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. 2014 Sep 2;20(3):471-82.
doi: 10.1016/j.cmet.2014.06.002. Epub 2014 Jul 10.

Xbp1s in Pomc neurons connects ER stress with energy balance and glucose homeostasis

Kevin W Williams  1 , Tiemin Liu  2 , Xingxing Kong  3 , Makoto Fukuda  4 , Yingfeng Deng  5 , Eric D Berglund  6 , Zhuo Deng  7 , Yong Gao  8 , Tianya Liu  9 , Jong-Woo Sohn  2 , Lin Jia  2 , Teppei Fujikawa  2 , Daisuke Kohno  10 , Michael M Scott  11 , Syann Lee  2 , Charlotte E Lee  2 , Kai Sun  5 , Yongsheng Chang  12 , Philipp E Scherer  13 , Joel K Elmquist  14
Affiliations

Xbp1s in Pomc neurons connects ER stress with energy balance and glucose homeostasis

Kevin W Williams et al. Cell Metab. .

Abstract

The molecular mechanisms underlying neuronal leptin and insulin resistance in obesity and diabetes remain unclear. Here we show that induction of the unfolded protein response transcription factor spliced X-box binding protein 1 (Xbp1s) in pro-opiomelanocortin (Pomc) neurons alone is sufficient to protect against diet-induced obesity as well as improve leptin and insulin sensitivity, even in the presence of strong activators of ER stress. We also demonstrate that constitutive expression of Xbp1s in Pomc neurons contributes to improved hepatic insulin sensitivity and suppression of endogenous glucose production. Notably, elevated Xbp1s levels in Pomc neurons also resulted in activation of the Xbp1s axis in the liver via a cell-nonautonomous mechanism. Together our results identify critical molecular mechanisms linking ER stress in arcuate Pomc neurons to acute leptin and insulin resistance as well as liver metabolism in diet-induced obesity and diabetes.

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Figures

Figure 1
Figure 1
Body weight and metabolic assessment of male WT and PIXs mice on HFD. Body weight curve of (A) male PIXs mice (*p<0.05). Body fat composition: whole body volume (B), visceral (C), and subcutaneous (D). Male PIXs mice display (E) increased hepatic triglyceride and cholesterol, (F) Increased VO2, (G) increased VCO2, (H) decreased RER (I) increased heat production, and (J) increased ambulatory activity. Error bars indicate SEM. (K) Leptin induced hypophagia was observed at 1, 2, 4, and 6 after refeeding. PIXs mice exhibited increased hypophagia in response to pharmacological administration of leptin at 4 and 6 hours after refeeding. (n=10 per group; *p<0.05). (Note: mice used in (E-I) were age-matched male littermates (8 weeks of age), and had comparable body weight and lean mass. (for F-J, n=14-16 per group; *p<0.05).
Figure 2
Figure 2
PIXs mice express increased thermogenic markers in BAT and iWAT. Weight matched PIXs mice were fed HFD DOX enriched diet for 2 weeks. qPCR was performed to examine the relative expression Ppargc1α, Prdm16, UCP1, Cidea, Dio2, and Elovl6 which are known genes associated with heat production in (A) BAT and (B) iWAT. *, p < 0.05. Brown adipose tissue (BAT) from (C) WT and (D) PIXs mice was imaged by light microscopy after hematoxylin-eosin staining. Inguinal white adipose tissue from (E) WT and (F) PIXs mice was imaged by light microscopy after UCP-1 immunohistochemistry. Bars, 100μm. (note C and D are the same scale as E and F).
Figure 3
Figure 3
Blood glucose and insulin levels of male WT and PIXs mice on chow diet. Blood glucose and insulin levels were measured from WT and PIXs mice fed chow diet without dox (Panel A), fed on a chow diet without dox then fasted overnight (Panel B), fed a chow diet with dox for 1 week (Panel C), and fed chow diet with dox for 1 week then fasted overnight (Panel D). Blood glucose and insulin levels were decreased in mice fed or fasted after 1 week on a chow diet with dox (n=10-15 per group; *p<0.05).
Figure 4
Figure 4
Improved glucoregulation in mice which constitutively express Xbp1s in Pomc neurons. (A) Basal and clamp blood glucose levels during the hyperinsulinemic-euglycemic experiment. (B) Exogenous glucose infusion rate (GIR) needed to clamp blood glucose. (C and D) Endogenous rates of glucose appearance (endo Ra) and disappearance (Rd) were determined using a constant infusion of [3-3H]glucose and Steele's steady-state calculations. (E) Body weight of PIXs mice on day of experiment. (n=7)
Figure 5
Figure 5
Regulation of Xbp1s and GalE in the arcuate nucleus and the liver. (A) Relative mRNA expression of Xbp1s and GalE in the liver and the arcuate nucleus of mice fasted (18h) and mice re-fed (2h) after fasting (18h). (B) Relative mRNA expression of Xbp1s as well as GalE, Edem1, Erdj4, and Bip, which are which are known Xbp1s target genes, in the arcuate nucleus from PIXs and WT mice fed either non-Dox or Dox containing diet. (C-D) Relative mRNA expression of Xbp1s, GalE, Edem1, Erdj4, Bip, Atf4, and Atf6 in FACS-Pomc neurons. (C) Data represents from WT mice fasted (18h) and WT mice re-fed (2h) after fasting (18h). (D) Data represents PIXs and WT mice chronically fed Dox containing diet. (E) qPCR was performed on mice chronically fed HF-Dox diet to examine the relative expression XBP1s as well as erdj4, erdeml, chop, and bip which are which are known Xbp1s target genes in (L) liver. *P < 0.05 compared with control. A-E. Fold change relative to 18S mRNA; Error bars indicate SEM.
Figure 6
Figure 6
ER stress blunts the leptin and insulin activity in the Arcuate nucleus (A) Blots represent changes in the protein levels for leptin-induced phospho-STAT3 and phospho-eif2α in response to ER stress. (B) Quantitative densitometry for protein expression of leptin-induced pSTAT3 in control and ER stress activators. (C) ER stress blunts leptin-induced pSTAT3 immunoreactivity in the arcuate nucleus of the hypothalamus. (*P < 0.05, values are means ±SEM from 3-6 independent experiments, error bars indicate SEM) (D) Blots represent changes in the protein levels for insulin-induced phospho-AKT and phospho-eif2α in response to ER stress. (E) Quantitative densitometry for the protein expression of insulin-induced pAKT.
Figure 7
Figure 7
ER stress blunts the leptin-induced activation and the insulin-induced inhibition of Pomc neurons. (A) 1. Brightfield illumination of Pomc-hrGFP::Lepr-cre::tdtomato neuron from PLT mice. 2. and 3. The same neuron under FITC (hrGFP) and Alexafluor 594 (tdtomato) illumination. 4. Complete dialysis of Alexa Fluor 350 from the intracellular pipette. 5. Merge image illustrates colocalization of hr-GFP, tdtomato, and Alexa Fluor 350 indicative of a Pomc neuron which expresses Leprs. Control (above). Electrophysiological study demonstrates a Pomc-hrGFP::Lepr-cre::tdtomato (green/red) neuron that depolarized in response to leptin. Below demonstrates a current clamp recording of a separate Pomc-hrGFP::Lepr-cre::tdtomato (green/red) neuron in which ER stress blunted the leptin induced depolarization. (B) Histogram demonstrating that multiple activators of ER stress blunted the leptin-induced activation of Pomc neurons (n= 8-15 per group). Deletion of either Ptp1b or Socs3 restores the leptin-induced excitation of arcuate Pomc neurons after ER stress induction. Similarly, constitutive expression of Xbp1s in Pomc neurons restores the leptin-induced excitation of arcuate Pomc neurons after ER stress induction. *P < 0.05. Error bars indicate SEM. (C) 1. Brightfield illumination of Pomc-hrGFP neuron from PLT mice. 2. and 3. The same neuron under FITC (hrGFP) and Alexafluor 594 (tdtomato) illumination. 4. Complete dialysis of Alexa Fluor 350 from the intracellular pipette. 5. Merge illustrates colocalization of hr-GFP and Alexa Fluor 350 indicative of a Pomc neuron which does not expresses Leprs. Control (above): Electrophysiological study demonstrates a Pomc-hrGFP (green) neuron is hyperpolarized in response to insulin. Below: A separate Pomc-hrGFP (green) neuron in which ER stress blunts the insulin induced hyperpolarization. (D) Histogram illustrating that chemical activation of ER stress blunts the insulin-induced inhibition of arcuate Pomc neurons (n= 8-18 per group). Deletion of either Ptp1b or Socs3 restores the insulin-induced inhibition of arcuate Pomc neurons after ER stress induction. *P < 0.05, Error bars indicate SEM. (E) Relative mRNA expression of Socs3 and Ptp1b in organotypic slices following pretreatment with ER stress activators. (F) Relative mRNA of Ptp1b and Socs3 in the arcuate nucleus from PIXs and WT mice fed HFD-Dox. (*P < 0.05, values are means ±SEM from 3-6 independent experiments, error bars indicate SEM)
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