Influence of Daytime LED Light Exposure on Circadian Regulatory Dynamics of Metabolism and Physiology in Mice

Robert T Dauchy; David E Blask; Aaron E Hoffman; Shulin Xiang; John P Hanifin; Benjamin Warfield; George C Brainard; Murali Anbalagan; Lynell M Dupepe; Georgina L Dobek; Victoria P Belancio; Erin M Dauchy; Steven M Hill

doi:10.30802/AALAS-CM-19-000001

. 2019 Oct;69(5):350–373. doi: 10.30802/AALAS-CM-19-000001

Influence of Daytime LED Light Exposure on Circadian Regulatory Dynamics of Metabolism and Physiology in Mice

Robert T Dauchy ^1,^*, David E Blask ¹, Aaron E Hoffman ², Shulin Xiang ¹, John P Hanifin ⁴, Benjamin Warfield ⁴, George C Brainard ⁴, Murali Anbalagan ¹, Lynell M Dupepe ³, Georgina L Dobek ³, Victoria P Belancio ¹, Erin M Dauchy ⁵, Steven M Hill ¹

PMCID: PMC6807725 PMID: 31540584

Abstract

Light is a potent biologic force that profoundly influences circadian, neuroendocrine, and neurobehavioral regulation in animals. Previously we examined the effects of light-phase exposure of rats to white light-emitting diodes (LED), which emit more light in the blue-appearing portion of the visible spectrum (465 to 485 nm) than do broad-spectrum cool white fluorescent (CWF) light, on the nighttime melatonin amplitude and circadian regulation of metabolism and physiology. In the current studies, we tested the hypothesis that exposure to blue-enriched LED light at day (bLAD), compared with CWF, promotes the circadian regulation of neuroendocrine, metabolic, and physiologic parameters that are associated with optimizing homeostatic regulation of health and wellbeing in 3 mouse strains commonly used in biomedical research (C3H [melatonin-producing], C57BL/6, and BALB/c [melatonin-non-producing]). Compared with male and female mice housed for 12 wk under 12:12-h light:dark (LD) cycles in CWF light, C3H mice in bLAD evinced 6-fold higher peak plasma melatonin levels at the middark phase; in addition, high melatonin levels were prolonged 2 to 3 h into the light phase. C57BL/6 and BALB/c strains did not produce nighttime pineal melatonin. Body growth rates; dietary and water intakes; circadian rhythms of arterial blood corticosterone, insulin, leptin, glucose, and lactic acid; pO₂ and pCO₂; fatty acids; and metabolic indicators (cAMP, DNA, tissue DNA ³H-thymidine incorporation, fat content) in major organ systems were significantly lower and activation of major metabolic signaling pathways (mTOR, GSK3β, and SIRT1) in skeletal muscle and liver were higher only in C3H mice in bLAD compared with CWF. These data show that exposure of C3H mice to bLAD compared with CWF has a marked positive effect on the circadian regulation of neuroendocrine, metabolic, and physiologic parameters associated with the promotion of animal health and wellbeing that may influence scientific outcomes. The absence of enhancement in amelatonic strains suggests hyperproduction of nighttime melatonin may be a key component of the physiology.

Abbreviations: bLAD, blue-enriched LED light at day; CWF, cool white fluorescent; dpm, disintegrations per minute; GSK3β; glycogen synthase kinase 3β; ipRGC, intrinsically photosensitive retinal ganglion cell; LED, light-emitting diode; mTOR, mechanistic target of rapamycin; SCN, suprachiasmatic nuclei; SIRT1, nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1; TFA, total fatty acid

Light and lighting protocols in laboratory animal facilities have long been a concern to biomedical researchers and animal care personnel alike. All life varies in its capacity to use visible and nonvisible photic energy. Alternating cycles of light and darkness entrain the master biologic clock, located within the suprachiasmatic nucleus (SCN) in the anterobasal hypothalamus of the brain.^10,50 A small set of cells within the retina of the eyes called the intrinsically sensitive retinal ganglion cells (ipRGC) contain the photopigment melanopsin, which mediates photobiologic responses in circadian rhythms of metabolism and physiology.^{1,3,6,18,33,45,48,53,72} Melanopsin ipRGC detect light quanta mostly in the blue-appearing portion of the visible spectrum (465 to 485 nm), and photic information is transmitted to the SCN through the retinohypothalamic tract. The SCN regulates the nighttime pineal gland production of the circadian neurohormone melatonin (N-acetyl-5-methoxytryptamine) that results in high nighttime and low daytime levels.³¹ The daily melatonin signal contributes to the temporal coordination of normal behavioral and physiologic functions associated with the promotion of animal health and wellbeing.^3,56 Indeed, as humans age, maintenance of a robust melatonin rhythm, which signifies a strong signal from the SCN, is associated with improved health.⁶¹ Therefore, exposure to light in an intensity-, duration-, and wavelength- (that is, spectrum) dependent manner is essential to regulation of the SCN.⁹ Variations in any of these parameters at a given time of day influences endogenous circadian nighttime melatonin levels and may lead to various age- and disease-related health problems, including metabolic syndrome, obesity, and cancer.^{7-9,20,21,28,31,48,56-59}

Recently, we demonstrated that nighttime plasma melatonin levels in rats maintained in daytime light-emitting diode (LED) lighting, enriched in light in the blue-appearing portion of the visible spectrum (bLAD), were 7-fold higher than those in rats maintained in broad-spectrum cool white fluorescent (CWF) lighting.^23,24 Furthermore, the melatonin duration extended 2 to 3 h into the light phase, due to the augmented production of melatonin in the night. In addition, we recently determined that athymic nude mice (Crl:NU(NCr)-Foxn1^nu), used to support the development of human tumors for our tissue-isolated nude rat model (Crl:NCI-Foxn1^rnu),^7,8,21,22 are circadian complete (melatonin-producing) and respond in a similar fashion to bLAD as do nude rats (male and female).²³ Most notable was the finding that, in both male and female nude rats and mice exposed to bLAD, compared with daytime full-spectrum CWF light, nighttime melatonin levels were 6- to 7-fold higher, thus significantly inhibiting metabolism, signal transduction activity, and growth of human tumors.^21,22

In some commonly used strains of mice, however, nighttime pineal melatonin production is impaired or nonexistent.⁴³ Melatonin is produced in the pineal gland by N-acetylation of the precursor serotonin by akylaryl N-acetyltransferase (AANAT) followed by O-methylation by hydroxyindole-O-methyltransferase (HIOMT).⁴⁴ Despite some original controversy regarding the C57BL/6 strain,^27,70 it is now generally accepted that the inbred C57BL/6 and BALB/c mouse strains do not produce nighttime pineal melatonin, as both strains are missing active NAT and HIOMT from the pineal gland;^43,69,71 in contrast, the C3H strain expresses both enzymes and displays a robust nighttime pineal melatonin surge.^43,69 C57BL/6 mice have a point mutation in the AANAT gene, resulting in a truncated protein with little-to-no enzyme activity.⁵⁹ One of the aims of our current study was to determine whether, in the absence of the normal nighttime melatonin signal, bLAD lighting may nonetheless influence circadian rhythms of metabolism and physiology in the C57BL/6 and BALB/c strains and, ultimately, animal health and wellbeing.

Institutions around the world are rapidly transitioning to the emerging LED technology due primarily to improved efficiency, lower heat production, markedly longer operating life, and overall cost savings.^{23,26,35,40,54} The American Medical Association has released 2 public health reports on the effects of nighttime LED lighting on humans and the environment.^16,17 The reports reviewed current research on the biologic and behavioral effects of light. The most recent report covers primarily nighttime LED outdoor street lighting in the community as it pertained to visibility glare, visual impairment due to stray light, and the putative environmentally disruptive effects of high-intensity LED light (including blue spectrum light) on humans and nocturnal animals.¹⁶ The 2016 American Medical Association report elicited some response and controversy from the lighting and energy communities.^41,68

The 8th edition of the Guide for the Care and Use of Laboratory Animals,³⁹ as well as several prior editions, focuses primarily on retinopathy concerns in rodents relative to vivarium lighting. Little to no mention is made regarding circadian regulation by light and lighting protocols in the brief section devoted to animal facility lighting. In addition, little information is available concerning the potential influence of different types of lighting such as LED lighting on animal health and wellbeing. Furthermore, scientific consensus on the methods for quantifying and reporting light stimuli in experimental studies has occurred only recently.⁴⁸ Following these recommended practices will facilitate comparing results across investigations. This consensus was adapted into a balloted report and distributed internationally.¹⁴ More recently this consensus has been developed into an international standard.¹⁵ Therefore, our laboratory aims to report as thoroughly as possible all light measurements necessary for replication of experimental conditions, enabling comparison of results across investigations, including comprehensive spectral power distribution measurements, which are currently the most appropriate and accepted methodology available in the lighting industry.^11,14,15,48

Increasingly, our 24/7 global society is exposed to primarily broad-spectrum light at night (LAN) and bLAD in the workplace and home environment—one of the most profound environmental changes to occur since the advent of the electric light bulb 130 y ago.^16,40 Prior to this invention and the onset of the industrial revolution, our global societies were exposed primarily to blue-enriched environmental light during daytime and, if any light at night, long-spectrum (yellow to red) nighttime light (oil lamps, candles, wood fires) through thousands of years of evolution.⁵⁴ Even before animal life evolved on our planet there existed a night–day (blue-enriched light) cycle.^10,48,54 It is understandable, then, how the light:dark cycle has had an impact on our development and evolution over the millennia. Evidence strongly suggests that the use of the more natural daytime blue-enriched LED light in animal facilities, compared with broad-spectrum incandescent and CWF light, has a marked positive effect on the circadian regulation of behavioral, neuroendocrine, metabolic, and physiologic parameters associated with the promotion of animal health and wellbeing and thereby influences scientific outcomes.²³ In our previous studies,^23,24 using a new light metric⁴⁸ to assess our findings, we provided compelling evidence that animals exposed to bLAD compared with CWF lighting displayed characteristics of enhanced health and wellbeing.

With these concerns in mind, the overall goal of this study was to examine the influence of bLAD light, as compared with broad-spectrum CWF light, on metabolic and physiologic measures of laboratory animal health and wellbeing. Specifically, we examined the hypothesis that the spectral characteristics (wavelength) of bright LED light during the light phase, compared with standard CWF lighting, not only amplifies the normal circadian nocturnal melatonin signal but also alters the circadian regulation of plasma measures of metabolism and physiology in these important strains of mice to the fields of laboratory animal science and biomedical research.

Materials and Methods

Animals, housing conditions, and diet.

Male and female inbred mice (C3H [Charles River Lab strain code 025; n = 60 male; n = 60 female]; C57BL/6 [CRL strain code 027; n = 60 male; n = 60 female]; and, BALB/c inbred [CRL strain code 028 n = 60 male; n = 60 female]; age, 3 to 4 wk of age) used in this study were purchased from Charles River (Wilmington, MA). Animals were maintained in an AAALAC-accredited facility in accordance with the Guide for the Care and Use of Laboratory Animals.³⁹ All procedures for animal use were approved by the Tulane University IACUC. In addition, this study adheres to NIH principles and guidelines regarding strict application of the scientific method to ensure robust and unbiased experimental design, methodologies, analysis, rigor and reproducibility, and transparency in reporting.¹³

Mice were maintained as described following in autoclaved cages containing hardwood maple bedding (no. 7090, Sanichips, Harlan Teklad, Madison, WI; 3 bedding changes weekly). To ensure that all animals remained infection-free from both bacterial and viral agents, serum samples from sentinel animals were tested quarterly and during the course of this study by using Multiplex Fluorescent Immunoassay 2 (IDEXX Research Animal Diagnostic Laboratory, Columbia, MO), as described previously.^19-23 All mice were free of the following pathogens: mouse hepatitis virus, minute virus of mice, mouse parvovirus, mouse norovirus, Theiler murine encephalomyelitis virus, mouse rotavirus, Sendai virus, pneumonia virus of mice, reovirus 3, lymphocytic choriomeningitis virus, ectromelia virus, mouse adenovirus types 1 and 2, mouse polyoma virus, mouse cytomegalovirus, Encephalitozoon cuniculi, cilia-associated respiratory bacillus, Clostridium piliforme, and Mycoplasma pulmonis. In addition, mice were negative for Heliobacter spp. (real-time PCR analysis of feces) and free of endo- and ectoparasites. Throughout this study, animals were assessed daily for food and water intake and body weight changes. Food spillage and water evaporation has been reported to be less than 0.1 g per mouse daily and 0.2 mL per 48 h, respectively, for these strains of mice and were therefore not taken into account here.⁴ Eight independent determinations of this diet revealed that it contained 4.72 g total fatty acid (TFA) per 100 g of diet composed of 0.78% myristic (C14:0), 13.68% palmitic (C16:0), 1.05% palmitoleic (C16:1n7), 3.62% stearic (C18:0), 20.58% oleic (C18:1n9), 52.10% linoleic (C18:2n6), 7.82% γ-linolenic, and 0.18% arachidonic (C20:4n6) acids. Minor amounts of other FA comprised 0.19%. Conjugated linoleic acids (CLAs) and trans FAs were not found. More than 90% of the TFA was in the form of triglycerides; more than 5% was in the form of free fatty acids.

Caging, lighting, and spectral transmittance measurements.

After a 1-wk acclimation period, mice were randomized, taking into account strain and sex, into 2 designated groups of control (standard cool white fluorescent lighting; CWF) and experimental (blue-enriched LED lighting at daytime, bLAD) in standard translucent laboratory mouse cages; the study timeline was 12 wk. Standard mouse cages (polycarbonate translucent clear, catalog no. N10; 7.5 in. × 11.5 in. × 5 in.; wall thickness, 0.10 in.), wire bar lids (catalog no. N10SS), and microfilter tops (catalog no. N10NBT) used in this study were purchased from Ancare (Bellmore, NY). The SPF mice were maintained in environmentally controlled rooms (25 °C; 50% to 55% humidity) with 12:12-h diurnal lighting (lights on, 0600). The CWF control animal room was lighted with a series of 2 overhead luminaires containing 4 standard soft, cool-white (2700 lm; 4100 correlated color temperature) fluorescent lamps per ballast (F32T8TL841, model 272484, Alto II Collection T8 [diameter, 1 in.; tubular] 32 W, 48 in., series 800, Philips, Somerset, NJ). The experimental LED animal room was lighted with a series of 2 overhead luminaires containing 4 Philips LED lamps, high in emission of blue-appearing portion of the visible spectrum (465 to 485 nm; 2650 lm, >5000 correlated color temperature) lamps per ballast (model 9290011242, 12T8/AMB/48, T8, 12 W, 48 in., Philips). Photo images, along with radiometric and photometric values, of the study lamps were reported previously.^22-24 Animal rooms were completely devoid of light contamination during the dark phase.^7,8,19-24

Daily during the course of this experiment, the animal room was monitored for normal light-phase lighting intensity at 1 m above the floor in the center of the room (at rodent eye level) and outside, from within, and at the front of the animal cages. Irradiance measures were recorded by using a radiometer–photometer (IL-1400A, International Light Technologies, Peabody, MA) with a silicon diode detector head (catalog no. SEL033) with a wide-angle input optic (catalog no. W6849) and a filter (catalog no. F23104) that provided a flat response across the visible spectrum. Illuminance measures used a silicon diode detector head (catalog no. SEL033) with a wide-angle input optic (catalog no. W10069) and a filter (catalog no. Y23104) to provide a photopic illuminance response. The meter and associated optics were calibrated annually, as described previously.^19-23 Each day and at the same time (0800), prior to light intensity measurements for that day, all cages on the static rack shelf were rotated one position to the right (placed at an identical, premeasured distance apart) in the same horizontal plane; the cage at position 6 (last position at far right on the shelf) was moved to position 1 (first position at far left on the shelf) on the next lower shelf. Although there were no significant differences in light intensity, as measured outside and from within the front of each cage at each of the 36 positions, the daily cage shift further ensured uniformity of intensity of ocular light exposure and accounted for the effects of any unforeseen subtle differences due to the position on the rack shelf.

Under current convention, when discussing human and laboratory animal environments, the term lux is employed, indicating the amount of light falling on a surface that stimulates the mammalian eye during the daytime, that is, the perceived brightness to the eye (photometric values). Measures of lux are appropriate for human daytime vision but are not appropriate for quantifying light stimuli that regulates circadian, neuroendocrine or neurobehavioral physiology in animals or humans.^10,31,48 Consequently, radiometric values of irradiance (μW/cm²) were measured in the cages by using the radiometric detector described earlier. Irradiance quantifies the radiant power of light over a defined bandwidth of electromagnetic energy.⁴⁸ Given these standards, the light stimuli in the investigation reported here are presented in terms of both lux and μW/cm² for ease of understanding.

Spectral power measurements.

The lamps were installed in an overhead T8 assembly in a lightproof room, and then the spectral characteristics of each light source were measured separately by using a handheld spectroradiometer (ASD FieldSpec, ASD, Boulder, CO) with a cosine receptor attachment, as described previously.^19,21-23

Calculation of effective rod, cone, and melanopsin photoreceptor illuminances.

To calculate the effective rodent rod, cone, and melanopsin photoreceptor illuminances, the light sources were entered into a Toolbox worksheet, which is a software model for rodent photoreception freely available online.⁵² The spectral power distributions for the experiments shown here were imported into the worksheet in 1-nm increments between 325 and 782 nm. The Toolbox lists the rodent spectral range as extending to 298 nm, outside the range of the spectroradiometer used in this study. According to Toolbox instructions, values between 298 and 325 nm were manually changed to 0.

Tissue collection.

After a total of 12 wk (84 d) under the lighting regimens described earlier, animals were prepared for tissue collection (whole blood, liver, left retroperitoneal fat pad, left quadriceps femoris skeletal muscle, heart, lungs, kidneys, duodenum, brain, testes, ovaries) over the next 36-h period at 6 circadian times (1200, 1600, 2000, 2400, 0400, and 0800) during an 12:12-h light:dark cycle. All procedures were conducted using asceptic techniques. Briefly, mice were anesthetized with ketamine (80 mg/kg) and xylazine (9.80 mg/kg) intraperitoneally by using a 1-mL, 25-gauge tuberculin syringe (Becton Dickenson; Franklin Lakes, NJ). After the appropriate plane of anesthesia (lowered respiration rate; pedal reflex test) was achieved, the mouse was then placed on a heating pad in the surgical area, with the head facing the surgeon. The neck area was shaved and cleaned to remove hair, and a small incision (1 cm) was made to expose the right jugular vein and carotid artery. A small amount of [³H]thymidine (0.05 µCi in 20 µL sterile saline) was injected into the jugular vein and allowed to circulate for a period of 1 min. Next, the carotid artery was ligated, and arterial whole blood was allowed to flow freely into a collection minivial as the mouse was exsanguinated. A portion of the arterial blood collected was immediately measured for glucose, lactate, pO_2, pCO₂, pH, Hct, and Hgb by using an VetScan iSTAT1 Analyzer and iSTAT1 G4⁺ (no. 600-9000-25) and G8⁺ (no. 600-9001-25) cartridges (Abaxis, Union City, CA). Values for glucose and lactate are reported as mg/dL and mmol/L, and for pO₂ and pCO₂ as mm Hg, respectively. Minimal detection levels for pH, pO₂, pCO_2, glucose, and lactate values were 0.01, 0.1 mm Hg, 0.1 mm Hg, 0.2 mg/dL and 0.01 mmol/L, respectively.

Finally, all remaining tissues were rapidly removed from the euthanized mouse and snap-frozen under liquid nitrogen for future analysis. During dark phase, this process is completed under low-intensity (less than 35 lx) red safety light, as previously described.^7,8,19-23 The entire procedure required approximately 5 min per mouse. Remaining arterial whole blood samples were centrifuged and serum samples frozen (–20 °C) for future analysis.

Melatonin analysis.

Arterial plasma melatonin levels were measured by using a melatonin ¹²⁵I radioimmunoassay kit (catalog no. 01-RK-MEL2, Alpco, Salem, NH; lot no. 1429.18, prepared by Bühlmann Laboratories, Schönenbuch, Switzerland) and analyzed by using a Cobra 5005 Automated Gamma Counter (Packard, Palo Alto, CA), as previously described.^7,8,19-23 The minimal detection level for the assay was 1 to 2 pg/mL plasma.

FA extraction and analysis.

Arterial plasma TFA, as well as tissue TFA, were extracted from 0.1 mL arterial and venous samples after the addition of heptadecanoic acid (C17:0), methylated, and analyzed through gas chromatography as previously described.^7,8,19-20 Values for TFA represent the sum of the 7 major fatty acids (myristic, palmitic, palmitoleic, stearic, oleic, linoleic, and arachidonic acids) in the blood plasma. Tissue TFA in control and experimental groups were extracted from 0.2 mL of 20% homogenates, as previously described.^7,8,19-23 The minimal detectable limit for the assay was 0.05 μg/mL.

ELISA analysis of corticosterone, insulin, and leptin.

Arterial plasma samples were prepared in duplicate for measurement of corticosterone (catalog no. 55-CORMS-E01, mouse/rat, protocol version 09/06/17, Alpco), insulin (catalog no. 80-INSMSH-E01, E10, mouse, high range, protocol version 09/14/17, Alpco), and leptin (catalog no. 22-LEPMS-E01, mouse/rat, protocol version 030112, Alpco) by using chemiluminescent ELISA diagnostic kits. Samples were measured by using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nM. Detection sensitivities for corticosterone, insulin, and leptin plasma analyses were respectively 6.1 ng/mL, 0.52 ng/mL, and 10 pg/mL, and the coefficient of variation for all assays was less than 8.0%.

Determination of [³H]thymidine incorporation into tissue DNA, DNA content, and tissue cAMP content.

[³H]thymidine incorporation into tissue DNA, DNA content, and metabolic tissue levels of cAMP (ELISA, GE Lifesciences, Piscataway, NJ) were determined as described previously.^7,8,19-23

Western blot measurement of tissue phosphorylated kinases.

Excised animal organs were extracted and analyzed for signal transduction activity (that is, Akt/mTOR, GSK3β, and SIRT1), as previously described.^7,8,19-23

Statistical analysis.

Unless otherwise noted, all data are presented as the mean ± 1 SD (Figure 2 through 10: n = 5 male and n = 5 female mice per time point per strain; Tables 1 through 8: n = 30 male and 30 female mice for each strain in each CWF and bLAD experimental group; n = 360 mice total). The nonparametric JTK_CYCLE algorithm,³⁶ as implemented in scripts for the R software package (R version 3.1.0; http//openwetware.org/wiki/HughesLab:JTK Cycle), was used to determine the statistical significance of 24-h cycling for each analyte, with adjustments for multiple comparison. This algorithm also was used to determine phase (time of peaks) and amplitude of cycling. Statistical differences between mean values in group bLAD compared with the control CWF group at each circadian time point were assessed by using an unpaired Student test. A P value of less than 0.05 was considered to indicate a significant difference compared with the baseline values within each group. Statistical differences among group means were determined by using one-way ANOVA followed by the Bonferroni multiple comparison test. Student tests and one-way ANOVA followed by Bonferroni posthoc testing were all performed by using Prism 5 (GraphPad Software, La Jolla, CA).

Figure 10. — Circadian alterations in [³H]thymidine into skeletal muscle DNA in (A) C3H, (B) C57BL/6, and (C) BALB/c mice male maintained on either CWF control (male, solid black circles; female, solid amber squares) or experimental bLAD (male, solid blue triangles; female, solid green inverted triangles) lighting conditions. Data are plotted twice to better demonstrate rhythmicity. Dark bars represent dark-phase lighting conditions from 1800 to 0600. Concentrations with asterisks are different (P < 0.05) from those without asterisks.

Table 1.

Calculated radiometric values, photopic illuminances v(λ), and mouse photopigment illuminances (α-opic lux) of the polychromatic LED and fluorescent light sources as transmitted through the cage^48,55

	Radiometric and photometric values (325–780 nm inclusive)			Rodent retinal photopigment weighted illuminances (α-opic lux)
	Photon flux (cm²/s)	Irradiance (µW/cm²)	Photopic illuminance (lux)	S cone	Melanopsin ipRGC	Rod	M cone
Cage transmittance, bLAD	5.62E+14	203	646	29	437	478	502
Cage transmittance, CWF	5.10E+14	182	657	20	305	373	416

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Table 8.

Effects of cool white fluorescent and blue-enriched LED daytime lighting on tissue DNA levels (mg/g tissue) in C3H, C57BL/6, and BALB/c mice.

	C3H				C57BL/6				BALB/c
	CWF		bLAD		CWF		bLAD		CWF		bLAD
Tissue	M	F	M	F	M	F	M	F	M	F	M	F
Liver	2.91 ± 0.03	2.94 ± 0.09	3.02 ± 0.04 a^,b	3.00 ± 0.07 a^,b	2.95 ± 0.10 c^,d	3.03 ± 0.06 a	2.87 ± 0.13	2.72 ± 0.11^a-g	2.91 ± 0.12 h	3.09 ± 0.13 a^,b^,g-i	2.80 ± 0.11 c^,d^,f^,j	3.03 ± 0.08 a^,b^,e^,g-i,k
Fat	1.13 ± 0.07	1.04 ± 0.02	1.25 ± 0.04 a^,b	1.07 ± 0.03^a-c	1.28 ± 0.05 a^,b	1.26 ± 0.01 a^,b^,d	1.12 ± 0.04 b^,c^,e^,f	1.14 ± 0.02 c^,e^,f	1.16 ± 0.04 c^,d	1.27 ± 0.01 a^,b^,d^,g-i	1.04 ± 0.02 a^,c^,e-i,j	1.11 ± 0.03 b^,c^,e^,f^,j
Muscle	2.74 ± 0.11	2.53 ± 0.09 a	3.19 ± 0.10 a^,b	2.97 ± 0.08^a-c	3.07 ± 0.1 a^,b^,c	3.03 ± 0.09 a^,b	2.91 ± 0.10 a^,b^,e	2.70 ± 0.08 b^,c-e,g	3.11 ± 0.12 a^,b^,g^,h	2.90 ± 0.06^a-c,h^,i	2.91 ± 0.09 a^,b^,c^,e^,i	2.82 ± 0.07^c-f,i
Heart	3.01 ± 0.09	2.96 ± 0.04	3.35 ± 0.10 a^,b	3.08 ± 0.05 b^,c	3.02 ± 0.03^b-d	3.03 ± 0.06	3.00 ± 0.10	3.023 ± 0.06 c	3.11 ± 0.07 b	3.10 ± 0.04 b^,c	2.87 ± 0.12 c^,d^,h^,i^,j	3.06 ± 0.08 b^,c^,k
Lung	1.99 ± 0.04	1.98 ± 0.03	2.18 ± 0.05 a^,b	2.08 ± 0.04^a-c	2.23 ± 0.06 a^,b^,d	2.18 ± 0.05 a^,b^,d	1.97 ± 0.03^c-f	2.57 ± 0.11^a-c,e-g	2.22 ± 0.04 a^,b^,g	2.22 ± 0.06 a^,b^,d^,g	2.01 ± 0.05 e^,f^,h^,I,j	2.12 ± 0.09 b^,c
Kidney	2.76 ± 0.04	2.86 ± 0.03	2.97 ± 0.03 a^,b	2.92 ± 0.04^a-c	3.10 ± 0.08^a-c,d	2.90 ± 0.04 a^,b^,e	2.92 ± 0.06 a^,e	2.85 ± 0.05 a^,c	3.20 ± 0.04 a^,b^,d^,h	3.00 ± 0.06^a-d,f^,g-h	3.06 ± 0.04^a-c,d^,f-i	2.93 ± 0.07 a^,b^,e^,i-k
Gut	3.09 ± 0.04	3.14 ± 0.03	3.35 ± 0.06 a^,b	3.32 ± 0.04^a-c	3.21 ± 0.03^a-c,d	3.09 ± 0.08 c^,d^,e	3.01 ± 0.11^b-e	3.02 ± 0.04^b-e	3.37 ± 0.07^a-h	3.31 ± 0.05 a^,b^,f^,-h	2.95 ± 0.08 a^,b^,d-i,j	3.09 ± 0.06^c-e,I,j
Brain	1.35 ± 0.04	1.95 ± 0.06 a	2.05 ± 0.08 a^,b	2.13 ± 0.06^a-c	2.14 ± 0.05 a^,b	1.93 ± 0.05 a^,c-e	2.02 ± 0.08 a^,d^,e^,f	1.83 ± 0.08^a-f	2.09 ± 0.05 b^,f^,h	2.05 ± 0.04 a^,f^,h	2.06 ± 0.08 a^,b^,f^,h	1.90 ± 0.07 a^,c-e,i^,k
Testes	2.66 ± 0.03	_	2.85 ± 0.02 a	_	2.95 ± 0.03 a^,c	_	2.94 ± 0.09 c^,d	_	2.92 ± 0.05 a^,c	_	2.95 ± 0.04 a^,c	_
Ovary	_	2.70 ± 0.06	_	2.78 ± 0.05	_	2.90 ± 0.03 b^,d	_	2.70 ± 0.04 f	_	2.88 ± 0.03 b^,d^,h	_	2.76 ± 0.10 f^,j

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Values represent means ± 1 SD (n = 30/group; that is, 30 males and 30 females in each lighting environment per strain).

P < 0.05 compared with value for C3H mice in male CWF control group.

P < 0.05 compared with value for C3H mice in female CWF control group.

P < 0.05 compared with value for C3H mice in male bLAD experimental group.

P < 0.05 compared with value for C3H mice in female bLAD experimental group.

P < 0.05 compared with value for C57BL/6 mice in male CWF control group.

P < 0.05 compared with value for C57BL/6 mice in female CWF control group.

P < 0.05 compared with value for C57BL/6 mice in male bLAD experimental group.

P < 0.05 compared with value for C57BL/6 mice in female bLAD experimental group.

ⁱ

P < 0.05 compared with value for BALB/c mice in male CWF control group.

P < 0.05 compared with value for BALB/c mice in female CWF control group.

P < 0.05 compared with value for BALB/c mice in male bLAD experimental group.

Results

Animal-room illumination and spectral comparisons.

Animal room illumination during light phase at the center each room and at 1 m above the floor (with the detector facing upward toward the luminaires) varied minimally (n = 240 measurements). Illuminance and irradiance values were 500.78 ± 2.65 lx (204.40 ± 1.08 µW/cm²) in the control CWF room and 494.76 ± 7.11 lx (201.1 ± 2.89 µW/cm²) in the experimental bLAD room. Measurements of photometric illuminance (lux) and radiometric irradiance (μW/cm²) within the cages of each group are a result of a mean of the values taken at 6 locations within the cages with the detector facing forward (front, center, and rear on right and left sides of cage) at all cage positions to accurately account for actual ocular light levels within the cages independent of the animal location. Average interior ocular light levels showed little to no intercage or intergroup variability (n = 216 measurements per cage per rack) and are reported here for the CWF and bLAD groups, respectively, as 72.50 ± 3.52 lx (29.59 ± 1.44 µW/cm²) and 65.14 ± 6.91 lx (26.05 ± 2.76 µW/cm²). Normalized spectral power distributions of the T8 lights used in this study, as transmitted through the cage, are shown in Figure 1. The fluorescent lamp shows signature peaks in the appropriate wavelengths (545 and 612 nm) for this type of light. The LED lamp shows a standard blue LED phosphor spectral power distribution with a peak at 448 nm. In terms of total energy in the 465- to 485-nm range, the LED lamp had 65% higher emissions than the CWF lamp. Table 1 provides the calculated photon flux, irradiances, and weighted rodent photopigment illuminances for both lights used, as transmitted through the cage. The data illustrate that, although photon flux and irradiance are relatively similar in the 2 lights, there are marked differences in stimulation of the rodent photoreceptors. In terms of the potential light stimulation to the melanopsin-containing ipRGC, the LED lamp had more than 43% higher emissions than the CWF lamp. In addition, compared with the CWF lamp, the LED lamp had 45%, 28%, and 21% higher emissions for potential light stimulation of the S cones, rods, and M cones, respectively.

Figure 1. — Normalized spectral power distributions of the polychromatic blue-enriched LED (blue) and fluorescent lamp (red) light as transmitted through a standard polycarbonate, translucent laboratory mouse cage.

Animal food and water intakes and body growth measurements.

Food and water intakes and body growth rates differed significantly (P < 0.05) between the male and female mice in all strains during the course of the study (Table 2). These parameters differed significantly (P < 0.05) in C3H male and female mice maintained in the CWF compared with the bLAD lighting regimen but not in C57BL/6 or BALB/c mice (n = 90 measurements/group). On the basis of grams of intake per 100 g body weight²² in either CWF or bLAD lighting environments, female mice consumed approximately 4.6% ± 0.9% more food daily in both C3H and C57BL/6 strains and 14.8% ± 2.2% more food daily in the BALB/c strain. There were no within-sex differences in food consumption in the C57BL/6 and BALB/c strains maintained in either the CWF or bLAD lighting environments. However, for the C3H mouse strain, male and female mice in the CWF group consumed 7.1% ± 0.1% and 7.2% ± 0.9% more food daily, as compared with bLAD groups. In terms of milliliters per 100 g of body weight daily water consumption,² in either CWF or bLAD lighting environments, female mice consumed 0.7% ± 0.1% more water daily in the C3H strain and 14.3% ± 3.8% more water daily in the C57BL/6 and BALB/c strains, compared with male mice (n = 90 measurements/group). Within-sex daily water consumption was significantly (P < 0.05) greater in C57BL/6 (males, 14.0% ± 0.1%; females, 7.0% ± 0.1%) mice maintained in CWF compared with bLAD lighting environments and lower in both C3H (males, 2.4% ± 0.1%; females, 3.9% ± 0.2%) and BALB/c (males, 1.6% ± 0.3%; females, 2.4% ± 0.3%) mice.

Table 2.

Effects of CWF and bLAD lighting on food intake (g /100 g body weight daily), water intake (mL/100 g body weight daily), and animal growth rates (g/d) in C3H, C57BL/6, and BALB/c male and female mice during maintenance phase

	C3H				C57BL/6				BALB/c
	CWF		bLAD		CWF		bLAD		CWF		bLAD
	M	F	M	F	M	F	M	F	M	F	M	F
Food	19.88 ± 0.17	20.93 ± 0.13^a	18.56 ± 0.09^a,b	19.55 ± 0.06^a-c	21.09 ± 0.23^a,c,d	22.02 ± 0.22^a-e	21.44 ± 0.18^a-d,f	22.18 ± 0.26^a-g	19.26 ± 0.12^a-h	22.46 ± 0.16^a-g	19.66 ± 0.19^b-h	22.10 ± 0.17^a-e,g,i,k
Water	19.11 ± 0.06	19.13 ± 0.10	19.62 ± 0.07^a,b	19.87 ± 0.11^a-c	18.49 ± 0.25^a-d	20.82 ± 0.29^a-e	16.22 ± 0.24^a-f	19.46 ± 0.30^a-g	16.67 ± 0.19^a-h	18.64 ± 0.13^a-e,g	16.93 ± 0.13^b-f	19.08 ± 0.17^c-g,i-k
Growth	0.220 ± 0.035	0.150 ± 0.0180^a	0.111 ± 0.001^a,b	0.071 ± 0.001^a-c	0.146 ± 0.020^a,c,d	0.113 ± 0.021^a,b,d,e	0.147 ± 0.014^a,c,d,f	0.099 ± 0.010^a-g	0.120 ± 0.020^a-e,g,h	0.074 ± 0.017^a-i	0.113 ± 0.021^a-e,h-j	0.070 ± 0.011^a-i,k

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F, female; M, male

Values represent means ± 1 SD (n = 30/group; that is, 30 males and 30 females in each lighting environment per strain)

P < 0.05 compared with value for C3H mice in male CWF control group.

P < 0.05 compared with value for C3H mice in female CWF control group.

P < 0.05 compared with value for C3H mice in male bLAD experimental group.

P < 0.05 compared with value for C3H mice in female bLAD experimental group.

P < 0.05 compared with value for C57BL/6 mice in male CWF control group.

P < 0.05 compared with value for C57BL/6 mice in female bLAD experimental group

P < 0.05 compared with value for BALB/c mice in male CWF control group.

P < 0.05 compared with value for BALB/c mice in male experimental control group.

Animal growth rates (grams daily) revealed marked differences in both sexes (males gained more than females) of animals maintained in bLAD compared with CWF lighting environments in the C3H strain but not the C57BL/6 or BALB/c strains. Male and female C3H mice grew at a rate of 49.6% ± 0.8% and 52.7 ± 0.5% more slowly in bLAD compared with CWF lighting (P < 0.05), respectively, during the maintenance phase. Final body weights at time of the tissue harvest were 26.3 ± 1.8 g for males and 23.3 ± 1.2 g for females in the C3H strain, 28.9 ± 1.4 g for males and 25.0 ± 1.5 g for females in the C57BL/6 strain, and 29.3 ± 1.5 g for males and 22.3 ± 2.2 g for females in the BALB/c strain for control CWF groups, as compared with 23.3 ± 1.2 g for males and 19.9 ± 1.2 g for females in the C3H strain, 29.9 ± 1.2 g for males and 24.0 ± 0.7 g for females in the C57BL/6 strain; and 29.0 ± 1.0 g for males and 21.8 ± 0.9 g for females in the BALB/c strain for experimental bLAD groups.

Plasma melatonin levels.

Circadian rhythms in concentrations of plasma melatonin for the 3 mouse strains in CWF and LED lighting are shown in Figure 2. In each strain, plasma melatonin levels did not differ significantly between male and female mice, so values were combined. The overall patterns of daily plasma melatonin level rhythms was similar for both CWF and bLAD groups in the respective strains of mice: low during daytime (less than 4 pg/mL) and significantly (P < 0.001) higher during the dark phase, with peak levels occurring between 2400 and 0400 and decreasing to a nadir between 1200 and 1600 in C3H mice and consistently low in both the C57BL/6 and BALB/c strains throughout the 24-h day (Table 3). Significant differences in either the peak height and phase duration of the nocturnal melatonin signal between the CWF and bLAD groups were present only in the C3H mice. In the C3H strain, melatonin levels in the bLAD group began to rise rapidly after the onset of the dark phase, reaching nearly 70% of the peak of those in the CWF group (control) by 2000. However, the peak dark-phase melatonin level for C3H mice in the bLAD group (that is, at 2400) was nearly 6-fold higher (P < 0.0001) than that in control mice at the same time point. Arterial plasma melatonin levels remained more than 11-fold higher (P < 0.05) in bLAD compared with CWF, even at 2 to 3 h after the onset of light phase, and did not reach normal daytime levels (less than 10 pg/mL) until 1200. The integrated mean levels of melatonin over the 24-h period for bLAD mice (951.1 pg/mL) were more than 4-fold higher than those of animals in CWF (216.4 pg/mL; P < 0.001).

Table 3.

Summary of JTK_CYCLE analysis for mice maintained in control CWF and experimental bLAD lighting environments

	Estimated Peak Phase^a		Phase Shift^b (hrs)	Amplitude^a		Fold Change^b	Q-value for Circadian Cycling^a
	CWF	bLAD		CWF	bLAD		CWF	bLAD
C3H mice
Corticosterone	18:00	18:00	0	3.72E-05	2.27E-05	1.64	NS	2.85E-02
Glucose	14:00	14:00	0	5.41	4.90E-05	1.10E+05	4.55E-05	8.29E-11
Insulin	2:00	4:00	+2	1.94	2.20	−1.13	7.11E-08	1.46E-04
Lactate	12:00	18:00	+6	0.00	5.80E-02	—	1.29E-03	3.05E-04
Leptin	22:00	24:00	+2	0.67	0.51	1.32	7.11E-08	2.30E-10
Melatonin	02:00	02:00	0	36.94	113.06	−3.06	8.29E-11	2.60E-14
pCO₂	NE^d	22:00	—	NE^e	0.64	—	8.20E-04	NS
pO₂	14:00	14:00	0	7.78	3.89	2.00	2.97E-10	1.86E-06
TFA	04:00	04:00	0	1328.09	982.88	1.35	2.43E-22	2.43E-22
C57BL/6 mice
Corticosterone	1800	NE^d	—	2.18E–05	NE^e	—	4.42E–02	2.42E–01
Glucose	1400	1400	0	5.05	5.24	−1.04	2.77E–05	5.22E–05
Insulin	0200	0200	0	1.91	1.90	1.00	2.22E–09	1.32E–09
Lactate	1400	1400	0	0.04	0.03	1.17	7.40E–03	1.49E–02
Leptin	2200	2200	0	0.70	0.70	1.00	7.11E–08	7.11E–08
Melatonin	NE^d	1000	—	NE^e	0.16	—	NS	NS
pCO₂	1400	1400	0	9.86E–06	0.16	−1.66E+04	3.95E–03	5.97E–03
pO₂	1400	1400	0	8.49	7.78	1.09	1.41E–12	1.09E–13
TFA	0400	0400	0	719.97	700.74	1.03	1.33E–20	4.73E–21
BALB/c mice
Corticosterone	NE^d	1800	—	NE^e	3.23E–05	—	NS	1.91E–02
Glucose	1400	1400	0	5.47	5.87	−1.07	4.55E–05	3.78E–05
Insulin	0200	0200	0	1.88	1.87	1.01	7.04E–10	9.00E–10
Lactate	NE^d	1400	—	NE^d	0.03	—	1.44E–02	5.24E–03
Leptin	0200	0200	0	0.60	0.66	−1.11	2.07E–11	6.13E–12
Melatonin	1000	0200	+8	0.34	37.32	−109.47	NS	6.82E–13
pCO₂	1400	1400	0	5.00E–15	0.07	−1.42E+13	5.97E–03	2.29E–03
pO₂	1400	1400	0	7.78	8.49	−1.09	2.06E–10	2.99E–14
TFA	0400	0400	0	1156.38	1074.34	1.08	2.43E–22	2.92E–17

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NS, not significant

Phase-, amplitude-, and multiple-testing-adjusted P value (Q) estimated by using JTK_CYCLE analysis with a fixed 24-h period and original units described in the text.

Phase difference and fold change are for the bLAD group relative to the the CWF group. A decrease in bLAD amplitude with regard to CWF is a negative value; an increase is represented by a positive value.

NE, not estimated (JTK_CYCLE analysis did not converge on a single peak phase or amplitude; a value was not provided)

Plasma measures of TFA.

Circadian rhythms in the concentrations of arterial blood plasma TFA were measured in mice with free access to the food (Figure 3). As a rule, the integrative mean of plasma TFA for each strain over a 24-h day (mg/mL) was C3H > BALB/c > C57BL/6. The plasma pattern of lipid levels in the experimental bLAD mice followed that of the control animals in all strains, similar to that reported earlier in rats.^19,21,22 Daily levels in TFA between male and female mice were significantly (P < 0.001)different in all 3 strains (Figure 3 A through C) in both CWF and bLAD groups. Total TFA areas assessed over the 24-h day for the C3H within-sex curves (Figure 3 A) were 14.17 ± 0.20 mg/mL for males and 10.79 ± 0.14 mg/mL for females in CWF groups and 10.76 ± 0.14 mg/mL for males and 8.39 ± 0.12 mg/mL for females in bLAD groups. Total TFA areas assessed over the 24-h day for the C57BL/6 (Figure 3 B) and BALB/c (Figure 3 C) mouse curves shown for CWF compared with bLAD revealed no within-sex differences and were, respectively, 7.89 ± 0.10 mg/mL for males and 7.92 ± 0.10 mg/mL for females compared with 7.32 ± 0.10 mg/mL for males and 7.44 ± 0.10 mg/mL for females in the C57BL/6 strain, respectively, and 12.68 ± 0.20 mg/mL for males and 12.81 ± 0.20 mg/mL compared with 11.53 ± 0.15 mg/mL for males and 11.58 ± 0.15 mg/mL for females in the BALB/c strain, respectively. Circadian cycling was evident for both CWF and bLAD groups in all strains of mice, with a severely dampened amplitude during daytime (Table 3).

Arterial blood glucose, lactate, acid-gas levels.

Figure 4 depicts the combined values for male and female 24-h rhythms in levels of arterial blood glucose, lactate, pO₂, and pCO₂ in C3H (Figure 4 A), C57BL/6 (Figure 4 B), and BALB/c (Figure 4 C) mice. Phase shifts were determined by comparing the peak values (acrophases) between the mice in bLAD (experimental) and CWF lighting regimens. A ‘phase advance’ was defined as a shift in a group peak level to an earlier time (for example, from 1600 to 1200), whereas a ‘phase delay’ was defined as a shift in a group to a later time (for example, from 0800 to 1200), as compared with control values. Daily rhythms for arterial glucose and lactate concentrations (Figure 4 A and B) were equivalent between CWF and bLAD groups for all strains, with peaks for both constituents occurring at 1200 and 2000 and bathyphase occurring between 2400 and 0400 (Table 3). In contrast to the C57BL/6 and BALB/c strains, however, the integrative mean over the 24-h day for C3H control mice was higher compared with that in the bLAD experimental group. C3H mice were the only strain that displayed significant differences in the arterial blood chemistries measured in the study; the average arterial blood glucose and lactate concentrations calculated over the 24-h day were 916 ± 3 mg/dL and 7.1 ± 0.1 mmol/L, respectively, for the control group and 828 ± 2 mg/dL and 6.84 ± 0.03 mmol/L, respectively, for the experimental group (P < 0.0006). The integrative mean arterial blood pO₂ and pCO₂ concentrations calculated over the 24-h day were 977 ± 3 mm Hg and 202 ± 1 mm Hg for the control group and 944 ± 2 mm Hg and 190 ± 0.3 mm Hg for the experimental group (P < 0.007). In C57BL/6 mice, the average arterial blood glucose and lactate concentrations calculated over the 24-h day were 996 ± 2 mg/dL and 7.8 ± 0.1 mmol/L for the control group and 997 ± 3 mg/dL and 7.81 ± 0.04 mmol/L for the experimental group. The integrative mean arterial blood pO₂ and pCO₂ concentrations calculated over the 24-h day were 974 ± 2 mm Hg and 202 ± 1 mm Hg for the control group and 970 ± 2 mm Hg and 202 ± 1 mm Hg for the experimental group. In BALB/c mice, the average arterial blood glucose and lactate concentrations calculated over the 24-h day were 998 ± 3 mg/dL and 7.80 ± 0.01 mmol/L for the control group and 1003 ± 3 mg/dL and 7.84 ± 0.01 mmol/L for the experimental group. The integrative mean arterial blood pO₂ and pCO₂ concentrations calculated over the 24-h day were 973 ± 2 mm Hg and 203 ± 1 mm Hg for the control group and 973.3 ± 1.1 mm Hg and 202.3 ± 0.6 mm Hg for the experimental group.

Arterial blood pH and O₂ saturation, remained relatively constant for both sexes of all strains of both CWF and bLAD groups over the 24-h day, at 7.43% ± 0.17% and 99.2% ± 0.2%, respectively (n = 360). Cumulative mean arterial Hct values were 45.8% ± 0.20% for males and 44.0 ± 0.30% for females in the C3H strain, 51.7% ± 0.20% for males and 51.0% ± 0.30% for females in the C57BL/6 strain, and 45.7% ± 0.20% for males and 44.9% ± 0.20% for females in the BALB/c strain. Mean arterial Hgb values (n = 60 per sex) were 15.7 ± 0.2 g/dL for males and 15.1 ± 0.1 g/dL for females in the C3H strain, 15.9 ± 0.20 g/dL for males and 16.3 ± 0.3 g/dL for females in the C57BL/6 strain, and 13.5 ± 0.2 g/dL for males and 14.7 ± 0.3 g/dL for females in the BALB/c strain.

Plasma measures of corticosterone, insulin, and leptin.

Figure 5 depicts 24-h rhythms in concentrations of arterial blood plasma corticosterone in male and female C3H, C57BL/6, and BALB/c mice (Table 3). Plasma corticosterone levels revealed clear differences between the CWF and bLAD groups, with regard to integrative concentrations, and the amplitude of both the primary and secondary peaks was significantly (P < 0.05) lower in the bLAD group than in the CWF group, with the general trend following C57BL/6 > BALB/c > C3H (female > male). Values for arterial plasma corticosterone in all mouse strains of both groups began to increase after 1200 (P < 0.05), with a major peak value occurring at 1600 (secondary peak; P < 0.05) in the experimental and control groups, decreasing to a low value at 2000 (P < 0.05) for both CWF and bLAD groups in all strains and sexes. A second minor peak occurred in both CWF and bLAD groups at 0400 (but was higher in control CWF mice), decreasing to a nadir at 0800 for both groups. Integrated plasma corticosterone concentrations calculated over the 24-h day (male/female) in C57BL/6, BALB/c, and C3H strains, respectively, were 1069.5 ± 47.3 ng/mL for male mice and 2640.5 ± 114.2 ng/mL for female mice, 852.0 ± 37.5 ng/mL for male mice and 2462.5 ± 107.7 ng/mL for female mice, and 665.0 ± 29.2 ng/mL for male mice and 1663.5 ± 72.8 ng/mL for female mice in the CWF animals, as compared with 1153.5 ± 50.5 ng/mL for male mice and 2524.0. ± 110.5 ng/mL for female mice, 890.0 ± 39.0 ng/mL for male mice and 2515.5 ± 110.2 ng/mL for female mice, and 540.0 ± 20.2 ng/mL for male mice and 1221.5 ± 53.4 ng/mL for female mice, respectively, in bLAD animals.

Plasma concentrations of insulin (Figure 6) showed clear intergroup differences with regard to 24-h rhythms and integrative levels in male and female C3H, C57BL/6, and BALB/c mice (Table 3). Plasma insulin levels revealed clear differences between the CWF and bLAD groups, with regard to integrative concentrations, and the amplitude of both the primary and secondary peaks was significantly lower (P < 0.05) in the bLAD group than in the CWF group, with the general trend following C57BL/6 > C3H > BALB/c (male > female). Values for arterial plasma insulin in all mouse strains of both groups began to increase sharply after 1600 (P < 0.05), with a major peak value occurring at 2000 for both CWF and bLAD groups in all strains and sexes (P < 0.001), decreasing gradually throughout the night phase and into the light phase to a nadir at 1200 (P < 0.001). Integrated plasma insulin concentrations calculated over the 24-h day in C57BL/6, BALB/c, and C3H strains, respectively, were 5.56 ± 0.49 ng/mL for male mice and 2.43 ± 0.17 ng/mL for female mice, 5.16 ± 0.45 ng/mL for male mice and 2.31 ± 0.16 ng/mL for female mice, and 5.29 ± 0.53 ng/mL for male mice and 2.53 ± 0.16 ng/mL for female mice in the CWF groups, as compared with 5.72 ± 0.37 ng/mL for male mice and 2.41 ± 0.18 ng/mL for female mice, 5.33 ± 0.49 ng/mL for male mice and 2.28 ± 0.17 ng/mL for female mice, and 3.22 ± 0.34 ng/mL for male mice and 1.31 ± 0.13 ng/mL for female mice, respectively, in bLAD groups. Integrated plasma leptin concentrations calculated over the 24-h day in C57BL/6, C3H, and BALB/c mice, respectively, were 5.56 ± 0.49 ng/mL for male mice and 2.43 ± 0.17 ng/mL for female mice, 5.16 ± 0.45 ng/mL for male mice and 2.31 ± 0.16 ng/mL for female mice, and 5.29 ± 0.53 ng/mL for male mice and 2.53 ± 0.16 ng/mL for female mice in the CWF animals, as compared with 5.72 ± 0.37 ng/mL for male mice and 2.41 ± 0.18 ng/mL for female mice, 5.33 ± 0.49 ng/mL for male mice and 2.28 ± 0.17 ng/mL for female mice, and 3.22 ± 0.34 ng/mL for male mice and 1.31 ± 0.13 ng/mL for female mice, respectively, in bLAD animals.

Plasma concentrations of leptin (Figure 7) revealed clear intergroup differences with regard to 24-h rhythms (Table 3) and integrative levels in male and female C3H, C57BL/6, and BALB/c mice. For male and female mice in groups (C3H > BALB/c > C57BL/6) in either CWF or bLAD lighting environments (female > male), values for arterial plasma leptin began to increase 2 h after the onset of the dark phase (P < 0.05), with peak levels occurring at 2400 and gradually decreasing to a nadir at 1200 (P < 0.05) in controls. Integrated plasma leptin concentrations calculated over the 24-h day in C3H, BALB/c, and C57BL/6 strains, respectively, were 39.18 ± 2.02 ng/mL for male mice and 47.84 ± 2.60 ng/mL for female mice, 27.45 ± 1.87 ng/mL for male mice and 37.23 ± 2.18 ng/mL for female mice, and 21.45 ± 1.79 ng/mL for male mice and 30.88 ± 2.15 ng/mL for female mice in the CWF animals, as compared with 23.90 ± 1.69 ng/mL for male mice and 31.30 ± 2.04 ng/mL for female mice, 26.95 ± 1.99 ng/mL for male mice and 38.15 ± 2.15 ng/mL for female mice, and 21.18 ± 1.89 ng/mL for male mice and 31.90 ± 2.11 ng/mL for female mice, respectively, in bLAD animals. Again, circadian cycling for all 3 neurohormones was evident in all strains, with the general trend toward high levels near the end of the light phase and markedly dampened during dark phase (Table 3).

Major tissue organ weight, total fatty acid, protein, and cAMP content.

Table 4 depicts mean weights (in grams) of excised organs (liver, left retroperitoneal fat pad, left quadricep femoris muscle, heart, lungs, kidneys, small intestine, brain, testes, and ovaries) at the time of harvest (age, 12 wk) from male and female C3H, C57BL/6, and BALB/c mice maintained in either CWF control or bLAD experimental groups. With the exception of the fat pad and brain, organs collected from male mice were significantly (P < 0.05) heavier than those of female mice, proportional to the final body weights in these animals. Overall, normalizing for sex, strain, and final body weights, the combined (male and female) weight for all 10 major organs in the CWF control group was 7.89% ± 0.01% and 7.96 ± 0.01% higher, respectively, than that of the bLAD experimental group in the C3H mouse strain (P < 0.05). This was not the case with either the C57BL/6 or BALB/c strains, in which this value did not differ significantly between lighting conditions. In general, organ weight was proportional to the final body weight (male > female) for each strain (C3H > C57BL/6 > BALB/c), and the cumulative tissue mass for all tissues collected were 4.69 ± 0.01 g for male mice and 4.13 ± 0.004 g for female mice, 4.49 ± 0.01 g for male mice and 3.93 ± 0.01 g for female mice, and 4.00 ± 0.01 g for male mice and 3.91 ± 0.004 g for female mice, respectively, in the control CWF group, as compared with 4.34 ± 0.01 g for male mice and 3.83 ± 0.004 g for female mice; 4.40 ± 0.01 g for male mice and 3.84 ± 0.01 g for female mice, and 4.27 ± 0.01 g for male mice and 3.78 ± 0.01 g for female mice, respectively, in the experimental bLAD group.

Table 4.

Effects of CWF and bLAD lighting on animal organ wet weight (g) of liver, retroperitoneal fat pad (1), quadricep femoris muscle (1), heart, lung (2), kidney (2), gut, brain, testes (2), and ovary (2) tissues in C3H, C57BL/6, and BALB/c male and female mice.

	C3H				C57BL/6				BALB/c
	CWF		bLAD		CWF		bLAD		CWF		bLAD
Tissue	M	F	M	F	M	F	M	F	M	F	M	F
Liver	1.870 ± 0.038	1.650 ± 0.015^a	1.728 ± 0.032^a,b	1.502 ± 0.013^a-c	1.606 ± 0.026^b,c,d	1.466 ± 0.021^a,c,d,e	1.512 ± 0.013^a-c,e,f	1.368 ± 0.026^a-g	1.610 ± 0.022^a-d,f,g,h	1.472 ± 0.019^a-e,g,h,i	1.506 ± 0.025^a-e,h,i	1.350 ± 0.038^a-g,i,j,k
Fat	0.060 ± 0.009	0.123 ± 0.008^a	0.047 ± 0.006^a,b	0.103 ± 0.004^a-c	0.054 ± 0.006^b,d	0.120 ± 0.006^a,c,d,e	0.055 ± 0.008^b,d,f	0.121 ± 0.004^a,c,d,e,g	0.057 ± 0.010^b,d,f,h	0.122 ± 0.0 07^a,c,d,e,g,i	0.055 ± 0.005^b,d,f,h,i	0.121 ± 0.002^{a,c,d,e,g,i,k}
Muscle	0.179 ± 0.003	0.167 ± 0.002^a	0.187 ± 0.002^a,b	0.176 ± 0.002^b,c	0.168 ± 0.001^a,c,d	0.133 ± 0.002^a-e	0.168 ± 0.001^a,c,d,f	0.134 ± 0.002^a-e,g	0.170 ± 0.001^a-h	0.153 ± 0.001^a-i	0.171 ± 0.001^a-h,j	0.155 ± 0.001^a-i,k
Heart	0.131 ± 0.001	0.115 ± 0.001^a	0.124 ± 0.001^a,b	0.112 ± 0.001^a-c	0.211 ± 0.001^a-d	0.172 ± 0.002^a-e	0.212 ± 0.002^a-c,d,f	0.173 ± 0.001^a-e,g	0.151 ± 0.001^a-h	0.122 ± 0.001^a,b,d-i	0.151 ± 0.001^a,-h,j	0.122 ± 0.002^a,b,d-i,k
Lung	0.188 ± 0.001	0.180 ± 0.001^a	0.179 ± 0.001^a,b	0.175 ± 0.001^a-c	0.191 ± 0.001^a-d	0.181 ± 0.001^a,c,d,e	0.191 ± 0.001^a-d,f	0.181 ± 0.001^a,d,e,g	0.191 ± 0.001^a-d,f,h	0.180 ± 0. 001^a,d,e,g,i	0.191 ± 0.001^a-c,d,f,h,j	0.179 ± 0.002^a,d,e,g,i,k
Kidney	0.460 ± 0.002	0.276 ± 0.001^a	0.412 ± 0.002^a,b	0.251 ± 0.002^a-c	0.424 ± 0.002^a-d	0.266 ± 0.002^a-e	0.425 ± 0.001^a-d,f	0.267 ± 0.002^a-e,g	0.444 ± 0.002^a-g	0.285 ± 0.001^a-i	0.445 ± 0.001^a-h,j	0.285 ± 0.002^a-i,k
Gut	1.219 ± 0.007	1.192 ± 0.0.009^a	1.116 ± 0.007^a,b	1.085 ± 0.007^a-c	1.192 ± 0.008^a,c,d	1.165 ± 0.006^a-e	1.194 ± 0.005^a,c,d,f	1.166 ± 0.006^a-e,g	1.200 ± 0.005^a-d,f,h	1.180 ± 0.003^a-i	1.201 ± 0.008^a-d,f,h,j	1.181 ± 0.005^a-i,k
Brain	0.421 ± 0.002	0.425 ± 0.002^a	0.416 ± 0.002^a,b	0.420 ± 0.001^b,c	0.416 ± 0.001^a,b,d	0.421 ± 0.002^a-c,e	0.413 ± 0.002^a-c,f	0.419 ± 0.001^b,e,g	0.376 ± 0.002^a-h	0.386 ± 0.001^a-i	0.371 ± 0.001^a-j	0.383 ± 0.001^a-i,k
Testes	0.160 ± 0.020	_	0.134 ± 0.024^a	_	0.233 ± 0.020^a,c	_	0.230 ± 0.030^a,c	_	0.180 ± 0.023^a,c,e,g	_	0.183 ± 0.019^a,c,e,g	_
Ovary	_	0.0051 ± 0.0008	_	0.0044 ± 0.0006^b	_	0.0073 ± 0.0027^b,d	_	0.0074 ± 0.0019^b,d	_	0.0056 ± 0.0017^d,f,h	_	0.0056 ± 0.0014^d,f,h