Introduction
Obesity and type 2 diabetes are increasing globally at an unprecedented rate across all ages and sexes. Although a genetic basis for obesity susceptibility is undisputed, only a small proportion of the BMI variation within the population can be explained by known genetic variants, suggesting there is an interaction between genetic factors and the environment [
1]. Evidence from clinical and experimental studies shows that the risk of developing many non-communicable diseases can be influenced by the early-life environment [
2,
3]. Numerous studies have shown that low birthweight is associated with increased risk of developing impaired glucose tolerance and, subsequently, type 2 diabetes [
4,
5]. Furthermore, it is well established that the accelerated growth that often follows low birthweight, as well as accelerated postnatal growth alone, are important risk factors for type 2 diabetes and obesity [
6,
7]. Indeed, the combination of low birthweight with rapid weight gain, particularly if the baby crosses growth percentiles, is strongly linked to developing type 2 diabetes and obesity later in life [
8,
9].
Insulin signalling is essential for maintaining whole-body energy homeostasis. The main sites of insulin action in the periphery are muscle and adipose tissue, where insulin increases glucose uptake; and liver, where insulin decreases glucose production. As recognised more recently, insulin signalling in the central nervous system is essential for maintaining energy homeostasis [
10]. The hypothalamus is the primary site of insulin action in the brain [
11]. In particular, agouti-related peptide/neuropeptide Y and pro-opiomelanocortin (POMC) neurons in the arcuate nucleus (ARC), and steroidogenic factor 1 (SF1) neurons in the ventromedial nucleus (VMH), have been shown to be important for hypothalamic insulin signalling [
11,
12]. Neurons in the paraventricular (PVH) nucleus are also capable of sensing insulin [
13], although the relevance of these neurons in maintaining glucose homeostasis is unknown.
Infants born small for gestational age (SGA) display defective insulin signalling in peripheral tissues, which may contribute to their increased type 2 diabetes risk [
14,
15]. We have previously reported defects in the insulin signalling pathway in peripheral tissues of rodents that underwent IUGR followed by catch-up growth [
16,
17] and similar differences in adipose and muscle biopsies from low-birthweight humans [
14,
18]. Disrupted central insulin signalling, particularly in the ARC, causes defects in energy homeostasis, including disrupted glucose homeostasis and obesity [
19]. Therefore, altered central insulin signalling may underlie the phenotypes reported in individuals that experience IUGR followed by catch-up growth. However, there are no studies that have yet addressed whether low birthweight followed by accelerated postnatal growth leads to central insulin resistance. This is not feasible to address in humans but can be analysed using animal models, which may also be used to determine whether relationships are causal.
Therefore, the aim of the current study was to examine whether murine offspring subjected to IUGR followed by postnatal catch-up growth show altered central insulin sensitivity, and to investigate the underlying molecular pathways mediating any programmed effects.
Discussion
We used a rodent model to show that animals that undergo IUGR followed by rapid postnatal catch-up growth display whole-body insulin resistance in adulthood. This is associated with peripheral insulin resistance, as demonstrated by the results of the hyperinsulinaemic–euglycaemic clamps. Our findings show, for the first time, that suboptimal nutrition during early life can also lead to central insulin resistance, and identify a novel mechanism in the ARC that may underpin this. Previous rodent models have shown that raising neonates in small litters disrupts energy homeostasis and causes insulin resistance in neuronal cultures [
24]. Therefore, the phenotype in recuperated offspring could be due either to the low birthweight caused by IUGR or to the accelerated neonatal growth caused by the small litter rearing, or indeed a combination of the two (as in the human situation where most low birthweight babies will experience postnatal catch-up growth).
Recuperated offspring had higher glucose levels during the GTT, indicating decreased glucose tolerance, in agreement with other rodent and larger mammal models of nutritional programming [
25‐
28]. This glucose intolerance is likely attributed to reduced insulin sensitivity, as these mice also required a lower GIR to maintain blood glucose during the hyperinsulinaemic–euglycaemic clamp. We have previously reported that recuperated offspring show molecular defects in expression of key components of the insulin signalling pathway in peripheral tissues [
16,
17], thus this may contribute to the insulin resistance shown in the clamp. Despite no changes in total body weight or adiposity, recuperated offspring showed an increase in epididymal fat mass. This is in agreement with previous reports from human studies that infants born SGA have higher central adiposity, despite a comparable BMI to infants born at a normal weight [
29]. Recuperated offspring demonstrated reduced EE, which may predispose them to an increase in body weight, as well as total adiposity, as they get older. Our data from indirect calorimetry also suggest different fuel usage in recuperated offspring as their RER was lower, indicating that fats are preferentially used as a substrate for energy generation compared with carbohydrates.
In this study, control mice were sensitive to the anorectic effects of ICV insulin; food intake was significantly reduced 2 and 4 h after food presentation when insulin was administered into the lateral ventricle. This is in agreement with several studies showing the anorectic effects of central insulin administration, either intranasally [
30] or directly to the brain via injection [
31‐
33]. However, there was no effect on food intake in recuperated mice, showing that these animals were resistant to the anorectic effects of exogenous insulin and suggesting central insulin resistance. Studies in both rodents and humans have shown that central insulin resistance contributes to the pathology of whole-body insulin resistance [
10,
34]. Therefore, the central insulin resistance observed in recuperated offspring could contribute to the whole-body insulin resistance observed.
Although central insulin resistance is associated with obesity and type 2 diabetes [
35,
36], it is usually thought of as a consequence of chronic hyperinsulinaemia and obesity rather than a cause. However, it has been shown in rodents fed a high-fat diet (HFD) that resistance to ICV insulin can occur within a matter of days and before an increase in adiposity [
37]. In this previous study of the effects of an acute HFD, central insulin resistance was associated with elevated NEFA. NEFA are able to cross the blood–brain barrier and activate inflammatory pathways, resulting in hypothalamic insulin resistance [
38]. Interestingly, in our study, recuperated offspring develop central insulin resistance despite being maintained on a regular chow diet and in the absence of elevated NEFA levels. The presence of insulin resistance in the absence of frank obesity, elevated NEFA or hyperinsulinaemia raises the possibility that changes to insulin sensitivity in these mice are programmed during early life. There is evidence from human studies that the fetal brain responds to changes in maternal glucose in insulin-sensitive but not insulin-resistant mothers, suggesting that fetuses of insulin-resistant mothers are themselves insulin resistant in utero [
39]. Furthermore, babies from obese pregnancies display insulin resistance at birth [
40]. Therefore, it is possible that in circumstances of altered metabolic state in the mother during pregnancy, insulin resistance programmed during the perinatal period is causative of further metabolic dysfunction, rather than being a consequence of it.
Our previous studies in adipose tissue and muscle from recuperated offspring [
16,
17] and from humans with a low birthweight [
14] have demonstrated changes in expression of key insulin signalling proteins. One of the biggest effects is observed on levels of the p110β catalytic subunit of PI3K. In humans and rodents, the reduced expression occurs at the post-transcriptional level, with no differences being observed at the mRNA level. Here, for the first time, we demonstrate that this protein is also present at substantially reduced levels in the ARC of recuperated animals. In addition to reduced p110β, we observed an increase in serine phosphorylation of IRS-1.
Ptpn1 mRNA levels were also increased in the ARC from recuperated animals. Whole-body or neuronal deletion of
Ptpn1 improves insulin sensitivity [
19]. Therefore, all of these changes could contribute to the lack of anorectic response to ICV insulin in recuperated offspring. As intact insulin signalling in the ARC is essential for maintaining energy homeostasis, ARC insulin resistance also likely contributes strongly to the peripheral insulin resistance observed in the hyperinsulinaemic–euglycaemic clamps in recuperated offspring. A recent study has shown that insulin signals through POMC neurons to cause browning of white adipose tissue and therefore increased EE [
41]. Abrogation of insulin signalling by overexpression of PTP1B in the ARC inhibits this process. Conversely, mice with a global knockout of
Ptpn1 have increased EE [
42]. Therefore, the increased
Ptpn1 expression we observed in the ARC may also contribute to the decreased EE that we observed in recuperated offspring.
The specific increase in
Ptpn1 expression in the ARC but not in other hypothalamic areas examined suggests selective insulin resistance. Selective ARC resistance to leptin has been reported previously as a consequence of diet-induced obesity [
43]. To our knowledge, there have not been reports of selective insulin resistance within the different nuclei of the hypothalamus. However, selective hypothalamic insulin resistance has been reported in humans, as obese men show responses in cognitive areas but not hypothalamic areas of the brain after intranasal insulin administration [
44].
We have previously shown that, compared with control animals, recuperated animals have a reduced lifespan and show signs of accelerated cellular ageing [
45]. Central insulin responsiveness is decreased with ageing [
46], which may in part be due to increased negative regulation of insulin signalling transduction. The expression of PTP1B, as well as other phosphatases that negatively regulate insulin signalling, is increased with ageing and has been suggested as an underlying mechanism of age-associated insulin resistance [
47‐
49]. The increase in
Ptpn1 expression in the ARC and central insulin resistance could therefore be further examples of accelerated ageing in mice that have undergone IUGR followed by catch-up growth.
In conclusion, our results show that in mice, undernutrition during the in utero period followed by rapid neonatal catch-up growth causes central insulin resistance and peripheral insulin resistance. This has important implications for the long-term health of an individual. A recent study has shown that humans with central insulin resistance have a reduced capacity to lose weight by lifestyle intervention [
50]. Furthermore, it is unclear whether primary treatments for improving insulin sensitivity, such as thiazolidinediones and metformin, can cross the blood–brain barrier and improve brain insulin sensitivity. Therefore, humans with central insulin resistance have a reduced capacity for metabolic improvements via both lifestyle and pharmaceutical interventions. These findings in mice, if extrapolated to humans, suggest that individuals exposed to a suboptimal early environment may be less responsive to currently available interventions.