Loss of the Circadian Peak of Dopamine Release at the Biological Clock is Associated with the Onset of Seasonal Insulin Resistance / Glucose Intolerance Animals on the cusp of Summer-Fall transition recovery 4 days Guide cannula inserted at the SCN area HPLC analysis 24 hour microdialysis of free living animals Daily Profiles of Dopamine Extracellular Metabolite (HVA) and Serotonin Extracellular Metabolite (5-HIAA) in Hypothalamic Clock (SCN) of Freely Behaving Seasonally Glucose Tolerant (●) and Seasonally Glucose Intolerant (o) Hamsters Figure 1 Luo S, Luo J, Cincotta AH. Suprachiasmatic nuclei monoamine metabolism of glucose tolerant versus intolerant hamsters. Neuroreport. 1999;10(10):2073-7.

Destruction of Dopaminergic Projections to SCN of Insulin Sensitive Animals Induces Insulin Resistance Summer animals in the summer condition Glucose Tolerance Test recovery 4 days SCN dopaminergic neuron lesion via neurotoxin maintain on regular diet for 16 days Glucose Tolerance Test Loss of this circadian peak of dopaminergic activity at the SCN induces severe insulin resistance Figure 2 Luo S, Luo J, Meier AH, Cincotta AH. Dopaminergic neurotoxin administration to the area of the suprachiasmatic nuclei induces insulin resistance. Neuroreport. 1997;8(16):3495-9.

○ glucose-intolerant, freely moving ● glucose tolerant, freely moving Norepinephrine (MHPG) and Serotonin (5- HIAA) VMH Extracellular Levels are Elevated in Seasonally Glucose-Intolerant Hamsters Animals in the Fall-Winter condition Cannula placement at VMH Recovery for 4 days 24 hour microdialysis of free living animals HPLC analysis ○ glucose-intolerant, freely moving ● glucose tolerant, freely moving Time of Day (hours) Increased norepinephrine at the VMH elicits the counter-regulatory response to high glycemia Two-way ANOVA with repeated measures indicates significant difference of 5-HIAA and MHPG between the two groups (p < 0.05). Beverly JL, de Vries MG, Beverly MF, Arseneau LM. Norepinephrine mediates glucoprivic-induced increase in GABA in the ventromedial hypothalamus of rats. Am J Physiol Regul Integr Comp Physiol. 2000;279(3):R990-6. Borg WP, Sherwin RS, During MJ, Borg MA, Shulman GI. Local ventromedial hypothalamus glucopenia triggers counterregulatory hormone release. Diabetes 1995;44:180–184. Borg MA, Sherwin RS, Borg WP, Tamborlane WV, Shulman GI: Local ventromedial hypothalamus glucose perfusion blocks counterregulation during systemic hypoglycemia in awake rats. J Clin Invest 1997;99:361–365. Steffens AB, Damsma G, van der Gugten J, Luiten PG. Circulating free fatty acids, insulin, and glucose during chemical stimulation of hypothalamus in rats. Am J Physiol. 1984;247(6 Pt 1):E765-71. Steffens AB, Flik G, Kuipers F, Lotter EC, Luiten PG. Hypothalamically-induced insulin release and its potentiation during oral and intravenous glucose loads. Brain Res. 1984;301(2):351-61. Luo S, Meier AH, Cincotta AH. Bromocriptine reduces obesity, glucose intolerance and extracellular monoamine metabolite levels in the ventromedial hypothalamus of Syrian hamsters. Neuroendocrinology. 1998 Jul;68(1):1-10. Figure 3

Chronic Ventromedial Hypothalamic Infusion of Norepinephrine and Serotonin in Summer Insulin Sensitive Animals Promotes Insulin Resistance and Glucose Intolerance Animals in Summer condition surgery to insert VMH cannula and initiate Serotonin and Norepinephrine infusion Infuse NE and S for 5 weeks Glucose Tolerance Test Figure 4 Luo S, Luo J, Cincotta AH. Chronic ventromedial hypothalamic infusion of norepinephrine and serotonin promotes insulin resistance and glucose intolerance. Neuroendocrinology. 1999;70(6):460-5.

Chronic Infusion of Norepinephrine into the VMH of Normal Young Insulin Sensitive SD rats Induces the Obese Glucose-Intolerant Hypertensive State Young insulin sensitive female Sprague-Dawley rats at 10 weeks of age Surgery to insert VMH cannula and initiation of unilateral Norepinephrine infusion Infuse for 3-4 weeks Measure metabolic parameters throughout the infusion period Plasma Glucose Plasma Norepinephrine P<0.01 Plasma Insulin Plasma Glucagon Plasma Epinephrine P<0.05 Plasma Leptin Increased VMH NE tone is not merely an association of, but rather a contributing factor to, the metabolic syndrome *P<0.05 and **P<0.01 Cincotta AH, Luo S, Zhang Y, Liang Y, Bina KG, Jetton TL, Scislowski PW. Chronic infusion of norepinephrine into the VMH of normal rats induces the obese glucose-intolerant state. Am J Physiol Regul Integr Comp Physiol. 2000 Feb;278(2):R435-44. Luo S, Ezrokhi M, Trubitsyna Y, Li Y, Cincotta AH. Elevation of Norepinephrine (NE) Activity at the Ventromedial Hypothalamus (VMH) of Normal Rats Induces the Obese Hypertensive Insulin Resistant State without Altering Feeding. Diabetes 2015;64(Suppl1):A540 Figure 5

Chronic infusion of norepinephrine into the VMH of normal rats induces the obese glucose-intolerant state Young insulin sensitive female Sprague-Dawley rats at 10 weeks of age Surgery to insert VMH cannula and initiation of unilateral Norepinephrine infusion Infuse for 3-4 weeks Measure metabolic parameters throughout the infusion period Effects of unilateral VMH Norepinephrine (NE:) or vehicle (○) infusions Food Consumption Body Weight Retroperitoneal Fat Pad Whitening of brown fat Vehicle infusion Norepinephrine infusion Glucose tolerance test Basal Lipolysis isoproterenol-stimulated lipolysis in isolated adipocytes of rats treated with VMH NE for 4 (■), 9 (▲), or 14 () days or vehicle (○). Plasma TG 14C=Glucose incorporation into lipid Respiratory quotient *P<0.05 and **P<0.01 Increased VMH NE tone is not merely an association of, but rather a contributing factor to, the metabolic syndrome Figure 6 Cincotta AH, Luo S, Zhang Y, Liang Y, Bina KG, Jetton TL, Scislowski PW. Chronic infusion of norepinephrine into the VMH of normal rats induces the obese glucose-intolerant state. Am J Physiol Regul Integr Comp Physiol. 2000 Feb;278(2):R435-44.

Timed Daily Administration of Bromocriptine Reduces (Normalizes) Elevated NE and Serotonin Release at the VMH of Insulin Resistant Syrian Hamsters and SHR Rats Winter 14 week old male Syrian hamsters GTT Separate glucose tolerant from glucose intolerant Microdialysis on freely moving animals 16 week old male hypertensive insulin resistant SHR rats or normotensive insulin sensitive Wistar rats Timed daily bromocriptine treatment for 2 weeks at the onset of locomotor activity Microdialysis on freely moving animals Time of day (hour) VMH 5-HIAA VMH MHPG Time of day (hour) VMH 5-HIAA VMH MHPG 5-HIAA SHR rats exhibited elevated VMH NE release relative to Wistar normal controls (P < 0.0001). Timed daily bromocriptine administration significantly reduced VMH MHPG of SHR rats compared with vehicle treated SHR rats (P < 0.0001) In glucose- tolerant hamsters, the average daily VMH extracellular levels of 5-HIAA and MHPG were 48 and 44% lower, respectively, than in glucose-intolerant hamsters Two-way ANOVA with repeated measures indicates significant difference of 5-HIAA and MHPG between the two groups (p<0.05) MHPG SHR rats exhibited significantly lower extracellular VMH HVA content compared with normal Wistar rats (p < 0.001) Daily profiles of 5-HIAA (A), and MHPG (B) in microdialysate samples from VMH of freely moving glucose tolerant () and glucose-intolerant (○) hamsters Luo S, Meier AH, Cincotta AH. Bromocriptine reduces obesity, glucose intolerance and extracellular monoamine metabolite levels in the ventromedial hypothalamus of Syrian hamsters. Neuroendocrinology. 1998 Jul;68(1):1-10. Ezrokhi M, Luo S, Trubitsyna Y, Cincotta AH . Neuroendocrine and metabolic components of dopamine agonist amelioration of metabolic syndrome in SHR rats. Diabetology & Metabolic Syndrome 2014; 6:104 Figure 7

Circadian Timed Bromocriptine Treatment Reduces Free Fatty Acids Mobilization and Plasma Triglycerides in vivo in Winter Obese Insulin Resistant Hamsters Obese, insulin resistant, hyperinsulinemic Syrian hamsters Timed daily IP Bromocriptine administration for 9-10 weeks Lipolysis, plasma TG and FFA measured in vivo Light period Figure 8 Cincotta AH, MacEachern TA, Meier AH. Bromocriptine redirects metabolism and prevents seasonal onset of obese hyperinsulinemic state in Syrian hamsters. Am J Physiol. 1993; 264(2 Pt 1):E285-93

Circadian Timed Bromocriptine Treatment Increases Whole Body Protein Turnover in Winter Obese Insulin Resistant Syrian Hamsters Obese, insulin resistant, hyperinsulinemic Syrian hamsters Timed daily IP Bromocriptine administration for 9-10 weeks Protein turnover rate measured in vivo Figure 9 Cincotta AH, MacEachern TA, Meier AH. Bromocriptine redirects metabolism and prevents seasonal onset of obese hyperinsulinemic state in Syrian hamsters. Am J Physiol. 1993; 264(2 Pt 1):E285-93

Circadian Timed Bromocriptine Treatment Reduces Plasma Glucose, Insulin, Leptin, Norepinephrine, Systolic and Diastolic Blood Pressure in Hypertensive Insulin Resistant SHR Rats Hypertensive, obese, insulin resistant male 16 week old SHR rats, healthy Wistar rats as control Timed daily IP Bromocriptine administration for 2 weeks Blood pressure, liver enzymes and metabolic parameters measurements   P<0.02   P=0.007 P<0.003 P=0.0002   P=0.03 P=NS P=0.001   P<0.0001   P<0.001 P<0.001 P<0.001   P<0.001 P<0.001 P=0.001 SHR rats treated with vehicle control   SHR rats treated with Timed Daily Bromocriptine Wistar rats Figure 10 Ezrokhi M, Luo S, Trubitsyna Y, Cincotta AH. Neuroendocrine and metabolic components of dopamine agonist amelioration of metabolic syndrome in SHR rats. Diabetology & Metabolic Syndrome 2014; 6:104

Circadian-Timed BC/SKF Treatment Reduces PVN Levels of NPY and CRH * * *P<0.05 Figure 11 Bina KG, Cincotta AH. Dopaminergic agonists normalize elevated hypothalamic neuropeptide Y and corticotropin-releasing hormone, body weight gain, and hyperglycemia in ob/ob mice. Neuroendocrinology. 2000;71(1):68-78.

Western Diet / Stress / Sleep Wake Alterations Induce A, B, and C Schematic of Dopamine – Clock Interactions in the Regulation of Peripheral Fuel Metabolism Western Diet / Stress / Sleep Wake Alterations Induce A, B, and C Refs. 1, 20, 23, 27, 28 Timed Daily Dopamine Agonist Administration Reverses A, B and C A B C = increases = decreases = results in = stimulates Abbreviations: 1. CRH: Corticotropin Releasing Hormone 2. eNOS: Endothelial Nitric Oxide Synthase 3. FFA: Free Fatty Acids 4. NPY: Neuropeptide Y 5. PVN: Paraventricular Nucleus 6. SCN: Suprachiasmatic Nucleus 7. SNS: Sympathetic Nervous System 8. TG: Triglycerides 9. VMH: Ventromedial Hypothalamus Figure 12 Raskin P, Cincotta AH. Expert Review of Endocrinology & Metabolism 2016;11(2):113-48.