Obesity-induced hepatic steatosis is mediated by endoplasmic reticulum stress in the subfornical organ of the brain (original) (raw)
Acute chemical induction of brain ER stress promotes hepatic steatosis independent of body weight, food intake, or adiposity. While ER stress in the CNS is emerging as a key controller of metabolic processes, the extent to which it is involved in NAFLD remains unknown. To investigate this, we first examined the effects of selective induction of ER stress in the brain on hepatic parameters. Adult C57Bl/6 mice on normal chow were preimplanted with an intracerebroventricular (ICV) cannula in the lateral ventricle, and the chemical ER stress inducer thapsigargin was subsequently administered ICV once daily over 3 days. Consistent with previous reports (21, 24), this approach evoked robust UPR activation in the CNS (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.90170DS1), particularly within forebrain and hindbrain regions. Short-term ICV administration of thapsigargin resulted in hepatomegaly (~25% increase in liver weight; Figure 1A) and an almost 4-fold increase in liver triglycerides (Figure 1B). In addition, histological examination of the liver revealed substantial steatosis (Figure 1C), as represented by vacuolation and lipid droplet accumulation in hepatocytes, in mice treated with ICV thapsigargin. In contrast, induction of brain ER stress did not influence body weight, food intake, or regional adipose tissue mass (Figure 1, D–G). Importantly, ICV thapsigargin did not alter hepatic ER stress markers (Supplemental Figure 2), demonstrating that these findings are due to UPR activation in the brain and not in the liver. This indicates that acute brain ER stress can cause hepatic steatosis, independent of changes in body weight, food intake, adiposity, and hepatic ER stress.
Short-term induction of brain ER stress promotes hepatic steatosis independent of changes in body weight, food intake, or adiposity. Liver mass (A) and hepatic triglyceride levels (B) following 3 days of daily intracerebroventricular (ICV) vehicle or thapsigargin (to induce brain ER stress) administration. n = 6–10. (C) H&E staining of the liver in mice that underwent ICV vehicle or thapsigargin dosing. Representative of n = 4. Scale bar: 100 μm. Body weight (D), food intake (E), and cumulative food intake (F) at baseline and during 3 days of daily ICV vehicle or thapsigargin administration. Regional adipose tissue mass (G) following 3 days of ICV vehicle or thapsigargin. n = 6–10. *P < 0.05 vs. ICV vehicle with two-tailed unpaired _t_-test or 2-way repeated measures ANOVA. Box-and-whisker plots represent the median (line within box), upper and lower quartile (bounds of box), and maximum and minimum values (bars).
Short-term reduction in brain ER stress rescues obesity-induced hepatic steatosis without affecting body weight, food intake, or adiposity. Next, we asked if chronic brain ER stress, such as that which occurs in DIO, contributes to fatty liver. In C57Bl/6 mice with DIO induced by a high-fat diet (HFD) for 15 weeks, we utilized short-term ICV administration of taurosdeoxycholic acid (TUDCA), an ER chemical chaperone that reduces brain ER stress (21, 22, 24). As expected, significant elevations in liver weight (i.e., hepatomegaly; Figure 2A) were observed following HFD feeding. Remarkably, obesity-induced hepatomegaly was restored to normal chow levels following ICV TUDCA over 3 days (Figure 2B). Moreover, while HFD-fed animals treated with ICV vehicle demonstrated profound elevations in liver triglycerides and hepatic lipid vesicle accumulation, 3-day ICV brain administration of TUDCA reduced HFD-induced hepatic steatosis and restored liver histology to a normal appearance (Figure 2, B and C). Importantly, ICV TUDCA did not alter mRNA levels of hepatic ER stress markers in DIO or normal-chow mice (Supplemental Figure 3A). As anticipated, HFD resulted in significant elevations in body weight and regional adipose tissue mass, but reducing brain ER stress via ICV TUDCA over 3 days did not influence these parameters or food intake (Figure 2, D–G). Collectively, these findings demonstrate that CNS ER stress underlies hepatic steatosis during obesity, and this effect is dissociated from body weight, adiposity, food intake, and hepatic ER stress.
Short-term reductions in brain ER stress reduce obesity-induced hepatic steatosis, hypertension, and tachycardia independent of body weight, food intake, or adiposity. Liver mass (A) and hepatic triglyceride levels (B) following 3 days of daily intracerebroventricular (ICV) administration of the ER chemical chaperone TUDCA (to reduce ER stress) or vehicle in normal chow–fed and HFD-fed mice. n = 6–8. (C) H&E staining of the liver in mice that underwent ICV vehicle or TUDCA dosing. Representative of n = 4. Scale bar: 100 μm. Body weight (D), food intake (E), and cumulative food intake (F) in normal chow–fed and HFD-fed mice at baseline and during 3 days of ICV administration of TUDCA or vehicle. Regional adipose tissue mass (G) following 3 days of ICV vehicle or TUDCA administration. n = 6–8. Radiotelemetric measurements of mean arterial blood pressure (H) and heart rate (I) at baseline and during daily ICV TUDCA or vehicle administration. The respective change in blood pressure and heart rate is shown on the right (H and I). #P < 0.05 vs. normal chow groups; *P < 0.05 vs. high fat ICV TUDCA. One-way or two-way repeated measures ANOVA. Box-and-whisker plots represent the median (line within box), upper and lower quartile (bounds of box), and maximum and minimum values (bars).
Short-term reductions in brain ER stress rescue hypertension and tachycardia in DIO mice. Activation of the UPR in the CNS is also emerging as an important contributor to cardiovascular diseases, including hypertension (21, 24). In this context, we and others have previously shown that acute induction of brain ER stress (ICV thapsigargin) increases arterial blood pressure (21, 24). Since obesity is associated with the development of hypertension (25, 26), we also determined the contribution of brain ER stress to obesity-induced hypertension. Using radiotelemetric analyses, HFD-fed animals were found to have significant elevations in arterial pressure, as well as tachycardia (Figure 2, H and I). ICV administration of TUDCA over 3 days lowered arterial pressure and heart rate in DIO mice, a response that was evident within 24–48 hours, with blood pressure and heart rate in ICV TUDCA–treated DIO mice returning to normal chow levels within 72 hours (Figure 2, H and I). Thus, brain ER stress contributes to obesity-induced hypertension, an abnormality that commonly occurs in concert with NAFLD (27, 28).
Obesity elicits robust UPR activation and ER ultrastructural abnormalities in the SFO. We subsequently sought to determine the neural regions involved in the CNS ER stress–mediated responses in DIO mice. In light of our findings of ER stress–mediated cardiovascular and metabolic effects (Figure 2, A–C, H, and I), we focused our attention on the sensory circumventricular SFO. This tiny forebrain CNS nucleus located at the base of the lateral ventricle is highly vascularized and lacks a blood-brain-barrier (Figure 3A), unlike the vast majority of brain regions, including the hypothalamus (29–31). This unique feature makes the SFO highly susceptible to ER stress during obesity due to its constant exposure to circulating metabolites, hormones, and inflammatory mediators. Well known for its role in fluid and cardiovascular homeostasis, an emerging body of evidence suggests that the SFO may also be intricately involved in metabolic control and serve as a central integrator to couple metabolic and cardiovascular responses (30, 32, 33).
Obesity elicits robust UPR activation and ER ultrastructural alterations in the SFO. (A) Schematic highlighting the location of the SFO at the base of the lateral ventricle (LV). (B) Real-time PCR measurements of ER stress biomarkers p58IPK, CHOP, and XBP1s from micropunches of the SFO in normal chow–fed or HFD-fed mice. n = 5. Two brains pooled per sample. (C) Western blot and quantitative summary of the ER chaperones GRP78 and PDI in SFO homogenates from normal chow–fed and HFD-fed mice. n = 4. Two brains pooled per sample. *P < 0.05 vs. normal chow with a two-tailed unpaired _t_-test. Box-and-whisker plots represent the median (line within box), upper and lower quartile (bounds of box), and maximum and minimum values (bars). (D) Representative electron micrographs of rough ER (arrows) in SFO neurons of a normal chow–fed (left) and HFD-fed mouse (right). The electron micrographs are representative of 20–28 neuronal and dendritic fragments evaluated from 3 mice in each group. Scale bar = 2 μm
We first performed gene expression of the ER stress indicators p58IPK, CHOP, and the spliced isoform of X-box-binding protein-1 (XBP1s) in micropunches of the SFO. Near 2-fold elevations in all UPR biomarkers examined were found in the SFO (Figure 3B), indicative of robust ER stress in this circumventricular region during DIO. Based on these findings, further examination of the UPR in the SFO was conducted. In response to UPR activation, ER chaperones including GRP78 and protein disulfide isomerase (PDI) were upregulated in the SFO, indicative of an attempt to counter HFD-induced ER stress (Figure 3C). Collectively, these findings indicate significant activation of the UPR in the SFO in response to HFD feeding.
In addition to activation of the UPR, ER stress results in ultrastructural alterations in the rough ER, including swelling and ribosomal detachment (24, 34). Therefore, electron microscopy was utilized to evaluate the ER ultrastructure in neuronal perikarya and large dendrites of the SFO in mice that were fed normal chow or HFD. The rough ER was graded as of normal appearance if: (i) substantial lengths of rough ER membrane were present; (ii) the rough ER membranes were parallel and showed no bloating; and (iii) substantial numbers of ribosomes were attached to the rough ER. Neuronal fragments were graded as showing ER stress if: (i) substantial lengths of rough ER were absent; (ii) the membranes of the rough ER were not parallel, and bloated segments or saccule-like fragments were present; and (iii) few or no ribosomes were seen attached to the rough ER. SFO rough ER from lean animals exhibited flat, tube-like cisternae with dense ribosomal attachment (Figure 3D, top). In contrast, ER in the SFO of obese animals appeared bloated or had saccule-like fragments with ribosomal detachment evident (Figure 3D, bottom). Thus, in addition to marked activation of the UPR, obesity is associated with alterations in rough ER morphology in SFO neurons.
SFO-ER stress mediates obesity-induced hepatic steatosis independent of changes in body weight, food intake, or adiposity. Our findings presented in Figures 1–3 indicate that brain ER stress mediates hepatic steatosis and that HFD feeding elicits marked ER stress in the circumventricular SFO. Interestingly, short-term ICV administration of TUDCA reduced ER stress in the SFO of HFD-fed mice, as indicated by a 35%–40% reduction in UPR markers (Supplemental Figure 3B), suggesting that SFO-ER stress is functionally linked to hepatic steatosis in the context of obesity. To more specifically interrogate the SFO, we utilized an approach to genetically reduce ER stress selectively in the SFO. An effective means to prevent and rescue ER stress is via overexpression of the ER chaperone GRP78, which prevents downstream UPR activation (24, 35, 36). In this context, we recently developed an adenoviral vector that allows for overexpression of the full-length murine sequence of GRP78 (AdGRP78) (24). This vector includes an ER retention signal (KDEL) — which results in targeting of GRP78 to the ER, as demonstrated previously (24) — and is highlighted with transfection and immunohistochemical evaluation of Neuro2A neuronal cells (Supplemental Figure 4). Using this genetic approach, mice that had been fed normal chow or HFD for 15 weeks (starting at 6 weeks of age) underwent stereotaxic SFO–targeted AdGRP78 or control AdLacZ microinjections and were monitored for 5 weeks after adenoviral delivery. HFD-fed control vector mice demonstrated significantly higher body weights than normal chow counterparts, and targeted overexpression of GRP78 in the SFO did not influence body weight in obese or lean animals (Figure 4A). Similarly, food intake over the 5-week study period was not different between control and AdGRP78-treated mice (Figure 4, B and C). Upon sacrifice, evaluation of regional adipose tissue mass revealed significant HFD-induced elevations in s.c. and visceral white adipose tissue, as well as subscapular brown fat mass (Figure 4D). These increases in adipose tissue were not altered by delivery of GRP78 to the SFO. Importantly, this lack of influence on body weight, food intake, and adiposity was not due to lack of functionality of the viral vector, as reductions in gene expression of UPR markers following SFO-targeted AdGRP78 were found, in concert with significant overexpression of the transgene (Supplemental Figure 5). Moreover, targeting of AdGRP78 to the SFO did not alter ER stress in other metabolic control regions, including the ventromedial hypothalamus and arcuate nucleus, highlighting that this approach was selective for the SFO (Supplemental Figure 5). These findings indicate that reducing ER stress in the SFO during established obesity does not influence body weight, food intake, or adiposity.
Selective reductions in SFO-ER stress rescue obesity-induced hepatic steatosis, independent of body weight, food intake, or adiposity. Body weight (A), food intake (B), and cumulative food intake (C) in normal chow–fed and HFD-fed mice following chronic SFO-targeted viral overexpression of the ER chaperone GRP78 (AdGRP78) or control vector (AdLacZ). Regional adipose tissue (D) mass 5 weeks after SFO-targeted AdGRP78 or AdLacZ. Representative liver images in HFD-fed mice (E), as well as liver mass (F) and hepatic triglyceride levels (G) in HFD-fed and normal chow–fed mice following SFO-targeted AdGRP78 or AdLacZ. n = 6–8. H&E staining (H) of the liver in mice 5 weeks after SFO-targeted AdGRP78 or AdLacZ. Representative of n = 4. Scale bar: 100 μm. Radiotelemetric measurements of mean arterial blood pressure (I) and heart rate (J) following adenoviral overexpression of GRP78, or control vector, in the SFO. #P < 0.05 vs. normal chow groups; *P < 0.05 vs. high fat AdLacZ. One-way or two-way repeated measures ANOVA. Box-and-whisker plots represent the median (line within box), upper and lower quartile (bounds of box), and maximum and minimum values (bars).
As shown in Figure 4E, livers from obese mice treated with the control vector were enlarged with a pale yellowish color. In sharp contrast, livers from SFO-targeted AdGRP78 obese animals exhibited a normal dark red color. In line with this, HFD feeding elicited significant hepatomegaly, which was rescued by selectively reducing ER stress in the SFO (Figure 4F). Measurement of hepatic triglycerides revealed significant steatosis in response to HFD feeding, and overexpression of GRP78 in the SFO of obese animals reduced liver triglycerides to normal chow levels (Figure 4G). This SFO-ER stress–associated influence on hepatic steatosis was also reflected at the histological level, with lipid droplet accumulation in the liver in response to HFD feeding being abrogated following targeted reductions in ER stress with AdGRP78 in the SFO (Figure 4H). No changes in liver weight or steatosis were found in normal chow mice treated with SFO-targeted AdGRP78 (Figure 4, F–H). Importantly, liver GRP78 mRNA and protein expression were not increased in normal chow or HFD mice following SFO-targeted AdGRP78 (Supplemental Figure 6), underscoring that the robust hepatic effects were due to SFO overexpression of GRP78. Collectively, these findings indicate that selectively reducing obesity-induced ER stress in the SFO rescues hepatic steatosis and hepatomegaly, independent of changes in body weight, food intake, or adiposity.
SFO-ER stress does not contribute to the maintenance of obesity-induced hypertension and tachycardia. As presented and discussed in Figure 2, H and I, short-term administration of the ER chemical chaperone TUDCA to globally reduce brain ER stress demonstrated a role for CNS UPR in the maintenance of obesity-induced hypertension and tachycardia. However, these studies did not permit selective evaluation of the brain regions involved. The SFO is crucial for cardiovascular regulation, and ER stress in this nucleus has been shown to underlie the development of angiotensin-II–mediated hypertension (24). Given this, along with our finding of SFO-ER stress–mediated hepatic steatosis (Figure 4, E–H), we reasoned that SFO-ER stress may also contribute to hypertension and tachycardia in DIO mice. Consistent with previous reports (21, 37) and Figure 2, H and I, 24-hour radiotelemetry recordings revealed significant elevations in arterial pressure and heart rate in DIO mice compared with lean, normal chow–fed counterparts (Figure 4, I and J). Surprisingly, adenoviral overexpression of GRP78 targeted to the SFO did not alter arterial pressure or heart rate in HFD-fed mice (Figure 4, I and J), suggesting that ER stress in this brain nucleus is not involved in obesity-induced cardiovascular changes. These data reveal a striking selectivity in the pathological consequence of SFO-ER stress in DIO, with a robust contribution to hepatic steatosis but a lack of an effect on arterial pressure, heart rate, food intake, body weight, and adiposity.