Nicotinic acid timed to feeding reverses tissue lipid accumulation and improves glucose control in obese Zucker rats[S]
2016; Elsevier BV; Volume: 58; Issue: 1 Linguagem: Inglês
10.1194/jlr.m068395
ISSN1539-7262
AutoresTobias Kroon, Tania Baccega, Arne Olsén, Johan Gabrielsson, Nicholas D. Oakes,
Tópico(s)Adipose Tissue and Metabolism
ResumoNicotinic acid (NiAc) is a potent inhibitor of lipolysis, acutely reducing plasma free fatty acid (FFA) concentrations. However, a major FFA rebound is seen during rapid NiAc washout, and sustained exposure is associated with tolerance development, with FFAs returning to pretreatment levels. Our aim was to find a rational NiAc dosing regimen that preserves FFA lowering, sufficient to reverse nonadipose tissue lipid accumulation and improve metabolic control, in obese Zucker rats. We compared feeding-period versus fasting-period NiAc dosing for 5 days: 12 h subcutaneous infusion (programmable, implantable mini-pumps) terminated by gradual withdrawal. It was found that NiAc timed to feeding decreased triglycerides in liver (−47%; P < 0.01) and heart (−38%; P < 0.05) and reduced plasma fructosamine versus vehicle. During oral glucose tolerance test, plasma FFA levels were reduced with amelioration of hyperglycemia and hypertriglyceridemia. Furthermore, timing NiAc to feeding resulted in a general downregulation of de novo lipogenesis (DNL) genes in liver. By contrast, NiAc timed to fasting did not reduce tissue lipids, ameliorate glucose intolerance or dyslipidemia, or alter hepatic DNL genes. In conclusion, NiAc dosing regimen has a major impact on metabolic control in obese Zucker rats. Specifically, a well-defined NiAc exposure, timed to feeding periods, profoundly improves the metabolic phenotype of this animal model. Nicotinic acid (NiAc) is a potent inhibitor of lipolysis, acutely reducing plasma free fatty acid (FFA) concentrations. However, a major FFA rebound is seen during rapid NiAc washout, and sustained exposure is associated with tolerance development, with FFAs returning to pretreatment levels. Our aim was to find a rational NiAc dosing regimen that preserves FFA lowering, sufficient to reverse nonadipose tissue lipid accumulation and improve metabolic control, in obese Zucker rats. We compared feeding-period versus fasting-period NiAc dosing for 5 days: 12 h subcutaneous infusion (programmable, implantable mini-pumps) terminated by gradual withdrawal. It was found that NiAc timed to feeding decreased triglycerides in liver (−47%; P < 0.01) and heart (−38%; P < 0.05) and reduced plasma fructosamine versus vehicle. During oral glucose tolerance test, plasma FFA levels were reduced with amelioration of hyperglycemia and hypertriglyceridemia. Furthermore, timing NiAc to feeding resulted in a general downregulation of de novo lipogenesis (DNL) genes in liver. By contrast, NiAc timed to fasting did not reduce tissue lipids, ameliorate glucose intolerance or dyslipidemia, or alter hepatic DNL genes. In conclusion, NiAc dosing regimen has a major impact on metabolic control in obese Zucker rats. Specifically, a well-defined NiAc exposure, timed to feeding periods, profoundly improves the metabolic phenotype of this animal model. Lipid accumulation in peripheral nonadipose tissues has been shown to be a major driver of insulin resistance, nonalcoholic steatohepatitis, and dyslipidemia (1Shulman G.I. Ectopic fat in insulin resistance, dyslipidemia, and cardiometabolic disease.N. Engl. J. Med. 2014; 371: 1131-1141Crossref PubMed Scopus (422) Google Scholar, 2Lomonaco R. Bril F. Portillo-Sanchez P. Ortiz-Lopez C. Orsak B. Biernacki D. Lo M. Suman A. Weber M.H. Cusi K. Metabolic impact of nonalcoholic steatohepatitis in obese patients with type 2 diabetes.Diabetes Care. 2016; 39: 632-638Crossref PubMed Scopus (84) Google Scholar, 3Krauss R.M. Lipids and lipoproteins in patients with type 2 diabetes.Diabetes Care. 2004; 27: 1496-1504Crossref PubMed Scopus (492) Google Scholar). Circulating lipids, including plasma free fatty acids (FFAs) and TGs, are important sources of the intracellular lipid pool in muscle and liver (4Frayn K.N. Arner P. Yki-Jarvinen H. 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FFA increases in the fasting state, and it has recently been suggested that bedtime dosing might limit NiAc efficacy by triggering powerful counter-regulatory mechanisms (20Guyton J.R. Campbell K.B. Lakey W.C. Niacin: risk benefits and role in treating dyslipidemias..in: Garg A. In Dyslipidemias, Contemporary, Endocrinology. Springer, New York2015: 439-452Crossref Scopus (1) Google Scholar). It is not unreasonable that bedtime dosing might also be involved in the above-mentioned glucose metabolic impairments. Furthermore, an insulin-NiAc synergy on antilipolysis (10Kroon T. Kjellstedt A. Thalen P. Gabrielsson J. Oakes N.D. Dosing profile profoundly influences nicotinic acid's ability to improve metabolic control in rats.J. Lipid Res. 2015; 56: 1679-1690Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) would favor mealtime over bedtime dosing. We have been interested in determining whether a rational NiAc dosing regimen exists that maximizes FFA lowering and metabolic control. To achieve this, both tolerance and FFA rebound need to be minimized. Recently we tried to reduce tolerance by interspacing abruptly terminated NiAc exposures with drug holidays. This approach was partially successful, retaining acute NiAc-induced FFA lowering and insulin sensitivity but without delivering significant peripheral tissue lipid unloading (10Kroon T. Kjellstedt A. Thalen P. Gabrielsson J. Oakes N.D. Dosing profile profoundly influences nicotinic acid's ability to improve metabolic control in rats.J. Lipid Res. 2015; 56: 1679-1690Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). This absence of tissue lipid lowering may have reflected a failure to lower average FFA levels over the 24 h period due to a combination of FFA rebound and insufficient containment of tolerance. We speculated that abrupt infusion termination, in the context of NiAc's short plasma half-life [∼2 min in the rat (21Ahlstrom C. Kroon T. Peletier L.A. Gabrielsson J. Feedback modeling of non-esterified fatty acids in obese Zucker rats after nicotinic acid infusions.J. Pharmacokinet. Pharmacodyn. 2013; 40: 623-638Crossref PubMed Scopus (11) Google Scholar)], may exacerbate the FFA rebound. Furthermore, our initial study was performed without timing NiAc exposure to the feeding/fasting periods. In the present study, we used a preclinical animal model of peripheral tissue lipid overload-induced insulin resistance, the obese Zucker rat (22Wallenius K. Kjellstedt A. Thalen P. Lofgren L. Oakes N.D. The PPARalpha/gamma agonist, tesaglitazar, improves insulin mediated switching of tissue glucose and free fatty acid utilization in vivo in the obese Zucker rat.PPAR Res. 2013; 2013: 305347Crossref PubMed Scopus (19) Google Scholar). Our aim was to find a NiAc dosing regimen that would preserve robust FFA lowering sufficient to reverse nonadipose tissue lipid accumulation and improve metabolic control. In study I, we address the issue of FFA rebound during NiAc withdrawal. Acute metabolic responses to either rapid or gradual NiAc withdrawal in the basal fasting or glucose-infused situation were defined. The aim was to identify the protocol with the lowest FFA rebound for deployment in study II. In study II, we investigated the metabolic consequences of dosing NiAc to feeding versus fasting using a 12 h time-shifted but otherwise identical administration protocol. The results demonstrate a remarkable influence of dosing regimen on metabolic control. Experimental procedures were approved by the local Ethics Committee for Animal Experimentation (Gothenburg region, Sweden). Male obese fa/fa Zucker rats (Charles River) were housed in groups of five in an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility with environmental control (20–22°C, relative humidity 40–60% and 12 h light-dark cycle) with free access to water and rodent chow (R70; Laktamin AB, Stockholm, Sweden). At 12–13 weeks of age, animals were weighed, and a tail vein blood sample was taken for determination of glycated hemoglobin (A1cNOW+ PTS Diagnostics, Indianapolis, IN). Animals with glycated hemoglobin <6.2% (44 mmol/mol) were admitted to the studies. Surgical procedures were performed as previously described (10Kroon T. Kjellstedt A. Thalen P. Gabrielsson J. Oakes N.D. Dosing profile profoundly influences nicotinic acid's ability to improve metabolic control in rats.J. Lipid Res. 2015; 56: 1679-1690Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). In brief, for subcutaneous NiAc/saline administration, a programmable mini-pump (iPrecio SMP200 Micro Infusion Pump; Primetech Corporation, Tokyo, Japan) was implanted subcutaneously via a dorsal skin incision. For study II animals only, a catheter was placed in the right jugular vein, exteriorized at the nape of the neck, and plugged for later use to allow blood sampling. Subcutaneous analgesic injections (buprenorphine, Temgesic, 1.85 µg/kg; RB Pharmaceuticals Ltd, Berkshire, UK) were given postoperatively and once daily for 2 days. Animals were housed individually until study completion, with a 3 day recovery period before treatment start. General health status, body weight, and food intake were monitored and recorded daily. Fresh NiAc (pyridine-3-carboxylic acid; Sigma-Aldrich, St. Louis, MO) infusion solution was prepared to a concentration of ∼350 mM using procedures described in (10Kroon T. Kjellstedt A. Thalen P. Gabrielsson J. Oakes N.D. Dosing profile profoundly influences nicotinic acid's ability to improve metabolic control in rats.J. Lipid Res. 2015; 56: 1679-1690Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Vehicle, for control animals, consisted of sodium chloride solutions at equimolar concentrations. Study I was performed to address the issue of FFA rebound during NiAc withdrawal. Specifically, the influence of two potentially important factors were examined: 1) the rate of plasma NiAc decay upon acute drug withdrawal, either rapidly (NiAc-Off) or gradually (NiAc-Stp-Dwn), and 2) the metabolic status at the time of withdrawal (i.e., either during fasting state or glucose infusion), a situation that partially mimics the fed state. Metabolic responses were assessed in overnight-fasted, anesthetized obese Zucker rats. The results of study I were used to select the NiAc termination protocol (NiAc-Off vs. NiAc-Stp-Dwn) and the metabolic state (fed vs. fasted) in which NiAc was to be terminated in study II. Study II was performed to test the metabolic consequences of NiAc dosing timed to either fasting (daytime) or feeding (nighttime). Chronically jugular catheterized obese Zucker rats, with food freely available during nighttime only (to entrain defined periods of feeding and fasting in these hyperphagic animals), were treated for 5 days with NiAc (NiAc Day vs. NiAc Night). Acute experiments were then performed in the conscious state. Metabolic control was assessed using an oral glucose tolerance test (OGTT). The study I protocol is diagrammed in Fig. 1 (top panel). The day before the acute study, food was removed at 17:00, and at 01:00 the preprogrammed implanted pump began infusing NiAc at a constant rate (170 nmol/min/kg, corresponding to 29.1 µl/hr/kg) for 12 h. At 09:30 animals were anesthetized and surgically prepared with jugular and carotid catheters as previously described (10Kroon T. Kjellstedt A. Thalen P. Gabrielsson J. Oakes N.D. Dosing profile profoundly influences nicotinic acid's ability to improve metabolic control in rats.J. Lipid Res. 2015; 56: 1679-1690Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). We hypothesized that a gradual withdrawal of NiAc during washout would attenuate FFA rebound. To test this, we compared a NiAc washout exposure that delayed the plasma NiAc decay toward in vivo IC50 for FFA lowering by several hours versus abrupt termination. A specific step-down infusion protocol needed to produce this profile was identified with the aid of a previously developed model describing acute NiAc pharmacokinetics and FFA response (21Ahlstrom C. Kroon T. Peletier L.A. Gabrielsson J. Feedback modeling of non-esterified fatty acids in obese Zucker rats after nicotinic acid infusions.J. Pharmacokinet. Pharmacodyn. 2013; 40: 623-638Crossref PubMed Scopus (11) Google Scholar). The step-down NiAc infusion rates were 88.9, 58.3, 43.7, 34.0, 24.3, 17.0, and 9.7 nmol/min/kg. After a postsurgery stabilization period of at least 1.5 h, blood sampling was initiated at 12:00. At 12:30, half the animals (Glu+ groups) began to receive an intravenous glucose infusion based on lean body mass (lbm) (20.6 mg/min/kglbm, corresponding to 41.2 µl/min/kglbm) delivered using an external syringe pump (Pump 11 Elite Series; Harvard Apparatus, Cambridge, MA). The remaining animals did not receive glucose (Glu− groups). At 13:00, NiAc infusion was either programmed to switch off (NiAc-Off) or to decrease in a stepwise manner (see above), with final switch-off at 16:30 (NiAc-Stp-Dwn). All NiAc protocols were matched with saline-infused controls. Blood samples were drawn at 12:00, 12:30, 12:45, 13:00, 13.15, 13:30, 13:45, 14:00, 14:30, 15:00, 15:30, 16:00, 16.30, 16:45, 17:00, 17:15, 17.30, and 18.00. Samples (30–150 µl, with total loss less than 5% of blood volume) were collected in potassium-EDTA tubes and centrifuged. Plasma was stored at −80°C pending analysis for NiAc, FFAs, glucose, and insulin. The study II protocol is diagrammed in Fig. 1 (bottom panel). During 5 days of treatment, food was freely available during the 12 h dark period only (lights on at 07:00). A daily NiAc exposure profile, with gradual stepwise decline, was timed either to fasting (NiAc Day group) or feeding (NiAc Night group), including a 12-h drug holiday period. Daily NiAc dosing profiles commenced at 06:00 for NiAc Day and at 18:00 for NiAc Night, beginning with an 8.5 h constant infusion at 170 nmol/min/kg followed by a 3.5 h step-down protocol as described for study I. All NiAc protocols were matched with saline infused controls. In the morning of day 5, to enable blood sampling in unrestrained animals, the jugular catheter was unplugged and connected to a swivel system as previously described (10Kroon T. Kjellstedt A. Thalen P. Gabrielsson J. Oakes N.D. Dosing profile profoundly influences nicotinic acid's ability to improve metabolic control in rats.J. Lipid Res. 2015; 56: 1679-1690Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). After a 2.5-h adaptation period, blood sampling was initiated at 13:00. Samples were drawn at 13:00, 14:00, 14:30, 15:00, 15:30, 16:00, 16:30, 17:00, 17:30, 18:00, and 18:45. Corresponding to the timing of breaking the fast (19:00), animals received an OGTT (4.1 g/kglbm, 8.2 ml/kglbm). Blood samples were taken at 7, 15, 30, 45, 60, 90, and 120 min after glucose load. Samples (30–150 µl, with total loss <5% of blood volume) were collected in potassium-EDTA tubes and centrifuged. Plasma was stored at −80°C pending analysis for NiAc, FFAs, glucose, insulin, fructosamine, and TG. After the last blood sample was taken, animals were anesthetized with isoflurane, and tissues (liver, heart, and epididymal adipose tissue) were dissected and snap frozen in liquid nitrogen and stored at −80°C pending TG and mRNA analysis. Plasma concentrations of NiAc, clinical chemistry biomarkers, and tissue TG content were measured according to previously described methods (10Kroon T. Kjellstedt A. Thalen P. Gabrielsson J. Oakes N.D. Dosing profile profoundly influences nicotinic acid's ability to improve metabolic control in rats.J. Lipid Res. 2015; 56: 1679-1690Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) with the exception of plasma glucose and fructosamine (Horiba ABX, Montpellier, France). Area under the concentration-time curves (AUCs) for plasma NiAc, FFAs, insulin, glucose, and TG were calculated by trapezoidal approximation using GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, CA). RNA was extracted, and cDNA was prepared as previously described (10). Gene expression was determined by quantitative RT-PCR using a QuantStudio7 Flex system (Applied Biosystems, Foster City, CA). Premade primer/probe TaqMan assays (Applied Biosystems) were used for the following genes (alias/gene: Applied Biosystems assay ID): carbohydrate-responsive element-binding protein (ChREBP/Mlxipl: Rn00591943_m1), sterol regulatory element binding protein-1c (SREBP-1c/Srebf1: Rn01495769_m1), acetyl-CoA carboxylase 1 (ACC1/Acaca: Rn00573474_m1), fatty acid synthase (FAS/Fasn: Rn00569117_m1), fatty acid elongase 6 (Elovl6: Rn01522302_g1), stearoyl-CoA desaturase-1 (SCD1: Rn00594894_g1), PPARγ2 (Pparg: Rn00440945_m1), cluster of differentiation 36/fatty acid translocase (CD36/Fat: Rn01442640_g1), fatty acid binding protein-4 (FABP4/Albp: Rn00670361_m1), and perilipin-1 (PLIN1: Rn00558672_m1). The housekeeping gene ribosomal protein large P0 (RPLP0; also known as 36B4) was used for normalization of gene expression data; forward (5′-AAA TCT CCA GAG GTA CCA TTG AAA TC-3′) reverse (5′-GCT GGC TCC CAC CTT GTC T-3′). Lean body mass (lbm) was estimated from body weight as previously described (23Oakes N.D. Thalen P. Hultstrand T. Jacinto S. Camejo G. Wallin B. Ljung B. Tesaglitazar, a dual PPAR{alpha}/{gamma} agonist, ameliorates glucose and lipid intolerance in obese Zucker rats.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 289: R938-R946Crossref PubMed Scopus (33) Google Scholar). Statistical significance was evaluated using one-way ANOVA followed by Tukey's multiple comparisons test, performed using GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, CA) with comparisons between groups for repeatedly measured variables based on AUC estimates. For study I, the primary objective was to determine whether gradual NiAc termination induced a smaller FFA rebound (measured by FFA AUC) compared with abrupt NiAc termination. Using variability estimates obtained previously for abrupt NiAc withdrawal in obese Zucker rats [coefficient of variation (CV)] for FFA AUC, 9.4%) (10Kroon T. Kjellstedt A. Thalen P. Gabrielsson J. Oakes N.D. Dosing profile profoundly influences nicotinic acid's ability to improve metabolic control in rats.J. Lipid Res. 2015; 56: 1679-1690Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), a minimum sample size of five per group was calculated to be required to detect a 20% difference in FFA AUC between two of the six means with 80% power at the 0.05 level of significance (24Sokal Robert R. James R.F. Biometry: The Principles and Practice of Statistics in Biological Research.. WIH Freeman, New York1969Google Scholar). Study I also compared the effects of NiAc termination on plasma NiAc, glucose, and insulin AUCs. For study II, the primary objective was to determine whether NiAc timed to feeding could induce a substantial reversal of liver TG accumulation compared with control animals with hepatic steatosis. Using historical data from obese Zucker rats (CV for hepatic lipid content, 32%) (25Oakes N.D. Thalen P.G. Jacinto S.M. Ljung B. Thiazolidinediones increase plasma-adipose tissue FFA exchange capacity and enhance insulin-mediated control of systemic FFA availability.Diabetes. 2001; 50: 1158-1165Crossref PubMed Scopus (158) Google Scholar), a minimum sample size of eight per group was calculated to be required to detect a 50% reduction in liver TG between two of the three means with 80% power at the 0.05 level of significance. Secondary endpoints of key interest from the efficacy standpoint were metabolic responses to a glucose tolerance test, especially plasma glucose and TG. Based on historical meal challenge data in obese Zucker rats (CVs for plasma glucose and TG AUCs, 4 and 9%, respectively) (23Oakes N.D. Thalen P. Hultstrand T. Jacinto S. Camejo G. Wallin B. Ljung B. Tesaglitazar, a dual PPAR{alpha}/{gamma} agonist, ameliorates glucose and lipid intolerance in obese Zucker rats.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 289: R938-R946Crossref PubMed Scopus (33) Google Scholar), it was estimated that group sizes of n = 8 would be adequate, having at least an 80% chance of detecting reductions at the 0.05 level of 17% and 35% or more for glucose and TG AUCs, respectively. Several other secondary measurements were also measured in study II, with estimated variability based on similar experiments in the range of the above-mentioned measurements: food intake, body weight, fructosamine, heart TG content, tissue mRNA levels (see Materials and Methods), and AUCs for NiAc and insulin. P < 0.05 was considered statistically significant. Throughout, results are reported as mean ± standard error of the mean (SEM). The target plateau plasma NiAc concentration of ∼1 µM was successfully achieved in all NiAc-infused groups (Fig. 2A, B). After preprogrammed initiation of the step-down protocol at 13:00, plasma NiAc concentrations declined gradually in the NiAc-Stp-Dwn groups, taking ∼3.8 h to reach acute in vivo IC50 for FFA lowering (∼0.07 µM) versus ∼1.2 h for abrupt termination in the NiAc-Off groups. During this period (13:00–18:00), NiAc-Stp-Dwn groups had significantly increased NiAc exposures (∼3-fold vs. the corresponding NiAc-Off group) (Fig. 2C). In control animals (saline infused), endogenous NiAc levels were below the detection limit (6 nM). Upon abrupt NiAc withdrawal (NiAc-Off groups), a FFA rebound was observed in both no-glucose (Glu−) and glucose-infused (Glu+) groups (Fig. 2D, E), with FFA AUC greater than corresponding saline-infused control groups (P < 0.01 and P < 0.001, respectively) (Fig. 2F). Upon gradual NiAc withdrawal in the Glu− groups, the FFA AUC was unexpectedly higher in the NiAc-Stp-Dwn versus the corresponding NiAc-Off group (P < 0.001) (Fig. 2F), despite a ∼3-fold higher NiAc exposure (P < 0.001) (Fig. 2C). By contrast, in the Glu+ groups, NiAc step-down successfully attenuated the FFA AUC versus abrupt withdrawal (P < 0.05) (Fig. 2F). Abrupt NiAc withdrawal induced a pronounced insulin rebound in the no-glucose group (Fig. 2G), where insulin AUC was profoundly raised versus NiAc-Stp-Dwn group (P < 0.01) (Fig. 2I). The marked hyperinsulinemia provides a likely explanation for the lower FFA exposure in the NiAc-Off versus NiAc-Stp-Dwn groups (Fig. 2F). In the Glu+ groups, over the whole study period (13:00–18:00), the glucose-induced hyperinsulinemia was unaffected and was of similar magnitude in both Off and Stp-Dwn groups (Fig. 2I). Abrupt termination of NiAc had remarkably little influence on plasma glucose levels in the no-glucose situation (Fig. 2J) despite the impressive changes in FFA and insulin levels. There was no difference in the glucose AUC between NiAc-Off and NiAc-Stp-Dwn groups. In the glucose-infused state, gradual and abrupt NiAc withdrawal worsened glucose control similarly (Fig. 2K), with glucose AUC significantly elevated versus saline control for both NiAc groups (P < 0.001) (Fig. 2L). Gradual NiAc withdrawal during glucose loading displayed the lowest FFA AUC versus all other NiAc groups. Thus, to minimize FFA rebound, gradual NiAc withdrawal should occur during the fed state in study II. Pump/catheter implantation did not result in reduced food intake or body weight loss during the 3 day recovery period (day −2 to 0) (Fig. 3). Coincident with treatment start (day 0) until termination, food was restricted to nighttime only. Initial dips in food intake and body weight were fully compensated by the end of the 5 day treatment period. Importantly, food intake and body weight trajectories were practically identical in all groups (Fig. 3). Target plateau plasma NiAc concentrations were similar at ∼1 µM in both NiAc-infused groups (Fig. 4A). After preprogrammed initiation of the step-down protocol at 14:30, plasma NiAc levels declined gradually in the Day group. Overall, the NiAc exposure results confirm reliable pump performance according to preprogramming. In control animals, endogenous NiAc levels were below the detection limit (6 nM). In the NiAc Day group, FFA AUC(13:00–19:00) was higher versus Night and Saline groups (P < 0.05) (Fig. 4D) despite having the highest NiAc AUC(13:00–19:00). In the Night group, during the last half of the drug holiday, FFA AUC(13:00–19:00) was similar to the Saline group. As expected, in the Saline group the oral glucose load at 19:00 reduced FFA levels (Fig. 4C). Strikingly, Day versus Night dosing had opposite effects on FFA levels duri
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