Steve C. Fordahl

Obesity Alters Adipose Tissue Macrophage Iron Content and Tissue Iron Distribution

Adipose tissue (AT) expansion is accompanied by the infiltration and accumulation of AT macrophages (ATMs), as well as a shift in ATM polarization. Several studies have implicated recruited M1 ATMs in the metabolic consequences of obesity; however, little is known regarding the role of alternatively activated resident M2 ATMs in AT homeostasis or how their function is altered in obesity. Herein, we report the discovery of a population of alternatively activated ATMs with elevated cellular iron content and an iron-recycling gene expression profile. These iron-rich ATMs are referred to as MFehi, and the remaining ATMs are referred to as MFelo. In lean mice, ~25% of the ATMs are MFehi; this percentage decreases in obesity owing to the recruitment of MFelo macrophages. Similar to MFelo cells, MFehi ATMs undergo an inflammatory shift in obesity. In vivo, obesity reduces the iron content of MFehi ATMs and the gene expression of iron importers as well as the iron exporter, ferroportin, suggesting an impaired ability to handle iron. In vitro, exposure of primary peritoneal macrophages to saturated fatty acids also alters iron metabolism gene expression. Finally, the impaired MFehi iron handling coincides with adipocyte iron overload in obese mice. In conclusion, in obesity, iron distribution is altered both at the cellular and tissue levels, with AT playing a predominant role in this change. An increased availability of fatty acids during obesity may contribute to the observed changes in MFehi ATM phenotype and their reduced capacity to handle iron.

Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain

Unlike other essential trace elements (e.g., zinc and iron) it is the toxicity of manganese (Mn) that is more common in human populations than its deficiency. Data suggest alterations in dopamine biology may drive the effects associated with Mn neurotoxicity, though recently gamma-aminobutyric acid (GABA) has been implicated. In addition, iron deficiency (ID), a common nutritional problem, may cause disturbances in neurochemistry by facilitating accumulation of Mn in the brain. Previous data from our lab have shown decreased brain tissue levels of GABA as well as decreased (3)H-GABA uptake in synaptosomes as a result of Mn exposure and ID. These results indicate a possible increase in the concentration of extracellular GABA due to alterations in expression of GABA transport and receptor proteins. In this study weanling-male Sprague-Dawley rats were randomly placed into one of four dietary treatment groups: control (CN; 35mg Fe/kg diet), iron-deficient (ID; 6mg Fe/kg diet), CN with Mn supplementation (via the drinking water; 1g Mn/l) (CNMn), and ID with Mn supplementation (IDMn). Using in vivo microdialysis, an increase in extracellular GABA concentrations in the striatum was observed in response to Mn exposure and ID although correlational analysis reveals that extracellular GABA is related more to extracellular iron levels and not Mn. A diverse effect of Mn exposure and ID was observed in the regions examined via Western blot and RT-PCR analysis, with effects on mRNA and protein expression of GAT-1, GABA(A), and GABA(B) differing between and within the regions examined. For example, Mn exposure reduced GAT-1 protein expression by approximately 50% in the substantia nigra, while increasing mRNA expression approximately four-fold, while in the caudate putamen mRNA expression was decreased with no effect on protein expression. These data suggest that Mn exposure results in an increase in extracellular GABA concentrations via altered expression of transport and receptor proteins, which may be the basis of the neurological characteristics of manganism.

Waterborne Manganese Exposure Alters Plasma, Brain, and Liver Metabolites Accompanied by Changes in Stereotypic Behaviors

Overexposure to waterborne manganese (Mn) is linked with cognitive impairment in children and neurochemical abnormalities in other experimental models. In order to characterize the threshold between Mn-exposure and altered neurochemistry, it is important to identify biomarkers that positively correspond with brain Mn-accumulation. The objective of this study was to identify Mn-induced alterations in plasma, liver, and brain metabolites using liquid/gas chromatography-time of flight-mass spectrometry metabolomic analyses; and to monitor corresponding Mn-induced behavior changes. Weanling Sprague-Dawley rats had access to deionized drinking water either Mn-free or containing 1g Mn/L for 6 weeks. Behaviors were monitored during the sixth week for a continuous 24h period while in a home cage environment using video surveillance. Mn-exposure significantly increased liver, plasma, and brain Mn concentrations compared to control, specifically targeting the globus pallidus (GP). Mn significantly altered 98 metabolites in the brain, liver, and plasma; notably shifting cholesterol and fatty acid metabolism in the brain (increased oleic and palmitic acid; 12.57 and 15.48 fold change (FC), respectively), and liver (increased oleic acid, 14.51 FC; decreased hydroxybutyric acid, -14.29 FC). Additionally, Mn-altered plasma metabolites homogentisic acid, chenodeoxycholic acid, and aspartic acid correlated significantly with GP and striatal Mn. Total distance traveled was significantly increased and positively correlated with Mn-exposure, while nocturnal stereotypic and exploratory behaviors were reduced with Mn-exposure and performed largely during the light cycle compared to unexposed rats. These data provide putative biomarkers for Mn-neurotoxicity and suggest that Mn disrupts the circadian cycle in rats.

Manganese exposure inhibits the clearance of extracellular GABA and influences taurine homeostasis in the striatum of developing rats

Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mn-induced increase in extracellular GABA (GABA(EC)) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn-exposure. NA led to a 2-fold increase in GABA(EC) of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn-exposure, and Mn significantly increased (3)H-Taurine uptake after 3-min exposure in primary rat astrocytes. These data suggest that Mn increases GABA(EC) by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain.

Cocaine Self-Administration Produces Long-Lasting Alterations in Dopamine Transporter Responses to Cocaine

Cocaine addiction is a debilitating neuropsychiatric disorder characterized by uncontrolled cocaine intake, which is thought to be driven, at least in part, by cocaine-induced deficits in dopamine system function. A decreased ability of cocaine to elevate dopamine levels has been repeatedly observed as a consequence of cocaine use in humans, and preclinical work has highlighted tolerance to cocaines effects as a primary determinant in the development of aberrant cocaine taking behaviors. Here we determined that cocaine self-administration in rats produced tolerance to the dopamine transporter-inhibiting effects of cocaine in the nucleus accumbens core, which was normalized following a 14 or 60 d abstinence period; however, although these rats appeared to be similar to controls, a single self-administered infusion of cocaine at the end of abstinence, even after 60 d, fully reinstated tolerance to cocaines effects. A single cocaine infusion in a naive rat had no effect on cocaine potency, demonstrating that cocaine self-administration leaves the dopamine transporter in a “primed” state, which allows for cocaine-induced plasticity to be reinstated by a subthreshold cocaine exposure. Further, reinstatement of cocaine tolerance was accompanied by decreased cocaine-induced locomotion and escalated cocaine intake despite extended abstinence from cocaine. These data demonstrate that cocaine leaves a long-lasting imprint on the dopamine system that is activated by re-exposure to cocaine. Further, these results provide a potential mechanism for severe cocaine binge episodes, which occur even after sustained abstinence from cocaine, and suggest that treatments aimed at transporter sites may be efficacious in promoting binge termination following relapse. SIGNIFICANCE STATEMENT Tolerance is a DSM-V criterion for substance abuse disorders. Abusers consistently show reduced subjective effects of cocaine concomitant with reduced effects of cocaine at its main site of action, the dopamine transporter (DAT). Preclinical literature has shown that reduced cocaine potency at the DAT increases cocaine taking, highlighting the key role of tolerance in addiction. Addiction is characterized by cycles of abstinence, often for many months, followed by relapse, making it important to determine possible interactions between abstinence and subsequent drug re-exposure. Using a rodent model of cocaine abuse, we found long-lasting, possibly permanent, cocaine-induced alterations to the DAT, whereby cocaine tolerance is reinstated by minimal drug exposure, even after recovery of DAT function over prolonged abstinence periods.

High-Fat-Diet-Induced Deficits in Dopamine Terminal Function Are Reversed by Restoring Insulin Signaling

Systemically released insulin crosses the blood-brain barrier and binds to insulin receptors on several neural cell types, including dopaminergic neurons. Insulin has been shown to decrease dopamine neuron firing in the ventral tegmental area (VTA), but potentiate release and reuptake at dopamine terminals in the nucleus accumbens (NAc). Here we show that prolonged consumption of a high fat diet blocks insulins effects in the NAc, but insulins effects are restored by inhibiting protein tyrosine phosphatase 1B, which supports insulin receptor signaling. Mice fed a high fat diet (60% kcals from fat) displayed significantly higher fasting blood glucose 160 mg/dL, compared to 101 mg/dL for control-diet-fed mice, and high-fat-diet-fed mice showed reduced blood glucose clearance after an intraperitoneal glucose tolerance test. Using fast scan cyclic voltammetry to measure electrically evoked dopamine in brain slices containing the NAc core, high-fat-diet-fed mice exhibited slower dopamine reuptake compared to control-diet-fed mice (2.2 ± 0.1 and 2.67 ± 0.15 μM/s, respectively). Moreover, glucose clearance rate was negatively correlated with Vmax. Insulin (10 nM to 1 μM) dose dependently increased reuptake rates in control-diet-fed mice compared with in the high-fat-diet group; however, the small molecule insulin receptor sensitizing agent, TCS 401 (300 nM), restored reuptake in high-fat-diet-fed mice to control-diet levels, and a small molecule inhibitor of the insulin receptor, BMS 536924 (300 nM), attenuated reuptake, similar to high-fat-diet-fed mice. These data show that a high-fat diet impairs dopamine reuptake by attenuating insulin signaling at dopamine terminals.

Manganese accumulation in membrane fractions of primary astrocytes is associated with decreased γ-aminobutyric acid (GABA) uptake, and is exacerbated by oleic acid and palmitate

Manganese (Mn) exposure interferes with GABA uptake; however, the effects of Mn on GABA transport proteins (GATs) have not been identified. We sought to characterize how Mn impairs GAT function in primary rat astrocytes. Astrocytes exposed to Mn (500 μM) had significantly reduced (3)H-GABA uptake despite no change in membrane or cytosolic GAT3 protein levels. Co-treatment with 100 μM oleic or palmitic acids (both known to be elevated in Mn neurotoxicity), exacerbated the Mn-induced decline in (3)H-GABA uptake. Mn accumulation in the membrane fraction of astrocytes was enhanced with fatty acid administration, and was negatively correlated with (3)H-GABA uptake. Furthermore, control cells exposed to Mn only during the experimental uptake had significantly reduced (3)H-GABA uptake, and the addition of GABA (50 μM) blunted cytosolic Mn accumulation. These data indicate that reduced GAT function in astrocytes is influenced by Mn and fatty acids accumulating at or interacting with the plasma membrane.

Amphetamine Reverses Escalated Cocaine Intake via Restoration of Dopamine Transporter Conformation

Cocaine abuse disrupts dopamine system function, and reduces cocaine inhibition of the dopamine transporter (DAT), which results in tolerance. Although tolerance is a hallmark of cocaine addiction and a DSM-V criterion for substance abuse disorders, the molecular adaptations producing tolerance are unknown, and testing the impact of DAT changes on drug taking behaviors has proven difficult. In regard to treatment, amphetamine has shown efficacy in reducing cocaine intake; however, the mechanisms underlying these effects have not been explored. The goals of this study were twofold; we sought to (1) identify the molecular mechanisms by which cocaine exposure produces tolerance and (2) determine whether amphetamine-induced reductions in cocaine intake are connected to these mechanisms. Using cocaine self-administration and fast-scan cyclic voltammetry in male rats, we show that low-dose, continuous amphetamine treatment, during self-administration or abstinence, completely reversed cocaine tolerance. Amphetamine treatment also reversed escalated cocaine intake and decreased motivation to obtain cocaine as measured in a behavioral economics task, thereby linking tolerance to multiple facets of cocaine use. Finally, using fluorescence resonance energy transfer imaging, we found that cocaine tolerance is associated with the formation of DAT-DAT complexes, and that amphetamine disperses these complexes. In addition to extending our basic understanding of DATs and their role in cocaine reinforcement, we serendipitously identified a novel therapeutic target: DAT oligomer complexes. We show that dispersion of oligomers is concomitant with reduced cocaine intake, and propose that pharmacotherapeutics aimed at these complexes may have potential for cocaine addiction treatment. SIGNIFICANCE STATEMENT Tolerance to cocaines subjective effects is a cardinal symptom of cocaine addiction and a DSM-V criterion for substance abuse disorders. However, elucidating the molecular adaptions that produce tolerance and determining its behavioral impact have proven difficult. Using cocaine self-administration in rats, we link tolerance to cocaine effects at the dopamine transporter (DAT) with aberrant cocaine-taking behaviors. Further, tolerance was associated with multi-DAT complexes, which formed after cocaine exposure. Treatment with amphetamine deconstructed DAT complexes, reversed tolerance, and decreased cocaine seeking. These data describe the behavioral consequence of cocaine tolerance, provide a putative mechanism for its development, and suggest that compounds that disperse DAT complexes may be efficacious treatments for cocaine addiction.