Light modulates glucose and lipid homeostasis via the sympathetic nervous system

Abstract

Light is an important environmental factor for vision and for diverse physiological and psychological functions. Light can also modulate glucose metabolism. Here, we show that in mice, light is critical for glucose and lipid homeostasis by regulating the sympathetic nervous system, independent of circadian disruption. Light deprivation from birth elicits insulin hypersecretion, glucagon hyposecretion, lower gluconeogenesis, and reduced lipolysis by 6 to 8 weeks in male, but not female, mice. These metabolic defects are consistent with blunted sympathetic activity, and indeed, sympathetic responses to a cold stimulus are substantially attenuated in dark-reared mice. Further, long-term dark rearing leads to body weight gain, insulin resistance, and glucose intolerance. Notably, metabolic dysfunction can be partially alleviated by 5 weeks exposure to a regular light-dark cycle. These studies provide insight into circadian-independent mechanisms by which light directly influences whole-body physiology and better understanding of metabolic disorders linked to aberrant environmental light conditions.

Fig. 1.. Dark rearing from birth results in increased insulin secretion in male mice at 6 to 8 weeks.

(A) Mice were reared in constant darkness (DD) or in a 12:12 light-dark (LD) cycle from postnatal day 0 (P0). White, subjective day for LD mice; dark gray, subjective night; light gray, subjective day for DD mice. (B and C) Representative actograms showing intact endogenous circadian rhythms in LD (B) or DD (C) mice, as assessed by locomotor activity. (D) Total locomotor activity is comparable between LD and DD mice measured by the total number of infrared monitor crossings per day. n = 10 for LD and 9 for DD mice, unpaired t test. (E) Total food intake (g/day) is similar between LD and DD mice. n = 8 for each group, unpaired t test. (F and G) 6 to 8 weeks of dark rearing increases plasma insulin levels in male mice in GSIS test. Area under the curve (AUC) of (F) is shown in (G). n = 11 for each group, two-way ANOVA, Sidak’s multiple-comparisons tests for (F), and unpaired t test for (G). (H) Fasting plasma insulin levels are elevated in male DD mice at 6 to 8 weeks. n = 10 LD and 9 DD mice in fed condition, n = 25 LD and 22 DD mice in fasting condition, unpaired t test. (I and J) Glucose tolerance is mildly impaired in male DD mice after 6 to 8 weeks of dark rearing. AUC of (I) is shown in (J). n = 8 LD and 9 DD mice, two-way ANOVA, Sidak’s multiple-comparisons tests for (I) and unpaired t test for (J). (K and L) Insulin sensitivity is unaffected by dark rearing for 6 to 8 weeks. Area over the glucose curve of (K) is shown in (L). n = 10 LD and 8 DD mice. Two-way ANOVA, Sidak’s multiple-comparisons tests for (K) and unpaired t test for (L). Results are means ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001; n.s, not significant.

Fig. 2.. Dark-reared mice show defects in glucagon secretion, gluconeogenesis, and plasma NEFA levels.

(A and B) Glucagon secretion tests. Male DD mice show decreased glucagon secretion in response to insulin-induced hypoglycemia. AUC of (A) is shown in (B). Results are means ± SEM with n = 9 mice for LD and 7 for DD. *P < 0.05 and **P < 0.01, two-way ANOVA, Sidak’s multiple-comparisons tests for (A) and unpaired t test for (B). (C and D) Pyruvate tolerance tests show decreased gluconeogenesis in male DD mice after pyruvate injections. AUC of (C) is shown in (D). Results are means ± SEM with n = 8 mice for LD and 10 for DD. *P < 0.05, **P < 0.01, and ***P < 0.001, two-way ANOVA, Sidak’s multiple-comparisons tests for (C) and unpaired t test for (D). (E and F) Lipolysis tests. Plasma nonesterified fatty acids (NEFA) levels measured by milliequivalents per liter (mEq/liter) are elevated after fasting in both LD and DD animals (E). However, the fold increase in fasting plasma NEFA levels compared to the fed state is lower in DD animals (F). NEFA fold change of each animal is calculated by dividing the fasting level by the fed level. Results are means ± SEM with n = 11 mice for LD and 9 for DD. *P < 0.05 and ***P < 0.001, two-way ANOVA, Sidak’s multiple-comparisons tests for (E) and unpaired t test for (F).

Fig. 3.. Sympathetic responses are attenuated in dark-reared mice.

(A to D) c-Fos immunostaining in the celiac-superior mesenteric ganglion complex (CG-SMG) of LD and DD mice. RT, room temperature; cold, 4°C for 1 hour. Scale bars, 100 μm. (E) Quantification of c-Fos–positive sympathetic neurons in CG-SMG from LD and DD mice at RT and in response to cold exposure (4°C, 1 hour). The numbers of c-Fos–positive neurons are similar between 6- to 8-week-old mice raised in LD and DD at RT. Cold exposure significantly increases number of c-Fos–positive neurons in mice reared in LD, but not, in DD. Data are presented as means ± SEM with n = 5 mice for LD and 6 for DD at RT; n = 10 mice for LD and 9 for DD under cold exposure. **P < 0.01; n.s, not significant, two-way ANOVA, Sidak’s multiple-comparisons tests. (F) Circulating norepinephrine (NE) levels of LD and DD mice. Cold exposure (4°C, 2 hours) significantly increases circulating NE in LD, but not DD, animals. Basal NE levels at RT are similar between LD and DD mice. Data are means ± SEM for n = 4 mice for LD and 5 mice for DD. **P < 0.01, two-way ANOVA, Sidak’s multiple-comparisons tests. (G) Glucose-stimulated insulin secretion (GSIS) tests in DBH-Cre;TeNT^fl/+ mice. Plasma insulin levels are elevated in DBH-Cre;TeNT^fl/+ mice compared to littermate controls (TeNT ^fl/+) at 6 to 8 weeks of age. Results are means ± SEM with n = 6 control and four mutant mice. *P < 0.05 and ***P < 0.001, unpaired t test. (H) Basal and GSIS in isolated islets. Secreted insulin normalized to total insulin content is similar between islets isolated from DBH-Cre;TeNT^fl/+ mice and litter-mate controls (TeNT ^fl/+). Results are means ± SEM for islets isolated from n = 7 control and 6 mutant mice. ***P < 0.001, unpaired t test.

Fig. 4.. Dark-reared animals develop insulin resistance, glucose intolerance, and gain in body weight with age.

(A and B) Insulin sensitivity tests. Dark-reared male mice become insulin resistant by 3 months. Data are represented as percentages (%) of the blood glucose level at time “0.” Area over the glucose curve of (A) is shown in (B). The 100% blood glucose level at time 0 is set as the baseline. Results are means ± SEM for n = 9 mice for LD and 8 for DD. **P < 0.01, two-way ANOVA, Sidak’s multiple-comparisons tests for (A) and unpaired t test for (B). (C and D) Glucose tolerance tests. Dark-reared male mice develop glucose intolerance at 6 to 8 months. AUC of (C) is shown in (D). Results are means ± SEM with n = 8 mice for LD and 10 for DD. *P < 0.05 and **P < 0.01, two-way ANOVA, Sidak’s multiple-comparisons tests for (C) and unpaired t test for (D). (E) Body weight of LD and DD mice. Dark rearing causes increased body weight with age in male mice. Results are means ± SEM with n = 6 to 32 mice for LD and 8 to 20 mice for DD. **P < 0.01, ***P < 0.001, and ****P < 0.0001, two-way ANOVA, Sidak’s multiple-comparisons tests. (F) Weight of epididymal white adipose tissue (WAT) of LD and DD mice. Dark-reared male mice accumulate WAT compared to mice raised in LD cycle at 6 to 8 months measured by percentage of the epididymal WAT weight normalized by the body weight. Results are means ± SEM for n = 5 each group. **P < 0.01, unpaired t test. (G and H) H&E staining shows that adipocytes are larger in DD animals (H) compared to LD (G) at 6 to 8 months. Scale bars, 100 μm. (I) Quantification of average adipocyte size (μm²) in LD versus DD animals. Results are means ± SEM for n = 3 mice for each group. *P < 0.05, unpaired t test.

Fig. 5.. Exposing dark-reared animals to a regular light/dark cycle partially alleviates metabolic defects.

(A) Schematic diagram showing that mice were reared in DD from P0 for 10 months and then moved to a regular 12:12 LD light condition for another 5 weeks. Metabolic tests were performed immediately before, and after, the 5 weeks of LD exposure. Light gray, subjective day for DD phase; dark gray, subjective night; white, subjective day for LD phase. (B) Body weights were unaffected by 5 weeks of LD exposure in animals that were reared in DD for 10 months. Results are means ± SEM for n = 17 mice. n.s, not significant, paired t test. (C) Fasting glucose levels were reduced after 5 weeks of LD exposure in mice dark-reared for 10 months. Results are means ± SEM for n = 9 mice. *P < 0.05, paired t test. (D and E) Glucose tolerance was improved after 5 weeks of exposure to a regular LD cycle. AUC of (D) is shown in (E). Results are means ± SEM for n = 9 mice. **P < 0.01; two-way ANOVA, Sidak’s multiple-comparisons tests for (D) and paired t test for (E). (F and G) Insulin sensitivity was improved by 5 weeks of LD exposure. Data are represented as percentages (%) of the blood glucose level at time “0.” Area over the glucose curve of (F) is shown in (G). The 100% blood glucose level at time 0 is set as the baseline. Results are means ± SEM for n = 7 mice. *P < 0.05 and **P < 0.01, two-way ANOVA, Sidak’s multiple-comparisons tests for (F) and unpaired t test for (G).