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 6-8 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 significantly 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 inform new approaches for understanding metabolic disorders linked to aberrant environmental light conditions.

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

(A) Schematic showing the experimental paradigm for animals reared in constant darkness (DD) from birth (Postnatal day 0, P0), compared to control animals reared in a 12:12 light-dark (LD) cycle. White, subjective day for control mice; dark grey, subjective night; light grey, subjective day for dark-reared mice. Metabolic tests were performed at 6–8 weeks, 3 months and 6–8 months after birth. (B) Representative actogram showing intact endogenous circadian rhythms in dark-reared mice, as assessed by locomotor activity. Activity is counted by the times that animal crosses the monitor in every 15 seconds. (C) Total locomotor activity is comparable between LD and DD mice. The total activity of an animal is measured by the total number of infrared monitor crossings per day. Results are means ± s.e.m with n=10 for LD and 9 for DD mice. n.s, not significant, unpaired t-test. (D) Total food intake (g/day) is similar between LD and DD mice. Results are means ± s.e.m with n=8 for each group. n.s, not significant, unpaired t-test. (E, F) Glucose-stimulated insulin secretion (GSIS) test. 6–8 weeks of dark-rearing increases plasma insulin levels in male mice. Area under the curve (AUC) of (E) is shown in (F). Data are as means ± s.e.m with n=11 for each group. *p<0.05; **p<0.01; ***p<0.001, two-way ANOVA, Sidak’s multiple comparisons tests for (E) and unpaired t-test for (F). (G) Plasma insulin levels. Fasting plasma insulin levels are elevated in male DD mice at 6–8 weeks. Data are means ± s.e.m for n=10 LD and 9 DD animals in fed condition, n=25 LD and 22 DD animals in fasting condition. *p<0.05, n.s, not significant, unpaired t-test. (H, I) Glucose tolerance tests. Glucose tolerance is mildly impaired in male DD mice after 6–8 weeks of dark rearing. AUC of (H) is shown in (I). Results are means ± s.e.m for n=8 LD and 9 DD mice. * p<0.05; **p<0.01, two-way ANOVA, Sidak’s multiple comparisons tests for (H) and n.s, not significant, unpaired t-test for (I). (J, K) Insulin sensitivity tests. Insulin sensitivity is unaffected by dark rearing for 6–8 weeks. Data are represented as percentages (%) of the blood glucose level at time “0”. Area over the glucose curve of (J) is shown in (K). The 100% blood glucose level at time 0 is set as the baseline. Results are means ± s.e.m with n=10 LD and 8 DD mice. Two-way ANOVA, Sidak’s multiple comparisons tests for (J) and n.s, not significant, unpaired t-test for (K).

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

(A, B) Glucagon secretion tests. Male DD mice show decreased glucagon secretion in response to insulin-induced hypoglycemia. Area under the curve (AUC) of (A) is shown in (B). Results are means ± s.e.m with n=9 mice for LD and 7 for DD. *p<0.05, **p<0.01, two-way ANOVA, Sidak’s multiple comparisons tests for (A) and unpaired t-test for (B). (C, D) Pyruvate tolerance tests show decreased gluconeogenesis in male DD mice after pyruvate injections. AUC of (C) is shown in (D). Results are means ± s.e.m with n=8 mice for LD and 10 for DD. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA, Sidak’s multiple comparisons tests for (C) and unpaired t-test for (D). (E, F) Lipolysis tests. Plasma non-esterified fatty acids (NEFA) levels measured by milliequivalents per liter (mEq/L) 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 ± s.e.m with n=11 mice for LD and 9 for DD. *p<0.05, ***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-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 hr. 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 hr). The numbers of c-Fos-positive neurons are similar between 6–8-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 ± s.e.m 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 hr) significantly increases circulating NE in LD, but not DD, animals. Basal NE levels at room temperature are similar between LD and DD mice. Data are means ± s.e.m for n=4 mice for LD and 5 mice for DD. **p<0.01 n.s, not significant, 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 litter-mate controls (TeNT^fl/+) at 6–8 weeks of age. Results are means ± s.e.m with n=6 control and 4 mutant mice. *p<0.05, ***p<0.001, n.s, not significant, unpaired t-test. (H) Basal and glucose-stimulated insulin secretion 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 ± s.e.m for islets isolated from n= 7 control and 6 mutant mice. ***p<0.001, n.s, not significant, unpaired t-test.

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

(A, 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 ± s.e.m 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, D) Glucose tolerance tests. Dark-reared male mice develop glucose intolerance at 6–8 months. Area under the curve (AUC) of (C) is shown in (D). Results are means ± s.e.m with n=8 mice for LD and 10 for DD. *p<0.05; **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 ± s.e.m with n=6–32 mice for LD and 8–20 mice for DD. ** p<0.01; *** p<0.001; **** 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 6–8 months measured by percentage of the epididymal WAT weight normalized by the body weight. Results are means ± s.e.m for n=5 each group. ** p<0.01, unpaired t-test. (G, H) H&E staining shows that adipocytes are larger in DD animals (H) compared to LD (G) at 6–8 months. Scale bar: 100 μm. (I) Quantification of average adipocyte size (μm²) in LD versus DD animals. Results are means ± s.e.m 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 grey, subjective day for DD phase; dark grey, 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 ± s.e.m 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 ± s.e.m for n=9 mice. *p<0.05, paired t-test. (D, E) Glucose tolerance was improved after 5 weeks of exposure to a regular LD cycle. Area under the curve (AUC) of (D) is shown in (E). Results are means ± s.e.m for n=9 mice. **p<0.01; two-way ANOVA, Sidak’s multiple comparisons tests for (D) and paired t-test for (E). (F, 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 ± s.e.m for n=7 mice. * p<0.05; **p<0.01, two-way ANOVA, Sidak’s multiple comparisons tests for (F) and unpaired t-test for (G).