abstract: Hypoglycemia stimulates counterregulatory hormone release to restore euglycemia. This protective response is diminished by recurrent hypoglycemia, limiting the benefits of intensive insulin treatment in patients with diabetes. We previously reported that EphA5 receptor-ephrinA5 interactions within the ventromedial hypothalamus (VMH) influence counterregulatory hormone responses during acute hypoglycemia in nondiabetic rats. In this study, we examined whether recurrent hypoglycemia alters the capacity of the ephrinA5 ligand to activate VMH EphA5 receptors, and if so, whether these changes could contribute to pathogenesis of defective glucose counterregulation in response to a standard hypoglycemic stimulus. The expression of ephrinA5, but not EphA5 receptors within the VMH, was reduced by antecedent recurrent hypoglycemia. In addition, the number of synaptic connections was increased and astroglial synaptic coverage was reduced. Activation of VMH EphA5 receptors via targeted microinjection of ephrinA5-Fc before a hyperinsulinemic hypoglycemic clamp study caused a reduction in the glucose infusion rate in nondiabetic rats exposed to recurrent hypoglycemia. The increase in the counterregulatory response to insulin-induced hypoglycemia was associated with a 150% increase in glucagon release (*P* \< 0.001). These data suggest that changes in ephrinA5/EphA5 interactions and synaptic plasticity within the VMH, a key glucose-sensing region in the brain, may contribute to the impairment in glucagon secretion and counterregulatory responses caused by recurrent hypoglycemia.
author: Barbara Szepietowska; Tamas L. Horvath; Robert S. SherwinCorresponding author: Robert S. Sherwin, <email@example.com>.
date: 2014-03
references:
title: Role of Synaptic Plasticity and EphA5-EphrinA5 Interaction Within the Ventromedial Hypothalamus in Response to Recurrent Hypoglycemia

Frequent episodes of acute hypoglycemia represent the principal obstacle to achieving optimal glycemic control during insulin treatment in patients with type 1 diabetes and long-standing type 2 diabetes (1,2). This problem is further magnified by the loss of an appropriate counterregulatory response to hypoglycemia that results as a consequence of frequent episodes iatrogenic insulin-induced hypoglycemia (3,4). The molecular mechanisms underlying this phenomenon remain uncertain but are likely to involve the key brain glucose–sensing region, the ventromedial hypothalamus (VMH) (5,6).

We have previously reported that local stimulation of VMH EphA5 receptors by microinjection of ephrinA5-Fc or ephrinA5 overexpression increased, whereas knockdown of VMH ephrinA5 reduced, counterregulatory responses to hypoglycemia. Furthermore, overexpression of VMH ephrinA5 transiently increased local glutamate concentrations, whereas ephrinA5 knockdown produced profound suppression of VMH interstitial fluid glutamine concentrations in the basal state and during hypoglycemia. These data suggest that the activation of VMH EphA5 receptors by ephrinA5 may act in concert with β-cell Eph receptor forward signaling to restore glucose homeostasis during acute hypoglycemia via alterations in glutamate/glutamine cycling (7,8).

Within the central nervous system, Eph receptors and their ligands, the ephrins, play a key role in cell-to-cell communication as well as in synaptic structure and function. Eph receptors function as transmembrane receptor tyrosine kinases and are divided by sequence similarity and ligand affinity into an A and a B subclass. Their ligands, the ephrins, are also divided into an A and a B subclass: the A subclass is tethered to the cell membrane by a glycosylphosphatidylinositol anchor, and members of the B subclass have a transmembrane domain and as a short cytoplasmic region. For the most part, A-type receptors bind to most or all A-type ligands, and B-type receptors bind to most or all B-type ligands (9). As for many other receptor tyrosine kinases, ligand-binding induces "forward signaling," mostly through phosphotyrosine-mediated pathways. However, ephrins can also signal into their host cell—referred to as "reverse signaling'"(10). Eph receptors and their ephrin ligands are present in the adult brain and are specifically enriched in glutamate excitatory synapses (11). Moreover, Eph receptor tyrosine kinases are mainly expressed in synaptic terminals where they influence synaptic plasticity via binding to ephrins found on astrocytic processes that surround the synapse or on neuronal synapses (12,13).

Several observations suggest that changes in hypothalamic synaptic plasticity may play a significant role in the regulation of energy balance. For example, peripheral signals, such as leptin, ghrelin, and estrogen, induce synaptic adaptations that serve as dynamic regulators of neuronal activity in the arcuate nucleus, the hypothalamic center for feeding control (14,15). Whether hypoglycemia per se induces local changes in the VMH affecting both neuronal synapses and/or surrounding glia cells is unknown, but such alterations could potentially modulate neurotransmission within VMH, thereby altering brain glucose sensing. This study tests whether recurrent hypoglycemia alters EphA5 receptor–ephrinA5 interactions within the VMH, which might contribute to diminished activation of counterregulatory responses to acute hypoglycemia.

# Research Design and Methods

## Animals

Male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 300–350 g were individually housed in the Yale Animal Resource Center in temperature- (22–23°C) and humidity-controlled rooms. Animals were fed rat chow (Prolab 3000; Agway, Syracuse, NY) and water ad libitum and were acclimatized to a 12-h light-dark cycle. The Yale University Institutional Animal Care and Use Committee approved the experimental protocols. Different sets of animals were used for the clamp and electron microscopy (EM) experiments described below.

## Vascular and Stereotaxic Surgery

Animals were anesthetized ∼7–10 days before study and underwent aseptic surgery in which vascular catheters were implanted into the left carotid artery for blood sampling and into the right jugular vein for infusion, as previously described (8). These catheters were tunneled subcutaneously and exteriorized at the back of the neck between the scapulae. Wound staples were used to close the incision. For stereotaxic surgery, animals were placed into a stereotaxic frame (David Kopf Instruments, Tujunga, CA), and stainless steel guide cannulas (Plastics One, Roanoke, VA) were inserted into the brain and secured in place with screws and dental acrylic (coordinates from Bregma: anteroposterior −2.6 mm, mediolateral ± 0.8 mm, and dorsoventral −8.0) for microinjection.

## Recurrent Hypoglycemia Protocol

Rats received intraperitoneal injections of insulin (10 units/kg) for 3 consecutive days. After each injection, food was withheld to allow plasma glucose to fall into the hypoglycemic range (30–50 mg/dL); throughout, animals were monitored with tail vein glucose measurements every 30 min using the AlphaTRAK rodent glucometer (Abbott Animal Health, Chicago, IL) to ensure sustained hypoglycemia and to avoid glucose reduction sufficient to cause seizure activity. At the end of this period, the rats were given free access to food again. Control rats received an injection of 0.9% saline under the same conditions.

## Microinjection of EphrinA5-Fc or Control-Fc

On the morning of the study, awake, overnight-fasted rats were connected to infusion pumps ∼90 min before the start of the experiment and then left undisturbed to recover from handling stress. After the recovery period, 22-gauge microinjection needles (Plastics One), designed to extend 1 mm beyond the tip of the guide cannula, were inserted bilaterally through the guide cannula into each VMH region. Rats then received a microinjection of recombinant human ephrinA5-Fc or recombinant human IgG1-Fc (control-Fc protein; both R&D Systems, Minneapolis, MN; catalog number: 374-EA-200) in a concentration of 0.3 μg/μL dissolved in artificial extracellular fluid delivered at a rate of (0.1 µL/min) over 60 min (dose: 1.8 μg for each side). After the microinjection, needles were left in place for 30 min before being removed. Immediately thereafter, a hyperinsulinemic hypoglycemic clamp study was performed. These compounds have been previously administered into the central nervous system in in vivo studies (16) and in vitro in brain slices (17), without adverse effects. In addition, ephrinA5-Fc has been shown to specifically bind EphA5 receptors (18).

## Hyperinsulinemic-Hypoglycemic Clamp

A primed continuous infusion of 20 mU ⋅ kg^–1^ ⋅ min^–1^ insulin (Humulin R; Eli Lilly, Indianapolis, IN) was given, and a variable infusion of 20% dextrose was adjusted at 5- to 10-min intervals based on glucose measurements (Analox Instruments, Lunenburg, MA) designed to maintain plasma glucose at 50 mg/dL from 30 to 90 min (19). Additional blood was drawn at baseline and at 30, 60, and 90 min for measurement of insulin, glucagon, epinephrine, and norepinephrine. Rats were killed at study termination, and the probe position was confirmed histologically.

## Hormone and Neurotransmitter Analyses

Plasma catecholamine measurements were analyzed by high-performance liquid chromatography using electrochemical detection, and plasma insulin and glucagon concentrations were determined by radioimmunoassay (Linco, St. Charles, MO).

## Immunoblot Analysis

Frozen tissue micropunches from VMH and control regions were homogenized in buffer containing 1% NP40, 150 mmol/L NaCl, 50 mmol/L Tris (pH 7.4), 1 mmol/L Na~3~VO~4~, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor (Roche Diagnostics) using a plastic pestle and ultrasonicator. Protein content was assessed with the Bradford protein assay. Protein samples were fractioned under reducing conditions on SDS-9% PAGE (Bio-Rad). After electrophoresis, proteins were electroblotted onto nitrocellulose membranes, blocked with 5% nonfat dry milk in PBS, probed with first antibody (α-tubulin; Cell Signaling, cat. 2125S; ephrin-A5; R&D Systems, cat. AF3743) and EpA5 receptor (EphA5; Sigma-Aldrich, cat. P8651), and incubated with the appropriate secondary antibody conjugated to peroxidase by horseradish peroxidase–linked protein A (1:2000; Sigma-Aldrich). The immunoblots were developed using an enhanced chemiluminescence detection system (Amersham Biosciences).

## Electron Microscopy

Briefly, animals were perfused with paraformaldehyde fixative and ultrathin brain sections were cut on a Leica ultramicrotome, collected on Formvar-coated single-slot grids, and analyzed with a Tecnai 12 Biotwin EM (FEI). Glia coverage of the cell membrane of random VMH cells was performed using ImageJ software. EM photographs (original magnification ×11,500) were used to first measure the perimeter of each VMH neuron analyzed, followed by determination of the amount of membrane covered by glia (in nanometers). Results are reported as glia coverage/perimeter of the VMH neurons.

Characteristics of synaptic contacts were defined, as previously described (20). They were collected from serial sections of the cell membrane of random VMH neurons. Synapse characterization was performed at original magnification ×20,000, and quantitative measurements were performed at original magnification ×11,500. Results are reported as synapses number/perimeter of the VMH neurons (21).

## Statistics

Data are expressed as the means ± SEM. Analysis was performed by one-way ANOVA or the Student *t* test, as appropriate. Statistical analysis was then performed by two-way ANOVA for repeated measures, followed by post hoc analysis using GraphPad Prism 4.0 software (GraphPad Software, Inc., San Diego, CA). *P* \< 0.05 was considered statistically significant.

# Results

## Recurrent Hypoglycemia and EphrinA5 Expression

As shown in Fig. 1, rats exposed to recurrent hypoglycemia for 3 days exhibit a 25% reduction in ephrinA5 expression in the VMH (Fig. 1A and B). In contrast, no significant change in the expression of the EphA5 receptor in the VMH was detected (Fig. 1C and D).

## Stimulation of VMH EphA5 Receptors in Rats Exposed to Recurrent Hypoglycemia

To assess the biological consequences of the reduction of ephrinA5 expression, we microinjected the Eph receptor agonist, ephrinA5-Fc, into the VMH before conducting a hyperinsulinemic-hypoglycemic clamp study in rats previously exposed to three episodes of insulin-induced hypoglycemia. Schematic representation of the experimental protocol is presented in Fig. 2A. Body weight and plasma levels of glucose, insulin, glucagon, epinephrine, and norepinephrine were indistinguishable at baseline and immediately after completion of the VMH microinjection of ephrinA5-Fc or control-Fc (Table 1). Subsequently, during the hypoglycemic clamp study, plasma glucose (Fig. 2B) and insulin (Fig. 2C) were indistinguishable between the two groups. EphrinA5-Fc delivery, however, significantly reduced within 15 min the glucose infusion rate required to maintain hypoglycemia (Fig. 2D). This was accompanied by a rapid 150% (*P* \< 0.001) increase in glucagon release (Fig. 2E). As was observed in our previous study (8) in rats not exposed to antecedent hypoglycemia, neither plasma epinephrine (Fig. 2F) nor norepinephrine (Fig. 2G) responses to hypoglycemia were significantly altered by VMH delivery of ephrinA5-Fc compared with the control-Fc microinjection.

Characteristics for the rats with recurrent hypoglycemia at baseline and after microinjection of ephrinA5-Fc in the VMH

![](1140tbl1)

## Effect of Recurrent Hypoglycemia on Glia Ensheetment and Synaptic Input Organization

Next, we assessed if recurrent hypoglycemia affected the VMH synaptic organization and glia ensheetment in rats exposed for 3 days to recurrent hypoglycemia compared with controls, a model we have previously shown suppresses hypoglycemic counterregulation (22). Figure 3A and B compares representative EMs of glia ensheetment in random VMH neuron perikarya in control and recurrent hypoglycemic rats. The rats exposed to recurrent hypoglycemia exhibited reduced glial coverage of neurons (*P* \< 0.001) (Fig. 3C) and, as a result, more total synaptic contacts in random VMH neurons (*P* \< 0.05) (Fig. 3D).

# Discussion

The current study demonstrates that exposure of nondiabetic rats to recurrent hypoglycemia for 3 consecutive days diminishes ephrinA5 expression within the VMH in association with reduced glial coverage of VMH neurons and synaptic remodeling. In addition, targeted VMH delivery of the EphA5 receptor ligand ephrinA5-Fc enhances glucose counterregulation and glucagon release in rats exposed to recurrent hypoglycemia. These findings are consistent with the possibility that recurrent hypoglycemia diminishes EphA5 receptor forward signaling in the VMH, which in turn reduces the magnitude of glucagon secretion.

Signaling via the EphA/ephrinA receptor system has been reported to regulate neuron-astrocyte interactions that cause rapid changes in synaptic structural and functional plasticity (17,23). It has been proposed that the loss of ephrinA alters astrocytic-neuronal contacts (24), whereas application of ephrinA3-Fc or endogenous ephrin induces rapid growth of the astrocytes processes and growth of new filopodia (17). In addition, activation of EphA4 by ephrinA3 has been shown to induce spine retraction (25). The current finding that recurrent hypoglycemia decreases ephrinA5 expression and produces diminished glia coverage and more synaptic connections within the VMH raises the question of a possible relationship. The fact that bypassing ephrinA5 using a targeted VMH ephrinA5-Fc microinjection can increase counterregulatory responses in animals exposed to recurrent hypoglycemia is in keeping with the hypothesis that reduced VMH ephrinA5 expression might induce alterations in VMH synaptic plasticity that in turn contribute to the development of disordered glucose counterregulation. However, a direct link between the observed changes in ephrinA5 and in glia coverage as well as synaptic connection rearrangements remains to be established in future studies.

Previous studies have reported rapid changes in synaptic network connectivity and glia morphology in the VMH in response to alterations in energy substrate bioavailability (15,26–30). Acute hypoglycemia has been shown to alter synaptophysin expression, findings consistent with a rapid alternation in synaptic morphology (31,32), whereas insulin-deficient diabetic rats display a decrease in the number of hypothalamic astrocytes as a consequence of increased death and decreased proliferation (33). Given that diabetes and recurrent glucose deprivation are both accompanied by impaired counterregulation (22), these observations are consistent with the hypothesis that synaptic connectivity and the function of glia in the VMH play a significant role in supporting the neurotransmission required for proper counterregulatory responses to acute hypoglycemia.

It is noteworthy that the principal effect of VMH microinjection of the ephrinA5 ligand on hypoglycemia-induced counterregulatory hormone release was on glucagon, whereas recurrent hypoglycemia normally leads to a suppression of glucagon and epinephrine levels in nondiabetic animals. These findings are consistent with previous studies in mice showing that knockdown of VGLUT2 (vesicular glutamate transporter) selectively in SF1 VMH neurons predominately inhibited the secretion of glucagon in response to acute hypoglycemia (34). It should be noted, however, that alterations in EphA receptor/ephrinA signaling appear to influence epinephrine responses as well. Using a targeted gene expression manipulation approach to chronically alter VMH ephrinA5 expression, we observed that overexpression of ephrinA5 in the VMH stimulates, whereas targeted VMH knockdown of ephrinA5 inhibits, epinephrine as well as glucagon responses to acute hypoglycemia. These effects also appear to be mediated by alterations in glutamate–glutamine cycling (8). In this study, we did not include a control group not subjected to recurrent hypoglycemia, and thus, the extent that acute delivery of the EphA receptor agonist restored the glucagon response to hypoglycemia in rats exposed to recurrent hypoglycemia cannot be determined. However, results from previous studies from our laboratory using a similar hypoglycemic clamp protocol show the effect of the EphA receptor agonist on glucagon responses appears to be at least as great as the suppressive effect of recurrent hypoglycemia on glucagon levels (8).

Given that the EphA5/ephrinA5 system is mainly localized on glutamatergic synapses (12,25), it is intriguing to speculate that the stimulation of glucagon produced by the EphA5 receptor agonist in the current study is most likely mediated by the augmented VMH glutamatergic neurotransmission. The maintenance of VMH glutamate neurotransmission during acute hypoglycemia is supported by the transport of glutamate into astrocytes, resulting in the production of glutamine for delivery to neurons and glutamate–glutamine cycle activation (35). Previous studies have shown that astrocyte synaptic coverage is linked to glutamate clearance and the activation of metabotropic glutamate receptors (36), and this has been proposed to alter synaptic and astroglia organization, and in turn, neurotransmission (37,38). Thus, the reduced glia coverage of the VMH neurons observed in the current study in rats exposed to recurrent hypoglycemia may have produced a deficit in astroglial function to support proper glutamate neurotransmission during hypoglycemia. Interestingly, this was associated with more neuronal synaptic contacts in the VMH (Fig. 3D), which appeared to be in large part symmetric and thus potentially γ-aminobutyric acid (GABA) inhibitory in nature (21). An increase in the VMH GABA tone has been shown to be an important contributor to the development of impaired glucose counterregulation in response to recurrent hypoglycemia (39).

Taken together, our data demonstrate that recurrent hypoglycemia alters neuron-glia plasticity in VMH nuclei and diminishes ephrinA5 ligand expression within the VMH. It is thus possible decreased ephrin-induced activation of Eph receptors in VMH glutamate neurons may contribute to the impairment in glucose counterregulation in response to recurrent antecedent hypoglycemia.

## Article Information

**Acknowledgments.** The authors are grateful to Wanling Zhu, from R.S.S.'s laboratory, for performing animal surgeries; Aida Groszman, Maria Batsu, Codruta Todeasa, Ralph J. Jacob, from the Yale Center for Clinical Investigation Core Laboratory of the Yale School of Medicine, and Jan Czyzyk, from the University of Rochester Medical Center School of Medicine and Dentistry, for assisting with the protein expression experiments; and Erzsebet Borok, from the Yale Program in Integrative Cell Signaling and Neurobiology of Metabolism, for excellent technical support and assistance.

**Funding.** This work was supported by research grants from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (20495), the Juvenile Diabetes Research Foundation, and the NIDDK-supported Diabetes Endocrinology Research Center.

**Duality of Interest.** No potential conflicts of interest relevant to this article were reported.

**Author Contributions.** B.S. designed the study, performed animal surgery and studies, analyzed data, and wrote the manuscript. T.L.H. designed EM experiments. R.S.S. designed the study, reviewed data, and revised the manuscript. B.S. and R.S.S. are the guarantors of this work, and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

**Prior Presentation.** This study was presented at the 70th Scientific Sessions of the American Diabetes Association, Orlando, FL, 25–29 June 2010.

# References


date: 2017-08
title: WHO urges action against HIV drug resistance threat

20 JULY 2017 \| GENEVA - WHO alerts countries to the increasing trend of resistance to HIV drugs detailed in a report based on national surveys conducted in several countries. The Organization warns that this growing threat could undermine global progress in treating and preventing HIV infection if early and effective action is not taken.

The WHO HIV drug resistance report 2017 shows that in 6 of the 11 countries surveyed in Africa, Asia and Latin America, over 10% of people starting antiretroviral therapy had a strain of HIV that was resistant to some of the most widely used HIV medicines. Once the threshold of 10% has been reached, WHO recommends those countries urgently review their HIV treatment programmes.

# HIV drug resistance report 2017

"Antimicrobial drug resistance is a growing challenge to global health and sustainable development," said Dr Tedros Adhanom Ghebreyesus, WHO Director-General. "We need to proactively address the rising levels of resistance to HIV drugs if we are to achieve the global target of ending AIDS by 2030."

HIV drug resistance develops when people do not adhere to a prescribed treatment plan, often because they do not have consistent access to quality HIV treatment and care. Individuals with HIV drug resistance will start to fail therapy and may also transmit drug-resistant viruses to others. The level of HIV in their blood will increase, unless they change to a different treatment regimen, which could be more expensive – and, in many countries, still harder to obtain.

Of the 36.7 million people living with HIV worldwide, 19.5 million people were accessing antiretroviral therapy in 2016. The majority of these people are doing well, with treatment proving highly effective in suppressing the HIV virus. But a growing number are experiencing the consequences of drug resistance.

WHO is therefore issuing new guidelines to help countries address HIV drug resistance. These recommend that countries monitor the quality of their treatment programmes and take action as soon as treatment failure is detected.

"We need to ensure that people who start treatment can stay on effective treatment, to prevent the emergence of HIV drug resistance," said Dr Gottfried Hirnschall, Director of WHO's HIV Department and Global Hepatitis Programme. "When levels of HIV drug resistance become high we recommend that countries shift to an alternative first-line therapy for those who are starting treatment."

Increasing HIV drug resistance trends could lead to more infections and deaths. Mathematical modelling shows an additional 135 000 deaths and 105 000 new infections could follow in the next five years if no action is taken, and HIV treatment costs could increase by an additional US\$ 650 million during this time.

Tackling HIV drug resistance will require the active involvement of a broad range of partners. A new five-year Global Action Plan calls on all countries and partners to join efforts to prevent, monitor and respond to HIV drug resistance and to protect the ongoing progress towards the Sustainable Development Goal of ending the AIDS epidemic by 2030. In addition, WHO has developed new tools to help countries monitor HIV drug resistance, improve the quality of treatment programmes and transition to new HIV treatments, if needed.

The WHO HIV drug resistance report 2017 was co-authored by the Global Fund to Fight AIDS, Tuberculosis and Malaria, and the Centers for Disease Control and Prevention, USA.

"This new report shows a worrying picture of increasing levels of HIV drug resistance and, if unchecked, it will be a major risk to program impact," said Dr Marijke Wijnroks, Interim Executive Director of the Global Fund. "We strongly recommend implementing WHO recommendations for early warning indicators and HIV drug resistance surveys in every national plan for antiretroviral therapy, and to consider funding them through Global Fund grants or reprogramming."

Dr Shannon Hader, Director of CDC's Division of Global HIV and Tuberculosis, US Centers for Disease Control and Prevention, added: "The new report pulls together key HIV drug resistance survey findings from across the globe that, taken together with other national-level data, confirm we must be forward-thinking in our efforts to combat resistance: scaling up viral load testing, improving the quality of treatment programs, and transitioning to new drugs like dolutegravir."

Dr. Hader continued, stating that "Overall high rates of viral suppression across three recent national Population-based HIV Impact Assessments showed that present first-line regimens remain largely effective. However, special attention to populations at risk for higher resistance, such as pediatrics, adolescents, pregnant women and key populations, will be critical to target more urgent interventions. We call on the global community for continued vigilance and responsiveness."

Available from: <http://www.who.int/mediacentre/news/releases/2017/hiv-drug-resistance/en/>


abstract: Decades of research in rodent models has shown that early postnatal overnutrition induces excess adiposity and other components of metabolic syndrome that persist into adulthood. The specific biologic mechanisms explaining the persistence of these effects, however, remain unknown. On postnatal day 1 (P1), mice were fostered in control (C) or small litters (SL). SL mice had increased body weight and adiposity at weaning (P21), which persisted to adulthood (P180). Detailed metabolic studies indicated that female adult SL mice have decreased physical activity and energy expenditure but not increased food intake. Genome-scale DNA methylation profiling identified extensive changes in hypothalamic DNA methylation during the suckling period, suggesting that it is a critical period for developmental epigenetics in the mouse hypothalamus. Indeed, SL mice exhibited subtle and sex-specific changes in hypothalamic DNA methylation that persisted from early life to adulthood, providing a potential mechanistic basis for the sustained physiological effects. Expression profiling in adult hypothalamus likewise provided evidence of widespread sex-specific alterations in gene expression. Together, our data indicate that early postnatal overnutrition leads to a reduction in spontaneous physical activity and energy expenditure in females and suggest that early postnatal life is a critical period during which nutrition can affect hypothalamic developmental epigenetics.
author: Ge Li; John J. Kohorst; Wenjuan Zhang; Eleonora Laritsky; Govindarajan Kunde-Ramamoorthy; Maria S. Baker; Marta L. Fiorotto; Robert A. WaterlandCorresponding author: Robert A. Waterland, <firstname.lastname@example.com>.[^1]
date: 2013-08
references:
title: Early Postnatal Nutrition Determines Adult Physical Activity and Energy Expenditure in Female Mice

Environmental influences on the development of body weight regulatory mechanisms may be an important factor in the worldwide obesity epidemic (1,2). Evidence in humans indicates that overnutrition during early postnatal life can permanently alter body weight regulation, increasing susceptibility to obesity throughout life (3,4). Accordingly, various animal models have been developed to explore the effects of infant overnutrition on lifelong obesity risk. Artificial feeding of rodent pups by intragastric cannula provides clear evidence for sustained effects of early postnatal overnutrition (5) but requires raising newborn rodents in isolation, which itself has long-term consequences. Overfeeding dams during lactation, with a high-fat diet, for example, could indirectly overnourish pups. Indeed, two recent rodent studies (6,7) report that the obesogenic effect of maternal high-fat diet occurs specifically during the suckling period. Pups from high-fat-fed dams are not consistently heavier at weaning, however (8,9), indicating that maternal overnutrition does not reliably induce early postnatal overnutrition.

In the rodent small litter model of early postnatal overnutrition (10), offspring from several litters born on the same day are randomized and fostered to either normal size (control \[C\]) or small litters (SL). Suckling in SL is naturalistic, easy to implement, and consistently induces early postnatal overnutrition, providing an apt model in which to study potential long-term effects of infantile overnutrition by excessive formula feeding (11). The early postnatal exposure induces elevated body weight and adiposity that persists to adulthood (10–12), with concomitant increases in plasma insulin (11,13) and leptin concentrations (13) and impaired glucose tolerance (11,13,14). It remains unresolved, however, whether the sustained increase in adiposity of adult SL rodents results from increased energy intake or decreased energy expenditure (13,15). Moreover, the fundamental mechanisms by which the metabolic effects of SL exposure persist to adulthood are unknown.

Environmental influences on developmental epigenetics (16,17) provide a likely mechanism. Epigenetic mechanisms regulate mitotically heritable alterations in gene expression potential that are not caused by changes in DNA sequence (18) and are known to play key roles in brain development (19). DNA methylation, the most stable epigenetic modification (20), is a likely mechanism to explain effects that persist for a lifetime (2). Given its central role in regulating food intake and energy expenditure (21), the hypothalamus is an obvious tissue in which to explore a potential epigenetic basis for induced alterations in body weight regulation. We therefore set out to determine *1*) whether the persistently elevated adiposity of SL mice is caused by increased food intake or decreased energy expenditure, and *2*) whether early postnatal overnutrition causes persistent changes in hypothalamic epigenetic regulation that may perpetuate altered body weight regulation.

# RESEARCH DESIGN AND METHODS

For the litter size studies, virgin FVB/NJ females (The Jackson Laboratory) were mated with FVB/NJ males at age 8 weeks. In each batch, 14–15 mating pairs were set up on the same day; four independent batches of mice were studied over the course of 2 years. On postnatal day 1 (P1), pups from all litters born on the same day (P0) were weighed, sexed, and pooled randomly. Only pups from a birth litter size of 6–12 were included. Foster dams received either four (SL) or nine (C) pups. There were two females and two males in each SL and four to five females and males in each C litter. Litter assignment was performed systematically to balance body weight at P1. At P21, offspring from both groups were weaned onto a fixed-formula, soy protein–free diet (2020X; Harlan Teklad); females were housed two to five per cage, and males were housed individually. Body composition, food intake, energy expenditure, and physical activity were measured at P21–P25 and approximately P180. The P21 vs. P0 methylation-specific amplification and microarray hybridization (MSAM) comparisons used female C57BL/6J mice, and the pyrosequencing validation studies were performed in C57BL/6J and FVB/NJ mice of both sexes (The Jackson Laboratory). Sex was confirmed by PCR amplification of *Sry*. All applicable institutional and governmental regulations concerning the ethical use of animals were followed during this research. The protocol was approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. All mice were housed in a temperature-controlled facility (22°C), provided free access to food and water, and maintained on a 12-h light cycle.

## Body composition.

Body composition was determined by quantitative magnetic resonance (EchoMRI-100; Echo Medical Systems) according to the manufacturer's instructions.

## Food intake, energy expenditure, and physical activity.

Prior to metabolic study, mice were acclimatized to single housing in Comprehensive Laboratory Animal Monitoring System (CLAMS) cages (Columbus Instruments, Columbus, OH) for 3 days. Mice were subsequently transferred to calorimetry cages for 4 days, during which food intake, energy expenditure (by indirect calorimetry), and physical activity were monitored in real time (22). Only data from the last 3 days (6:00 a.m. to 6:00 a.m.) were used. Upon weaning (P21), mice were housed in a normal cage for 1 day before being introduced to the CLAMS cages. Hence, the metabolic measurements did not commence until P25.

## Genome-scale DNA methylation profiling.

MSAM was performed as previously described (23,24), using a starting quantity of 0.5 µg genomic DNA. In both MSAM experiments, two independent cohybridizations (biological replicates) were performed on a custom 2×105k array (Agilent Technologies). The array includes 90,694 probes covering 86% (23,742) of the 27,675 potentially informative *Sma*I*/Xma*I intervals between 200 bp and 2 kb in the mouse genome (average 3.8 probes per interval) (25). Fold change and *P* value were calculated as mean and median, respectively, of all probes in each interval; "hits" were called for fold change \>2 or \<0.5 and *P* \< 0.0001 in both cohybridizations. Gene Ontology analysis was performed using the Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GOrilla) (26), in each case using the potentially informative genes on the array as the background set. *Sma*I*/Xma*I cut sites were annotated by RefGene according to NCBI36/mm8 mouse genome (Feb 2006 release), according to the schema in [Supplementary Fig. 2](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1). Relevant details of the MSAM experiments and the hybridization data are available in the GEO database: P21 vs. P0, [www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32475](http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32475); and SL vs. C, P180, [www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32477](http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32477).

## Quantitative analysis of DNA methylation.

Site-specific analysis of CpG methylation was performed by bisulfite pyrosequencing (23,24). For validation of MSAM hits, pyrosequencing assays were designed to cover both informative *Sma*I*/Xma*I sites when possible. A hit was considered validated if either assay showed a substantial methylation difference in the same direction as in MSAM. Sensitivity and linearity of each pyrosequencing assay were confirmed by running methylation standards (27). Pyrosequencing primers and annotations of the CpG sites examined are listed in [Supplementary Tables 3 and 4](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1), respectively.

## Gene expression profiling and analysis.

Total hypothalamic RNA was isolated by the RNeasy kit (Qiagen). RNA quality control, cRNA preparation, labeling, and microarray hybridization were conducted as previously described (28). cRNA was hybridized onto Illumina MouseWG6 v2 Expression BeadChips (Illumina) following the manufacturer's protocol. Signal intensities extracted from Illumina Genome Studio software were preprocessed using LUMI (29) and the R statistical package (<http://www.r-project.org/>), including probes with a detection *P* value ≤0.05 in at least half of the samples. These 20,791 probe signals were then quantile normalized. To test for effects of group, sex, and group × sex, we performed two-way ANOVA using Genomics Suite Software, version 6.6 β (Partek). Contrasts were applied to all groups to identify differentially expressed transcripts, using an α level of 0.05 after Benjamini-Hochberg false discovery rate adjustment. Network analyses were performed using IPA (Ingenuity Systems). Relevant details of the expression microarray experiment and the hybridization data are available in the GEO database: [www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE40616](http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE40616).

## Quantitative analysis of gene expression.

Expression levels of specific genes were determined by quantitative PCR. Total hypothalamic RNA was isolated by the RNeasy kit (Qiagen), and cDNA was synthesized using M-MLV Reverse Transcriptase (Promega) with random primers (Life Technologies). Gene expression levels were determined with either TaqMan (Life Technologies) or Sybr Green (Life Technologies), using the 2^−ΔΔCt^ method (assay details provided in [Supplementary Table 6](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)). In all cases, *ActB* was used as an endogenous control.

## General statistical methods.

Enrichment of *Sma*I*/Xma*I cut sites relative to genomic regions was analyzed by χ^2^ test. Group differences in body weight at P1 were analyzed by two-tailed *t* test. Body composition data at P25 and P180 were analyzed by ANOVA (SAS Proc Mixed). Body weight data at P21, P60, P120, and P180 were analyzed by repeated-measures ANOVA (SAS Proc Mixed, compound symmetry covariance structure) with age in the repeated effect. For the CLAMS studies, hourly measurements of food intake, energy expenditure, and activity counts for three consecutive days were averaged into one 24-h record for each mouse. Repeated-measures ANOVA used the full power of these time-series data while recognizing the nonindependence of the 24 multiple measures within each mouse. Analysis of food intake, energy expenditure, and physical activity were performed both with and without lean mass and fat mass included as independent variables to adjust for group differences in body size and composition (30). Group differences in DNA methylation by pyrosequencing were analyzed by repeated-measures ANOVA, with CpG site as the repeated effect ([Supplementary Table 2](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)). Loci that showed significant group effects on methylation but no significant group × age interaction were considered persistently altered by SL suckling. Requiring the same group difference, in the same direction, at both ages (in independent sets of mice) affords substantial protection against type 1 error; these analyses were therefore not otherwise adjusted for multiple testing. Akaike information criterion (AIC) model selection by adjusted *R*^2^ (SAS Proc Reg) was performed based on individual average dark-period energy expenditure and physical activity data.

# RESULTS

## Early postnatal overnutrition reduces adult energy expenditure in females.

We used the SL mouse model (Fig. 1A) to study persistent effects of overnutrition during the suckling period. We studied four independent batches (groups of litters cross-fostered at one time) over 2 years, including offspring from 24 C and 26 SL litters total. Consistent with previous studies, SL mice were heavier at P21 and remained so into adulthood (*P* \< 0.0001 in both females and males) (Fig. 1B). Although the increase in adult body weight was modest, effects on body composition were substantial. Both male and female SL adults had 50% higher fat mass and percent body fat compared with C mice (*P* \< 0.005 in all comparisons) (Fig. 1C). There were no group differences in lean mass. Clearly, suckling in a small litter induces persistent changes in regulatory mechanisms that affect adult body composition.

To determine whether these changes involve alterations in food intake and/or energy expenditure, we used metabolic cages to simultaneously monitor food intake, energy expenditure, and voluntary physical activity. In an attempt to identify persistent metabolic differences that might explain the sustained group differences in adiposity, we performed the metabolic measurements shortly after weaning (P25) and in adulthood (P180). (Again, these data represent four batches of mice studied over the course of 2 years.) After appropriate least squares normalization for lean mass and fat mass (30), food intake of SL mice tended, surprisingly, to be slightly lower than that of C mice at both P25 and P180 (Fig. 2A), but these differences were not statistically significant. Energy expenditure (normalized for lean mass and fat mass \[30\]) was nearly identical between SL and C mice at P25 (Fig. 2B). At P180, however, energy expenditure of SL females was significantly lower than that of C females (*P* = 0.002); this group difference was significant during both the light and dark periods. Resting metabolic rate was estimated as the lowest average energy expenditure within 1 h for each mouse. After least squares normalization for lean mass and fat mass, female mice showed no group differences in resting metabolic rate at either age. Resting metabolic rate of SL males, however, was higher at P25 (*P* = 0.02) and lower at P180 (*P* = 0.03) relative to C males. There were no group differences in respiratory exchange ratio. Group differences in voluntary physical activity, again normalized for lean mass and fat mass, were consistent with those in energy expenditure: none were found at P25, but SL females were significantly less active than C females at P180, specifically during the dark period (group × light interaction *P* = 0.0009) (Fig. 2C). It is noteworthy that adult SL females were less physically active even after including body weight and body composition in the model; hence, their lower activity was not caused by their excess adiposity. (For comparison, unnormalized data on food intake, energy expenditure, and physical activity are shown in [Supplementary Fig. 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1).) Including physical activity in the model for energy expenditure of P180 females drastically reduced the significance of the SL effect (from *P* = 0.002 to *P* = 0.01), suggesting that physical activity explains much of the group difference in energy expenditure. Together, these data indicate that the persistent alterations in energy balance of female SL mice are due not to excess food intake but, rather, to reduced energy expenditure. The sex specificity of this effect may be related to the male-specific decline of physical activity with age ([Supplementary Fig. 1*C*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)).

## Extensive epigenetic development occurs in the early postnatal hypothalamus.

Ontogenic periods when epigenetic mechanisms are being established or undergoing maturation constitute critical periods during which environmental influences can cause persistent changes in epigenetic regulation (31,32). To determine whether the suckling period might be a critical period for developmental epigenetics in the hypothalamus, we tested for changes in hypothalamic DNA methylation. We used MSAM, which is based on sequential digestion of genomic DNA with the methylation-sensitive and -insensitive isoschizomers *Sma*I and *Xma*I (23,33). Two independent P21 vs. P0 MSAM cohybridizations (incorporating a dye swap) were performed.

Using stringent criteria validated in our previous studies (24,33), 868 *Sma*I*/Xma*I intervals changed methylation from P0 to P21. Only 31 intervals lost methylation (Fig. 3A), and the genomic distribution of associated *Sma*I*/Xma*I cut sites was not different from that on the array (Fig. 3B and [Supplementary Fig. 2](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)). Methylation increased at 837 intervals (Fig. 3A); associated cut sites were significantly underrepresented at promoters (*P* \< 0.0001) and overrepresented in introns (*P* \< 0.0001) (Fig. 3B). In a larger number of P0 and P21 mice, we used bisulfite pyrosequencing (23,33) to measure P0–P21 changes in hypothalamic CpG methylation at 10 intervals identified by MSAM; all 10 validated (100%) (7 are shown in Fig. 4). Additionally, since the P21 vs. P0 MSAM studies were performed in C57BL/6J mice, we confirmed (at a subset of loci) that these methylation changes also occur in both sexes of FVB/NJ mice (the strain used for the litter size studies) ([Supplementary Fig. 3](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)).

Functional significance of the developmental changes in DNA methylation was evaluated by gene ontology analysis. Relative to all potentially informative genes on the array, no enriched ontologies were found for the few genes associated with intervals that lost methylation. Genes that gained methylation, however, were significantly enriched for 20 biologic process categories (Fig. 3C); of these, 17 are explicitly related to development, including neurodevelopmental processes such as axon guidance and neuron differentiation. Postnatal development of the mouse hypothalamus clearly involves functionally important epigenetic changes. This may be a critical period during which environment can influence these processes, with long-term consequences.

## Early postnatal overnutrition causes persistent and sex-specific alterations in hypothalamic DNA methylation and gene expression.

We therefore examined DNA methylation differences among SL and C hypothalami. Intrigued by the large group differences in energy expenditure and physical activity in P180 females, we used MSAM to compare hypothalamic DNA methylation of SL and C females at P180. Two independent SL vs. C cohybridizations were performed, with each hypothalamic DNA sample pooled from five females drawn from different foster litters. The results, however, provided no evidence of persistent group differences in hypothalamic DNA methylation.

Reasoning that DNA methylation changes in SL mice might be too subtle to detect by MSAM, we used bisulfite pyrosequencing to examine a panel of candidate genes. Since genomic regions undergoing methylation change from P0 to P21 are most likely to show persistent effects of overnutrition during this period (2), most of the genomic regions that we selected were those identified in our P21 vs. P0 MSAM experiments. In addition to 10 of those already validated (Fig. 4), we examined six hits near genes previously reported to change expression in hypothalamus from P0 to P21 (34) and two showing interindividual variation in DNA methylation. Additionally, promoters of a few genes critical to hypothalamic function and development (*Agrp, Fto*, and *Pomc*) were included. In total, 24 loci (named according to the nearest gene) were selected ([Supplementary Table 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)).

Of these, 15 showed no DNA methylation differences between SL and C hypothalamus at P25 ([Supplementary Fig. 4](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1) and [Supplementary Table 2](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)) and were not examined at P180. The remaining nine loci were examined at both P25 and P180 ([Supplementary Fig. 5](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1) and [Supplementary Table 2](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)). Considering this an exploratory data analysis, we set an α-level of 0.1 for main effects and 0.2 for interactions. The initial model including both sexes showed significant main effects for age but not group (SL vs. C); interestingly, however, four loci showed significant group × sex interactions ([Supplementary Table 2](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)). We therefore performed sex-specific analyses. In females, *AK145544*, *Aqp4*, and *Nolz1* and in males *Gadd45b* showed main effects of group that did not differ by age (no group × age interaction). Plots of average site-specific methylation at these loci (Fig. 5) illustrate subtle but persistent differences in DNA methylation. Since most of these changes were found in females, we used quantitative real-time RT-PCR to measure gene expression of *AK145544*, *Aqp4*, and *Nolz1* in hypothalamus of P180 females but found no significant group differences. To test whether differences in methylation and expression at these loci could explain individual variation in adult physical activity or energy expenditure, we applied the AIC (35) to identify the best model for each. In addition to methylation and expression of the three genes, SL group membership, lean mass, and fat mass were included as potential explanatory variables. Physical activity was not significantly predicted by any model. Remarkably, however, the best model for energy expenditure ([Supplementary Fig. 6](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)) included expression of all three genes and methylation at *Nolz1* and *Aqp4* and predicted 55% of the variation in P180 energy expenditure in females (*P* = 0.025). Hence, group differences in DNA methylation and gene expression at these loci, though subtle, may explain some of the observed alterations in energy balance.

As a complementary approach to identify genes with persistent alterations in epigenetic regulation, we profiled gene expression in P180 hypothalamus of SL and C mice. Three males and three females were studied in each group (12 arrays total). The results showed strong effects of sex (Fig. 6A), with 342 transcripts showing significant differences (false discovery rate \<0.05). Although none of the group or group × sex effects survived multiple testing correction, there was an enrichment of low *P* value probes in the group × sex analysis (Fig. 6A), suggesting subtle sex-specific effects. We therefore performed gene ontology analysis on the 37 and 732 transcripts with at least a 20% group difference (*P* \< 0.05) in females and males, respectively. (The reference list comprised the 14,628 transcripts significantly expressed in hypothalamus.) No enriched gene ontology terms were found in females. In males, however, for both the 381 genes upregulated and the 351 downregulated in SL hypothalamus, the foremost biological process related to formation of neuronal projections. Examination of the gene ontology terms associated with the genes comprising these enrichments (Fig. 6B and [Supplementary Table 5](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)) suggests a subtle shift in expression profile that may favor neuronal remodeling in the hypothalamus of adult SL males. Additionally, in an analysis of gene networks associated with the expression changes in male hypothalamus ([Supplementary Fig. 7](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)), two of the top three networks were related to cellular development and nervous system development. These networks are centered on Atn1 and dynein, respectively (both regulators of neurodegeneration), again supporting potential alterations of neuronal remodeling in SL males.

# DISCUSSION

Here we showed that early postnatal overnutrition, known to permanently increase body weight and adiposity, also reduces voluntary physical activity and energy expenditure in adult females. These physiological changes were associated with persistent alterations in hypothalamic DNA methylation at specific loci. Overall, these findings provide support for the hypothesis that early postnatal overnutrition causes subtle but widespread changes in hypothalamic epigenetic regulation that persist to influence adult energy balance.

Our study addresses a key outstanding question: whether the persistently altered energy balance of SL rodents is due to increased food intake or decreased physical activity. Previous studies reported increased food intake (13,36) and energy expenditure (37) in adult SL rodents. Those conclusions, however, were based on nonnormalized data, disregarding the altered weight and body composition of SL rodents. Here, we used least squares means to appropriately adjust expenditure and intake data for body weight and composition (30). Compared with C mice of the same weight and body composition, adult SL mice were not hyperphagic (Fig. 2A). Their energy expenditure, however, again compared with C mice of the same weight and body composition, was lower (Fig. 2B), significantly so in females. Hence, our data provide strong evidence that reduced energy expenditure, not increased food intake, explains the increased adiposity of female adult SL mice.

In addition to food intake and energy expenditure, however, there are other determinants of energy balance, such as nutrient absorption, which were not measured in this study. Also, it is possible that group differences in central regulation of food intake may have been unmasked if mice were provided a highly palatable diet (38). Other than physical activity, we did not measure additional determinants of energy expenditure, such as brown adipose tissue activity. These shortcomings may explain why the excess adiposity of male SL mice occurred without measurable differences in physical activity or energy expenditure. (Notably, a recent study found age-associated alterations in thermogenic capacity of brown adipose tissue in male SL rats \[39\], consistent with our finding that resting metabolic rate is increased at P21 but decreased at P180 in male SL mice.) In females, the reduced energy expenditure was largely explained by reduced physical activity (Fig. 2C). In an earlier report in rats, prenatal undernutrition likewise caused persistent reductions in locomotor activity, most prominently in females (40). Given the worldwide trends of decreasing physical activity (41), it is crucial to determine whether, in humans as in rodents, nutrition during early life modulates voluntary physical activity for a lifetime.

Despite its importance in central regulation of food intake and energy expenditure (21), our understanding of the molecular mechanisms driving functional development of the hypothalamus remains limited. Mouse hypothalamic development continues into early postnatal life, a critical period for formation of leptin-sensitive neuroanatomic projections that function in energy homeostasis (42) and major alterations of hypothalamic gene expression (34). Here, we have shown for the first time that during this same period widespread changes in DNA methylation—mostly increases—are underway. The association of these methylation increases with genes involved in neural development (Fig. 3C) suggests a process of postnatal epigenetic maturation. Because projections from the arcuate nucleus of the hypothalamus to other brain regions form prenatally in primates but postnatally in rodents (43), it is often proposed that hypothalamic development during the suckling period in the mouse is comparable with that in a third-trimester human. It is currently unknown, however, whether and when the epigenetic maturation we have documented in the postnatal mouse occurs in humans. Moreover, our findings that postnatal overnutrition leads to a decrease in physical activity in female mice raises the question of whether the mouse is a good model for physical activity in humans. Although we currently have only a rudimentary understanding of the neurobiological regulation of spontaneous physical activity (44), the hypothalamus and other brain regions are known to be involved, as are several highly conserved neuropeptides including cholecystokinin, corticotrophin-releasing hormone, leptin, and orexins.

It was recently reported that SL rats have alterations in hypothalamic DNA methylation (45,46). Those studies, however, assessed DNA methylation only at P21. Our data therefore provide the first evidence that early postnatal overnutrition induces persistent epigenetic changes in the hypothalamus. Additionally, unlike previous studies on related models (45–48), rather than focus on single CpG sites we performed integrated analysis of all CpG sites represented in each assay because *1*) regional changes in DNA methylation are more likely to affect gene expression and *2*) concordant changes at multiple adjacent sites are less likely to occur by chance. Notably, contrary to the previous report of increased DNA methylation (at 2 of 20 CpG sites measured) at the *Pomc* promoter in the hypothalamus of P21 SL rats (45), our methylation assay spanning five nearby CpG sites found no SL vs. C differences at P25 ([Supplementary Fig. 4](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)).

We developed the strategy of examining genomic regions undergoing DNA methylation changes from P0 to P21, based on the conjecture that these changes may be susceptible to environmental influences. Indeed, 4 of 21 regions undergoing P0–P21 DNA methylation change showed evidence of persistent methylation differences between SL and C mice, supporting the utility of this approach. Hence, the ∼900 loci that we report that undergo postnatal methylation changes may provide useful candidate regions for future studies of environmental influences on hypothalamic developmental epigenetics.

With the potential exception of *AK145544*, all four genes with persistent changes in hypothalamic DNA methylation in SL hypothalamus play important roles in neural development or function (49–51). At each of these genes, the methylation change in SL mice was modest (Fig. 5); the cumulative effect of such changes at hundreds or thousands of genes, however, could be considerable. This interpretation is supported by the AIC model selection ([Supplementary Fig. 6](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)), which included as significant predictors of adult energy expenditure expression of three and methylation at two of the genes that we identified, in most cases with *F* values comparable with that of lean body mass.

The results of transcriptional profiling in P180 hypothalamus mirrored our DNA methylation analyses in detecting subtle, widespread, and sex-specific alterations in gene expression. Analyzing the corpus of genes with potentially altered expression in male SL hypothalamus identified highly significant gene ontology enrichments pertaining to regulation of neuronal projections (Fig. 6). The adult rodent hypothalamus maintains significant synaptic plasticity (52); our data suggest that early postnatal overnutrition in males may persistently augment this capability. Adult mice that become obese owing to a high-fat diet, conversely, appear to have reduced hypothalamic neurogenesis (53).

All the potential explanatory effects we found—changes in energy expenditure, physical activity, DNA methylation, and gene expression—were sex specific. The long-term consequences of early life exposures have long been recognized to differ by sex (16). Our findings of sexual dimorphism in the epigenetic responses to early postnatal environment suggest that nutrition may interact with the epigenetic mechanisms regulating hormone-dependent sexualization of the neonatal hypothalamus (54). In fact, the sex differences found here might provide an answer as to why the lower physical activity in SL females arose only in adulthood. In male mice, physical activity declined with age in both groups, but in females this decline was seen only in SL mice ([Supplementary Fig. 1*C*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1306/-/DC1)). Our results may suggest, therefore, that postnatal overnutrition is leading to masculinization of the CNS pathways that regulate age-related changes in physical activity.

Encouraged by earlier studies that gained insights into hypothalamic developmental epigenetics (55), we too studied DNA methylation in whole hypothalamus. The interpretation of our data is therefore complicated by the heterogeneity of the hypothalamus at both the regional and the cellular level. The hypothalamus is comprised of distinct regions, or "nuclei," with specialized functions, gene expression patterns (21), and epigenetic regulation (56). Additionally, the nervous system includes diverse cell types; the simplest classification distinguishes neurons and glia, which are epigenetically distinct (57,58). To improve our understanding of how early postnatal overnutrition causes persistent changes in regulation of body weight and body composition, it will be advantageous to characterize epigenetic effects within specific hypothalamic nuclei and specific cell types. For example, based on our current data we cannot exclude the possibility that the persistent alterations in DNA methylation that we identified represent a shift in the proportion of hypothalamic cell types rather than induced alterations in epigenetic regulation within specific cell types. Moreover, since early postnatal life is a critical period for not only epigenetic but also neuroanatomic development (42), studying these processes in an integrated fashion will likely be necessary to gain a clear understanding of how early postnatal nutrition affects the establishment of hypothalamic body weight regulation.

## ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK081557) and the U.S. Department of Agriculture (USDA) (CRIS 6250-51000-055) (to R.A.W.). Body composition and CLAMS studied were performed in the Mouse Metabolic Research Unit at the USDA/Agricultural Research Service (ARS) Children's Nutrition Research Center, which is supported by funds from the USDA ARS.

No potential conflicts of interest relevant to this article were reported.

G.L. and J.J.K. performed experiments and wrote the manuscript. W.Z. and E.L. performed experiments. G.K.-R. performed bioinformatic analyses. M.S.B. performed experiments. M.L.F. provided critical guidance on experimental procedures and edited the manuscript. R.A.W. designed the study and wrote the manuscript. R.A.W. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors thank Adam Gillum (USDA/ARS Children's Nutritional Research Center \[CNRC\]) for assistance with the figures and Firoz Vohra (USDA/ARS CNRC) for assistance with the CLAMS studies.

# REFERENCES

[^1]: G.L. and J.J.K. contributed equally to this study.


abstract: Cardiovascular autonomic neuropathy (CAN) is associated with increased mortality in diabetes. Since CAN often develops in parallel with diabetic nephropathy as a confounder, we aimed to investigate the isolated impact of CAN on cardiovascular disease in normoalbuminuric patients. Fifty-six normoalbuminuric, type 1 diabetic patients were divided into 26 with (+) and 30 without (−) CAN according to tests of their autonomic nerve function. Coronary artery plaque burden and coronary artery calcium score (CACS) were evaluated using computed tomography. Left ventricular function was evaluated using echocardiography. Blood pressure and electrocardiography were recorded through 24 h to evaluate nocturnal drop in blood pressure (dipping) and pulse pressure. In patients +CAN compared with −CAN, the CACS was higher, and only patients +CAN had a CACS \>400. A trend toward a higher prevalence of coronary plaques and flow-limiting stenosis in patients +CAN was nonsignificant. In patients +CAN, left ventricular function was decreased in both diastole and systole, nondipping was more prevalent, and pulse pressure was increased compared with −CAN. In multivariable analysis, CAN was independently associated with increased CACS, subclinical left ventricular dysfunction, and increased pulse pressure. In conclusion, CAN in normoalbuminuric type 1 diabetic patients is associated with distinct signs of subclinical cardiovascular disease.
author: Ulrik Madvig Mogensen; Tonny Jensen; Lars Køber; Henning Kelbæk; Anne Sophie Mathiesen; Ulrik Dixen; Peter Rossing; Jannik Hilsted; Klaus Fuglsang KofoedCorresponding author: Ulrik Madvig Mogensen, <email@example.com>.
date: 2012-07
references:
title: Cardiovascular Autonomic Neuropathy and Subclinical Cardiovascular Disease in Normoalbuminuric Type 1 Diabetic Patients

Cardiovascular autonomic neuropathy (CAN) is a severe complication of diabetes associated with increased morbidity and mortality (1,2). CAN results from damage to the autonomic nerve fibers to the heart, and an early manifestation is a decrease in heart rate variation (HRV) during deep breathing (3). CAN is present in 25% of type 1 diabetic patients but is often overlooked (1,3).

The increased mortality associated with CAN appears to be partly explained by coexistence with other long-term complications of diabetes (4), and since especially nephropathy and CAN generally develop in parallel, the relative contribution of CAN on mortality has been difficult to identify (5). However, some studies have found CAN to be associated with sudden cardiac death (6) and to be an independent predictor of mortality (7,8). Different mechanisms through which CAN may promote mortality have been suggested including silent myocardial ischemia, silent myocardial infarction, impaired respiratory response to hypoxia, intraoperative cardiovascular lability, and fatal arrhythmia due to QT prolongation (2,5,9–11). However, the mechanism remains unclear (12,13).

A noninvasive evaluation of coronary artery stenosis can be performed with high diagnostic accuracy using multislice computed tomography (MSCT) coronary angiography (14). MSCT also allows assessment of coronary artery calcification, which can be quantified as a coronary artery calcium score (CACS). Coronary artery calcium deposit has been shown to be a strong predictor of future cardiovascular events (15–17). Other markers of increased cardiovascular risk have recently been introduced: left ventricular dysfunction evaluated with tissue Doppler imaging (TDI) (18,19), a reduction in nighttime blood pressure drop (20,21), and increased arterial pulse pressure (22) are all independently associated with increased cardiovascular risk.

The aim of this study was to investigate the potential association between CAN and subclinical coronary atherosclerosis in addition to other prognostic markers of cardiovascular disease. For avoidance of the confounding effect of diabetic nephropathy, only patients with normal albumin excretion rates (albuminuria) were included.

# RESEARCH DESIGN AND METHODS

Patients with long-lasting type 1 diabetes, persistent normoalbuminuria, and no history or symptoms of cardiac disease (23) were recruited from the outpatient clinic cohort of type 1 diabetic patients at Steno Diabetes Center and the Diabetes Unit, Rigshospitalet. Inclusion criteria were type 1 diabetes according to American Diabetes Association criteria of at least 10 years' duration, age between 18 and 75 years, and HbA~1C~ \<10%. Exclusion criteria were albuminuria (urinary albumin-to-creatinine ratio \>30 mg/g; elevated *S*-creatinine \>120 μmol/L), untreated hypertension (\>140/85 mmHg), and electrocardiographic signs or clinical symptoms of heart disease.

In the study population registry of 3,000 type 1 diabetic patients, autonomic testing had previously been performed in 350 patients owing to clinical suspicion of autonomic neuropathy, and 123 of these patients were eligible according to inclusion/exclusion criteria. CAN testing was repeated in all patients included in the current study.

From this group, informed consent to participate in the study was obtained in 60 randomly selected patients during their visit to the outpatient clinic. Patients were divided into two groups according to the outcome of four autonomic function tests: HRV during deep breathing, Valsalva ratio, lying-to-standing test, and blood pressure response to standing up. CAN was defined as two or more abnormal tests (24), and age-normative values were used to define abnormality (25,26). Of the included 60 patients, 26 had CAN, 30 were without CAN, and 4 had inconclusive tests.

All patients underwent MSCT and transthoracic echocardiography (TTE). In order to decrease heart rate for optimized image quality, all patients were given 5 mg ivabradine orally on the evening before and on the morning of cardiac imaging. Ivabradine was selected to reduce heart rate because β-adrenoceptor blockade was considered relative contraindicated in this population. MSCT and TTE were performed on the same day, and 24-h blood pressure and Holter monitoring was performed simultaneously after a few days. Information on retinopathy was retrieved from the records of the patients, as retinopathy status is registered at least once a year. Vibratory perception threshold (V) was evaluated using biothesiometry (Bio-Medical Instrument Company, Newbury, OH) as an assessment of distal neuropathy. Blood and urine samples were collected and analyzed with standardized clinical chemistry methods including measurements of N-terminal pro-brain natriuretic peptide (NT-proBNP), high-sensitivity C-reactive protein, and cystatin in addition to routine blood tests to exclude signs of other diseases or neuropathies.

Coronary artery disease evaluated with MSCT was the primary outcome measure. Other parameters were included as confounding factors in the analysis.

All measurements and analysis were performed with the investigator blinded to the CAN status of the patients. The study was conducted in accordance with the Declaration of Helsinki II and approved by the local ethics committee (protocol number H-4-2009-091). All patients gave written informed consent.

## Cardiovascular autonomic neuropathy tests.

Age-normative values were used to define abnormality in all tests (25,26). HRV was assessed in previously trained patients who were asked to breathe deeply at a rate of six breaths per minute while being monitored on a (50 mm/s) 12-lead electrocardiography. The maximum and minimum heart rates during each breathing cycle were measured, and the mean difference of six cycles was calculated.

The lying-to-standing heart rate ratio was determined after at least 5 min rest in the supine position, and HRV was determined by calculating the maximal-to-minimal heart rate ratio: the longest R-R interval, measured around the 30th beat after standing up, to the shortest R-R interval, measured around the 15th beat after standing up.

The Valsalva test consisted of forced exhalation into a mouthpiece with a pressure of 40 mmHg for 15 s, and the ratio of the maximum R-R after the maneuver to the minimum R-R during the maneuver was calculated. The test was performed three times, and the mean value of the ratios was used.

Orthostatic hypotension was defined as a decrease in systolic blood pressure (SBP) of 30 mmHg when changing from supine to the upright position. Measurements were taken every minute for at least 3 min.

## MSCT.

All examinations were performed using a Toshiba Aquillon One 320 Volume scanner (Toshiba Medical Systems, Tokyo, Japan) according to the recommendations of the vendor and analyzed on dedicated software (Vitrea 2; Vital Images).

First, a prospective low-dose calcium scan without contrast enhancement was performed followed by coronary angiography using a prospective protocol. For the calcium score, slices of 3 mm were acquired using prospective electrocardiogram-gated axial scanning (27). We infused 80–100 mL intravenous contrast agent (Visipaque 320; GE Healthcare) (according to body weight) with a flow rate of 5 mL/s, followed by a saline chaser (50 mL). Image acquisition was initiated automatically at a density threshold of 180 Hounsfield units (HU) in the descending aorta. The scan parameters were 320 × 0.5 mm detector collimation and 100–120 kV tube voltage depending on BMI (threshold 30 kg/m^2^). Rotation time was between 350 and 375 ms depending on the heart rate. Owing to the relatively high heart rate (25 patients with heart rate \>65 bpm), the scanners (sureCardio software) frequently required two gantry rotations. Median radiation dose for nonconstrast and for angiography scans was 1.2 mSv (range 0.9–2.6) and 4.3 mSv (1.9–15.3), respectively. For the coronary computed tomography angiography, 0.5-mm slices were obtained. The images were reconstructed at 0.5-mm slices with an increment of 0.25 mm, thereby giving true isotropic voxels of 0.5 mm (28).

## Coronary calcium scoring.

Coronary calcium deposit was identified as a dense area in the coronary artery exceeding the threshold of 130 HU. Areas were automatically registered by the software and were manually assigned the corresponding coronary arteries. Total mass of the coronary calcium deposit and the Agatston score were determined for each patient. CACS was compared with reference values on an individual level according to age and sex (29,30).

## Coronary stenosis and plaque composition.

Based on computed tomography coronary angiography recordings, the presence of coronary atherosclerosis was evaluated by visual inspection of all coronary arteries. Degree of coronary stenosis was classified according to previously published guidelines (31): absence of plaque; minimal, plaque with \<25% stenosis; mild, 25–49% stenosis; moderate, 50–69% stenosis; severe, 70–99% stenosis; and occluded.

## Echocardiography.

Each patient underwent examination with a Philips IE with a S5 transducer with two-dimensonal and TDI in the left lateral decubitus using standard parasternal short axis and apical four-chamber, two-chamber, and long-axis views. Data were analyzed with commercially available software (Excelera; Philips).

Evaluation of the left ventricular ejection fraction was performed by the modified Simpsons biplane method. Left ventricular mass index was calculated as the anatomic mass divided by body surface area of the patient.

Pulsed-wave Doppler at the apical position was used to record mitral inflow between the tips of the mitral leaflets. Peak velocities of early (E) and atrial (A) transmitral flow velocities and deceleration time of the early transmitral flow were measured, and the E-to-A ratio was calculated. All valves were examined to exclude significant valvular disease.

With TDI, peak systolic (s′), early diastolic (e′), and late diastolic (a′) velocities were measured in all six mitral annular positions. Ratios of E to e′, e′ to a′, and e′ to (a′ × s′) (eas index), were calculated as measures of left ventricular filling pressures, diastolic performance, and combined systolic and diastolic performance (19).

## Ambulatory 24-h blood pressure recording.

Measurements were performed on the nondominant arm with a properly calibrated Blood Pressure Monitor System 90217 from Space Laboratories (Washington, DC). Blood pressure measurements were validated with an automatic oscillometric device. The examination was not considered valid if \>30% of the measurements were missing. The SBP, diastolic blood pressure (DBP), and heart rate were measured automatically every 20 min during daytime (between 0600 and 2200 h) and once every hour during nighttime (between 2200 and 0600 h) for 24 consecutive hours. Participants were instructed to go about their normal daily conditions; however they were advised not to rest in bed during the day or exercise heavily and to abstain from caffeine beverages and tobacco.

Analyses were performed with dedicated software (Spacelabs ABP 92506 report management system). Blood pressure dipping was defined as an average reduction in SBP and DBP ≥10% from day to night (32).

## Ambulatory 24-h electrocardiography.

Ambulatory electrocardiography recordings were obtained with a 12-lead Rozinn RZ153+. The electrocardiography recordings were analyzed with dedicated software and reviewed by highly trained observers. Time domain analysis was performed in order to verify the difference in autonomic function in patients with CAN (26,33). Measures included SD of R-R intervals during a 22-h period, SD of the average R-R interval in all 5-min recordings, and mean of the SD of all filtered R-R intervals for all 5-min segments over 22 h.

## Statistics.

All analyses were performed with SAS 9.2 (SAS Institute, Cary, NC). The χ^2^ or Fisher exact test was used for dichotomous variables and the Wilcoxon rank-sum test for continuous variables. Dichotomous variables are listed as percentages. Data of continuous variables are presented as median (range).

A multivariable logistic regression model was created with CAN status as the dependent variable and including sex, age, diabetes duration, HbA~1C~, total cholesterol, and smoking as variables considered important to coronary artery disease, and the corresponding *P* values for the independent association of CAN with a given variable are presented in all tables. Univariate linear regression models were created to identify independent predictors of increased calcium score, and these variables were included stepwise in a multiple logistic regression model.

To further compare groups of similar age, we performed a sensitivity analysis on 22 patients in each group. Excluded from this analysis were the four oldest patients with CAN (+CAN) and the eight youngest without CAN (−CAN) to obtain 22 matched pairs.

# RESULTS

## Patient characteristics.

Patients +CAN had a higher mean age and longer diabetes duration compared with patients −CAN (Table 1). The proportions of all other cardiovascular risk factors and recorded diabetes complications were similar in the two groups.

Clinical characteristics and laboratory results

![](1822tbl1)

Whereas +CAN tended to be associated with higher levels of high-sensitivity C-reactive protein than −CAN (median 1.1 mg/L \[range 0.2–13\] vs. 0.7 mg/L \[0.4–14.4\], respectively, *P* = 0.06), levels of NTproBNP and cystatin C were similar in +CAN and −CAN patients (10.3 μmol/L \[6.1–28.9\] vs. 18.6 μmol/L \[7.8–56.5\], *P* = 0.18, and 0.64 mg/L \[0.5–0.72\] vs. 0.67 mg/L \[0.45–1.2\], *P* = 0.16, respectively). Peripheral vibration threshold was significantly higher in patients +CAN compared with that in patients −CAN (23 V \[8–50\] vs. 17 V \[6–35\], *P* \< 0.01).

## MSCT.

The mean CACS was significantly higher in +CAN than in −CAN patients (Table 2). Categories of increasing CACS according to CAN status are illustrated in Fig. 1A. The proportions of patients having a CACS ≥400 were significantly higher in patients +CAN than in those −CAN. While nine patients +CAN were found in categories of CACS \>400, the highest CACS in −CAN was 312. Furthermore, a CACS \<10 was less common in patients +CAN than in patients −CAN.

Multislice computed tomography calcium scoring

![](1822tbl2)

With use of the CACS of each individual patient to find the corresponding percentile in a background population according to age and sex, the median percentile was significantly higher in patients +CAN compared with that in patients −CAN (median 92 \[range 0–99\] vs. 39 \[0–98\], respectively, *P* = 0.0205). Similarly, the proportion of patients with a CACS above the 95th percentile according to age and sex was significantly higher in patients +CAN compared with −CAN (13 \[72%\] vs. 5 \[17%\], *P* = 0.0077).

Coronary computed tomography angiography revealed focal coronary plaques in 38 (69%) patients. Obstructive lesions were found in 9 (16%) and occlusions in 1 (2%) patient.

Patients +CAN had a higher median number of plaque lesions than −CAN patients (three vs. one, respectively, *P* = 0.039) and a higher proportion of patients with more than seven plaque lesions (Fig. 1B) in univariate analysis. However, the prevalence of obstructive stenosis was not significantly higher (Fig. 1C).

Absence of elevated CACS did not exclude obstructive stenosis, as three patients had coronary atherosclerosis with a CACS of zero. Two of these patients had minimal plaques (\<25%), but one patient (+CAN) had a 70–99% noncalcified stenosis.

## Echocardiography.

All patients had a normal left ventricular ejection fraction. CAN status had no significant impact on left ventricular systolic function as measured by conventional echocardiography, but TDI measures of longitudinal systolic function (s′) were significantly lower in +CAN than in −CAN (Table 3).

Echocardiography findings

![](1822tbl3)

TDI measures of left ventricular diastolic function indicated higher filling pressures (E-to-e′ ratio) and impaired diastolic performance according to both e′ and e′-to-a′ ratio in +CAN compared with −CAN, whereas the eas index was not significantly different in the two groups (Table 3).

## Ambulatory blood pressure measurements.

Patients +CAN had higher SBP and lower DBP but mean arterial blood pressure similar to that in −CAN patients. Accordingly, the pulse pressure was significantly higher in patients +CAN compared with that in −CAN patients (Table 4).

Twenty-four-hour blood pressure and Holter measurements

![](1822tbl4)

The nocturnal drop in blood pressure was lower in +CAN patients compared with that in −CAN patients with respect to both SBP and DBP. The number of patients with abnormal dipping was significantly higher in +CAN patients compared with −CAN patients.

## Ambulatory electrocardiography.

Mean 24-h heart rate was independent of CAN status, but the maximal increase and decrease in heart rate were lower in +CAN compared with −CAN patients (Table 4).

The total number of ventricular extrasystoles (VES) was significantly higher in +CAN compared with −CAN patients. The majority of the VES were isolated, and the overall prevalence of consecutive VES was low and without difference between the groups. The number of patients having \>30 VES/h, which has been defined as frequent (32), were not significantly higher in +CAN than in the −CAN patients.

In the time domain analysis of HRV patients +CAN compared with −CAN had significantly lower SD of R-R intervals during a 22-h period (median 99 \[range 47–185\] vs. 152 \[99–208\], *P* = 0.0005) as well as SD of the average R-R interval in all 5-min recordings and mean of the SD of all filtered R-R intervals for all 5-min segments over 22 h (data not shown).

## Statistical analysis.

Variables independently associated with increased CACS from univariate analysis were DBP, pulse pressure, and measures of diastolic function (E-to-e′ ratio, interventricular septum diameter \[IVSD\], E, A, and left atrial volume). When all significant variables were included stepwise in a multiple logistic regression model, CAN remained an independent predictor of CACS (*P* = 0.0009) together with age (*P* = 0.0136), diabetes duration (*P* = 0.0010), and daytime DBP (*P* = 0.0269). Including BMI, pulse pressure, and/or urine albumin excretion in the model did not change the significance of any of the presented results.

## Sensitivity analysis.

A sensitivity analysis of 22 matched patient pairs (+CAN and −CAN) is presented in Table 5. In this analysis, there was no significant difference in age, sex, diabetes duration, HbA~1C~, or other cardiovascular risk factors between +CAN and −CAN patients. In this subset of patients, results regarding CACS, echocardiography, ambulatory blood pressure, and Holter monitoring did not differ from those of the entire study population (Table 5).

Findings of analysis of 22 patients with and without CAN of comparable mean age and diabetes duration

![](1822tbl5)

## Coexistence of CAN and markers of increased cardiovascular risk.

A higher proportion of patients +CAN had markers associated with increased cardiovascular mortality, including a CACS ≥400 (hazard ratio 2.99–3.24 for mortality \[15\]), abnormal dipping (2.16 for mortality \[21\]), a pulse pressure ≥62 mmHg (1.8 for mortality \[34\]), and a trend toward a higher proportion of patients with coronary artery stenosis ≥50% (41 for a composite end point of death, nonfatal myocardial infarction, and revascularization \[35\]) (Fig. 2).

# DISCUSSION

The current study demonstrates an association between the presence of CAN and several distinct signs of subclinical cardiovascular disease in type 1 diabetic patients with normal urinary albumin excretion rate. Patients with CAN were characterized by increased coronary calcium deposit, impaired left ventricular function, increased arterial pulse pressure, a higher prevalence of nondipping, and increased ventricular ectopia compared with patients without CAN.

Long-term type 1 diabetes carries an excess cardiovascular mortality (36), and nephropathy is a major contributing factor (37,38). Thus, even in asymptomatic patients a greater atherosclerotic plaque burden was demonstrated by magnetic resonance imaging in diabetic patients with albuminuria compared with patients with normal albumin excretion rate (39).

CAN has also been associated with high cardiovascular mortality (2). However, since nephropathy and CAN generally develop in parallel in type 1 diabetic patients, the relative contribution of CAN to cardiovascular disease has been difficult to identify (5). In an attempt to overcome this important confounder, the present CAN population was identified in a subset of type 1 diabetic patients characterized by long duration of diabetes yet with absence of nephropathy (i.e., normal albumin excretion rate).

CACS is a powerful predictor of clinical coronary artery disease (17). An association between CACS and reduced cardiac autonomic function has recently been reported in both type 1 (40–42) and type 2 (43) diabetes. Colhoun et al. (40) observed an association between CACS and CAN independent of age and triglycerides but not independent of BMI and SBP. By contrast, Rodrigues et al. (42) found an association between CAN and progression of CACS, independent of all confounders tested, including inflammatory markers. In the current study, we extended the evaluation of the coronary arteries with a combination of CACS and computed tomography angiography and included measurements of several other potential confounders, including left ventricular function and 24-h blood pressure.

CACS was markedly higher in patients +CAN, with a median CACS of 196 compared with a median CACS of 5 in patients −CAN. With use of MESA data as reference (29,30), 72% of patients +CAN had CACS similar to that in subjects in the upper 95th percentile according to age and sex, whereas this was only the case for 17% of patients −CAN. Likewise, the estimated arterial age for a person with CACS of 5 is 52 years (95% CI 48–56 years) (30,44), in agreement with the median age of patients –CAN. By contrast, a CACS of 196 corresponds to an estimated arterial age of 77 years (75–80), which is almost 20 years more than the actual median age of patients +CAN.

Despite a higher CACS among +CAN patients, computed tomography angiography did not reveal significant differences in the prevalence of obstructive stenoses. It could be speculated whether the increased calcification without increased luminal coronary plaque burden is due to a different localization of the calcified area relative to the intima and media. Surgical sympathectomy leads to arterial media calcification, a condition frequently found in the lower extremities in patients with diabetic neuropathy (45), and it has been suggested that arterial media calcification could be the result of autonomic denervation (40,46). Sympathetic denervation may cause dedifferentiation of vascular smooth muscle cells, and these alterations are associated with extracellular matrix production and migration to the intima—changes that are seen in atherosclerosis (40,47). Another suggested mechanism of how CAN could result in increased calcification is through loss of neuropeptides, involving biochemical pathways similar to those observed in studies on calcification processes in bone metabolism (46).

Data from the current study could be interpreted as supportive of the concept of increased media sclerosis. However, the relationship between CACS and luminal stenoses is known to be only modest (48), and a nonsignificant difference in the prevalence of stenoses does not necessarily imply that the calcification is differently located in the arterial wall. Furthermore, the existence of media sclerosis has been questioned (49).

Regardless of the location of the calcification, the difference in CACS could be caused by other confounding factors. Many cardiovascular parameters are interrelated, and associations could merely reflect that these conditions share common risk factors.

The presence of CAN was associated with a decreased systolic and diastolic function of the left ventricle. We found a decrease in s′ of approximately 1 cm/s in patients +CAN compared with patients −CAN. A similar decrease in s′ in a study of the background population corresponded to a hazard ratio of 1.35 for 5-year mortality (19), suggesting that the observed left ventricular dysfunction might have prognostic importance. Several echocardiographic measures of diastolic function were univariately associated with CACS. Blood pressure variables are other factors associated with CAN, CACS, and left ventricular function. However, CAN remained independently associated with CACS, even when these variables were included in multivariable models.

An association between CAN and nondipping has been attributed to impaired vagal activity during nighttime in patients with CAN (50). In the current study, nondipping was more prevalent in patients with CAN, but it was not an isolated phenomenon in these patients, since 37% of patients without CAN were also nondippers. Likewise, a recent study did not find CAN to be the main causal factor for nondipping in type 1 diabetes (51). Nonetheless, blood pressure levels at night were significantly higher in +CAN compared with patients −CAN, suggesting a relationship between CAN and nocturnal blood pressure regulation.

CAN was also found to be associated with increased pulse pressure. In addition to being associated with increased cardiovascular risk, arterial pulse pressure is considered an indirect marker of arterial stiffness (22). With use of other methods, arterial stiffness has been shown to contribute to left ventricular diastolic dysfunction (52) and has been suggested as a link between CAN and cardiovascular disease (53).

We did not observe a significant difference in mean corrected QT or in the prevalence of QTc prolongation. Very few studies have reported on ventricular ectopia associated with CAN. We found no signs of pathological arrhythmias, but CAN was associated with an increased number of isolated premature ventricular beats.

Though interrelationships are difficult to exclude, CAN remained independently associated with CACS even when differences in cardiovascular parameters were adjusted for in multivariable analysis and sensitivity analysis was applied, and CAN was found to be associated with markers of increased cardiovascular risk (Fig. 2).

This study had limitations. First, owing to stringent inclusion criteria and since autonomic dysfunction is a rare isolated complication in long-term diabetes (10), it was only possible to match patients for age, sex, and diabetes duration in a limited number of patients, and several of the variables measured are age dependent. However, both multivariable logistic regression models and sensitivity analysis of smaller groups of patients with no differences in demographics and cardiovascular risk factors were performed. In these analyses, all reported findings remained similar and significant. Second, investigations were only carried out on diabetic patients; reference values on the different measurements from a nondiabetic control group were therefore not available. Third, we did not have information on HRV on all patients in our registry. Many patients had HRV measured in previous studies, but some of the patients had HRV measured owing to clinical suspicion of CAN. We cannot exclude having captured a particular subgroup of patients being free of long-term diabetes complications not similar to the most common phenotype of diabetic patients, and our findings can only to a limited extent be extrapolated to the entire diabetic population.

In the analysis of ambulatory blood pressure measurements, a fixed method was used and not diary time. This could potentially have biased the results if differences in the sleep patterns between the two groups exist.

In the comparison between CACS and individual reference values from the MESA study, diabetes duration was a parameter that could not be accounted for. The longer diabetes duration in +CAN patients must be remembered when evaluating these data.

Confounding effects from differences in antihypertensive treatment and use of diuretics cannot be excluded, even though the differences were nonsignificant in demographics.

In conclusion, CAN was associated with several distinct signs of subclinical cardiovascular disease in type 1 diabetic patients with normoalbuminuria. These included increased coronary calcium deposit, subtle impairment of left ventricle systolic and diastolic function, increased arterial pulse pressure, a higher prevalence of nondipping, and marginally increased ventricular ectopia, and CAN was independently associated with CACS even with adjustment for confounding factors.

## ACKNOWLEDGMENTS

This study was supported by a grant from Arvid Nilssons Foundation. The MSCT Cardiac Research Unit, Rigshospitalet, is supported by a grant from the A.P. Møller og Hustru Chastine McKinney Møllers Fond til almene Formaal.

U.M.M. was supported by a scholarship from Novo Nordisk. No other potential conflicts of interest relevant to this article were reported.

U.M.M. researched data and wrote the manuscript. T.J. included patients, contributed to discussion, and reviewed the manuscript. L.K. researched data and reviewed the manuscript. H.K. reviewed the manuscript. A.S.M. screened patients. U.D. and P.R. reviewed the manuscript. J.H. contributed to discussion and reviewed the manuscript. K.F.K. researched data and reviewed the manuscript. U.M.M. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Parts of this study were presented in abstract form at the European Society of Cardiology Congress 2011, Paris, France, 27–31 August 2011, and at the 47th European Association for the Study of Diabetes Annual Meeting, Lisbon, Portugal, 12–16 September 2011.

The authors thank Tina Bock-Pedersen, MSCT Cardiac Research Unit, Rigshospitalet, for excellent technical assistance.

# REFERENCES