Dianne P. Figlewicz

Expression of receptors for insulin and leptin in the ventral tegmental area/substantia nigra (VTA/SN) of the rat

Recent studies have demonstrated that the metabolic hormones insulin and leptin can modulate behavioral performance in reward-related paradigms. However, specific anatomical substrate(s) within the CNS for these effects remain to be identified. We hypothesize that midbrain dopamine neurons, which have been implicated to be critical in the mediation of motivational and reward aspects of stimuli, contribute to these behavioral effects of insulin and leptin. As one approach to evaluate this hypothesis, we used double-labeling fluorescence immunohistochemistry to determine whether the midbrain dopamine neurons express insulin receptors or leptin receptors. Extensive co-expression of tyrosine hydroxylase (a marker for dopamine neurons) with both the insulin receptor and the leptin receptor was observed in the ventral tegmentum and substantia nigra. These findings suggest that midbrain dopamine neurons are direct targets of insulin and leptin, and that they participate in mediating the effects of these hormones on reward-seeking behavior.

Insulin and insulin-like growth factors in the CNS

Abstract Insulin has long been recognized as a major endocrine regulator of the uptake, cellular transport, and intermediary metabolism of small nutrient molecules such as amino acids, fatty acids, and glucose 1 . Adipose tissue and skeletal muscle are the classical major target of insulin action; the CNS, in contrast, has traditionally been considered to be largely insulin-insensitive. However, clues that insulin may have physiological functions in the CNS began to emerge in the 1960s, and an impressive body of literature has since accumulated about insulin in the CNS. Although insulin now regularly appears in litanies cataloging CNS peptides, its status as a CNS regulatory peptide remains obscure and elusive. The situation has become more clouded with the recent discovery that insulin-like growth-factors (IGFs) and their receptors are also present in the CNS. Since IGFs and insulin share similar primary amino acid structure, receptor binding, and biological activity 2 , any discussion about insulin as a CNS regulatory peptide must also consider the IGFs. In the present article, we summarize the status of insulin and IGFs as regulatory peptides in the CNS. Our choice of literature has been selective, with a focus on recent reports, controversial issues, and unsolved problems. Readers are referred to previous reviews for much of the earlier literature 1–5 .

Intraventricular insulin reduces food intake and body weight of lean but not obese zucker rats

Porcine insulin (2 mU/rat/day) and its saline vehicle were infused into the third cerebral ventricle of female lean or obese Zucker rats using 14-day osmotic minipumps. Lean rats receiving saline (N = 6) gained 14 +/- 3 g over the 14 days, whereas lean rats receiving insulin (N = 7) lost 12 +/- 4 g over the same interval (p less than 0.01). The average total food intake of the insulin-infused group was decreased by 14% (p less than 0.05) as compared with that of the saline-infused group. The decreased caloric consumption was adequate to account for the body weight loss. Insulin infusion had no effect on food intake or body weight of the obese rats relative to their saline-infused controls (change in body weight: saline (N = 5), -14 +/- 23 g; insulin (N = 7), +3 +/- 14 g). These results suggest that genetically obese Zucker rats have reduced sensitivity to insulin in the central nervous system. We propose that this phenomenon may participate in the development and maintenance of hyperphagia and obesity in these animals.

Localization of binding sites for insulin-like growth factor-I (IGF-I) in the rat brain by quantitative autoradiography

In vitro quantitative autoradiography was used to localize IGF-I binding sites in rat brain. Slide-mounted sections of frozen rat brain were incubated in 0.01 nM 125I[Thr59]IGF-I, alone or mixed with 10 nM unlabeled [Thr59]IGF-I or insulin, for 22 h at 4 degrees C and apposed to LKB Ultrofilm. Measurement of labeled [Thr59]IGF-I binding by computer digital image analysis of the autoradiographic images indicated that high affinity IGF-I binding sites are widely distributed at discrete anatomical regions of the brain microarchitecture. The highest concentration of specific binding sites was in the choroid plexus of the lateral and third ventricles. Unlabeled porcine insulin was less potent than unlabeled IGF-I in competing for binding sites on brain slices. Regions of the olfactory, visual, and auditory, as well as visceral and somatic sensory systems were labeled, in particular the glomerular layer of the olfactory bulb, the anterior olfactory nucleus, accessory olfactory bulb, primary olfactory cortex, lateral-dorsal geniculate, superior colliculus, medial geniculate, and the spinal trigeminal nucleus. High concentrations of IGF-I-specific binding sites were present throughout the thalamus and the hippocampus, (dentate gyrus, Ca1, Ca2, Ca3). The hypothalamus had moderate binding in the paraventricular, supraoptic, and suprachiasmatic nucleus. Highest binding in the hypothalamus was in the median eminence. The arcuate nucleus showed very low specific binding, approaching the levels found in optic chiasm and white matter regions. Layers II and VI of the cerebral cortex also had moderate IGF-I binding. The results suggest that the development and functions of brain sensory and neuroendocrine pathways may be regulated by IGF-I.

Obesity, diabetes and the central nervous system

Nearly 25 years ago, one of us wrote a collaborative review entitled “Obesity and Diabetes, the Odd Couple” [1]. At that time we pointed out that obesity was a risk factor for diabetes mellitus, despite the fact that most obese people do not have and never will develop diabetes. We also pointed out that obesity is associated with insulin resistance and hyperinsulinaemia, whereas in the non-obese, diabetes is characterized by hypoinsulinaemia and hyperglycaemia. We asked, how is it that such different syndromes interact so closely? We concluded that while these two syndromes are likely to have independent risk factors and mechanisms, their known interactions made it likely that shared metabolic defects are involved in their pathogenesis. We concluded that further studies into the pathophysiology of these syndromes were needed to explain this apparently paradoxical asso

Central insulin enhances sensitivity to cholecystokinin

Insulin acts in the brain to reduce food intake and body weight. Cholecystokinin (CCK) reduces meal size when administered peripherally. The purpose of these experiments was to examine their interaction. In Experiment 1, Long-Evans rats were infused with vehicle or insulin at doses from 0.5 to 2.0 mU/day into the third cerebral ventricles. Doses of 1.0 mU/day and higher caused reduced body weight. A dose of 0.5 mU/day was therefore taken to be subthreshold. In Experiment 2, rats receiving 0.5 mU/day of insulin intracerebroventricularly had greater suppression of 30-min meal size in response to intraperitoneal CCK-8 at doses from 0.25 to 8 mg/kg than did rats receiving intracerebroventricular saline. By itself, the insulin had no effect on body weight or meal size. However, a change of sensitivity to CCK by control rats over the course of the experiment clouded the interpretation. A third experiment was therefore conducted in which rats received an acute intracerebroventricular injection of insulin (0.1 mU) or saline 1 h prior to a 30-min meal, and IP CCK-8 (4 mg/kg) or saline immediately prior to the meal. As in Experiment 2, insulin, itself, had no effect on meal size but enhanced the anorexic effect of CCK. These results are consistent with the hypothesis that central insulin acts by altering sensitivity to satiety agents.

Insulin, leptin, and food reward: update 2008

The hormones insulin and leptin have been demonstrated to act in the central nervous system (CNS) as regulators of energy homeostasis at medial hypothalamic sites. In a previous review, we described new research demonstrating that, in addition to these direct homeostatic actions at the hypothalamus, CNS circuitry that subserves reward and motivation is also a direct and an indirect target for insulin and leptin action. Specifically, insulin and leptin can decrease food reward behaviors and modulate the function of neurotransmitter systems and neural circuitry that mediate food reward, i.e., midbrain dopamine and opioidergic pathways. Here we summarize new behavioral, systems, and cellular evidence in support of this hypothesis and in the context of research into the homeostatic roles of both hormones in the CNS. We discuss some current issues in the field that should provide additional insight into this hypothetical model. The understanding of neuroendocrine modulation of food reward, as well as food reward modulation by diet and obesity, may point to new directions for therapeutic approaches to overeating or eating disorders.

Localization of 125I-insulin binding sites in the rat hypothalamus by quantitative autoradiography.

In vitro autoradiography and computer video densitometry were used to localize and quantify binding of 125I-insulin in the hypothalamus of the rat brain. Highest specific binding was found in the arcuate, dorsomedial, suprachiasmatic, paraventricular and periventricular regions. Significantly lower binding was present in the ventromedial nucleus and median eminence. The results are consistent with the hypothesis that insulin modulates the neural regulation of feeding by acting at sites in the hypothalamus.

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.

Vitamin D deficiency in obese rats exacerbates nonalcoholic fatty liver disease and increases hepatic resistin and toll‐like receptor activation

Childhood obesity is associated with type 2 diabetes mellitus and nonalcoholic fatty liver disease (NAFLD). Recent studies have found associations between vitamin D deficiency (VDD), insulin resistance (IR), and NAFLD among overweight children. To further explore mechanisms mediating these effects, we fed young (age 25 days) Sprague‐Dawley rats with a low‐fat diet (LFD) alone or with vitamin D depletion (LFD+VDD). A second group of rats was exposed to a Westernized diet (WD: high‐fat/high‐fructose corn syrup) that is more typically consumed by overweight children, and was either replete (WD) or deficient in vitamin D (WD+VDD). Liver histology was assessed using the nonalcoholic steatohepatitis (NASH) Clinical Research Network (CRN) scoring system and expression of genes involved in inflammatory pathways were measured in liver and visceral adipose tissue after 10 weeks. In VDD groups, 25‐OH‐vitamin D levels were reduced to 29% (95% confidence interval [CI]: 23%‐36%) compared to controls. WD+VDD animals exhibited significantly greater hepatic steatosis compared to LFD groups. Lobular inflammation as well as NAFLD Activity Score (NAS) were higher in WD+VDD versus the WD group (NAS: WD+VDD 3.2 ± 0.47 versus WD 1.50 ± 0.48, P < 0.05). Hepatic messenger RNA (mRNA) levels of Toll‐like receptors (TLR)2, TLR4, and TLR9, as well as resistin, interleukins (IL)‐1β, IL‐4, and IL‐6 and oxidative stress marker heme oxygenase (HO)‐1, were higher in WD+VDD versus WD animals (P < 0.05). Logistic regression analyses showed significant associations between NAS score and liver mRNA levels of TLRs 2, 4, and 9, endotoxin receptor CD14, as well as peroxisome proliferator activated receptor (PPAR)γ, and HO‐1. Conclusion: VDD exacerbates NAFLD through TLR‐activation, possibly by way of endotoxin exposure in a WD rat model. In addition it causes IR, higher hepatic resistin gene expression, and up‐regulation of hepatic inflammatory and oxidative stress genes. (HEPATOLOGY 2012)