Jordan Dourmashkin

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 galanin : control by signals of fat metabolism

The peptide, galanin (GAL), is known to stimulate eating behavior, reduce energy expenditure and affect the release of metabolic hormones. Further, the activity of this peptide in the hypothalamus is modulated, in turn, by these hormones as well as by the ingestion of nutrients. The focus of this investigation is on signals related to nutrient metabolism that may also affect GAL production and, through these neurochemical events, control the ingestion of specific nutrients. Three experiments were performed in normal-weight male, Sprague-Dawley rats. In Experiment 1, the impact of food deprivation (24 and 48 h) was examined. Experiment 2 tested the effects of the compound, 2-deoxy-D-glucose (2-DG, 200 and 400 mg/kg), which blocks glucose utilization, whereas Experiment 3 studied mercaptoacetate (MA, 200 and 600 micromol/kg), which blocks fatty acid oxidation. Eating behavior was examined in some rats, whereas hypothalamic GAL activity was measured in others using radioimmunoassay, immunohistochemistry and in situ hybridization. Both food deprivation and MA (600 micromol/kg), but not 2-DG, affected GAL in the hypothalamus, in one specific area. This is the anterior parvocellular region of the paraventricular nucleus (aPVN), which has a dense concentration of GAL-containing neurons and terminals. GAL gene expression and peptide immunoreactivity in this area is enhanced by food deprivation; in contrast, it is reduced by injection of MA. Other hypothalamic sites with dense concentrations of GAL-containing neurons or fibers are unaffected by food deprivation or MA, and the antimetabolite 2-DG has no impact on GAL in any area. Behavioral measurements indicate that these shifts in GAL activity are accompanied by specific changes in eating behavior. Food deprivation which enhances aPVN GAL produces a marked increase in fat ingestion, whereas MA which reduces aPVN GAL causes a specific reduction in fat ingestion along with a stimulation of protein intake. In contrast, 2-DG preferentially enhances ingestion of carbohydrate. These findings suggest a possible relationship between GAL activity in the aPVN and the metabolic and behavioral processes of fat metabolism and ingestion.

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.

Neuropeptide Y in relation to carbohydrate intake, corticosterone and dietary obesity

Neuropeptide Y (NPY) is known to stimulate eating behavior and to be related to behavioral patterns of carbohydrate ingestion. The present report investigates this relationship further to: (1) characterize the specific NPY projection activated in different dietary paradigms; (2) understand associated changes in circulating hormones that may mediate dietary effects on NPY neurons; and (3) determine whether endogenous NPY in conditions with macronutrient diets can be linked to body fat. Male albino Sprague-Dawley rats were tested in two feeding paradigms, one in which the rats were given a choice of the macronutrients, carbohydrate, fat or protein, or the other involving a single diet varying in carbohydrate of fat content. These studies consistently demonstrated a close association between the ingestion of carbohydrate and NPY levels, specifically in the arcuate nucleus (ARC) and medial portion of the paraventricular nucleus (PVN) of the hypothalamus. In addition to revealing increased NPY activity in animals that naturally select high carbohydrate when given a choice of macronutrients, a single diet with 65% carbohydrate (10% fat), compared to a control diet with 45% carbohydrate (30% fat), significantly potentiates NPY gene expression and NPY-immunoreactivity, as determined by in situ hybridization and immunohistochemistry. A further lowering of carbohydrate to 15% has little effect on NPY. Studies of medial hypothalamic fragments in vitro also reveal enhanced NPY release from hypothalamic tissue taken from rats maintained on high-carbohydrate diet. Together with NPY, circulating corticosterone (CORT) levels are also highest in a high-carbohydrate condition and positively correlated with NPY in the ARC. An association between NPY and adiposity in these dietary conditions is indicated by significantly higher levels of NPY in the medial PVN in rats with high body fat, whether consuming a high-carbohydrate of high-fat diet. This evidence, linking NPY to carbohydrate intake and circulating CORT, suggests a role for this peptide in glucose homeostasis that is normally exhibited under conditions when carbohydrate stores are low. Disturbances in this homeostatic process, associated with hyperinsulinemia and higher levels of NPY, become evident with only a moderate rise in body fat on a high-carbohydrate as well as high-fat diet.

PVN galanin increases fat storage and promotes obesity by causing muscle to utilize carbohydrate more than fat

To understand the function of the feeding-stimulatory peptide, galanin (GAL), in eating and body weight regulation, the present experiments tested the effects of both acute and chronic injections of this peptide into the paraventricular nucleus (PVN) of rats. With food absent during the test, acute injection of GAL (300 pmol/0.3 microl) significantly increased phosphofructokinase activity in muscle, suggesting enhanced capacity to metabolize carbohydrate, and reduced circulating glucose levels. It also decreased beta-hydroxyacyl-CoA dehydrogenase activity in muscle, indicating reduced fat oxidation, while increasing circulating non-esterified fatty acids (NEFA) and lipoprotein lipase activity in adipose tissue (aLPL). Chronic PVN injections of GAL (300 pmol/0.3 microl/injection) versus saline over 7-10 days significantly stimulated daily caloric intake and increased the weight of four dissected fat depots by 30-40%. These effects, accompanied by elevated levels of leptin, triglycerides, NEFA and aLPL activity, were evident only in rats on a diet with at least 35% fat. Thus, by favoring carbohydrate over fat metabolism in muscle and reversing hyperglycemia, PVN GAL may have a function in counteracting the metabolic disturbances induced by a high-fat diet. As a consequence of these actions, GAL can promote the partitioning of lipids away from oxidation in muscle towards storage in adipose tissue.

Rapid changes in hypothalamic neuropeptide Y produced by carbohydrate-rich meals that enhance corticosterone and glucose levels

Prior studies have demonstrated that chronic consumption over several weeks of a high-carbohydrate (65%) diet, compared to a moderate-carbohydrate (45%) or low-carbohydrate (15%) diet, potentiates the expression, synthesis and release of hypothalamic NPY. This effect occurs specifically in neurons of the arcuate nucleus (ARC) which project to the paraventricular nucleus (PVN). In the present experiments, tests involving acute manipulations were conducted to determine whether such diet-induced changes in NPY can occur rapidly, perhaps within 1-2 h, and whether these effects can be linked to specific changes in circulating glucoregulatory hormones or glucose itself., In adult, albino rats maintained on lab chow, the acute manipulations included the presentation of either a high-carbohydrate, moderate-carbohydrate or high-fat diet for 90 min at the onset of the natural feeding cycle. They also involved manipulations of glucose itself, either through the ingestion of a glucose (20%) solution in a drinking tube or intraperitoneal injection of a glucose solution (10%). After a high-carbohydrate meal compared to a moderate-carbohydrate or high-fat meal, NPY gene expression examined via in situ hybridization is found to be significantly enhanced in the ARC. The high-carbohydrate meal also potentiates NPY immunoreactivity in the ARC and PVN but has little effect on NPY in other hypothalamic areas examined and actually causes a reduction in the feeding-stimulatory peptide, galanin, specifically in the PVN. The meal-induced increase in NPY is associated with specific endocrine patterns, as revealed by measurements in serum collected from trunk blood or from rats implanted with a chronic jugular catheter. After a high-carbohydrate meal, levels of glucose, together with corticosterone and insulin, are significantly elevated, while non-esterified fatty acids are reduced. A possible effect of circulating glucose on hypothalamic NPY is further suggested by the finding that the consumption or a single injection of a glucose solution at the onset of the feeding cycle similarly elevates NPY mRNA and peptide immunoreactivity in the ARC and PVN. These results demonstrate that hypothalamic NPY can change rapidly in response to dietary carbohydrate. They also suggest that this effect may be related to changes in circulating CORT as well as to the availability or utilization of glucose.

Increased Caloric Intake on a Fat‐Rich Diet: Role of Ovarian Steroids and Galanin in the Medial Preoptic and Paraventricular Nuclei and Anterior Pituitary of Female Rats

Previous studies in male rats have demonstrated that the orexigenic peptide galanin (GAL), in neurones of the anterior parvocellular region of the paraventricular nucleus (aPVN) projecting to the median eminence (ME), is stimulated by consumption of a high‐fat diet and may have a role in the hyperphagia induced by fat. In addition to confirming this relationship in female rats and distinguishing the aPVN‐ME from other hypothalamic areas, the present study identified two additional extra‐hypothalamic sites where GAL is stimulated by dietary fat in females but not males. These sites were the medial preoptic nucleus (MPN), located immediately rostral to the aPVN, and the anterior pituitary (AP). The involvement of ovarian steroids, oestradiol (E2) and progesterone (PROG), in this phenomenon was suggested by an observed increase in circulating levels of these hormones and GAL in MPN and AP with fat consumption and an attenuation of this effect on GAL in ovariectomised (OVX) rats. Furthermore, in the same four areas affected by dietary fat, levels of GAL mRNA and peptide immunoreactivity were stimulated by E2 and further by PROG replacement in E2‐primed OVX rats and were higher in females compared to males. Because both GAL and PROG stimulate feeding, their increase on a fat‐rich diet may have functional consequences in females, possibly contributing to the increased caloric intake induced by dietary fat. This is supported by the findings that PROG administration in E2‐primed OVX rats reverses the inhibitory effect of E2 on total caloric intake while increasing voluntary fat ingestion, and that female rats with higher GAL exhibit increased preference for fat compared to males. Thus, ovarian steroids may function together with GAL in a neurocircuit, involving the MPN, aPVN, ME and AP, which coordinate feeding behaviour with reproductive function to promote consumption of a fat‐rich diet at times of increased energy demand.