Median preoptic area neurons are required for the cooling and febrile activations of brown adipose tissue thermogenesis in rat.

Within the central neural circuitry for thermoregulation, the balance between excitatory and inhibitory inputs to the dorsomedial hypothalamus (DMH) determines the level of activation of brown adipose tissue (BAT) thermogenesis. We employed neuroanatomical and in vivo electrophysiological techniques to identify a source of excitation to thermogenesis-promoting neurons in the DMH that is required for cold defense and fever. Inhibition of median preoptic area (MnPO) neurons blocked the BAT thermogenic responses during both PGE2-induced fever and cold exposure. Disinhibition or direct activation of MnPO neurons induced a BAT thermogenic response in warm rats. Blockade of ionotropic glutamate receptors in the DMH, or brain transection rostral to DMH, blocked cold-evoked or NMDA in MnPO-evoked BAT thermogenesis. RNAscope technique identified a glutamatergic population of MnPO neurons that projects to the DMH and expresses c-Fos following cold exposure. These discoveries relative to the glutamatergic drive to BAT sympathoexcitatory neurons in DMH augment our understanding of the central thermoregulatory circuitry in non-torpid mammals. Our data will contribute to the development of novel therapeutic approaches to induce therapeutic hypothermia for treating drug-resistant fever, and for improving glucose and energy homeostasis.

Low-protein diet in puberty impairs testosterone output and energy metabolism in male rats.

We examined the long-term effects of protein restriction during puberty on the function of hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes in male rats. Male Wistar rats from the age of 30 to 60 days were fed a low-protein diet (4%, LP). A normal-protein diet (20.5%) was reintroduced to rats from the age of 60 to 120 days. Control rats were fed a normal-protein diet throughout life (NP). Rats of 60 or 120 days old were killed. Food consumption, body weight, visceral fat deposits, lipid profile, glycemia, insulinemia, corticosteronemia, adrenocorticotropic hormone (ACTH), testosteronemia and leptinemia were evaluated. Glucose-insulin homeostasis, pancreatic-islet insulinotropic response, testosterone production and hypothalamic protein expression of the androgen receptor (AR), glucocorticoid receptor (GR) and leptin signaling pathway were also determined. LP rats were hypophagic, leaner, hypoglycemic, hypoinsulinemic and hypoleptinemic at the age of 60 days (P < 0.05). These rats exhibited hyperactivity of the HPA axis, hypoactivity of the HPG axis and a weak insulinotropic response (P < 0.01). LP rats at the age of 120 days were hyperphagic and exhibited higher visceral fat accumulation, hyperleptinemia and dyslipidemia; lower blood ACTH, testosterone and testosterone release; and reduced hypothalamic expression of AR, GR and SOCS3, with a higher pSTAT3/STAT3 ratio (P < 0.05). Glucose-insulin homeostasis was disrupted and associated with hyperglycemia, hyperinsulinemia and increased insulinotropic response of the pancreatic islets. The cholinergic and glucose pancreatic-islet responses were small in 60-day-old LP rats but increased in 120-day-old LP rats. The hyperactivity of the HPA axis and the suppression of the HPG axis caused by protein restriction at puberty contributed to energy and metabolic disorders as long-term consequences.

Tonic inhibition of brown adipose tissue sympathetic nerve activity via muscarinic acetylcholine receptors in the rostral raphe pallidus.

KEY POINTS: A tonically active, muscarinic cholinergic inhibition of rostral raphe pallidus (rRPa) neurons influences thermogenesis of brown adipose tissue (BAT) independent of ambient temperature conditions. The tonically active cholinergic input to rRPa originates caudal to the hypothalamus. Muscarinic acetylcholine receptor (mAChR) activation in rRPa contributes to the inhibition of BAT sympathetic nerve activity (SNA) evoked by activation of neurons in the rostral ventrolateral medulla (RVLM). The RVLM is not the sole source of the muscarinic cholinergic input to rRPa. Activation of GABA receptors in rRPa does not mediate the cholinergic inhibition of BAT SNA. ABSTRACT: We sought to determine if body temperature and energy expenditure are influenced by a cholinergic input to neurons in the rostral raphe pallidus (rRPa), the site of sympathetic premotor neurons controlling thermogenesis of brown adipose tissue (BAT). Nanoinjections of the muscarinic acetylcholine receptor (mAChR) agonist, oxotremorine, or the cholinesterase inhibitor, neostigmine (NEOS), in the rRPa of anaesthetized rats decreased cold-evoked BAT sympathetic nerve activity (SNA, nadirs: -72 and -95%), BAT temperature (Tbat, -0.5 and -0.6°C), expired CO2 (Exp. CO2 , -0.3 and -0.5%) and heart rate (HR, -22 and -41 bpm). NEOS into rRPa reversed the increase in BAT SNA evoked by blockade of GABA receptors in rRPa. Nanoinjections of the mAChR antagonist, scopolamine (SCOP), in the rRPa of warm rats increased BAT SNA (peak: +1087%), Tbat (+1.8°C), Exp. CO2 (+0.7%), core temperature (Tcore, +0.5°C) and HR (+54 bpm). SCOP nanoinjections in rRPa produced similar activations of BAT during cold exposure, following a brain transection caudal to the hypothalamus, and during the blockade of glutamate receptors in rRPa. We conclude that a tonically active cholinergic input to the rRPa inhibits BAT SNA via activation of local mAChR. The inhibition of BAT SNA mediated by mAChR in rRPa does not depend on activation of GABA receptors in rRPa. The increase in BAT SNA following mAChR blockade in rRPa does not depend on the activity of neurons in the hypothalamus or on glutamate receptor activation in rRPa.

Glycinergic inhibition of BAT sympathetic premotor neurons in rostral raphe pallidus.

The rostral raphe pallidus (rRPa) contains sympathetic premotor neurons controlling thermogenesis in brown adipose tissue (BAT). We sought to determine whether a tonic activation of glycineA receptors (GlyAR) in the rRPa contributes to the inhibitory regulation of BAT sympathetic nerve activity (SNA) and of cardiovascular parameters in anesthetized rats. Nanoinjection of the GlyAR antagonist, strychnine (STR), into the rRPa of intact rats increased BAT SNA (peak: +495%), BAT temperature (TBAT, +1.1°C), expired CO2, (+0.4%), core body temperature (TCORE, +0.2°C), mean arterial pressure (MAP, +4 mmHg), and heart rate (HR, +57 beats/min). STR into rRPa in rats with a postdorsomedial hypothalamus transection produced similar increases in BAT thermogenic and cardiovascular parameters. Glycine nanoinjection into the rRPa evoked a potent inhibition of the cooling-evoked increases in BAT SNA (nadir: -74%), TBAT (-0.2°C), TCORE (-0.2°C), expired CO2 (-0.2%), MAP (-8 mmHg), and HR (-22 beats/min) but had no effect on the increases in these variables evoked by STR nanoinjection into rRPa. Nanoinjection of GABA into the rRPa inhibited the STR-evoked BAT SNA (nadir: -86%) and reduced the expired CO2 (-0.4%). Blockade of glutamate receptors in rRPa reduced the STR-evoked increases in BAT SNA (nadir: -61%), TBAT (-0.5°C), expired CO2 (-0.3%), MAP (-9 mmHg), and HR (-33 beats/min). We conclude that a tonically active glycinergic input to the rRPa contributes to the inhibitory regulation of the discharge of BAT sympathetic premotor neurons and of BAT thermogenesis and energy expenditure.

Dietary calcium supplementation in adult rats reverts brown adipose tissue dysfunction programmed by postnatal early overfeeding.

Brown adipose tissue (BAT) dysfunction is associated with obesity and its comorbidities, such as hypertension, and the improvement of BAT function seems important for obesity management. Here we investigated the effects of dietary calcium supplementation on BAT autonomic nerve activity, sympathoadrenal function and cardiovascular parameters in adult obese rats that were raised in small litters (SL group). Three days after birth, SL litters were adjusted to three pups to induce early overfeeding. The control group remained with 10 pups/litter until weaning (NL group). At PN120, the SL group was randomly divided into the following: rats fed with standard chow (SL) and rats fed with dietary calcium carbonate supplementation (SL-Ca, 10g/kg chow). Animals were killed either at PN120 or PN180. At both ages, SL rats had higher BAT autonomic nervous system activity, mass and adipocyte area, as well as increased heart rate and blood pressure (systolic and diastolic); 2 months of calcium supplementation normalized these parameters. At PN180 only, UCP1 and TRß1 in BAT were decreased in SL rats. These changes were also prevented by calcium treatment. Also at PN180, the SL group presented higher tyrosine hydroxylase and adrenal catecholamine contents, as well as lower hypothalamic POMC and MC4R contents. Calcium supplementation did not revert these alterations. Thus, we demonstrated that dietary calcium supplementation was able to improve cardiovascular parameters and BAT thermogenesis capacity in adult animals that were early overfed during lactation.

Cross-fostering reduces obesity induced by early exposure to monosodium glutamate in male rats.

Maternal obesity programmes a range of metabolic disturbances for the offspring later in life. Moreover, environmental changes during the suckling period can influence offspring development. Because both periods significantly affect long-term metabolism, we aimed to study whether cross-fostering during the lactation period was sufficient to rescue a programmed obese phenotype in offspring induced by maternal obesity following monosodium L-glutamate (MSG) treatment. Obesity was induced in female Wistar rats by administering subcutaneous MSG (4 mg/g body weight) for the first 5 days of postnatal life. Control and obese female rats were mated in adulthood. The resultant pups were divided into control second generation (F2) (CTLF2), MSG-treated second generation (F2) (MSGF2), which suckled from their CTL and MSG biological dams, respectively, or CTLF2-CR, control offspring suckled by MSG dams and MSGF2-CR, MSG offspring suckled by CTL dams. At 120 days of age, fat tissue accumulation, lipid profile, hypothalamic leptin signalling, glucose tolerance, glucose-induced, and adrenergic inhibition of insulin secretion in isolated pancreatic islets were analysed. Maternal MSG-induced obesity led to an obese phenotype in male offspring, characterized by hyperinsulinaemia, hyperglycaemia, hyperleptinaemia, dyslipidaemia, and impaired leptin signalling, suggesting central leptin resistance, glucose intolerance, impaired glucose-stimulated, and adrenergic inhibition of insulin secretion. Cross-fostering normalized body weight, food intake, leptin signalling, lipid profiles, and insulinaemia, but not glucose homeostasis or insulin secretion from isolated pancreatic islets. Our findings suggest that alterations during the lactation period can mitigate the development of obesity and prevent the programming of adult diseases.

Maternal flaxseed diet during lactation changes adrenal function in adult male rat offspring.

Flaxseed (Linum usitatissimum L.) has been a focus of interest in the field of functional foods because of its potential health benefits. However, we hypothesised that maternal flaxseed intake during lactation could induce several metabolic dysfunctions in adult offspring. In the present study, we aimed to characterise the adrenal function of adult offspring whose dams were supplemented with whole flaxseed during lactation. At birth, lactating Wistar rats were divided into two groups: rats from dams fed the flaxseed diet (FLAX) with 25% of flaxseed and controls dams. Pups received standard diet after weaning and male offspring were killed at age 180 days old to collect blood and tissues. We evaluated body weight and food intake during development, corticosteronaemia, adrenal catecholamine content, hepatic cholesterol, TAG and glycogen contents, and the protein expression of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), 11-ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and adrenaline ß2 receptor at postnatal day 180 (PN180). After weaning, pups from the FLAX group had a higher body weight (+10 %) and food intake (+10%). At PN180, the FLAX offspring exhibited higher serum corticosterone (+48%) and lower adrenal catecholamine ( - 23%) contents, lower glycogen ( - 30%), higher cholesterol (4-fold increase) and TAG (3-fold-increase) contents in the liver, and higher 11ß-HSD1 (+62%) protein expression. Although the protein expression of hypothalamic CRH was unaffected, the FLAX offspring had lower protein expression of pituitary ACTH ( - 34%). Therefore, induction of hypercorticosteronaemia by dietary flaxseed during lactation may be due to an increased hepatic activation of 11ß-HSD1 and suppression of ACTH. The changes in the liver fat content of the FLAX group are suggestive of steatosis, in which hypercorticosteronaemia may play an important role. Thus, it is recommended that lactating women restrict the intake of flaxseed during lactation.

Cis-9, trans-11 and trans-10, cis-12 CLA mixture does not change body composition, induces insulin resistance and increases serum HDL cholesterol level in rats.

Synthetic supplements of conjugated linoleic acid (CLA) containing 50:50 mixture of cis-9, trans-11 and trans-10, cis-12 CLA isomers have been commercialized in some places for reducing body fat. However the safety of this CLA mixture is controversial and in some countries the CLA usage as food supplement is not authorized. Changes in insulinemic control and serum lipids profile are potential negative effects related to consumption of CLA mixture. The present study aimed to evaluate the effects of a diet containing mixture of cis-9, trans-11 and trans-10, cis-12 CLA on prevention of obesity risk as well as on potential side effects such as insulin resistance and dyslipidemia in Wistar rats. Thirty male Wistar rats were randomly assigned to the following dietary treatments (n=10/group), for 60 days: Normolipidic Control (NC), diet containing 4.0% soybean oil (SO); High Fat-Control (HF-C), diet containing 24.0% SO; High Fat-synthetic CLA (HF-CLA), diet containing 1.5% of an isomeric CLA mixture (Luta-CLA 60) and 22.5% SO. Luta-CLA 60 (BASF) contained nearly 60% of CLA (cis-9, trans-11 and trans-10, cis-12 CLA at 50:50 ratio). The HF-CLA diet contained 0.3% of each CLA isomer. HF-CLA diet had no effect on dietary intake and body composition. HF-CLA-fed rats had lower levels of PPARγ protein in retroperitoneal adipose tissue, hyperinsulinemia compared to HF-C-fed rats, hyperglycemia compared to NC-fed rats while no differences in glycemia were observed between NC and HF-C groups, increased HOMA index and higher levels of serum HDL cholesterol. Thus, feeding rats with a high fat diet containing equal parts of cis-9, trans-11 and trans-10, cis-12 CLA isomers had no effect on body composition and induced insulin resistance. Despite HF-CLA-fed rats had increased serum HDL cholesterol levels, caution should be taken before synthetic supplements containing cis-9, trans-11 and trans-10, cis-12 CLA are recommended as a nutritional strategy for weight management.

Postnatal early overfeeding induces hypothalamic higher SOCS3 expression and lower STAT3 activity in adult rats.

Postnatal early overnutrition (EO) is a risk factor for future obesity and metabolic disorders. Rats raised in small litters (SLs) develop overweight, hyperphagia, hyperleptinemia, hyperinsulinemia and hypertension when adults. As obesity is related to hyperleptinemia, leptin resistance and metabolic syndrome, we aimed to investigate body composition, plasma hormone levels, glucose tolerance and the leptin signaling pathway in hypothalamus from early overfed animals at weaning and adulthood. To induce postnatal EO, we reduced litter size to three pups/litter (SL), and the groups with normal litter size (10 pups/litter) were used as control. Rats had free access to standard diet and water postweaning. Body weight and food intake were monitored daily, and offspring were killed at 21 (weaning) and 180 days old (adulthood). Postnatal EO group had higher body weight and total and visceral fat mass at both periods. Lean mass and serum high-density lipoprotein cholesterol (HDL-C) were higher at 21 days and lower at 180 days. Small litter rats presented higher levels of globulins at both periods, while albumin levels were higher at weaning and lower at adulthood. There was higher leptin, insulin and glucose serum concentrations at 21 days old, while no glucose intolerance was observed in adulthood. Leptin signaling pathway was unaffected at weaning. However, postnatal EO induced lower JAK2 and p-STAT3, and higher SOCS3 expression in adult animals, indicating central leptin resistance in adulthood. In conclusion, postnatal EO induces obesity, higher total and visceral fat mass, lower HDL-C and central leptin resistance in adult life.