Maternal metabolic homeostasis exerts long-term effects on the offspring’s health outcomes. Here, we demonstrate that maternal high-fat diet (HFD) feeding during lactation predisposes the offspring for obesity and impaired glucose homeostasis in mice, which is associated with an impairment of the hypothalamic melanocortin circuitry. Whereas the number and neuropeptide expression of anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP) neurons, electrophysiological properties of POMC neurons, and posttranslational processing of POMC remain unaffected in response to maternal HFD feeding during lactation, the formation of POMC and AgRP projections to hypothalamic target sites is severely impaired. Abrogating insulin action in POMC neurons of the offspring prevents altered POMC projections to the preautonomic paraventricular nucleus of the hypothalamus (PVH), pancreatic parasympathetic innervation, and impaired glucose-stimulated insulin secretion in response to maternal overnutrition. These experiments reveal a critical timing, when altered maternal metabolism disrupts metabolic homeostasis in the offspring via impairing neuronal projections, and show that abnormal insulin signaling contributes to this effect.

In contrast to humans, development of these hypothalamic neurocircuits in rodents is not completed at birth but continues until the third week of postnatal life. Whereas neuronal cell numbers are determined in utero, formation of functional neuronal networks with the ontogeny of axonal projections and synaptic connections occurs postnatally during the lactation phase (). Importantly, as a result of impaired maternal health, exposure to an altered developmental environment during both of these stages results in gross changes of these hypothalamic neurocircuits, including differential neuropeptide gene expression and altered hypothalamic neuronal cell numbers, as well as impaired formation of hypothalamic axonal projections (). However, differences in study design—in particular, in severity, duration, and onset of abnormal environmental cues ()—has made it challenging to clearly define (1) the exact timing requirements of metabolic insults to affect the metabolic fate of the offspring and (2) the specific molecular mechanisms underlying these persistent cellular changes. Thus, we aimed to establish a mouse model of metabolic programming that would allow us to identify the most sensitive period of hypothalamic neurocircuit development in response to maternal HFD feeding. Importantly, we further employed this model to delineate the distinct role of neuron-specific insulin signaling in mediating the lifelong predisposition for metabolic disorders in offspring of obese and/or hyperglycemic mothers ().

Formation of projection pathways from the arcuate nucleus of the hypothalamus to hypothalamic regions implicated in the neural control of feeding behavior in mice.

The sustained global rise in the prevalence of obesity and type 2 diabetes mellitus (T2DM) over the last decades increasingly affects young adults and children (). Thus, 15%–40% of pregnancies are complicated by maternal obesity and 3%–10% by maternal diabetes (). Several human epidemiological studies have demonstrated that maternal obesity and maternal diabetes and hyperglycemia—even independent of an elevated body mass index or genetic background—predispose the offspring to the development of metabolic disorders (). To date, little is known about the cellular and molecular mechanisms underlying this phenomenon known as “metabolic programming.” Nonetheless, a broad range of studies has demonstrated that an abnormal hormonal milieu during development triggers persistent changes in the function of hypothalamic neurocircuits, which physiologically regulate energy and glucose metabolism (). The hypothalamus integrates hormonal and nutritional signals from the periphery of the organism and conveys them into neuroendocrine and/or autonomic responses (). Key players in this neuronal network are the anorexigenic POMC and the orexigenic AgRP/neuropeptide Y (NPY)-coexpressing neurons (). These functionally antagonistic neuronal populations reside in the arcuate nucleus of the hypothalamus (ARH) and mediate their effects via second-order neurons mainly located in other key parts of the hypothalamus, such as the PVH, the dorsomedial nucleus of the hypothalamus (DMH), and the lateral hypothalamic area (LH) ().

High prevalence of type 2 diabetes and pre-diabetes in adult offspring of women with gestational diabetes mellitus or type 1 diabetes: the role of intrauterine hyperglycemia.

Sociodemographic correlates of the increasing trend in prevalence of gestational diabetes mellitus in a large population of women between 1995 and 2005.

Reciprocal neural connections between the hypothalamus and preganglionic motor neurons of the autonomic nervous system play an important role in the regulation of energy and glucose homeostasis (). Therefore, we aimed to identify changes in the autonomic tone in peripheral organs that could possibly be linked to the specific restoration of α-MSH axonal projections to the preautonomic compartment of the PVHin NCD/HFD POMCmice. Given the distinct rescue of glucose tolerance in POMC-specific IR-deficient NCD/HFD offspring in the absence of alterations in insulin sensitivity, we analyzed the parasympathetic innervation of pancreatic β cells by staining for vesicular acetylcholine transporter (vAChT) (). Strikingly, the number of vAChT-immunoreactive buttons per islet area was significantly reduced in NCD/HFD ctrl offspring but was rescued to NCD/NCD levels in NCD/HFD POMCmice ( Figure 7 A). In line with the decreased parasympathetic innervation of pancreatic β cells in NCD/HFD ctrl mice, glucose-stimulated insulin secretion was significantly decreased compared to NCD/HFD POMCoffspring ( Figure 7 B). Consistently, C-peptide levels were decreased 5 min after intravenous glucose injection in NCD/HFD ctrl offspring ( Figure 7 C). In contrast, this defect in insulin secretion in NCD/HFD ctrl mice was not seen upon L-arginine stimulation ( Figure 7 D) and was not associated with glucose-stimulated alterations in levels of free fatty acids (FFA) or glucagon-like peptide 1 (GLP-1) ( Figure S6 ). Moreover, neither maternal HFD feeding during lactation nor POMC-specific IR deficiency had any effect on the average pancreatic β cell mass or the average islet size of the pancreas ( Figure 7 E). Taken together, our results indicate that POMC-specific IR deficiency improves glucose-stimulated insulin secretion, presumably in part via modulation of the parasympathetic tone in offspring from postnatally HFD-fed mothers.

(A and B) (A) Serum glucagon-like peptide 1 (GLP-1) and (B) non-esterified free fatty acid (FFA) concentration under basal conditions and 5 min after intravenous glucose injection in NCD/HFD ctrl and NCD/HFD POMC ΔIR offspring at 15 weeks of age after a 16 hr fasting period (n = 7 vs. 11). Data are presented as mean ± SEM.

(E) Images and quantification of total β cell mass and average β cell islet size at 20 weeks of age (n = 6 vs. 5 vs. 4 vs. 5). Scale bar, 300 μm. Data are presented as mean ± SEM. ∗ p < 0.05 versus all other groups of offspring unless otherwise indicated.

(B–D) (B) Glucose-stimulated insulin secretion (n = 13 vs. 13), (C) C-peptide levels 0 and 5 min after glucose injection (n = 8 vs. 8) (see also Figure S6 for corresponding glucagon-like peptide 1 and free fatty acid concentrations), and (D) L-arginine-stimulated insulin secretion at 15 weeks of age (n = 7 vs. 7).

(A) Images and quantification of the parasympathetic marker vesicular acetylcholine transporter (vAChT, green) on pancreatic β cells (insulin, red) at 20 weeks of age (n = 6 vs. 5 vs. 8 vs. 6). Scale bar, 50 μm.

In contrast, AgRP fiber densities were significantly reduced in NCD/HFD offspring independent of their genotype in the PVH, DMH, LH, and most importantly, also the PVHat the age of 20 weeks ( Figures 6 A, 6B, S5 A, and S5B). Collectively, hyperinsulinemia in response to maternal HFD feeding during lactation impairs the axonal outgrowth of POMC neurons specifically to the preautonomic compartment of the PVH.

To decipher persistent hypothalamic cellular changes that are responsible for the metabolic rescue of glucose tolerance in NCD/HFD POMCmice, we analyzed fiber densities of ARH neurons to the distinct subcompartments of the PVH at 8 and 20 weeks of age, as well as to the DMH and LH at 20 weeks of age. Loss of the IR specifically on POMC neurons did not change α-MSH or AgRP fiber densities to any hypothalamic target site in NCD/NCD offspring at any age ( Figures 6 A and 6B , S5 A, and S5B). Moreover, maternal HFD feeding exclusively during lactation resulted in a decrease of α-MSH fiber density in the neuroendocrine PVHin young and old NCD/HFD animals ( Figure 6 A), as well as in the DMH ( Figure S5 A) and LH ( Figure S5 B), independent of their genotype. However, although NCD/HFD POMCmice displayed a similar decrease in α-MSH fiber density in the neuroendocrine PVHregion as did their control litter mates ( Figure 6 A), specific inactivation of the IR on POMC neurons protected against a decrease in the α-MSH fiber density in the preautonomic PVHcompartment, resulting in a persistent restoration of α-MSH fiber density in NCD/HFD POMCoffspring close to NCD/NCD levels both at 8 and 20 weeks of age ( Figure 6 B).

(A and B) Images and quantification of α-melanocyte-stimulating hormone (α-MSH) and agouti-related-peptide (AgRP) immunoreactive fibers at 20 weeks of age innervating (A) the dorsomedial nucleus of the hypothalamus (DMH; n α-MSH = 5 for all groups and n AgRP = 5 vs. 4 vs. 4 vs. 5); and (B) the lateral hypothalamic area (LH; n α-MSH = 4 vs. 5 vs. 5 vs. 5 and n AgRP = 5 vs. 4 vs. 5 vs. 5) in NCD/NCD ctrl, NCD/NCD POMC ΔIR , NCD/HFD ctrl and NCD/HFD POMC ΔIR offspring. White boxes indicate area of quantification. 3V = third ventricle, fx = fornix. Scale bar = 100 μm. Data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. all other groups of offspring, unless otherwise indicated.

White boxes indicate area of quantification. 3V, third ventricle. Scale bar, 100 μm. Data are presented as mean ± SEM.p < 0.05 versus all other groups of offspring unless otherwise indicated. See also Figure S5 for images and quantification of α-MSH and AgRP immunoreactive fibers innervating the dorsomedial nucleus of the hypothalamus (DMH) and the lateral hypothalamic area (LH) at 20 weeks of age.

Images and quantification of α-melanocyte-stimulating hormone (α-MSH) and agouti-related-peptide (AgRP) immunoreactive fibers innervating (A) the anterior neuroendocrine paraventricular nucleus of the hypothalamus (PVH ant ) at 8 (n α-MSH and n AgRP = 8 vs. 8 vs. 8 vs. 10) and 20 weeks of age (n = 5 for all groups) and (B) the posterior preautonomic PVH (PVH post ) at 8 (n α-MSH = 7 vs. 6 vs. 5 vs. 6 and n AgRP = 5 vs. 6 vs. 5 vs. 6) and 20 weeks of age (n = 5 for all groups).

However, when subjected to a glucose tolerance test (GTT), NCD/HFD ctrl mice displayed a pronounced glucose intolerance, which was rescued to NCD/NCD levels in NCD/HFD POMCoffspring ( Figure 5 G). Together, these results point to a distinct role for elevated neuronal insulin signaling in response to maternal postnatal HFD feeding in predisposing the offspring for an impaired glucose tolerance throughout lifetime.

Although maternal HFD feeding during lactation did not affect body weight, it significantly increased the glucose and insulin content of the milk in NCD/HFD mothers ( Figure S4 ), which was associated with a distinct hyperinsulinemia in the NCD/HFD offspring at 3 weeks of age ( Figure 1 E). Thus, we aimed to define the potential contribution of neuronal insulin signaling in impairing melanocortin projections in offspring of postnatally HFD-fed mothers. To this end, we specifically inactivated the insulin receptor (IR) from POMC neurons in NCD/NCD and NCD/HFD offspring by crossing female IR floxed/floxed (IR) mice with male IRmice expressing Cre recombinase under the control of the POMC promoter () and further subjected the mothers to our postnatal feeding paradigm. All groups of offspring were challenged with a HFD after 8 weeks of age, resulting in 4 groups of offspring differing in both the maternal diet during lactation and the presence (NCD/NCD ctrl and NCD/HFD ctrl) and absence (NCD/NCD POMCand NCD/HFD POMC) of IR expression on POMC neurons. Similar to our previous results, maternal HFD feeding exclusively during lactation did not alter body weight between NCD/NCD ctrl and NCD/HFD ctrl offspring on HFD and also had no effect on the respective POMC-specific IR-deficient offspring ( Figure 5 A). Despite showing no increases in body weight, NCD/HFD offspring developed greater adiposity independently of their genotype, as revealed by elevated body fat content ( Figure 5 B), increased perigonadal fat pad weight ( Figure 5 C), and elevated serum leptin levels ( Figure 5 D). Similarly, both measures of insulin sensitivity, i.e., the HOMA-IR ( Figure 5 E) and insulin tolerance tests (ITT) ( Figure 5 F), showed tendencies toward an impaired insulin sensitivity in both NCD/HFD ctrl and NCD/HFD POMCoffspring. Thus, POMC-specific inactivation of IR signaling affect neither adiposity nor the impaired insulin sensitivity in offspring from mothers fed a HFD exclusively during lactation.

Data are presented as mean ± SEM.p < 0.05,p < 0.01, andp < 0.001 versus all other groups of offspring unless otherwise indicated. See also Figure S4 for data on milk composition.

(D–G) (D) Fasted serum leptin levels (n = 9 vs. 9 vs. 9 vs. 14) and (E) homeostatic model assessment indices of insulin resistance (HOMA-IR; n = 6 vs. 10 vs. 10 vs. 14)) at 15 weeks, (F) insulin tolerance tests (ITT) at 14 weeks (n = 16 vs. 19 vs. 20 vs. 19), and glucose tolerance tests (GTT) at 15 weeks of age (n = 10 vs. 13 vs. 13 vs. 14).

(A–C) (A) Body weight (n = 10 vs. 13 vs. 14 vs. 14), (B) body fat content (n = 9 vs. 12 vs. 14 vs. 14), and (C) perigonadal fat pad weight (n = 10 vs. 12 vs. 14 vs. 14) at 20 weeks of age.

(A–E) (A) Maternal body weight changes between postnatal day 4 (P4) and P19 (n = 11 vs. 11), (B) glucose (n = 9 vs. 8), (C) insulin (n = 9 vs. 10) and (D) leptin levels (n = 9 vs. 10), as well as (E) non-esterified free fatty acids (FFA) content (n = 9 vs. 10) in the milk on P19 in mothers consistently exposed to a NCD during gestation and lactation (NCD/NCD) and mothers fed a HFD starting during lactation (NCD/HFD). Data are presented as mean ± SEM, ∗ p < 0.05. ∗∗ p < 0.01.

Considering that the strongest impact on metabolic fate of the offspring resulted from altering maternal diet selectively during lactation, the phase of hypothalamic neurocircuit development in which axonal projections are formed in rodents, we next analyzed the immunoreactivity of α-MSH- and AgRP-containing fibers in three of the main ARH downstream hypothalamic projection areas: the PVH, DMH, and LH. Of note, the PVH consists of distinct functional subcompartments that regulate neuroendocrine, behavioral, and autonomic responses to control energy and glucose homeostasis (). Neuroendocrine neurons, such as TRH neurons, reside mainly in the anterior two-thirds of the PVH (referred to as PVH), whereas preautonomic neurons are predominantly found in the posterior part of the PVH (referred to as PVH) (). Due to this distinct compartmentalization and the associated diverse functions of the PVH, we differentiated both the PVHand the PVHin our analysis. Quantification of the fiber density in the PVH Figure 4 A), the PVH Figure 4 B), the DMH ( Figure 4 C), and the LH ( Figure 4 D) revealed robust reductions in both the α-MSH and AgRP fiber densities in NCD/HFD offspring compared to NCD/NCD offspring. Thus, maternal HFD feeding results in a consistent decrease of ARH neuronal fiber densities in hypothalamic areas critically involved in the neuroendocrine and autonomic regulation of energy homeostasis, likely due to impaired axon formation in the offspring.

Schematics illustrating the localization in the CNS of the respective hypothalamic nuclei presented in the pictures were based on and modified from. Coordinates were adapted according to. White boxes indicate area of quantification. 3V, third ventricle; fx, fornix. Scale bar, 100 μm. Data are presented as mean ± SEM.p < 0.05,p < 0.01,p < 0.001 versus all other groups of offspring.

(A–D) Images and quantification of α-melanocyte-stimulating hormone (α-MSH) and agouti-related peptide (AgRP) immunoreactive fibers innervating (A) the anterior endocrine paraventricular nucleus of the hypothalamus (PVH ant ; n α-MSH = 6 vs. 7 and n AgRP = 7 vs. 7), (B) the posterior preautonomic PVH post (n α-MSH = 5 vs. 5 and n AgRP = 4 vs. 4), (C) the dorsomedial nucleus of the hypothalamus (DMH; n α-MSH = 7 vs. 7 and n AgRP = 4 vs. 5), and (D) the lateral hypothalamic area (LH; n α-MSH = 6 vs. 6 and n AgRP = 6 vs. 4) at 8 weeks of age.

Next, we analyzed whether maternal HFD feeding during lactation had an effect on the electrophysiological properties of POMC neurons in the offspring. Whole-cell and perforated patch-clamp recordings on POMCtransgenic NCD/NCD and NCD/HFD offspring indicated that maternal HFD feeding during lactation did not result in any differences in spontaneous firing frequency of POMC neurons ( Figure 3 F), POMC neuron resting membrane potential ( Figure 3 G), or the relative synaptic input onto POMC neurons ( Figure 3 H). Collectively, these data demonstrate that maternal HFD feeding exclusively during lactation permanently decreases anorexigenic TRH expression, which is a target of POMC and AgRP neurons of the ARH, without altering ARH neuropeptide gene expression, ARH neuronal cell number, neuropeptide processing of POMC to diacetylated α-MSH, and/or electrophysiological properties of POMC neurons.

Of note, AgRP can be modulated by but does not depend upon posttranslational modifications to decrease TRH expression in the PVH (). However, POMC has to undergo proprotein-convertase (PC)-1, -2, and carboxypeptidase (CPE)-mediated processing to generate the active neuropeptide α-melanocyte-stimulating-hormone (α-MSH), which exerts its anorexigenic functions, in part, via upregulation of TRH (). Thus, we investigated whether POMC processing might be impaired in NCD/HFD offspring. Hypothalamic mRNA expression of Pcsk1, Pcsk 2 (respectively, PC-1 and PC-2), and Cpe did not show any differences between groups of offspring ( Figure 3 D). Moreover, MALDI-TOF mass spectrometry of dissected ARH samples showed nearly identical peptide signals, including ions that are mass identical with products of the POMC precursor protein (i.e., α-MSH, diacetylated α-MSH, and joining peptide) () between NCD/NCD and NCD/HFD offspring ( Figure 3 E).

Agouti-related protein is posttranslationally cleaved by proprotein convertase 1 to generate agouti-related protein (AGRP)83-132: interaction between AGRP83-132 and melanocortin receptors cannot be influenced by syndecan-3.

Next, we focused on comparing NCD/NCD and NCD/HFD male offspring to define the molecular mechanism(s) underlying the obese and glucose-intolerant phenotype of NCD/HFD mice. First, we determined mRNA expression of hypothalamic neuropeptides critically involved in the regulation of energy and glucose homeostasis. Although there was no difference in the expression of ARH neuropeptide genes, i.e., Pomc, Agrp, and Npy ( Figure 3 A), mRNA expression of one of their anorexigenic downstream targets, thyrotropine-releasing hormone (Trh), which is predominantly but not exclusively expressed in the PVH (), was significantly lower in NCD/HFD offspring ( Figure 3 B). These experiments indicated that, in the absence of alterations of Pomc and Agrp expression in the ARH, expression of one of the melanocortin-effector pathways is impaired. To determine whether hypothalamic inflammation contributed to the impairment of the melanocortin circuitry, we analyzed mRNA expression of classical inflammatory markers in the hypothalamus. However, we could not detect differences in the hypothalamic expression of any of the genes analyzed between NCD/NCD and NCD/HFD offspring ( Figure S3 ). Next, we analyzed the effect of postnatal maternal HFD feeding on the cell number of ARH neurons by employing our postnatal feeding paradigm to female C57Bl/6 mice crossed to male transgenic mice expressing the enhanced green fluorescent protein (eGFP) under the transcriptional control of the POMC promoter (POMC) ()—and further, to females carrying a floxed Rosa26-tdTomato allele encoding red fluorescent protein (The Jackson Laboratories) crossed to male mice expressing Cre recombinase under the transcriptional control of the AgRP promoter () to generate AgRPmice. Consistent with unaltered Pomc– and Agrp/Npy expression, there was no difference in the number of eGFP-positive POMC or tdTomato-positive AgRP neurons between NCD/NCD and NCD/HFD offspring ( Figure 3 C).

Quantitative real-time PCR analysis of hypothalamic tumor necrosis factor (Tnf), interleukin 6 (Il6) and interleukin 1 beta (Il1b) mRNA expression of NCD/NCD and NCD/HFD offspring at 3 (n = 9 vs. 9) and 20 weeks (n = 7 vs. 6) of age on a NCD. Data are presented as mean ± SEM.

Data are presented as mean ± SEM.p < 0.05 versus all other groups at the same age. See also Figure S3 for hypothalamic mRNA expression of inflammatory markers at 3 and 20 weeks of age.

(E) MALDI-TOF mass spectra obtained by profiling extracts of the ARH at 20 weeks of age (n = 4 vs. 4). Prominent ion signals are labeled. (i) Comparison of mass fingerprints showing nearly identical ion signals, including ions that are mass identical with products of the POMC precursor (α-MSH, Di-Ac-MSH, joining peptide [JP]). Fragmentation experiments confirmed the sequences of all labeled peptides; the ion signal at 1622.81 (asterisk) is composed of two substances (including α-MSH). The arrow marks processed and biologically more potent diacetylated α-MSH. (ii) Isotopic pattern and signal intensity of diacetylated α-MSH before (lower traces) and after Stage Tip concentration. ii) MALDI-TOF/TOF fragment spectrum of diacetylated α-MSH purified and concentrated with Stage Tips. Y- and b-type fragment ions are labeled, which confirmed the amino acid sequence of di-acetylated α-MSH. (iii) Gel view of mass spectra (n = 4 each) from preparation of ARH and pituitary gland (Pit) demonstrating identical processing of Pomc-products in all samples.

(D) Quantitative real-time PCR analysis of hypothalamic proprotein convertase subtilisin/kexin type 1 (Pcsk1), proprotein convertase subtilisin/kexin type 2 (Pcsk2), and carboxypeptidase E (Cpe) mRNA expression at 3 (n = 9 vs. 11) and 20 weeks (n = 8 vs. 9) of age on NCD.

(C) Analysis of POMC (left) and AgRP (right) neurons in the arcuate nucleus of the hypothalamus (ARH) in POMC eGFP and AgRP tdTomato mice, respectively, at 8 weeks of age (n POMC = 3 vs. 3 and n AgRP = 4 vs. 5; scale bar, 100 μm).

(A and B) All of the following analyses were performed in NCD/NCD and NCD/HFD male offspring. Quantitative real-time PCR analysis of hypothalamic (A) pro-opiomelanocortin (Pomc), agouti-related peptide (Agrp), neuropeptide Y (Npy), and (B) thyrotropin-releasing hormone (Trh) mRNA expression at 3 (n = 9 vs. 11) and 20 weeks (n = 8 vs. 9) of age on NCD.

We first subjected all groups of offspring to a metabolic characterization. When male offspring were fed a NCD, only mice whose mothers were fed a HFD during lactation (NCD/HFD) displayed significantly increased body weight throughout their adult life compared to all other groups ( Figure 2 A). Consistently, NCD/HFD mice showed elevated body fat content ( Figure 2 B), increased perigonadal fat pad weight ( Figure 2 C), and elevated serum leptin levels ( Figure 2 D) compared to NCD/NCD mice. Moreover, NCD/HFD mice showed enhanced insulin resistance ( Figure 2 E) and glucose intolerance ( Figure 2 F) when compared to any other group of offspring. Notably, although most metabolic abnormalities were only seen in male NCD/HFD mice when exposed to a NCD, female offspring from NCD/HFD mothers displayed a similar obese phenotype only when challenged with a HFD after 8 weeks of age ( Figure S2 ). Taken together, exposure of mothers to a HFD exclusively during the lactation phase exerts the strongest effects on alterations in energy and glucose homeostasis in both male and female offspring.

(A–F) The following metabolic parameters were analyzed in all eight groups of female offspring. (A) Body weight (BW) on (i) normal chow diet (NCD) or (ii) high fat diet (HFD) after 8 weeks of age (n NCD = 11 vs. 9 vs. 5 vs. 6 and n HFD = 13 vs. 10 vs. 9 vs. 6), (B) body fat content (n NCD = 11 vs. 9 vs. 5 vs. 5 and n HFD = 13 vs. 10 vs. 8 vs. 5) and (C) perigonadal fat pad weight (n NCD = 11 vs. 9 vs. 5 vs. 5 and n HFD = 13 vs. 10 vs. 9 vs. 6) at 20 weeks of age, (D) fasted serum leptin levels (n NCD = 8 vs. 8 vs. 5 vs. 5 and n HFD = 9 vs. 8 vs. 8 vs. 5) and (E) homeostatic model assessment indices of insulin resistance (HOMA-IR) (n NCD = 8 vs. 8 vs. 5 vs. 5 and n HFD = 9 vs. 8 vs. 8 vs. 5) and (F) glucose tolerance tests (GTT) at 15 weeks of age on i) NCD and ii) HFD (n NCD = 10 vs. 9 vs. 4 vs. 5 and n HFD = 13 vs. 10 vs. 9 vs. 5). Data are presented as mean ± SEM, ∗ p < 0.05. ∗∗ p < 0.01. ∗∗∗ p < 0.001 versus all other groups within the same diet after the age of 8 weeks, if not indicated otherwise.

Data are presented as mean ± SEM.p < 0.05,p < 0.01,p < 0.001 versus all other groups within the same diet after 8 weeks of age unless otherwise indicated. See also Figure S2 for data on female offspring.

(A–F) (A) Body weight on (i) normal chow diet (NCD; n = 14 vs. 10 vs. 14 vs. 13) or (ii) high fat diet (HFD; n = 10 vs. 8 vs. 13 vs. 12), (B) body fat content and (C) perigonadal fat pad weight at 20 weeks (n NCD = 14 vs. 10 vs. 13 vs. 12 and n HFD = 10 vs. 8 vs. 12 vs. 11), (D) fasted serum leptin levels and (E) homeostatic model assessment indices of insulin resistance (HOMA-IR) at 15 weeks (n NCD = 13 vs. 8 vs. 11 vs. 9 and n HFD = 9 vs. 8 vs. 11 vs. 11) and (F) glucose tolerance tests (GTT) at 15 weeks of age on (i) NCD (n = 13 vs. 10 vs. 14 vs. 13) and (ii) HFD (n = 10 vs. 8 vs. 9 vs. 12).

To determine the most vulnerable time frame of hypothalamic neurocircuit development in response to maternal HFD feeding, we fed female C57Bl/6 virgin mice either a control normal chow diet (NCD) or a HFD for 8 weeks prior to gestation ( Figure S1 available online). HFD feeding for the period of 7 weeks resulted in moderately increased body weight ( Figure 1 A), elevated fasting blood glucose concentrations ( Figure 1 B), and an ∼7-fold increase in the homeostatic model assessment indices of insulin resistance (HOMA-IR) ( Figure 1 C), indicating that pregestational HFD feeding causes insulin resistance. During gestation, the mice were maintained on the same diet that they received during the pregestational period. At the day of birth (DOB), litter size was adapted to 6–7 pups per mother to assure the same quantitative nutritional availability for each litter, as small litter size increases the propensity for metabolic diseases in the offspring during adulthood (). At DOB, half of the NCD-fed mothers were exposed to a HFD (i.e., NCD/NCD and NCD/HFD), and half of the mothers fed a HFD pregestationally and during gestation were exposed to a NCD during the period of lactation (i.e., HFD/NCD and HFD/HFD). Interestingly, HFD exposure during lactation, independent of the prenatal maternal diet, resulted in a slight elevation of serum insulin concentrations in the mothers ( Figure 1 D) and in increased serum insulin levels in the offspring at 3 weeks of age ( Figure 1 E). After weaning, all offspring were fed a NCD until the age of 8 weeks, after which each group of offspring was divided into groups either exposed to a NCD or a HFD for the following 12 weeks, resulting in a total of 8 different groups of offspring that differed in the prenatal maternal diet, the postnatal maternal diet, and in the diet of the offspring after 8 weeks of age ( Figure S1 ).

NCD, normal chow diet; HFD, high-fat diet. Data are presented as mean ± SEM.p < 0.01,p < 0.001 versus all other groups within the same diet after week 8 if not indicated otherwise. See also Figure S1 for an overview of all experimental groups.

Female C57Bl/6 virgin mice were fed with either a normal chow diet (NCD) or a high-fat diet (HFD) from 4 to 11 weeks (wks) of age. Breedings with male C57Bl/6 mice were set up during the 11 th week of age. During gestation, mice were maintained on the same diet that they had received before. At the day of birth (DOB) of the offspring, litter size was reduced to 6-7 animals per mother and half of the NCD-fed mothers were exposed to a HFD, while half of the HFD-fed mothers were exposed to a NCD during lactation. After weaning, all offspring was exposed to a NCD until 8 weeks of age, after which each group of offspring was divided into groups either receiving a NCD or a HFD until 20 weeks of age. In total, this resulted in 8 groups of offspring differing in the prenatal and postnatal maternal diet and in the diet of the offspring after week 8. Light blue lines indicate NCD-feeding and dark blue lines indicate HFD-feeding.

Discussion

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Simerly R.B. Neonatal leptin exposure specifies innervation of presympathetic hypothalamic neurons and improves the metabolic status of leptin-deficient mice. The site specificity in the rescue of POMC axonal formation to the preautonomic compartment, but not to the neuroendocrine compartment of the PVH in POMC-specific IR-deficient offspring from postnatally HFD-fed mothers, might result from the cellular heterogeneity of this neuronal population. Only a subset of POMC neurons is insulin responsive, and this subpopulation of neurons is distinct from leptin-responsive POMC neurons in the ARH (). Thus, loss of IR signaling under hyperinsulinemic conditions might only lead to a beneficial effect on a subpopulation of POMC neurons, which might predominantly target the posterior part of the PVH. However, future studies will clearly have to address the mechanistic basis for the selective effect of IR signaling on projection formation to the PVH. Of note, Bouyer and Simerly have recently demonstrated a similar role for leptin in mediating site-specific axonal innervation of AgRP neurons to the preautonomic compartment of the PVH (). However, whether abnormal levels of insulin have identical effects on the axonal projections of AgRP neurons, as we described for POMC neurons, still requires further investigation.

Bouret et al., 2004b Bouret S.G.

Draper S.J.

Simerly R.B. Trophic action of leptin on hypothalamic neurons that regulate feeding. Bouyer and Simerly, 2013 Bouyer K.

Simerly R.B. Neonatal leptin exposure specifies innervation of presympathetic hypothalamic neurons and improves the metabolic status of leptin-deficient mice. Vickers et al., 2005 Vickers M.H.

Gluckman P.D.

Coveny A.H.

Hofman P.L.

Cutfield W.S.

Gertler A.

Breier B.H.

Harris M. Neonatal leptin treatment reverses developmental programming. Bonnin and Levitt, 2011 Bonnin A.

Levitt P. Fetal, maternal, and placental sources of serotonin and new implications for developmental programming of the brain. Grove and Cowley, 2005 Grove K.L.

Cowley M.A. Is ghrelin a signal for the development of metabolic systems?. Sasaki et al., 2013 Sasaki A.

de Vega W.C.

St-Cyr S.

Pan P.

McGowan P.O. Perinatal high fat diet alters glucocorticoid signaling and anxiety behavior in adulthood. Although we could show that POMC-specific inactivation of the IR rescues POMC axonal innervation of the preautonomic PVH, as well as glucose tolerance, and improves glucose-stimulated insulin secretion in offspring from postnatally HFD-fed mothers, loss of insulin signaling in POMC neurons neither ameliorated POMC axonal innervation of the neuroendocrine compartment of the PVH, the DMH, or the LH nor reduced the susceptibility to develop increased adiposity and insulin resistance. Thus, other mechanisms than abnormal insulin signaling in POMC neurons must play an important role in mediating metabolic programming in offspring from postnatally HFD-fed mothers. Indeed, maternal HFD feeding during lactation leads not only to increased levels of glucose and insulin, but also to elevated levels of leptin and free fatty acids in the milk ( Figure S4 ), presumably contributing to the strong metabolic impairments in offspring exposed to maternal HFD feeding during lactation. Accordingly, the crucial role of well-balanced leptin levels during hypothalamic neurocircuit development has been established in a series of outstanding studies (). Apart from leptin, abnormal levels of other hormones such as ghrelin, corticosterone, serotonin or elevated levels of free fatty acids in response to postnatal maternal HFD feeding might affect hypothalamic neurocircuit development and/or alter cellular plasticity in peripheral organs, thereby contributing to the predisposition of metabolic diseases (). Nevertheless, among all of the developmental factors that may act synergistically and antagonistically to shape neuronal circuitries, the present study highlights a critical role of insulin in impairing long-term organization of melanocortin projections within the hypothalamus under pathological conditions during development.