks after initiating the restricted diet. Therefore, we focused on defining the early time course of caloric restriction’s effects. To accomplish this, blood, liver and adipose tissue (subcutaneous and abdominal) were collected from 6-month-old C57Bl/6 mice that had undergone 0, 1, 4 or 16 weeks of calorie restriction (60% of ad libitum calories). We focused on liver because, while the main cause of neoplastic death of aging C57bl/6 mice is lymphoma not liver cancer, CR almost eliminates the incidence of liver tumors in this strain of mice whereas it has little effect on the rate of death due to lymphoma (Blackwell et al., 1995). For comparison to liver, another highly lipogenic tissue, white adipose tissue, was chosen, which itself has been suggested to play a key role in the calorie restriction phenotype (Wolf, 2006). Adipose tissue from two distinct anatomical sites was studied so that we could test the hypothesis that abdominal (epididymal) adipose tissue, which is more closely linked to pathology than subcutaneous adipose tissue, is resistant to calorie restriction-induced changes in gene expression (Lafontan and Berlan, 2003).

Section snippets

Animals

Mice (N = 36 C57Bl/6 males) were housed singly in the specific pathogen-free Shared Aging Rodent Facility at the Madison VA Geriatric Research, Education and Clinical Center, and provided a nonpurified diet (PLI 5001 [Purina Labs, St. Louis, MO]) and acidified water ad libitum for 3 weeks following weaning. From this time until 2 months of age, all 36 mice received 12 kcal/day of a semipurified diet (TD91349 [Teklad, Madison, WI]), which is ∼10% less than the average ad libitum intake. The

Results

Plasma values for glucose, insulin and IGF-1 measured at the time of sacrifice are shown in Fig. 2. Calorie restriction did not significantly affect plasma IGF-1 concentration at any of the time points. In contrast, plasma insulin levels were significantly lowered during the first week of calorie restriction, and this low level of insulin sustained at 4 and 16 weeks of calorie restriction. This fall in insulin occurred in parallel with a decline in glucose levels which became statistically

Liver

Calorie restriction extends maximal lifespan in animals largely by decreasing the rate of tumor formation (Dhahbi et al., 2004, Weindruch, 1996, Weindruch et al., 1986). In the liver, caloric restriction dramatically lowers the incidence of both spontaneously occurring tumors as well as tumors induced by a carcinogenic agent (Fu et al., 1994, Yoshida et al., 1999). These protective effects occur within the first few weeks of initiating a calorie restricted diet (Dhahbi et al., 2004). Our

Acknowledgements

The authors thank Dr. Richard Weindruch for his invaluable assistance in conducting this study. This work was supported by National Institute on Aging Grant AG-00908, National Heart Lung and Blood Institute Grant HL-07936 and a grant from the UW Comprehensive Cancer Center.

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