Hypothalamic perineuronal net assembly is required for sustained diabetes remission induced by fibroblast growth factor 1 in rats

Abstract

We recently showed that perineuronal nets (PNNs) enmesh glucoregulatory neurons in the arcuate nucleus (Arc) of the mediobasal hypothalamus (MBH)1, but whether these PNNs play a role in either the pathogenesis of type 2 diabetes (T2D) or its treatment remains unclear. Here we show that PNN abundance within the Arc is markedly reduced in the Zucker diabetic fatty (ZDF) rat model of T2D, compared with normoglycaemic rats, correlating with altered PNN-associated sulfation patterns of chondroitin sulfate glycosaminoglycans in the MBH. Each of these PNN-associated changes is reversed following a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) at a dose that induces sustained diabetes remission in male ZDF rats. Combined with previous work localizing this FGF1 effect to the Arc area2,3,4, our finding that enzymatic digestion of Arc PNNs markedly shortens the duration of diabetes remission following icv FGF1 injection in these animals identifies these extracellular matrix structures as previously unrecognized participants in the mechanism underlying diabetes remission induced by the central action of FGF1.

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Fig. 1: T2D-ZDF rats have reduced PNN structures in the Arc.
Fig. 2: T2D-ZDF rats exhibit abnormal hypothalamic CS/DS-GAG sulfation patterns.
Fig. 3: Effect of a single icv FGF1 injection on Arc PNN assembly and composition in ZDF rats.
Fig. 4: Impact of ChABC digestion of Arc PNNs on sustained blood glucose lowering induced by icv FGF1.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

The authors are grateful for the technical assistance provided by T. Harvey, M. Matsen, N. Acharya, H. Nguyen and B.A. Phan. Histochemical work was supported by the Cellular and Molecular Imaging Core (CMIC) and funding to K.M.A. and J.M.S. by the UW Diabetes Research Center (DRC) National Institute of Diabetes and Digestive and Kidney Diseases (NIH-NIDDK) sponsored grant P30DK017047. Mass spectrometry work was supported by University of Washington School of Pharmacy Mass Spectrometry Center. This work was also supported by NIH-NIDDK grants: F32DK122662 (to K.M.A.), R01DK101997 (to M.W.S.), R01DK089056 (to G.J.M.), R01DK083042 (to G.J.M. and M.W.S.) and K08DK114474 (to J.M.S.), the National Institute of General Medical Sciences (NIH-NIGMS) grant R01GM127579 (to M.G.), the National Institute on Aging (NIH-NIA) grant T32AG052354 (to A.F.L.), the National Institute of Neurological Disorders and Stroke Neurosurgeon Research Career Development Program (NRCDP) K12 Award K12NS080223 (to Z.M.), the Department of Defense Peer Reviewed Medical Research Program W81XWH-20-1-0250 and the Barrow Neurological Foundation Grant 18-0025-30-05 (both to Z.M.). This work was also supported by the NIH-NIDDK diseases–funded Nutrition Obesity Research Center (NORC; P30DK035816). Additional funding to support these studies was provided to J.M.S. by the UW Royalty Research Fund (RRF; A139339). Funding was also provided to M.W.S. by Novo Nordisk (CMS-431104) and to M.A.B. by the Novo Nordisk Foundation (NNF17OC0024328) and the Novo Nordisk Foundation Center for Basic Metabolic Research, which is an independent research center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation (NNF10CC1016515). Some figures were created with assistance from BioRender.com.

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Contributions

K.M.A., Z.M., J.M.S., M.A.B., A.F.L., W.A.B., T.N.W., M.G., G.J.M. and M.W.S. contributed to experimental design, data interpretation and manuscript preparation, with input from all authors. In vivo studies were completed by K.M.A.; additional in vivo studies were conducted by J.M.S. and J.M.B. Immunofluorescence of human hypothalamic tissue was completed by Z.M. and E.C.; K.M.A., A.F.L. and C.K.C. performed the hypothalamic immunofluorescence stereological mapping, quantitative data analyses and protein biochemical analyses. Mass spectrometry analysis was performed and evaluated by K.M.A., M.G. and A.F.L. Statistical analysis was performed by K.J.K. and K.M.A.

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Correspondence to Michael W. Schwartz.

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Support to M.W.S. was partly funded by Novo Nordisk. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Diabetic Wistar rats have reduced PNN CS-GAG structures in the arcuate nucleus.

Wistar rats were rendered diabetic by 2 wk of high fat diet (HFD) followed by a single injection of low-dose streptozotocin (STZ) (30 mg/kg sc) or citrate control and a, blood glucose and b, body weight were monitored for 24 d after treatment. Immunofluorescent detection of WFA (PNN CS-GAGs) and aggrecan (PNN CSPG) in coronal sections of rat hypothalamus (30 µm) from c, normoglycemic citrate Wistar controls and d, age-matched, hyperglycemic HFD/STZ Wistar rats. Scale bar: 200 µm. e, Quantification of the mean fluorescence intensity of PNNs averaged from medial hypothalamic sections (-2.2 to -2.8 mm posterior from bregma) for ArcM and ArcL areas from citrate and HFD/STZ Wistar rats and normalized to the citrate control mean fluorescence intensity averages. f, HFD/STZ Wistar rats exhibit no change in the relative percentages of CS disaccharides compared to age-matched, citrate controls. In vivo manipulation (a, b), immunofluorescence analyses (c-e), and CS/DS-GAG LC-MS2 analyses were performed on the same cohort of rats (n=8-9 rats/group; mean ± SEM). *P < 0.05, **P < 0.01, ****P < 0.0001, versus citrate controls; (a, b) Linear mixed-effects model analysis with Geisser-Greenhouse correction, and (e, f) Student’s t-test (unpaired, two-sided); (a) p=<0.0001, (b) p=0.0364, (e) WFA, ArcL p=0.0061; WFA, ArcM p=0.0467. Arc, arcuate nucleus; ArcM, medial Arc; ArcL, lateral Arc; s.c., subcutaneous; 3V, 3rd ventricle. Source data

Extended Data Fig. 2 Effect of a single icv injection of FGF1 on Arc PNN assembly in Wistar rats.

Immunofluorescence imaging of Arc PNN CS-GAGs in Wistar rats 24 h after icv treatment with FGF1 (3 µg) or vehicle, where the vehicle treated controls were pair-fed to the FGF1 group. Quantification of the mean fluorescence intensity of PNNs averaged from medial hypothalamic sections for total Arc (Arc-M+L) areas (n=5 rats/group; mean ± SEM). **P < 0.01, compared to icv vehicle controls; Student’s t-test (unpaired, two-sided); (A) p=0.0046. Arc, arcuate nucleus; ME, median eminence; 3V, 3rd ventricle. Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–12

Reporting Summary

Supplementary Video 1

PNNs in the visual cortex of normoglycaemic Wistar rats.

Supplementary Video 2

PNNs in the motor cortex of normoglycaemic Wistar rats.

Supplementary Video 3

PNNs in the Arc of normoglycaemic Wistar rats.

Supplementary Video 4

PNNs in the visual cortex of T2D-ZDF rats.

Supplementary Video 5

PNNs in the motor cortex of T2D-ZDF rats.

Supplementary Video 6

PNNs in the Arc of T2D-ZDF rats.

Source data

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Source Data Fig. 2

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 2

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Alonge, K.M., Mirzadeh, Z., Scarlett, J.M. et al. Hypothalamic perineuronal net assembly is required for sustained diabetes remission induced by fibroblast growth factor 1 in rats. Nat Metab (2020). https://doi.org/10.1038/s42255-020-00275-6

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