Abstract
Caloric restriction (CR) is the only environmental intervention with robust evidence that it extends lifespan and delays the symptoms of aging, but its mechanisms are incompletely understood. Based on the prolonged longevity of knockout models, it was hypothesized that the insulin-IGF pathway could be a target for developing a CR mimic. This study aimed to test whether CR has additive effects on glucose homeostasis and beta-cell function in mice with reduced insulin gene dosage. To study models with a range of basal insulin levels, wild-type C57BL/6J and mice on an Ins2 − / − background, were put on 8 weeks of 40% CR at various ages. Both male and female mice rapidly lost weight due to a reduced WAT mass. Glucose tolerance was improved and fasting glucose levels were reduced by CR in both wild type and 45- and 70-week-old Ins2 − / − mice. The effects of CR and reduced insulin on glucose tolerance were non-additive in 20-week-old mice. Interestingly, mice on CR generally exhibited an inability to further depress blood glucose after insulin injection, pointing to possible alterations in insulin sensitivity. In conclusion, our results demonstrate that CR can cause weight loss in the context of reduced insulin production, but that CR-improved glucose homeostasis does not occur near the ‘insulin floor’ in young mice. Collectively, these data shed further light on the relationships between CR, insulin and glucose homeostasis.
Introduction
As the median human lifespan rises with every generation, increasing our understanding of aging becomes more important. Despite significant research efforts to uncover approaches that delay the onset of age-related diseases, caloric restriction (CR) is still the only environmental intervention that can extend lifespan and improve health of most species (Fontana & Partridge 2015, Kapahi et al. 2017). Nearly 80 years following the first CR study, many theories have been proposed to be the underlying mechanism of caloric restriction (McCay et al. 1939, Masoro 2009). Among these mechanisms, a subset of these hypotheses involve hormonal and endocrine signaling, with important roles for elements of the nutrient-sensing pathway activity (Masoro 2005, 2009, Fontana et al. 2010a ). Similarly, experiments have extended longevity in mice and lower model organisms by reducing GH or IGF1/insulin signaling (Tatar et al. 2003, Mair & Dillin 2008, Fontana & Partridge 2015). The characteristic profile of CR-treated animals, such as reduced body weight, reduced GH and IGF1 levels, decreased plasma levels of insulin and glucose, reduced fertility and delayed puberty, are often found in animals with repression of the GH-IGF1 axis (Berryman et al. 2008, Anisimov & Bartke 2013). Based on these similarities, it is feasible that CR and reductions in the GH/IGF1 axis may increase lifespan through similar processes (Berryman et al. 2008). Several studies have looked whether CR has additive effects on lifespan in mice with mutations in the GH-IGF1 axis, but results remain inconclusive (Bartke et al. 2001, Masternak et al. 2004, Bonkowski et al. 2006, Gesing et al. 2014, Wiesenborn et al. 2017).
CR interventions often result in reduced body weight and reduced adiposity. It has been proposed that adipocytes modulate the pace of aging by secreting peptides like TNFα, adiponectin, leptin or angiotensin, factors that seem to promote aging when present in excess (Barzilai & Gupta 1999, Barzilai & Gabriely 2001, Okita et al. 2012). Previous research has shown that short-term CR reduces adipokine levels, improves insulin/IGF1 signaling and reduces reproductive investment (Mitchell et al. 2015). Notably, fat-specific knockout of the insulin receptor has been reported to extend mouse lifespan an effect associated with reduced adiposity with altered secretion of adipokines, including higher adiponectin and lower pro-inflammatory cytokines (Blüher et al. 2002, 2003). The complex effects of deleting insulin receptors in a single tissue, as well as caveats related to the reduction of IGF1 signaling through hybrid receptors (Belfiore et al. 2009), leave the role of insulin itself on metabolism and longevity ambiguous. Thus, the relationship between insulin and acute CR remains unclear.
One way to modify insulin signaling in vivo is the direct modulation of insulin gene dosage using combinations of null alleles (Mehran et al. 2012, Templeman et al. 2015, 2016, Dionne et al. 2016) For example, we have used mice that lack the ancestral Ins2 − / − gene, but compensate to maintain baseline levels of insulin secretion and approximately normal glucose homeostasis via the mouse-specific Ins1 gene (Duvillié et al. 1997, Leroux et al. 2000). When challenged with a high-fat diet, we have previously shown that these animals, which are genetically incapable of sustained hyperinsulinemia, exhibit long-term protection against diet-induced-obesity and eventually improved insulin sensitivity and prolonged lifespan (Mehran et al. 2012, Templeman et al. 2015, 2016, 2017). To investigate the relationship of insulin and the effects of CR, we have combined insulin gene manipulation with CR. In a recent study, we found that long-term caloric restriction reduced islet insulin content to the same extent as the removal of one Ins1 allele, and that the combination of CR and Ins1 heterozygosity were non-additive (Dionne et al. 2016). Interestingly, while CR-improved glucose homeostasis in Ins1 +/+ and Ins1 +/− mice, both sets of CR mice exhibited a paradoxical increase in age-associated adiposity in the absence of the Ins2 gene, despite lower overall body weight (Dionne et al. 2016). It remained unclear whether the increase in relative adiposity was due to the chronic nature of the CR employed or whether this was a direct effect observable after short-term CR.
In the present study, we employed a short-term CR intervention in normal mice and in mice with low insulin. After a short-term CR intervention, we studied wild-type mice, as well as both male and female Ins2 − / − mice at different ages. The outcomes of short-term CR depended on the context of normal or reduced insulin gene dosage, and we also noted differences between male and female Ins2 − / − mice.
Materials and methods
Experimental animals
The Ins2- and Ins1-mutant mice were created at INSERM by J. Jami and colleagues (Duvillié et al. 1997) and bred further to obtain Ins1 +/+ Ins2 − / − or Ins1 +/ − Ins2 − / − littermates with a mixed background (predominately C57BL/6J and 129 strains) (Templeman et al. 2017). Mice were housed in specific pathogen-free conditions on ventilated (50 air changes per hour), autowater Ehret mouse cage racks at ambient room temperature of 21°C. Animals were separated and housed individually a week prior to the start of the experiment, animals in the AL and CR group were matched based on body weight. Control animals that did not participate in the physiological experiments were group-housed until sacrifice. Male C57BL/6J (The Jackson Laboratory) mice were used as a wild-type reference strain, but are not direct controls for the other studies.
Mice were fed LM-485 chow (Teklad Diet Madison, WI, USA), either ad libitum or CR. CR was defined as 60% of average food intake of both genotypes from age-matched mice from an earlier cohort. Food was provided using an automated feeder (F14 Aquarium Fish Feeder, Fish Mate) that would drop weighted food pellets in three quasi-equal ‘meals’ during the dark phase. The meals were dispensed two, five and eight hours after initiation of dark phase of the 12-h light/dark cycle.
Animals were placed on 8 or 10 weeks of caloric restriction, starting at different life stages; adolescent mice (20 weeks old), adult mice (45 weeks old) and aged mice (60–70 weeks old). In order to allow adaptation to restricted feeding, the animals were fed one week with 90% food intake and a second week with 75% food intake and 60% food intake thereafter. Body weight was measured twice a week and animals were killed after 8 or 10 weeks. The night before killing, the meal times of the CR mice were shifted forward by 4 or 5 h so that mice were terminated within 1 h after feeding. All animal procedures were approved by the University of British Columbia Animal Care Committee, in accordance with the guidelines set out by the Canadian Council for Animal Care.
Physiological experiments
Two weeks before and after the CR treatment, blood was collected for analysis, blood glucose response to intraperitoneal administrations of glucose (2 g/kg) (GTT) or an insulin analog (0.75 U/kg of Humalog; Eli Lilly, Indianapolis, IN, USA) (ITT) was followed for 2 h using OneTouch Ultra2 glucose meters (LifeScan Canada Ltd, Burnaby, BC, Canada). A dose of 2 g/kg glucose was sufficient to induce glucose-stimulated insulin secretion, and we found that a dose of 0.75 U/kg insulin was most optimal to test insulin sensitivity. Higher doses of insulin were tested in pilot insulin tolerance test experiments but caused several of the mice to go into life-threatening hypoglycemia, requiring rescue with exogenous glucose (data not shown). Intraperitoneal glucose-stimulated (2 g/kg) insulin secretion (GSIS) was measured after mice were fasted for 4 h, initiated within 2 h after light turned on, to provide a postprandial state for glucose homeostasis measurements and blood sampling. Meal times of the CR mice were shifted forward by 4 or 5 h so that their final meal was given 1 h prior to fasting initiation. During the GSIS, blood from the saphenous vein was collected 3 times over 30 min. Plasma insulin levels were determined by mouse ultrasensitive insulin ELISA kit (Alpco Diagnostics, Salem, NH, USA), according to manufacturer’s instructions. Baseline subtracted area under the curve of both glucose and insulin, in IPGTT and GSIS studies, respectively, was calculated. Similarly, area over the curve was calculated starting at baseline glucose levels in the ITT experiments.
Islet isolation and insulin content determination
Islet isolation was performed using a 3G needle to inject 2 mL Liberase TL (1000 units/mL dissolved in 25 mL Hanks buffer containing 157 mM NaCl, 5.4 mM KCl, 4.2 mM NaH2PO4, 4.1 mM KH2PO4, 10 mM HEPES, 1 mM MgCl2 and 5 mM glucose (Salvalaggio et al. 2002). Incubation occurred for 7 min at 37°C to fully digest exocrine material before filtering the suspension with a 70 µm Nylon Falcon Cell Strainer. Islets were handpicked and cultured in RPMI medium (containing 100 U/mL penicillin, 100 μg/mL streptomycin, 10% FCS, pH of 7.4) at 37°C and 5% CO2 at saturated humidity overnight. Islet perifusions were performed as described (Dror et al. 2007) using 150 size-matched islets per group. Islets were equilibrated under basal (3 mM glucose) conditions and stimulated with either 15 mM glucose or 30 mM KCL conditions.
A total of 10 medium-sized islets were lysed in acid-ethanol buffer to determine insulin content. Sonicated samples were diluted to linear range of the mouse ultrasensitive insulin ELISA kit (Alpco Diagnostics) and insulin concentration determined according to manufacturer’s instructions.
Tissue analysis
At termination, gonadal (gWAT), subcutaneous (scWAT) and mesenteric (mWAT) fat were dissected, as well was interscapular brown adipose tissue (BAT). Tissues were weighed and flash-frozen in liquid nitrogen before being stored at −80°C or fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and cut into 5 μm thick sections that were stained by hematoxylin and eosin. Images were taken with identical exposure settings and time with Zeiss Axio Imager A1. Adipocyte size assessment was done by randomly measuring 100 adipocytes each mouse, using ImageJ.
Real-time qPCR
RNA was isolated from mouse fat tissues or ±100 islets using the RNEasy mini kit (Qiagen) with 1% β-mercaptoethanol for islets or phenol–chloroform extraction for adipose tissue with TRIzol (Invitrogen). Transcript levels of synthesized cDNA (Quanta Biosciences, Gaithersburg, MD, USA) were measured with SYBR green chemistry on a StepOnePlus Real-time PCR System (Applied Biosystems). Ct values were normalized by a standard curve and presented as relative expression of beta-actin (islets) or TBP (fat). qPCR primers are listed in Supplementary Table 1 (see section on supplementary data given at the end of this article).
Statistical analysis
Data presented are mean ± standard error of the mean (s.e.m.), and analyzed by ANOVA followed by Tukey or Bonferroni t-tests or by unpaired Student’s t-tests. Gene expression analysis was performed by multiple t-tests between the ad libitum-fed group and the CR group within each adipose depot. Significance was achieved when P ≤ 0.05 using GraphPad Prism 6.0.
Results
Effects of CR on insulin homeostasis and whole body fat storage
CR has been reported to improve metabolic health span and delay aging (Fontana et al. 2010a ), with decreased leptin, IGF1 and insulin levels, as well as improved glucose homeostasis, within 3 months (Mitchell et al. 2015). In contrast to results from a previous short-term CR study (Mitchell et al. 2015), in this study of short-term CR, 60 week-old male C57BL/6J mice showed no reduction in insulin homeostasis, shown by insulin content (Fig. 1A), ex vivo perifusion of isolated islets (Fig. 1B) and fasting insulin levels (Fig. 1C). However, 8 weeks of significantly improved glucose homeostasis, seen as a reduction of fasted glucose levels (Fig. 1D) and improved glucose tolerance measured after injections of 2 g/kg glucose (Fig. 1E). This improved glucose tolerance occurred despite a lack of observable differences in glucose-stimulated insulin secretion under the conditions we used to assess it (Fig. 1F). All mice with reduced glucose levels due to CR responded without the typical drop in glucose after an exogenous insulin injection of 0.75 U/kg (Fig. 1G). Collectively, these results suggested that despite no observed changes in the insulin secretion profile of CR-fed wildtype animals, their glucose tolerance is still improved.
Caloric restriction for 10 weeks improves metabolic health in male C57BL/6J mice by decreasing body weight and fasting glucose levels. The effects of CR vs ad libitum feeding on pancreatic islets insulin content (A), ex vivo glucose-stimulated (2 g/kg) insulin secretion (B), 4-h fasting insulin levels (C), 4-h fasting glucose levels (D), intraperitoneal glucose (2 g/kg) tolerance test following 4-h fast (E), intraperitoneal glucose-stimulated (2 g/kg) insulin secretion following 4-h fast (F), insulin (0.75 U/kg) tolerance test following 4-h fast (G), body weight (H) and composition (I); n = 7–10, together with fat composition (J) (representative pictures, magnified 100× scale bar = 200 μm) and quantitative adipocyte size assessment of scWAT (K), gWAT (L) and mWAT (M) of ad libitum and CR mice; n = 3, and altered mRNA expression within different fat depots (N); n = 7. Results are shown as mean ± s.e.m. Significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 between ad libitum vs CR animals. Gene expression data were analyzed by multiple t-tests between the ad libitum-fed group and the CR group within each adipose depot, with P < 0.05 as considered significant.
Citation: Journal of Endocrinology 237, 1; 10.1530/JOE-17-0505
We next conducted a thorough analysis of adipose tissue size, cellular morphology and gene expression in these ad libitum-fed or CR aged male C57BL/6J mice. Ten weeks of CR in these mice resulted in a significant reduction body weight (Fig. 1H), reflected in a reduced liver size and gonadal fat weight. Other major organs such as heart and kidney were not affected by the diet treatment (Fig. 1I). Histochemical analysis revealed no significant differences in adipocyte size within all different fat pads (representative pictures in Fig. 1J and quantitative adipocyte size assessment of scWAT, gWAT and mWAT, Fig. 1K, L and M respectively). The recently proposed hypothesis that CR promotes the development of functional beige fat (Fabbiano et al. 2016) was not confirmed within these aged male C57Bl-6J mice in either gWAT nor scWAT assessed mRNA markers of thermogenesis. If anything, there was a consistent trend toward a decrease in the expression of Ucp1, a mitochondrial membrane protein that contributes to non-shivering thermogenesis (Fig. 1N) (Ricquier & Bouillaud 2000). Interestingly, Insr expression of scWAT was significantly increased after a CR diet, suggesting the possibility of increased insulin sensitivity of this adipose depot. Interestingly, BAT size was not changed after CR in these mice, but changes were seen in the gene expression profile (Fig. 1N). Expression of Ucp1, Cox4 and Prdm16, genes involved in thermogenesis, tended to be decreased, as well as genes involved in lipogenesis and adipogenesis such as Pparg, Fas and Acaca (Fig. 1N). Therefore, based on our gene expression data with these C57BL/6J mice, we cannot attribute the beneficial effects of CR on energy homeostasis to increased thermogenic activation of either BAT or WAT. Collectively, these experiments suggest that even though whole-body insulin sensitivity does not appear improved by ITT in these CR C57BL/6J mice, individual tissues may still have altered responses to insulin.
Metabolic effects of CR in mice with reduced insulin dosage
In order to determine whether 8 weeks of CR and reduced insulin signaling have additive effects on glucose lowering, we next used male mice with an Ins2 − / − background. As expected, mice lacking the Ins2 gene have ~70% less islet insulin content compared to wildtype C57BL/6J animals (Fig. 2A). Despite their lower insulin reservoir, we have shown previously that these animals are healthy without significant impairment of glucose-insulin homeostasis (Leroux et al. 2000, Mehran et al. 2012, Templeman et al. 2015, 2016, Dionne et al. 2016). In the present study, we studied Ins2 − / − mice with and without 10 weeks of CR. In these mice, CR did not decrease insulin content in Ins2 − / − CR mice (Fig. 2A), in agreement with our previous investigation of lifelong CR wherein islet insulin content and beta-cell area were also not further reduced in female mice with 2 or 3 insulin alleles inactivated, at any age tested (Dionne et al. 2016). In the present study, fasting insulin levels seemed lower in CR Ins2 − / − mice, but the variability and bimodal distribution in the ad lib-fed mice meant that differences were not significant with this relatively low sample size (Fig. 2B). Nevertheless, as in our previous work, CR reduced lowered fasting glucose levels (Fig. 2C) and improved glucose tolerance despite secreting lower circulating insulin compared to the ad libitum-fed animals (Fig. 2D and E), suggesting that glucose uptake was increased independently of insulin alterations. Indeed, insulin tolerance tests, using exogenous insulin injection of 0.75 U/kg, revealed a flat pattern of lowered fasting glucose that was maintained for 120 min without the typical drop in glucose (Fig. 2F). This flat pattern can be interpreted as partial insulin resistance. Collectively, the findings that aged Ins2 − / − mice exhibit improved glucose tolerance were consistent with our observations of mice with a pure C57Bl/6 background. However, this improved glucose tolerance is not associated with improved whole-body insulin sensitivity under the conditions we tested.
Caloric restriction for 10 weeks improves glucose homeostasis in Ins2 − / − mice independent of insulin content. Reduced pancreatic islets insulin content in Ins2 − / − mice compared to wild type (A). The effects of CR on 4-h fasting insulin levels (B), 4-h fasting glucose levels (C), intraperitoneal glucose (2 g/kg) tolerance test following 4-h fast (D), intraperitoneal glucose-stimulated (2 g/kg) insulin secretion following 4-h fast (E), insulin (0.75 U/kg) tolerance test following 4-h fast (F) in Ins2 − / − mice by changes in body weight (G) composition (H); n = 8–10. Altered mRNA expression within different fat depots (I); n = 8–10. Results are shown as mean ± s.e.m. Significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 between ad libitum vs CR animals. Gene expression data was analyzed by multiple t-tests between the ad libitum-fed group and the CR group within each adipose depot, with P < 0.05 as considered significant.
Citation: Journal of Endocrinology 237, 1; 10.1530/JOE-17-0505
Histological and gene expression changes in WAT after CR
We next investigated fat pad characteristics and marker genes involved in adipose energy homeostasis in male Ins2 − / − mice. As expected, body weight dropped after 10 weeks of CR (Fig. 2G), reflected in significantly smaller gonadal and mesenteric fat pads and without affecting other organs like liver, heart and kidney (Fig. 2H). BAT size was unchanged after CR; however, genes involved in thermogenesis, like Ucp1 and Prdm16, were significantly downregulated. Decreased expression levels of lipogenic genes, like Fasn and Acaca, Ppargc1a (a coregulator of mitochondrial biogenesis) and apoptosis marker Ddit3 were also observed in the BAT of CR mice (Fig. 2I). Cidec expression was significantly increased in BAT of Ins2 − / − mice on a CR diet (Fig. 2I), in agreement with previous findings of Matsusue et al. (Matsusue 2010). Analysis of three different white adipose fat tissues at the levels of mRNA expression showed increased expression of Fasn and Acaca, genes that are both involved in lipogenesis. We also observed significantly increased levels of both Ppargc1a and Cidea gWAT (Fig. 2I), consistent with fat storage (Abreu-Vieira et al. 2015). Together, these data suggest that CR leads to an adaptation of WAT size and gene expression.
CR improves glucose homeostasis in female mice, independent of age or insulin dosage
Sex-specific regulation of glucose metabolism, insulin resistance and energy expenditure has been previously reported (Widdowson 1976, Valle et al. 2005, Varlamov et al. 2014, Mauvais-Jarvis 2015). Thus, we next assessed the effects of CR on female with low insulin gene dosage at 20, 45 and 70 weeks of age after 8 weeks of CR. We set out to test the hypothesis that short-term CR is still beneficial for energy metabolism during different life stages and might have different metabolic effects when comparing mice lacking 2 alleles vs mice lacking 3 insulin alleles. However, we did not observe statistically significant differences between Ins1 +/+:Ins2 − / − and Ins1 +/ − :Ins2 − / − mice in any of our tests (Supplementary Fig. 1). Therefore, the data were pooled to provide additional power to resolve the effects of CR in the context of low insulin (relative to C57BL/6J wild-type mice). In all three age groups, body weight dropped significantly (Fig. 3A). Compared to their original weight, the weight loss in the oldest females was slightly greater compared to the males at the same stage of life. Islets isolated from these mice had significantly reduced insulin content at all ages tested (Fig. 3B), in contrast to what we observed in the males (Figs 1A and 2A). We did not observe statistically significant differences in fasting insulin levels between CR mice and their controls (Fig. 3C), although mice on CR showed decreased fasting glucose levels at all ages tested (Fig. 3D). CR mice released less insulin in response to a glucose bolus at 20 weeks and 45 weeks of age, but not at 70 weeks of age (Fig. 3E). Interestingly, these CR mice had improved glucose tolerance at 45 and 70 weeks of age (Fig. 3F). At 20 weeks, the CR-induced improvement in glucose tolerance was not observed, demonstrating non-additivity in these young female mice with reduced insulin gene dosage (Fig. 3F). Insulin tolerance tests also revealed a similar pattern of apparent insulin resistance in the CR mice to what was observed in the males (Fig. 3G). Thus, our data demonstrate that in the context of reduced insulin gene dosage, short-term CR improves glucose homeostasis associated with paradoxical reduction in insulin secretion and insulin sensitivity. These phenomena are relatively consistent through the age range we tested.
CR-induced metabolic improvements are independent of age in female Ins2 − / − mice. The effects of CR vs ad libitum feeding on body weight (A), pancreatic islets insulin content (fed) (B), 4-h fasting insulin levels (C), 4-h fasting glucose levels (D), intraperitoneal glucose-stimulated (2 g/kg) insulin secretion following 4-h fast (E), intraperitoneal glucose (2 g/kg) tolerance test following 4-h fast (F) and insulin (0.75 U/kg) tolerance test following 4-h fast (G) in female Ins2 − / − mice (combined Ins1 +/ − and Ins1 +/+ genotypes) of different ages. Animals used; n = 10–16 (20 weeks), n = 18–20 (45 weeks) and n = 8–10 (70 weeks). Results are shown as mean ± s.e.m. Significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 between ad libitum and CR animals. LOD, limit of detection.
Citation: Journal of Endocrinology 237, 1; 10.1530/JOE-17-0505
Effects of CR on gene expression and depot size in BAT and WAT
In contrast to our findings in males, 45-week-old female mice showed more pronounced changes in WAT and BAT gene expression. All white fat depots measured (gonadal, subcutaneous and mesenteric) were significantly smaller (Fig. 4A and B), due to a decrease in adipocyte size (Fig. 4C, D, E and F). In the postprandial state, all fat depots of CR mice showed increased expression of lipogenic genes like Fas and Acaca consistent with the optimization of energy storage (Fig. 4G). Markers involved in browning of white adipose tissue were increased in the scWAT and mWAT depots, specifically Adrb3, Ppargc1a, Prmd16, Cidea and Cox4 expression. These genes would be predicted to stimulate Ucp1 activity and thermogenesis (Okita et al. 2012, Abreu-Vieira et al. 2015, Garcia et al. 2016). However, differences in Ucp1 expression were not significant (Fig. 4G). CR females also exhibited a slight increase in BAT mass, although this was also not statistically significant (Fig. 4A and B). Together, results from these female Ins2 − / − mice suggest a shift between white and brown adipocyte gene expression profiles.
CR-induced activation of BAT and browning of WAT in female Ins2 − / − mice. Body composition; n = 13–21 (A and B), representative pictures of fat composition, magnified 100× scale bar = 200 μm (C), adipocyte size assessment of scWAT (D), gWAT (E), mWAT (F); n = 5, and altered mRNA expression within different fat depots in female mice (combined Ins1 +/ − and Ins1 +/+ genotypes) aged 45 weeks; n = 7–8 (G). Results are shown as mean ± s.e.m. Significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.00 between ad libitum and CR animals. Gene expression data was analyzed by multiple t-tests between the ad libitum-fed group and the CR group within each adipose depot, with P < 0.05 as considered significant.
Citation: Journal of Endocrinology 237, 1; 10.1530/JOE-17-0505
Discussion
The aim of this study was to investigate the effects of short-term CR on glucose metabolism and adipose tissue in the contexts of age and genetically lowered insulin. One of the most consistent findings in animal models of CR is a significant improvement in glucose homeostasis (Colman et al. 2009, Fontana et al. 2010b ). Others have observed improvement in glucose tolerance, together with reduced levels of leptin, insulin and IGF1, after only 3 months of CR (Mitchell et al. 2015). In the present study, we confirmed the expected rapid improvement in glucose tolerance with CR, except in young female mice with reduced insulin gene dosage.
The interpretation of the role of circulating insulin on the effects of CR is complicated by the fact that fasting insulin levels were not dramatically different between the different strains of mice and at the different ages tested, despite a significant reduction in insulin content in the mice with reduced insulin gene dosage. Fasting insulin averaged 0.6–0.7 ng/mL in the male 60-week-old C57BL/6J mice regardless of CR. Male Ins2 − / − mice were able to compensate with increased insulin secretion such that fasting insulin averaged 0.75 ng/mL with ad libitum feeding and 0.5 ng/mL with CR. This compensation for reduced insulin gene dosage is consistent with previous studies, and we have found that this compensation is more robust in male mice (Leroux et al. 2000, Templeman et al. 2016, 2017). Consistent with this, female Ins2 − / − mice exhibited the lowest fasting insulin levels of any group in our investigation, averaging 0.2 ng/mL regardless of CR status at 20 weeks of age and increasing to 0.4 ng/mL by 70 weeks of age. Interestingly, it was only at the lowest circulating level in the 20-week-old female mice where CR did not further improve glucose tolerance. This non-additive effect suggests that CR and extreme insulin reduction act on glucose homeostasis via at least some common mechanisms in young mice. Other effects of CR, including the expected weight loss were maintained at all ages, indicating that the mechanisms controlling glucose homeostasis and weight loss in CR are distinct. The molecular mechanisms underlying these differences require further study and are beyond the scope of this investigation.
Our current study demonstrated beneficial effects of CR in aged mice, regardless of genotype. A previous study had suggested that the age when CR is started, the severity of restriction and the strain or genetic background of the animals determines the magnitude of life extension (Fontana & Klein 2007). Another study in mice demonstrated that initiating CR shortly after weaning (to age 6 months) caused a proportionate 30–60% increase in maximum life span, whereas a reduction in calorie intake started in adulthood extended maximum life span by only 10–20% (Weindruch & Walford 1982).
Furthermore, the physiological mechanisms by which CR improves glucose homeostasis remain to be fully elucidated. We did not observe differences in fasting insulin or glucose-stimulated insulin secretion in our wild-type mice as shown previously (Mitchell et al. 2015), suggesting a metabolic improvement independent of insulin secretion. The insulin tolerance tests changed the profile of blood glucose after a bolus insulin injection. Differences in metabolic parameters between our study and others could possibly be the result of our feeding protocol of 3 small meals during the dark phase, which reduces the time of fasting in the CR animals compared to standard protocol of 1 meal a day. In all cases, the significantly lower fasting glucose complicates the interpretation of these data, and we do not have data on counter-regulatory signals that are likely robust under these conditions. It should also be noted that the insulin tolerance test is not ideal for a full assessment of insulin sensitivity. Additional studies to further elucidate these phenomena will require hyperinsulinemic–euglycemic clamp experiments.
Our study identified possible sex differences in the metabolic response to short-term CR. We documented the expected rapid decrease in body weight and blood glucose levels after CR treatment, but found that it was more pronounced in females than in males. Some authors have argued that females conserve their energy more efficiently and are more resistant to CR because of their relative importance for reproduction and the survival of the species (Widdowson 1976, Valle et al. 2005). However, in our study, the female CR mice lose an equal percentage of their starting body weight. Female mice showed a reduction in their islets insulin content due to CR, which was not found in males, suggesting that female Ins2 − / − mice may be more susceptible to the effects of CR. BAT activity was diminished in male Ins2 − / − mice, whereas this tissue was still active in the female Ins2 − / − CR mice. Furthermore, upregulation of genes involved in lipogenesis and thermogenesis in scWAT are more pronounced in females. The molecular mechanisms underlying the sex differences in the metabolic responses to CR require further study. In female mice with reduced insulin, CR reduced the size of white adipocytes, which has been linked to alterations in altered adipokine secretion. Hypertrophic adipocytes, possessing more triglycerides, have been proposed to secrete less adiponectin and more pro-inflammatory cytokines, whereas small adipocytes are generally found to be more sensitive to insulin and act as a powerful buffer taking up free fatty acids during the postprandial period. For this reason, reducing adipocyte size by CR is considered beneficial for a healthy lifespan (Okita et al. 2012).
It has been proposed that the activation of BAT and the browning of WAT play important roles in the effects of CR. Originally, it was believed that WAT and BAT had distinct morphology and function, with WAT being a major source for triglyceride storage and adiponectin secretion to enhance insulin sensitivity, and BAT playing an important role in energy expenditure and thermogenesis (Saely et al. 2011, Okita et al. 2012). Lately, the distinction between BAT and WAT has become less rigid, with observations of browning of WAT (i.e. increased Ucp1 expression) under certain circumstances (Puigserver & Spiegelman 2003), including CR (Fabbiano et al. 2016) and strongly reduced circulating insulin (Mehran et al. 2012). Similarly, we found reduced adipocyte size and increased expression of some thermogenic genes in female Ins2 − / − mice with CR. We examined the expression levels of Ucp1 mRNA, which is critical for thermogenic activity (Puigserver et al. 1998, Puigserver & Spiegelman 2003) and is known to be positively correlated with metabolic inefficiency in overfeeding (Nedergaard et al. 2001). We did not observe an increase in Ucp1 mRNA, and in fact, there were robust reductions under some conditions. Nevertheless, we observed tendencies for increases in genes known to control Ucp1 expression and thermogenesis, such as Ppargc1a, Nrf1 and Cox4. The molecular mechanisms mediating the possible functional remodeling of adipose tissue in the context of short-term CR require further study. A previous study suggested that CR downregulated the mitochondrial electron transport chain but enhanced fatty acid biosynthesis in BAT, suggesting that in CR animals, BAT may change its function from an energy-consuming system to an energy reservoir system (Okita et al. 2012). It was suggested that CR promotes fatty acid biosynthesis and the metabolic process involving pyruvate, citrate, oxaloacetate and malate in both WAT and BAT based on increased ACLY (ATP citrate lyase) phosphorylation and upregulated Fasn mRNA levels for de novo fatty acid biosynthesis (Okita et al. 2012). A previous study from our group also found histological evidence for ‘whitening’ of BAT after long-term CR (Dionne et al. 2016). However, with the possible exception of upregulated expression of lipogenic genes in female Ins2 − / − mice, we did not find robust evidence for whitening of BAT with short-term CR in the present study.
In conclusion, we found that short-term CR promotes metabolic processes that are favorable for glucose homeostasis in C57BL/6J and Ins2 − / − mice. CR improves glucose tolerance, except in young mice with reduced insulin gene dosage, suggesting both insulin-independent and insulin-dependent mechanisms of CR that are age, and to some degree, sex dependent. The beneficial actions of CR are associated with both WAT and BAT remodeling toward increased thermogenesis. Understanding and harnessing mechanisms associated with CR may lead to additional ways to improve glucose and lipid homeostasis.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-17-0505.
Declaration of interest
The authors declare that they have no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
Work described in this study was supported by a CIHR grant to J D J.
Author contribution statement
M B D led the design, conduct and interpretation of experimental studies and wrote the manuscript. D A D and D F H assisted with design, conduct and interpretation of experimental studies, and edited the manuscript. J K K co-supervised M B D and edited the manuscript. J D J co-supervised M B D, conceived the studies, assisted with interpretation of experimental studies, edited the manuscript and is the guarantor of this work.
Acknowledgements
The authors thank Xiaoke Betty Hu for assistance with mouse husbandry and Farnaz Taghizadeh for her help with the islet isolation.
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