Guo-Qing Chang

Maternal High-Fat Diet and Fetal Programming: Increased Proliferation of Hypothalamic Peptide-Producing Neurons That Increase Risk for Overeating and Obesity

Recent studies in adult and weanling rats show that dietary fat, in close association with circulating lipids, can stimulate expression of hypothalamic peptides involved in controlling food intake and body weight. In the present study, we examined the possibility that a fat-rich diet during pregnancy alters the development of these peptide systems in utero, producing neuronal changes in the offspring that persist postnatally in the absence of the diet and have long-term consequences. The offspring of dams on a high-fat diet (HFD) versus balanced diet (BD), from embryonic day 6 to postnatal day 15 (P15), showed increased expression of orexigenic peptides, galanin, enkephalin, and dynorphin, in the paraventricular nucleus and orexin and melanin-concentrating hormone in the perifornical lateral hypothalamus. The increased density of these peptide-expressing neurons, evident in newborn offspring as well as P15 offspring cross-fostered at birth to dams on the BD, led us to examine events that might be occurring in utero. During gestation, the HFD stimulated the proliferation of neuroepithelial and neuronal precursor cells of the embryonic hypothalamic third ventricle. It also stimulated the proliferation and differentiation of neurons and their migration toward hypothalamic areas where ultimately a greater proportion of the new neurons expressed the orexigenic peptides. This increase in neurogenesis, closely associated with a marked increase in lipids in the blood, may have a role in producing the long-term behavioral and physiological changes observed in offspring after weaning, including an increase in food intake, preference for fat, hyperlipidemia, and higher body weight.

Distinct phenotypes of obesity-prone AKR/J, DBA2J and C57BL/6J mice compared to control strains

Objective:To characterize and compare three obesity-prone inbred strains, AKR/J, DBA/2J and C57BL/6J, to three control strains, C3H/HeJ, BALB/cByJ and C57L/J, selected based on their normal eating patterns and moderate weight gain on high-calorie diets.Methods and procedures:These six strains were examined at 5 weeks of age while still of normal body weight, and they were maintained for 1 day or 3 weeks on different feeding paradigms with macronutrient diets. Measurements were taken of macronutrient intake, body weight and body fat accrual, circulating hormones and metabolites, and the hypothalamic peptide, galanin.Results:The three control strains each selected a balanced diet with 50% carbohydrate and 15–25% fat when given a choice of macronutrients, and they had similar, normal range of scores for the measures of body weight, adiposity, the hormones, insulin and leptin, and the metabolites, glucose and triglycerides. When compared to this control baseline, the obesity-prone strains with similar total caloric intake to controls selected a diet with significantly more fat (30–40%) and less carbohydrate (<40%). They also had greater adiposity, with the largest differences detected for the AKR/J and DBA/2J strains. These two obesity-prone strains compared to control strains had elevated levels of insulin and leptin. They also had higher triglyceride levels and increased expression and levels of galanin in the hypothalamic paraventricular nucleus. A very different pattern was detected in the obesity-prone C57BL/6J strain, which exhibited a stronger preference for protein as well as fat, normal levels of insulin, leptin and triglycerides, hyperglycemia relative to all other strains, and a small increase in galanin.Conclusion:These comparisons to control strains revealed a distinct phenotype in the two obesity-prone strains, AKR/J and DBA/2J, which is very similar to that described in obesity-prone, outbred rats. They also identified a clearly different phenotype in the obesity-prone C57BL/6J strain.

Different forms of obesity as a function of diet composition

OBJECTIVE:To characterize the phenotype of obesity on a high-carbohydrate diet (HCD) as compared to a high-fat diet (HFD) or moderate-fat diet (MFD).METHODS AND PROCEDURES:In four experiments, adult Sprague–Dawley rats (275–300 g) were maintained for several weeks on a: (1) HFD with 50% fat; (2) balanced MFD with 25% fat; or (3) HCD with 10% fat/65% carbohydrate. Then, based on the amount of body fat accumulated in four dissected fat pads, the animals were subgrouped as lean (lowest tertile) or obese (highest tertile) and characterized with multiple measures.RESULTS:The obese rats of these diet groups, with 70–80% greater body fat than the lean animals, exhibited elevated levels of leptin and insulin and increased activity of lipoprotein lipase in adipose tissue (aLPL), with no change in muscle LPL. Characteristics common to the obese rats on the HFD or MFD, but not seen on the HCD, were hyperphagia, elevated circulating levels of triglycerides (TG), nonesterified fatty acids (NEFA) and glucose, and a significant increase in β-hydroxyacyl-CoA dehydrogenase (HADH) activity in muscle, reflecting its greater capacity to metabolize fat. This was accompanied by a significant increase in expression of the peptide, galanin (GAL), in the paraventricular nucleus (PVN), as measured by in situ hybridization and real-time quantitative PCR, and also in GAL peptide immunoreactivity. These measures of GAL were consistently, positively correlated with circulating TG levels and also with HADH activity in muscle. In contrast to these fat-associated changes, rats that became obese on an HCD maintained normal caloric intake and levels of TG, NEFA, and glucose. They also showed no change in PVN GAL mRNA or peptide. Instead, they exhibited a significant reduction in HADH activity compared to the lean animals, along with increased activity of phosphofructokinase in muscle, a key enzyme in glycolysis.CONCLUSION:Specific characteristics of obesity, including expression of hypothalamic peptides, are dependent upon diet composition. Whereas obesity on an HFD is associated with hyperphagia and elevated lipids, fat metabolism in muscle, and fat-stimulated peptides such as GAL, obesity on an HCD with a similar increase in body fat shows none of these characteristics and instead exhibits a metabolic pattern in muscle that favors carbohydrate over fat oxidation. These results suggest the existence of multiple forms of obesity with different underlying mechanisms that are diet dependent.

Acute high-fat diet paradigms link galanin to triglycerides and their transport and metabolism in muscle

To compare the effects of acute exposure to dietary fat to those of chronic exposure, Sprague-Dawley rats were given a high-fat diet (50% fat) or moderate-fat diet (25% fat) for 1 day, 2 h or 3 weeks. With measurements of various parameters, the high-fat diet for 21 days produced the expected changes of: (1) a significant increase in total caloric intake and dissected fat pad weights; (2) a rise in leptin and the metabolites, triglycerides (TG), non-esterified fatty acids and glucose; (3) an increase in muscle beta-hydroxyacyl-CoA dehydrogenase (HADH) and adipose lipoprotein lipase (aLPL) activity, along with a decrease in LPL activity in muscle (mLPL); and (4) elevated galanin (GAL) expression and peptide levels in the anterior region of the paraventricular nucleus (PVN), with no change in the arcuate nucleus. The acute 1-day or 2-h high-fat diet similarly increased circulating lipids, HADH activity and PVN GAL mRNA but stimulated rather than suppressed mLPL activity. These effects occurred in the absence of a change in total caloric intake, fat pad weights, and adipose-related measures, suggesting that they resulted more from the rise in dietary fat from 25% to 50% than from increased adiposity or hyperphagia. Moreover, PVN GAL mRNA in the different groups was consistently and positively correlated with the specific measures of TG levels and both HADH and mLPL activity, linking it to metabolic processes related to the transport and capacity for oxidation of TG in muscle, rather than adipose tissue.

Hypothalamic huntingtin-associated protein 1 as a mediator of feeding behavior

The hypothalamus responds to circulating leptin and insulin in the control of food intake and body weight. A number of neurotransmitters in the hypothalamus, including γ-aminobutyric acid (GABA), also have key roles in feeding. Huntingtin-associated protein 1 (Hap1) is expressed more abundantly in the hypothalamus than in other brain regions, and lack of Hap1 in mice leads to early postnatal death. Hap1 is also involved in intracellular trafficking of the GABAA receptor. Here, we report that fasting upregulates the expression of Hap1 in the rodent hypothalamus, whereas intracerebroventricular administration of insulin downregulates Hap1 by increasing its degradation through ubiquitination. Decreasing the expression of mouse hypothalamic Hap1 by siRNA reduces the level and activity of hypothalamic GABAA receptors and causes a decrease in food intake and body weight. These findings provide evidence linking hypothalamic Hap1 to GABA in the stimulation of feeding and suggest that this mechanism is involved in the feeding-inhibitory actions of insulin in the brain.

Model for predicting and phenotyping at normal weight the long-term propensity for obesity in Sprague-Dawley rats

Tests were conducted to determine whether weight gain or nutrient intake measures during the first week of exposure to a macronutrient diet can accurately predict an animals long-term propensity towards obesity. In multiple groups of normal-weight Sprague-Dawley rats (n=35-70/group), daily weight gain during the first 5 days on a high-fat diet (45-60% fat) was found to be strongly, positively correlated (r=+0.71 to r=+0.82) with accumulated body fat in 4 dissected depots after 4-6 weeks on the diet. This measure consistently identified obesity-prone (OP) rats which, relative to the obesity-resistant (OR) rats, were only slightly heavier (+15 g, 4%) and hyperphagic (+9 kcal, 8%) after 5 days but markedly heavier (+70g) with up to 2-fold greater fat mass after several weeks on the diet. Other dietary conditions and measures revealed weaker relationships to ultimate body fat accrual. The OP rats identified by their 5-day weight-gain score exhibited at this early stage clear disturbances characteristic of markedly obese rats. These included elevated leptin, insulin, triglycerides and glucose, along with increased lipoprotein lipase activity (LPL) in adipose tissue and galanin expression in the paraventricular nucleus. Most notable were significant reductions in muscle of LPL activity and ratio of beta-hydroxyacyl-CoA dehydrogenase to citrate synthase activity, indicating a decline in lipid transport and capacity of muscle to metabolize lipids. By occurring early with initial weight gain, these hypothalamic and metabolic disturbances in OP rats, favoring fat storage in adipose tissue over fat oxidation in muscle, may have causal relationships to long-term accumulation of body fat.

Ethanol intake increases galanin mRNA in the hypothalamus and withdrawal decreases it

Alcoholism can be viewed as a motivational disorder that results from alterations in brain systems for ingestive behavior. Therefore, it was hypothesized that alcohol intake might alter the expression of hypothalamic peptides that stimulate feeding. Earlier studies showed that hypothalamic injection of the feeding-stimulatory peptide, galanin (GAL), increases the release of dopamine (DA) in the nucleus accumbens (NAc), as does systemic alcohol, leading to a focus on GAL. Results of this study demonstrate the following: (1). Ethanol, injected daily (0.8 g/kg 10% v/v) for 7 days in male rats, markedly increased the expression of GAL but not of neuropeptide Y (NPY). This occurred in specific hypothalamic nuclei, namely the dorsomedial nucleus (DMN), paraventricular nucleus (PVN) and perifornical lateral hypothalamus (PLH). (2). Rats induced to drink ethanol ad libitum, by gradually increasing the concentration from 1% to 9% v/v without adding sugar or flavoring, exhibited a similar stimulation of GAL mRNA in the PVN and GAL immunoreactivity in the DMN and PVN. (3). Rats given increasing ethanol concentrations, with 12 h access starting 4 h into the dark cycle, had a mean blood alcohol concentration of 18 mg/dl and exhibited a similar increase in GAL expression in the DMN and PVN. (4) Withdrawal from the opioid effects of 9% ethanol, produced by injection of naloxone (3 mg/kg sc), reversed this ethanol effect by significantly reducing GAL expression in the DMN and PLH below baseline levels. These studies suggest a possible role for hypothalamic GAL in alcohol abuse.

Positive relationship between dietary fat, ethanol intake, triglycerides and hypothalamic peptides: Counteraction by lipid-lowering drugs

Studies in both humans and animals suggest a positive relationship between the intake of ethanol and intake of fat, which may contribute to alcohol abuse. This relationship may be mediated, in part, by hypothalamic orexigenic peptides such as orexin (OX), which stimulate both consumption of ethanol and fat, and circulating triglycerides (TGs), which stimulate these peptides and promote consummatory behavior. The present study investigated this vicious cycle between ethanol and fat, to further characterize its relation to TGs and to test the effects of lowering TG levels. In Experiment 1, the behavioral relationship between fat intake and ethanol was confirmed. Adult male Sprague-Dawley rats, chronically injected intraperitoneally with ethanol (1g/kg) and tested in terms of their preference for a high-fat diet (HFD) compared with low-fat diet (LFD), showed a significant increase in their fat preference, compared with rats injected with saline, in measures of 2h and 24h intake. Experiment 2 tested the relationship of circulating TGs in this positive association between ethanol and fat, in rats chronically consuming 9% ethanol versus water and given acute meal tests (25kcal) of a HFD versus LFD. Levels of TGs were elevated in response to both chronic drinking of ethanol versus water and acute eating of a high-fat versus low-fat meal. Most importantly, ethanol and a HFD showed an interaction effect, whereby their combination produced a considerably larger increase in TG levels (+172%) compared to ethanol with a LFD (+111%). In Experiment 3, a direct manipulation of TG levels was found to affect ethanol intake. After intragastric administration of gemfibrozil (50mg/kg) compared with vehicle, TG levels were lowered by 37%, and ethanol intake was significantly reduced. In Experiment 4, the TG-lowering drug gemfibrozil also caused a significant reduction in the expression of the orexigenic peptide, OX, in the perifornical lateral hypothalamus. These results support the existence of a vicious cycle between ethanol and fat, whereby each nutrient stimulates intake of the other. Within this vicious cycle, ethanol and fat act synergistically to increase TG levels, which in turn stimulate peptides that promote further consumption, and these phenomena are reversed by gemfibrozil, which lowers TG levels.

Cocaine- and amphetamine-regulated transcript in the arcuate nucleus stimulates lipid metabolism to control body fat accrual on a high-fat diet.

Previous studies have indicated a relationship between cocaine- and amphetamine-related transcript (CART) and leptin. The present study used quantitative PCR and in situ hybridization to examine this CART-leptin relationship in different animal models. With CART injection, the function of this pathway was also investigated. The results demonstrate that CART mRNA in the arcuate nucleus (ARC) was significantly increased in subjects fed a high-fat diet (HFD) compared to low-fat diet (LFD). It was also elevated in obese vs. lean rats and in normal-weight obesity-prone vs. obesity-resistant rats. In each group tested, CART mRNA in the ARC was positively correlated specifically with circulating levels of leptin. Its close association specifically with leptin was further supported by a stimulatory effect of this hormone on CART expression. This leptin-CART relationship in the ARC, in contrast, was less consistent or undetectable in the paraventricular nucleus and lateral hypothalamus. Central injection of CART peptide (55-102) increased circulating non-esterified fatty acid levels and decreased lipoprotein lipase activity in adipose tissue. These results suggest that, on a fat-rich diet, this leptin-CART pathway originating in the ARC inhibits excessive body fat accrual by causing a shift from lipid storage toward lipid mobilization.

Effect of Chronic Ethanol on Enkephalin in the Hypothalamus and Extra‐Hypothalamic Areas

BACKGROUND Ethanol may be consumed for reasons such as reward, anxiety reduction, or caloric content, and the opioid enkephalin (ENK) appears to be involved in many of these functions. Previous studies in Sprague-Dawley rats have demonstrated that ENK in the hypothalamic paraventricular nucleus (PVN) is stimulated by voluntary consumption of ethanol. This suggests that this opioid peptide may be involved in promoting the drinking of ethanol, consistent with our recent findings that PVN injections of ENK analogs stimulate ethanol intake. To broaden our understanding of how this peptide functions throughout the brain to promote ethanol intake, we measured, in rats trained to drink 9% ethanol, the expression of the ENK gene in additional brain areas outside the hypothalamus, namely, the ventral tegmental area (VTA), nucleus accumbens shell (NAcSh) and core (NAcC), medial prefrontal cortex (mPFC), and central nucleus of the amygdala (CeA). METHODS In the first experiment, the brains of rats chronically drinking 1 g/kg/d ethanol, 3 g/kg/d ethanol, or water were examined using real-time quantitative polymerase chain reaction (qRT-PCR). In the second experiment, a more detailed, anatomic analysis of changes in gene expression, in rats chronically drinking 3 g/kg/d ethanol compared to water, was performed using radiolabeled in situ hybridization (ISH). The third experiment employed digoxigenin-labeled ISH (DIG) to examine changes in the density of cells expressing ENK and, for comparison, dynorphin (DYN) in rats chronically drinking 3 g/kg/d ethanol versus water. RESULTS With qRT-PCR, the rats chronically drinking ethanol plus water compared to water alone showed significantly higher levels of ENK mRNA, not only in the PVN but also in the VTA, NAcSh, NAcC, and mPFC, although not in the CeA. Using radiolabeled ISH, levels of ENK mRNA in rats drinking ethanol were found to be elevated in all areas examined, including the CeA. The experiment using DIG confirmed this effect of ethanol, showing an increase in density of ENK-expressing cells in all areas studied. It additionally revealed a similar change in DYN mRNA in the PVN, mPFC, and CeA, although not in the NAcSh or NAcC. CONCLUSIONS While distinguishing the NAc as a site where ENK and DYN respond differentially, these findings lead us to propose that these opioids, in response to voluntary ethanol consumption, are generally elevated in extra-hypothalamic as well as hypothalamic areas, possibly to carry out specific area-related functions that, in turn, drive animals to further consume ethanol. These functions include calorie ingestion in the PVN, reward and motivation in the VTA and NAcSh, response-reinforcement learning in the NAcC, stress reduction in the CeA, and behavioral control in the mPFC.