The suppression of ghrelin signaling mitigates age-associated thermogenic impairment

Results

Obestatin increases UCP1 expression in HIB1B cells, but DAG has no effect

Our previous study showed that ghrelin inhibits adipogenesis and suppresses UCP1 expression in brown adipocyte HIB1B cells [34]. DAG and obestatin have been reported to have different physiological effects from ghrelin in several cell types and tissues [40, 41, 46, 47]. To assess the effects of DAG and obestatin on thermogenesis, we treated differentiated HIB1B cells with different concentrations of DAG or obestatin. Consistent with our previous report [34], 1 nM ghrelin inhibits UCP1 expression in differentiated HIB1B cells (Fig. 1A). In contrast, DAG had no effect on UCP1 expression even at high concentration (Fig. 1B), but obestatin increased UCP1 gene expression in a dose-dependent manner (Fig. 1C). Ghrelin and obestatin showed opposite effects on UCP1 expression in brown adipocytes (Fig. 1D), suggesting that ghrelin and obestatin may have opposing effects on thermogenesis.

Figure 1. Ghrelin and obestatin exhibit differential effects on UCP1 expression in differentiated brown adipocyte HIB1B cells. UCP1 expression in HIB1B cells treated with saline or different concentrations of ghrelin (A), des-acyl ghrelin, DAG (B), and obestatin (C). Summary of UCP1 expression in HIB1B cells treated with saline, 1 nM ghrelin, 10 nM DAG, or 10 nM obestatin (D). N = 9, and each assay was measured in triplicate. *p<0.05, Treatments vs. Controls.

Circulating ghrelin is increased in aging, but DAG and obestatin are not changed

To assess whether ghrelin, DAG and obestatin regulate thermogenesis and insulin resistance during aging, we measured circulating ghrelin, DAG, obestatin and insulin concentrations in young, middle-aged and old wild-type (WT) mice, under either fed or fasting conditions. Under fed condition, ghrelin concentration in old mice was significantly higher than young and middle-aged mice (Fig. 2A). Interestingly, fasting only increased ghrelin in young and middle-aged mice, but not old mice (Fig. 2A), suggesting that ghrelin regulation in old mice is less responsive to fasting. The concentrations of DAG and obestatin didn't change during aging (Fig. 2B and 2C); while fasting did not affect DAG (Fig. 2B), fasting significantly decreased obestatin in middle-aged and old mice (Fig. 2C). Elevated ghrelin in aging increases lipolysis and exacerbates insulin resistance [39]. Consistently, our data showed that fed insulin levels were higher in middle-aged and old mice relative to young mice, and fasting reduced insulin (Fig. 2D). The data support that increased ghrelin in old mice promotes age-associated insulin resistance, which may further impair BAT function in aging.

Figure 2. Plasma concentrations of active ghrelin, des-acylated ghrelin, and obestatin during aging. Active ghrelin (A), des-acyl ghrelin, DAG (B), obestatin (C), and insulin (D) of young, middle-aged and old WT mice under fed or fasting conditions. Young (4-months), middle-aged (10-months) and old (22-months) mice were used. The mice were fasted overnight. N = 10-12. *p<0.05, fed vs. fasting. #p<0.05, ## P<0.001, young vs. middle-aged or old.

GHS-R ablation attenuates age-associated decline of thermogenesis and activates insulin signaling in BAT

Similar to our previous reports [34, 45], energy expenditure of WT mice decreased with age in both light and dark cycles, and GHS-R ablation increased energy expenditure in middle-aged and old mice (Fig. 3A). GHS-R ablation did not alter food intake or physical activity in any age group, indicating that GHS-R exerts its effects primarily by regulating energy expenditure. We studied GHS-R expression in BAT of young, middle-aged and old mice, and found that Ghsr expression in BAT increased during aging (Fig. 3B).

Figure 3. Ghrelin receptor ablation attenuates age-associated decline of thermogenesis and enhances insulin signaling in BAT. (A) Energy expenditure of young, middle-aged and old WT and Ghsr^−/− mice normalized by lean body mass. (B) Ghsr expression in BAT of young, middle-aged and old WT mice. (C) BAT mass and percentage of BAT weight over body weight in young, middle-aged and old WT and Ghsr^−/− mice. (D) UCP1 expression in BAT of young, middle-aged and old WT and Ghsr^−/− mice. IR (E) and IRS-1 (F) expression in BAT of young, middle-aged and old WT and Ghsr^−/− mice. Young (4-months), middle-aged (10-months) and old (22-months) mice were used. N = 6-14. *p<0.05, **p<0.001, WT vs. Ghsr^−/−; #p<0.05, ##p<0.001, old or middle-aged vs. young.

Higher circulating ghrelin and increased GHS-R expression in BAT of aged mice suggest enhanced ghrelin signaling in BAT during aging, which may promote age-associated decline of thermogenesis. To further define the role of GHS-R in thermogenesis of BAT, we compared BAT mass and UCP1 expression in BAT of young, middle-aged and old WT and Ghsr^−/− mice. Although the absolute weight of BAT didn't change with age, BAT mass normalized to body weight decreased during aging in both WT and Ghsr^−/− mice (Fig. 3C). There was no significant difference in the ratio of BAT/body weight between WT and Ghsr^−/− mice in middle-aged and old mice, while the ratio of BAT/body weight in young Ghsr^−/− mice was higher than that of young WT mice (Fig. 3C). While UCP1 expression in BAT of WT mice declined during aging, higher UCP1 expression was detected in BAT of GHS-R ablated aged (middle-aged and old) mice, maintaining expression levels similar to that of young mice (Fig. 3D). These results suggest that GHS-R ablation improves thermogenesis through regulation of BAT activity, but not BAT mass.

It was reported that BAT regulates glucose homeostasis and insulin sensitivity [8, 9]. It has been shown that insulin signaling in BAT is activated by cold stress [48]. Our previous studies showed that aged Ghsr^−/− mice have improved insulin sensitivity [34]. Indeed, we detected lower expression of insulin receptor (IR) and insulin receptor substrate 1 (IRS-1) in BAT of old WT mice, but GHS-R ablation increased IR and IRS-1 expression in BAT (Fig. 3E and 3F). Together, these data suggest that GHS-R expression in BAT is increased during aging, and ablation of GHS-R attenuates age-associated decline of thermogenesis and insulin sensitivity in BAT.

Ablation of GHS-R activates thermogenic signaling and improves mitochondrial dynamics of BAT

BAT thermogenic capacity is determined by the availability of fuel substrate in BAT and UCP1 activity in mitochondria [3]. Protein kinase A (PKA) activates hormone-sensitive lipase (HSL), which is a predominant lipase for catecholamine-stimulated lipolysis in brown adipocytes [49]. Our data revealed that ablation of GHS-R stimulates PKA and HSL activation in BAT, indicative of stimulated thermogenic signaling (Fig. 4A). cAMP response element-binding protein (Creb) is a known downstream target of PKA; phosphorylation of Creb is known to increase the expression of mitochondrial respiratory chain proteins and UCP1 expression [50, 51]. As expected, Creb phosphorylation is increased in BAT of Ghsr^−/− mice (Fig. 4A). Consistently, our ex vivo lipolysis study showed that β-adrenergic agonist CL316243-treated brown adipocytes of Ghsr^−/− mice exhibited higher basal and stimulated glycerol release when compared with those of WT mice (Fig. 4B). These data together demonstrate that GHS-R ablation activates thermogenic signaling cascade and promotes lipolysis in BAT.

Figure 4. Ablation of GHS-R activates thermogenic signaling cascade and improves mitochondrial dynamics of BAT in aged mice. The representative data presented here were from 22 month-old WT and Ghsr^−/− mice. (A) Representative Western blots of key thermogenic regulators in BAT of aged WT and Ghsr^−/− mice. (B) Ex vivo lipolysis of BAT of aged WT and Ghsr^−/− mice, treated with or without 10 μM CL316243. (C) Representative Western blots show phosphorylated AMPK α (p-AMPK-α), total AMPK (t-AMPK-α) and UCP1 protein expression in BAT of old WT and Ghsr^−/− mice after 4 hours of cold exposure. (D) Expression of mitochondrial fission genes in BAT of aged WT and Ghsr^−/− mice. (E) Expression of mitochondrial fusion genes in BAT of aged WT and Ghsr^−/− mice. (F) Expression of mitochondrial complex biogenesis markers in BAT of aged WT and Ghsr^−/− mice. N = 6. *p<0.05, **p<0.001, WT vs. Ghsr^−/−; #p<0.05, basal vs. CL316243 treatment.

The 5′ AMP-activated protein kinase (AMPK), a key nutrient and energy sensor, is the master regulator of cellular energy metabolism in many tissues, including BAT [52, 53]. Cold exposure increases AMPK activity [52], and aging is associated with decreased AMPK activity [54]. To test whether GHS-R ablation in BAT activates AMPK, we investigated AMPK activity by studying protein levels of phosphorylated AMPK and total AMPK. Enhanced phosphorylated AMPK was detected in BAT of Ghsr^−/− mice, and was correlated with increased UCP1 (Fig. 4C). This result suggests that AMPK activity may facilitate GHS-R mediated thermogenic regulation.

Mitochondrial biogenesis and dynamics are critical for mitochondrial function [20], and mitochondrial dynamics have been suggested to play a critical role in the pathogenies of insulin resistance [55-57]. We thus studied expression of key mitochondrial dynamics genes in BAT of aged WT and Ghsr^−/− mice. The expression of Drp1, Fis1, OPA1, Mfn1, and Mfn2 was significantly increased in aged Ghsr^−/− mice (Fig. 4D and 4E). Consistently, the expression of subunits of mitochondrial respiratory chain complexes IV, cox2 and cox10 was increased in the BAT of Ghsr^−/− mice, indicative of increased mitochondrial activity (Fig. 4F). These data suggest that mitochondrial dynamics may also play a role in GHS-R mediated thermogenic regulation.

GHS-R regulates BAT thermogenesis through both central and peripheral mechanisms

We have evidence that GHS-R regulates thermogenic function, and ablation of GHS-R attenuates the age-associated decline of thermogenesis [34]. However, since the data were obtained from global GHS-R knockout mice, we could not determine with certainty whether the effect of GHS-R on thermogenesis is mediated through central SNS and/or peripheral brown adipocytes. SNS activation increases catecholamine (norepinephrine, NE) release at SNS endings in BAT, which in turn activates β3-AR in brown adipocytes. To determine the role of SNS-induced catecholamine release in GHS-R mediated thermogenesis, we assessed NE in the circulation and in BAT of WT and Ghsr^−/− mice. Since urinary cate-cholamines are less likely to be affected by handling stress [58], we measured NE concentration in urine of young, middle-aged and old WT and Ghsr^−/− mice. As expected, urinary NE decreased with age in WT mice, while GHS-R ablated mice maintained NE at a level comparable to that of young mice (Fig. 5A).

Figure 5. Ablation of GHS-R prevents age-associated decline of norepinephrine (NE) and enhances NE-responsiveness. NE concentrations in the urine (A) and NE levels in BAT (B) of young, middle-aged and old WT and Ghsr^−/− mice. Young (4-months), middle-aged (10-months) and old (22-months) mice were used. (C) β3-AR mRNA expression in BAT of 22 month-old WT and Ghsr^−/− mice. N = 6-10. *p<0.05, WT vs. Ghsr^−/−; #p<0.05, ##p<0.001, old or middle-aged vs. young.

Similarly, NE content in BAT of WT mice declined with age, while GHS-R ablation prevented NE decline in BAT of middle-age and old mice (Fig. 5B). Consistently, the expression of β3-AR in BAT was higher in old Ghsr^−/− mice than in old WT mice (Fig. 5C). Increased SNS-induced NE release and β3-AR expression in BAT of old Ghsr^−/− mice suggest that GHS-R ablation regulates BAT thermogenesis, at least in part, by activating central SNS-mediated thermogenic signaling pathway of SNS-NE-β3-AR.

To determine NE sensitivity of BAT, we studied NE-induced thermogenesis in old WT and Ghsr^−/− mice. O₂ consumption after NE injection was significantly higher in old Ghsr^−/− compared with old WT mice (Fig. 6A), suggesting that BAT of old Ghsr^−/− mice is more sensitive to NE stimulation. To assess whether GHS-R has direct effect in brown adipocytes, we further knocked down GHS-R in brown adipocytes (HIB1B) using shGHS-R. Transfection with shGHS-R resulted in 70% knockdown of GHS-R compared with scrambled (shScr) control (Fig. 6B). The expression of thermogenic marker UCP1 and adipogenic marker Peroxisome Proliferator-Activated Receptor γ (PPARγ) was significantly increased in isoproterenol-stimulated GHS-R knockdown HIB1B cells (Fig. 6B). These data suggest that GHS-R also directly inhibits thermogenic activity in brown adipocytes.

Figure 6. GHS-R directly regulates thermogenic activation in brown adipocytes. (A) Oxygen consumption of old WT and Ghsr^−/− mice after 1.5 mg/Kg subcutaneous injection of NE. N = 10-12. *p<0.05, WT vs. Ghsr^−/−. (B) Expression of Ghsr, UCP1 and PPARγ in shGHS-R knockdown HIB1B cells. shScr represents scramble shRNA; shGHS-R represents shRNA specific for GHS-R. N = 9. **p<0.001, shScr vs. shGHS-R.

Discussion

We previously showed that ghrelin decreases UCP1 expression in brown adipocyte HIB1B cells through GHS-R [34]. Current data suggest that obestatin increases UCP1 expression in HIB1B cells, while DAG has no effect. Thus, ghrelin and obestatin exert opposing effects on thermogenesis. Our data are in agreement with published reports showing that ghrelin decreases UCP1 expression in BAT [59], while obestatin attenuates the hypothermia of Ghrelin^−/− mice [60]. We observed improved thermogenesis in Ghsr^−/− mice, but not in Ghrelin^−/− mice [34, 45]. The absence of a thermogenic phenotype in Ghrelin^−/− mice may be explained by the fact that both ghrelin and obestatin are absent in Ghrelin^−/− mice; the opposing thermogenic effects of ghrelin and obestatin are eliminated, thus showing no thermogenic phenotype. On the other hand, Ghsr^−/− mice express both ghrelin and obestatin. Since GHS-R is exclusively activated by ghrelin, only ghrelin signaling is blocked in Ghsr^−/− mice, while obestatin signaling is intact. In Ghsr^−/− mice, the stimulatory effect of obestatin on thermogenesis is unopposed, which may explain the increased thermogenesis observed in Ghsr^−/− mice. Thus, the differential thermogenic phenotypes of Ghrelin^−/− and Ghsr^−/− mice are likely due to the opposing effects of ghrelin and obestatin on thermogenesis.

Fasting is associated with decreased energy expenditure, and UCP1 expression in brown fat decreases during fasting [61, 62]. Fasting is associated with increased ghrelin and decreased obestatin in the circulation [26, 41]. The changes of ghrelin and obestatin during fasting may contribute to the decrease of thermogenesis and energy expenditure. After meals, postprandial ghrelin and obestatin decrease, the ratio of ghrelin and obestatin may be involved in the regulation of diet-induced thermogenesis. Aging is associated with severely diminished thermogenic function [10, 11]. Here we found that circulating ghrelin is increased during aging. We previously reported that low levels of GHS-R expression are detectable in BAT [34]. Current data reveal GHS-R expression in BAT is increased during aging. Higher concentration of ghrelin has been shown to be associated with lower resting metabolic rate and postprandial thermogenesis in humans [63]. Ghrelin promotes fat deposition in white fat and suppresses sympathetic nerve activity in brown fat [64, 65]. Our new data show that thermogenic suppressor ghrelin increases during aging; conversely, thermogenic stimulator obestatin remains steady during aging, which may contribute to the age-associated decline of thermogenesis. Collectively, our data suggest that ghrelin signaling contributes to the age-associated thermogenic dysfunction, which in turn promotes obesity and insulin resistance in aging.

Our data further demonstrate that GHS-R regulates thermogenic signaling cascades in BAT. GHS-R ablation activates thermogenic signaling pathway PKA-Creb-UCP1, and lipolytic pathway PKA-HSL-UCP1. It has been reported that insulin resistance in BAT is associated with thermogenic defect in obese and diabetic rats [48, 66]. Age-associated mito-chondrial dysfunction is a well-known cause for insulin resistance [15-17]. It has been shown that acute cold exposure improves glucose clearance and insulin sensitivity of BAT by activating the PKA-HSL-Lipolysis pathway [48]. We observed decreased expression of IR and IRS-1 in BAT of old Ghsr^−/− mice, suggesting that GHS-R ablation improves insulin sensitivity of BAT, which then further promotes BAT thermogenic activation.

AMPK is the master regulator of cellular energy metabolism in BAT [52, 53]. It is well documented that activation of AMPK enhances PGC1α-dependent transcription, which senses energy availability and stimulates mitochondrial DNA and protein synthesis [53, 67]. Ghrelin has been shown to have differential effects on AMPK activity: stimulatory in hypothalamus and heart, but inhibitory in liver and WAT [68, 69]. Our previous study showed that GHS-R ablation increases mitochondrial content [34]. Our current data reveal that higher levels of phosphorylated AMPK in BAT of Ghsr^−/− mice are correlated with increased UCP1. This suggests that GHS-R ablation may activate AMPK to increase mitochondrial biosynthesis of BAT, thus enhancing thermogenesis.

Mitochondrial dynamics play a crucial role in mitochondrial function [20]. Abnormal mitochondrial structure has been observed i n obese and type 2 diabetic patients [22]. GHS-R deletion increased the expression of both fusion- and fission-related genes in BAT of aged Ghsr^−/− mice, indicative of an active state of mitochondrial dynamics and improved mitochondrial homeostasis. Improved mitochondrial homeostasis will enhance the sensitivity of mitochondria to FFA to further promote mitochondrial uncoupling. The data suggest that suppressing ghrelin signaling preserves youthful mitochondrial dynamics in BAT during aging. It has been shown that obesity induces Mfn2 deficiency which results in reduced mitochondrial activity, and Drp1 deletion leads to reduction of mtDNA content [24, 70]. We found that the expression of Mfn2 and Drp1 was increased in BAT of aged Ghsr^−/− mice, consistent with the increased thermogenic activity and mtDNA content detected in BAT of aged Ghsr^−/− mice [34]. In addition, the subunits of mitochondrial respiratory chain complexes IV, such as mtDNA-encoded cox2 and nuclear DNA-encoded cox10, were also increased in Ghsr^−/− mice, lending further support to the increased mitochondrial biogenesis. Taken together, we conclude that GHS-R ablation activates thermogenic signaling cascades and promotes lipolysis in BAT; GHS-R ablation enhances mitochondrial function of BAT by increasing mitochondrial biogenesis and stimulating mitochondrial dynamics.

Last, we explore the potential site(s) of action of GHS-R mediated thermogenesis. The phenotype we observed in Ghsr^−/− mice could result from GHS-R mediated effects in central and/or peripheral sites. Thyroid hormones are important regulators of thermogenesis [71]. However, serum T3 and T4 concentrations were comparable in WT and Ghsr^−/− mice [34], indicating that the elevated thermogenesis observed in old Ghsr^−/− mice is not due to changes in circulating thyroid hormones. Sympathetic activity is known to play a dominant role in thermogenic regulation [3, 4]. Aging is associated with decreased stress responsiveness, because of reduced circulating NE and impaired hypothalamic-pituitary-adrenal axis [72]. In the current study, we found that GHS-R ablation prevents age-associated decrease of NE in the circulation, suggesting that GHS-R mediated thermogenesis is likely regulated centrally via SNS-induced NE release. On the other hand, our data also showed that GHS-R ablation increases NE-induced O₂ consumption, indicative that BAT of Ghsr^−/− mice has increased sensitivity to NE stimulation; this suggests that GHS-R ablation may directly affect the thermogenic response of BAT. Indeed, we found that knockdown of GHS-R in HIB1B cells up-regulates the thermogenic mediator UCP1, suggesting that ghrelin signaling regulates thermogenesis directly in BAT. Together, our data suggest that ghrelin signaling regulates BAT ther-mogenesis via both central and peripheral mechanisms.

In summary, our studies show that ghrelin and GHS-R are important thermogenic regulators, and increased ghrelin signaling in BAT during aging contributes to the age-associated thermogenic impairment. GHS-R mediated thermogenesis in BAT is attributable to thermogenic signaling activation, insulin sensitivity, mitochondrial biogenesis and dynamics of BAT (Fig. 7). GHS-R likely regulates thermogenesis via both central mechanisms involving SNS-induced NE release and direct effect in BAT. Thus, GHS-R antagonists may serve as a unique class of drugs that can prevent/treat age-associated obesity and insulin resistance by enhancing thermogenesis.

Figure 7. Schematic diagram of GHS-R mediated thermogenic regulation in brown adipocytes. Our data suggest that ghrelin signaling may regulate thermogenesis in BAT via the following 4 independent and interconnected signaling pathways: 1) Ablation of GHS-R stimulates SNS-mediated NE release, which in turn induces β3-AR expression, subsequently activating thermogenic signaling cascades in BAT. This involves activation of thermogenic signaling pathway PKA-CREB-UCP1 and lipolytic pathway PKA-HSL-UCP1. 2) Ablation of GHS-R enhances insulin signaling in BAT, which improves insulin sensitivity of BAT and activates key thermogenic regulator PKA. 3) Ablation of GHS-R enhances AMPK activity in BAT, which increases DNA and protein synthesis of mitochondria, thus increasing mitochondrial biogenesis. 4) Ablation of GHS-R augments mitochondrial dynamics, enhancing both mitochondrial fission and fussion; this restores mitochondrial architecture and improves mitochondrial homeostasis. Improved mitochondrial homeostasis enhances the sensitivity of mitochondria to FFA to further promote mitochondrial uncoupling. Collectively, GHS-R ablation increases thermogenesis in BAT by activating thermogenic signaling, sensitizing insulin signaling, increasing mitochondrial biogenesis, and enhancing mitochondrial dynamics.

Author Contributions

LL designed and performed the experiments and wrote the manuscript. JHL, OYNB and XM performed experiments and edited the manuscript. XZ and DSH advised and critiqued the manuscript. YS designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgments

Indirect calorimetry was performed in the Mouse Metabolic Research Unit at the Children's Nutrition Research Center, Baylor College of Medicine. The authors acknowledge the expert assistance of Mr. Firoz Vohra and the MMRU Core Director Dr. Marta Fiorotto. We also thank Mr. Michael R. Honig at Houston's Community Public Radio Station KPFT for his excellent editorial assistance. This study was supported by USDA/CRIS grant ARS 6250-51000-059 (YS), American Heart Association (AHA) innovative grant 12IRG9230004 (YS) and Grant-in-Aid 14GRNT18990019 (YS), NIH RDK080306 (DSH), VA BX002006 (DSH), NIH training grant 1T32HD071839 (PI: Morey W. Haymond), and partly supported by the NIH-Diabetes and Endocrinology Research Center of Baylor College of Medicine (P30DK079638). This study was also supported by Macao Science and Technology Development Fund 042/2013/A2 (LL) and the Research Fund of University of Macau MRG015/LLG/2014/ICMS (LL).

Conflicts of Interest

The authors of this manuscript have no conflict of interests to declare.

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