Alfred J. Sipols

Effect of Intracerebroventricular Insulin Infusion on Diabetic Hyperphagia and Hypothalamic Neuropeptide Gene Expression

To test the hypothesis that diabetic hyperphagia results from insulin deficiency in the brain, diabetic rats (streptozotocin-induced) were given an intracerebroventricular (ICV) infusion of saline or insulin (at a dose that did not affect plasma glucose levels) for 6 days. Food and water intake were significantly increased in diabetic rats, but only food intake was affected by ICV insulin. Diabetic hyperphagia was reduced 58% by ICV insulin compared with ICV saline (P < 0.05) and was accompanied by a 69% increase in diabetes-induced weight loss (P < 0.05). To evaluate whether central nervous system (CNS) insulin deficiency affects expression of neuropeptides involved in food intake, in situ hybridization was done for neuropeptide Y (NPY), which stimulates feeding, in the hypothalamic arcuate nucleus and for cholecystokinin (CCK) and corticotropin-releasing hormone (CRH), which inhibit feeding, in the hypothalamic paraventricular nucleus. In diabetic rats, NPY mRNA hybridization increased 280% (P < 0.05), an effect reduced 40% by ICV insulin (P < 0.05). CCK mRNA hybridization increased 50% in diabetic rats (P < 0.05), a response reduced slightly by ICV insulin (P < 0.05), whereas CRH mRNA hybridization decreased 33% in diabetic rats (P < 0.05) and was unchanged by ICV insulin. The results demonstrate that CNS infusion of insulin to diabetic rats reduces both hyperphagia and overexpression of hypothalamic NPY mRNA. This observation supports the hypothesis that a deficiency of insulin in the brain is an important cause of diabetic hyperphagia and that increased hypothalamic NPY gene expression contributes to this phenomenon.

Intraventricular insulin and leptin reverse place preference conditioned with high-fat diet in rats

The authors hypothesized that insulin and leptin, hormones that convey metabolic and energy balance status to the central nervous system (CNS), decrease the reward value of food, as assessed by conditioned place preference (CPP). CPP to high-fat diet was blocked in ad-lib fed rats given intraventricular insulin or leptin throughout training and test or acutely before the test. Insulin or leptin given only during the training period did not block CPP. Thus, elevated insulin and leptin do not prevent learning a foods reward value, but instead block its retrieval. Food-restricted rats receiving cerebrospinal fluid, insulin, or leptin had comparable CPPs. Results indicate that the CNS roles of insulin and leptin may include processes involving memory and reward.

Modulation of food reward by adiposity signals

Extensive historical evidence from the drug abuse literature has provided support for the concept that there is functional communication between central nervous system (CNS) circuitries which subserve reward/motivation, and the regulation of energy homeostasis. This concept is substantiated by recent studies that map anatomical pathways, or which demonstrate that hormones and neurotransmitters associated with energy homeostasis regulation can directly modulate reward and motivation behaviors. Studies from our laboratory have focused specifically on the candidate adiposity hormones, insulin and leptin, and show that these hormones can decrease performance in behavioral paradigms that assess the rewarding or motivating properties of food. Additionally we and others have provided evidence that the ventral tegmental area may be one direct target for these effects, and we are currently exploring other potential anatomical targets. Finally, we are beginning to explore the interaction between adiposity signals, chronic maintenance diet of rats, and different types of food rewards to more closely simulate the current food environments of Westernized societies including the U.S. We propose that future studies of food reward should include a more complex environment in the experimental design that takes into account abundance and variety of rewarding foods, psychological stressors, and choices of reward modalities.

Differential effect of fasting on hypothalamic expression of genes encoding neuropeptide Y, galanin, and glutamic acid decarboxylase

The effect of caloric deprivation to stimulate hypothalamic neuropeptide Y (NPY) gene expression is hypothesized to represent a physiologically important adaptation in body weight homeostasis. To evaluate the specificity of this response, we used in situ hybridization histochemistry to measure hypothalamic expression of mRNA encoding NPY, galanin, and the two isoforms of glutamic acid decarboxylase (GAD67 and GAD65) in male Wistar rats either fed ad lib or deprived of food for 24 or 48 h. As expected, food deprivation for 24 and 48 h increased preproNPY mRNA levels in the arcuate nucleus by 43 +/- 13% (p = NS) and 127 +/- 29% (p < 0.05 vs. both fed and 24-h fasted groups) when compared to ad lib-fed controls, and hypothalamic preproNPY mRNA levels were significantly correlated to the percent change in body weight over the three groups of rats (r = -0.72; p < 0.05). In contrast, no significant effects of either 24 or 48 h of fasting were observed on hypothalamic levels of preprogalanin, GAD67, or GAD65 mRNA, and no relationship between percent change in body weight and expression of any of these mRNA species could be demonstrated. In conclusion, fasting increases preproNPY mRNA levels in the arcuate nucleus but does not alter expression of other hypothalamic mRNA species pertinent to feeding behavior. This supports the hypothesis that stimulation of NPY gene expression represents an important component of the hypothalamic response to caloric deprivation.

Insulin acts at different CNS sites to decrease acute sucrose intake and sucrose self-administration in rats.

Findings from our laboratory and others have demonstrated that the hormone insulin has chronic effects within the CNS to regulate energy homeostasis and to decrease brain reward function. In this study, we compared the acute action of insulin to decrease intake of a palatable food in two different behavioral tasks-progressive ratios sucrose self-administration and micro opioid-stimulated sucrose feeding-when administered into several insulin-receptive sites of the CNS. We tested insulin efficacy within the medial hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei, the nucleus accumbens, and the ventral tegmental area. Administration of insulin at a dose that has no chronic effect on body weight (5 mU) into the ARC significantly suppressed sucrose self-administration (75+/-5% of paired control). However, although the mu opioid DAMGO, [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin acetate salt, stimulated sucrose intake at all four CNS sites, the ventral tegmental area was the only sensitive site for a direct effect of insulin to antagonize acute (60 min) micro opioid-stimulated sucrose feeding: sucrose intake was 53+/-8% of DAMGO-induced feeding, when insulin was coadministered with DAMGO. These findings demonstrate that free feeding of sucrose, and motivated work for sucrose, can be modulated within unique sites of the CNS reward circuitry. Further, they support the interpretation that adiposity signals, such as insulin, can decrease different aspects of ingestion of a palatable food, such as sucrose, in an anatomically specific manner.

Intraventricular Insulin Enhances the Meal-Suppressive Efficacy of Intraventricular Cholecystokinin Octapeptide in the Baboon

Chronic intraventricular (IVT) insulin infusion suppresses food intake and body weight in the baboon. It has been hypothesized that one mechanism of this action may be enhancement of the effectiveness of satiety factors that regulate meal size. This hypothesis was supported by prior demonstration of a shift in the meal-suppressive effectiveness of cholecystokinin octapeptide (CCK-8) which was given intravenously. The authors tested the effectiveness of a near threshold dose of CCK-8 (25 ng/kg) given via the lateral ventricles (IVT) prior to a 30-min meal, while baboons were chronically infused with cerebrospinal fluid or insulin (100 microU/day) via the lateral ventricles. IVT CCK-8 infusion resulted in meal size changes of -44 +/- 7% and -75 +/- 9% in the absence and presence of insulin, respectively; this was observed in each of the three animals studied. These results provide further support for the hypothesis that IVT insulin can interact with other, meal-regulatory, peptides.

Immunocytochemical detection of insulin receptor substrate-1 (IRS-1) in rat brain: colocalization with phosphotyrosine

In peripheral insulin-sensitive tissues, insulin receptor substrate (IRS-1) undergoes tyrosine phosphorylation immediately after cells are stimulated by insulin or insulin-like growth factor-1 (IGF-1), and may function as a molecular link between insulin/IGF-1 receptor tyrosine kinases and enzymes regulating cell growth and metabolism. A fundamental question pertaining to insulin/IGF-1 action in the brain is whether IRS-1 is expressed by neurons. In this study, the distribution of cells containing immunoreactivity to IRS-1 in the brain was determined by immunocytochemistry with polyclonal IRS-1 antiserum, and compared to the localization of immunostaining for phosphotyrosine using polyclonal phosphotyrosine antiserum. The immunostaining results with ABC-peroxidase method and cryostat sections showed the presence of IRS-1 immunoreactivity in many neuron cell bodies throughout the rat forebrain, particularly in the habenula, cerebral cortex and piriform cortex. In the hypothalamus, IRS-1 immunostaining was present in neurons of the paraventricular nucleus, supraoptic nucleus, and arcuate nucleus. The choroid plexus stained intensely for IRS-1. The populations of cells that stained for IRS-1 also showed strong immunostaining for phosphotyrosine. Studies at the cellular level are needed to verify coexpression of IRS-1 and receptors for insulin or IGF-1 by the same neurons, as well as in cells of the choroid plexus. The present results are the first demonstration of IRS-1 expression by neurons in adult mammalian brain. These findings are consistent with the hypothesis that insulin and IGF-1 actions in the brain involve signal transduction mechanisms common to those found in peripheral tissues.

Effect of diet-induced obesity and experimental hyperinsulinemia on insulin uptake into CSF of the rat

We examined the hypothesis that the uptake of plasma insulin into cerebrospinal fluid (CSF) is saturable in two rat models. Dietary obese and control female Osborne Mendel rats received 24-h infusions of vehicle or insulin. CSF insulin levels in cafeteria- and chow-fed rats were comparable at all levels of plasma insulin (4.5 +/- 2.8, 7.6 +/- 2.4, and 23.9 +/- 6.4 microU/ml in cafeteria diet vs. 4.5 +/- 0.9, 6.8 +/- 1.1, and 17.0 +/- 4.0 microU/ml in chow rats). CSF insulin uptake as a percentage of plasma insulin decreased with increased plasma insulin in both groups. A similar relationship was observed in Wistar rats receiving 6-day infusions of vehicle or insulin (plasma insulin = 55 +/- 12 vs. 365 +/- 98 microU/ml; CSF/plasma insulin ratio = 0.022 +/- .007 vs. 0.013 +/- .006, respectively). Hyperinsulinemic Wistar rats did not demonstrate decreased brain capillary insulin binding vs. vehicle-infused controls. The results suggest that a saturable transport process contributes insulin transport into CSF in normal rats and that this process is not altered by moderate diet-induced obesity or hyperinsulinemia per se.

NPY and galanin in a hibernator : hypothalamic gene expression and effects on feeding

Neuropeptides such as neuropeptide Y (NPY) and galanin may play a role in regulating the pronounced seasonal changes in food intake shown by golden-mantled ground squirrels (Spermophilus saturatus). We used in situ hybridization histochemistry to localize the expression of NPY and galanin mRNA in the hypothalamus of normally feeding animals. NPY mRNA was abundantly expressed in the arcuate nucleus, while galanin mRNA was concentrated in both the arcuate nucleus and the dorsomedial nuclei. When NPY (0.1, 0.5, 2, and 8 micrograms) or galanin (0.1, 0.5, 2, and 8 micrograms) were injected into the third cerebral ventricle, food intake was significantly and dose-dependently increased over the subsequent 30 min. NPY stimulated significant increases in food intake for up to 2 h whereas galanins effect did not extend beyond 30 min. Our results suggest that hibernating and nonhibernating rodents share common neural substrates for the regulation of food intake. Seasonal modulation of these neural pathways may contribute to annual cycles of food intake in hibernating mammals.

Neuropeptide Y paradoxically increases food intake yet causes conditioned flavor aversions

Neuropeptides have been implicated in the short-term regulation of food intake and the long-term control of body weight. Previous studies have shown that central administration of neuropeptide Y (NPY), the most abundant of these peptides in the brain, produces robust increases of food intake. We now report that NPY, at doses that stimulate food intake when administered intraventricularly, also causes the formation of robust conditioned flavor aversions when given via the same cannula and at the same dose. This apparently paradoxical effect may be indicative of different populations of central NPY receptors having dissimilar effects on ingestive behaviors. The results also suggest that the use of conditioned aversions to investigate drug-induced malaise may not be appropriate when applied to ingestive behaviors.