Abstract

   In rodent models of type 2 diabetes (T2D), sustained remission of
   hyperglycemia can be induced by a single intracerebroventricular (icv)
   injection of fibroblast growth factor 1 (FGF1), and the mediobasal
   hypothalamus (MBH) was recently implicated as the brain area
   responsible for this effect. To better understand the cellular response
   to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes
   from the hypothalamus of diabetic Lep^ob/ob mice obtained on Days 1 and
   5 after icv injection of either FGF1 or vehicle. A wide range of
   transcriptional responses to FGF1 was observed across diverse
   hypothalamic cell types, with glial cell types responding much more
   robustly than neurons at both time points. Tanycytes and ependymal
   cells were the most FGF1-responsive cell type at Day 1, but astrocytes
   and oligodendrocyte lineage cells subsequently became more responsive.
   Based on histochemical and ultrastructural evidence of enhanced
   cell-cell interactions between astrocytes and Agrp neurons (key
   components of the melanocortin system), we performed a series of
   studies showing that intact melanocortin signaling is required for the
   sustained antidiabetic action of FGF1. These data collectively suggest
   that hypothalamic glial cells are leading targets for the effects of
   FGF1 and that sustained diabetes remission is dependent on intact
   melanocortin signaling.

   Subject terms: Gene expression analysis, Computational models,
   Neuroscience, Type 2 diabetes
     __________________________________________________________________

   In rodent models of type 2 diabetes, sustained remission of
   hyperglycemia can be induced by FGF1 action in the mediobasal
   hypothalamus. Here, the authors show that FGF1-injection is followed by
   marked changes in glial cell populations and that the sustained
   glycemic response is dependent on intact melanocortin signaling.

Introduction

   Type 2 diabetes (T2D) is among the most common and costly biomedical
   challenges confronting modern society^[80]1. Standard treatment of this
   disease involves daily dosing of one or more antidiabetic drugs and can
   be effective but fails to deliver adequate glycemic control to a
   majority of patients^[81]2. A compelling need for more effective
   treatment modalities therefore exists, and the brain has emerged as an
   important target for research in this area^[82]3.

   We recently reported that, in Lep^ob/ob mice and other rodent models of
   T2D, sustained diabetes remission can be achieved by a single
   intracerebroventricular (icv) injection of fibroblast growth factor 1
   (FGF1)^[83]4, and the hypothalamus has been identified as the brain
   area responsible for this FGF1 effect^[84]5. While this finding is in
   line with accumulating evidence supporting a key role for the
   hypothalamus in glucose homeostasis, our current understanding of
   glucoregulatory neurocircuitry is limited^[85]6,[86]7. Most of this
   work is focused on hypothalamic neurons with glucose-sensing
   properties^[87]8, but many glial cell types are also potential targets
   for the hypothalamic action of FGF1, since tanycytes and astrocytes are
   implicated in glucose homeostasis^[88]9,[89]10 and since glial cell
   responsiveness to FGF ligands is well documented^[90]5,[91]11,[92]12.
   Moreover, glia–neuron interactions can modify neuronal activity and
   remodel neurocircuit structure^[93]13–[94]15, effects of interest for
   understanding the long-lived nature of the FGF1 effect.

   As an unbiased approach to measuring gene expression within single
   cells in the hypothalamus^[95]16–[96]18, single-cell RNA sequencing
   (scRNA-seq) provides a useful strategy both for identifying
   FGF1-responsive cell types in this brain area and for characterizing
   their response to icv FGF1 administration at the transcriptional level.
   With this goal in mind, we performed this analysis across several time
   points following a single icv injection of FGF1 or vehicle in diabetic
   Lep^ob/ob mice. Using a combination of scRNA-seq and histological
   techniques, we report that glial responses to FGF1 were generally rapid
   in onset and accompanied by an activated morphology. We also identified
   Agrp neurons, key components of the melanocortin signaling pathway, as
   the most FGF1-responsive neuronal cell type at the transcriptional
   level, and in a series of in vivo studies, we show that sustained
   diabetes remission induced by icv FGF1 is dependent on intact
   melanocortin receptor signaling.

Results

Study design

   Consistent with previous studies^[97]4,[98]5,[99]11, icv delivery of
   FGF1 (3 µg) into the lateral ventricle of diabetic Lep^ob/ob
   mice induced a robust but transient anorectic response at Day 1, with
   normalization of blood glucose (BG) levels occurring by Day 5
   (Fig. [100]1a). To identify transcriptional correlates of these
   distinct responses to FGF1, we generated a series of RNA-seq datasets
   at Days 1, 5, and 42 (Fig. [101]1a) and adjusted for differences of
   food intake by pair-feeding of icv vehicle-injected controls to the
   intake of icv FGF1-injected mice.

Fig. 1. Study design and icv FGF1-induced temporal glia responses.

   [102]Fig. 1
   [103]Open in a new tab

   a Diabetic Lep^ob/ob mice received a single intracerebroventricular
   (icv) injection of FGF1 (3 µg) or vehicle at Day 0. Hypothalami were
   harvested on Days 1, 5, and 42 after icv injection. Vehicle-injected
   mice were pair-fed (PF) to the amount of food consumed by icv
   FGF1-injected Lep^ob/ob mice and morning blood glucose levels were
   monitored. Plots are represented as mean ± SEM, and source data are
   provided as a Source data file; FGF1 (n = 17), vehicle (n = 15). b
   Uniform manifold approximation and projection (UMAP) clustering of
   hypothalamic nuclei from Day 1 and Day 5 snRNA-seq datasets. Source
   data are available through the NCBI Gene Expression Omnibus (GEO;
   [104]GSE153551). c UMAP clustering from b colored according to
   treatment group and time of euthanasia. d UMAP clustering of
   hypothalamic non-neuronal cells from Day 1 and Day 5 snRNA-seq
   datasets. e Number of differentially expressed genes (DEGs) for
   non-neuronal subclusters at Day 1 identified through repeated
   downsampling (10 cells/group/cell type/iteration; 100 iterations),
   n = 3 mice/group. Data are presented as median with interquartile range
   (IQR) and summary statistics are provided as a Source data file. f Each
   bar represents the number of unique DEGs for one or a certain
   combination of cell types (pseudobulk approach, see “Methods”) from Day
   1 snRNA-seq dataset, n = 3 mice/group. g Associated Gene Ontology terms
   (biological process category; red), KEGG (blue) and REACTOME (green)
   pathways associated with DEGs in the indicated cell types. Point size
   indicates −log[10](p value) (Bonferroni adjusted). h Weighted gene
   co-expression network analysis (WGCNA) of tanycytes, ependymal cells,
   and astrocytes from Days 1 and 5 snRNA-seq datasets. Significantly
   changed gene modules (linear mixed-effects model; FDR < 0.05) are
   indicated with color and cell-specific expression is indicated by
   shape. Modules are plotted according to their β-values; Day 1
   (n = 3/group), Day 5 (n = 2/group). i Module plots of top 15 genes of
   sn-glia-M1–sn-glia-M3, which are upregulated on Day 1. Genes in red
   denote genes mentioned in the text. Astro astrocytes, Tany tanycytes,
   Epend ependymal cells, Olig oligodendrocytes, OPC oligodendrocyte
   precursor cell, COP committed to differentiate oligodendrocyte, VLMC
   vascular leptomeningeal cell.

   To transcriptionally characterize Days 1 and 5, we performed
   single-nucleus RNA-sequencing (snRNA-seq) and generated a dataset
   comprising 15,291 cells that clustered into 9 overall cell populations
   (Fig. [105]1b, c). For this dataset, we, on average, recovered 3258
   transcripts (unique molecular identifiers (UMIs)) and 1,792 unique
   genes per cell across all hypothalamic cell populations.

Tanycytes, ependymal, and astrocytes are highly responsive to icv FGF1

   To better characterize the response of hypothalamic tanycytes,
   astrocytes, and other glial cells Day 1 after icv FGF1 injection in
   diabetic Lep^ob/ob mice, we extracted all non-neuronal cells and
   re-clustered them to delineate glial subpopulations (Fig. [106]1d). To
   estimate which subpopulations were most affected one day after icv FGF1
   injection, differentially expressed genes (DEGs) were identified with a
   repeated downsampling approach to control for both cell number and
   between-sample variation^[107]19. In line with previous evidence that
   both tanycytes and astrocytes are highly responsive to
   FGF1^[108]4,[109]5,[110]11, robust transcriptomic responses were
   observed in tanycytes, ependymal cells, and, to a lesser degree,
   astrocytes (false discovery rate (FDR) < 0.1; Fig. [111]1e). Using a
   pseudobulk approach to identify DEGs (see “Methods”), our analysis of
   FGF1-induced transcriptional changes identified a core set of genes
   (n = 21) shared between tanycytes, ependymal cells, and astrocytes,
   suggesting a common response across these cell types (Fig. [112]1f,
   Supplementary Data [113]1). Furthermore, we tested for enrichment of
   genesets in the Gene Ontology biological process (GO:BP)^[114]20,
   REACTOME (REAC)^[115]21, and Kyoto Encyclopedia of Genes and Genomes
   (KEGG)^[116]22 databases (Fig. [117]1g; Supplementary Data [118]2).
   Tanycytes and ependymal cells showed strong upregulation of ribosomal
   pathways and downregulation of cilia activity, whereas astrocytes
   showed increased activity in processes involved in tissue remodeling
   compared to pair-fed, vehicle-treated mice.

   To further characterize both the biological pathways induced by FGF1
   across tanycytes, ependymal cells, and astrocytes and how these
   responses change over time, we performed weighted gene co-expression
   network analysis (WGCNA)^[119]23 on these glial subsets. Of the nine
   modules identified, three were significantly increased by icv FGF1
   at Day 1 (Fig. [120]1h). Modules sn-glia-M1 and sn-glia-M2 were
   increased by FGF1 to a similar extent across all three cell types,
   whereas sn-glia-M3 appeared to be more strongly upregulated in
   tanycytes than the other cell types (Fig. [121]1h; linear mixed-effects
   models; FDR < 0.05; Supplementary Data [122]3). Consistent with the
   ability of FGF1 to signal through the extracellular signal-related
   kinase 1/2 (ERK1/2) pathway^[123]5, the sn-glia-M1 module was
   significantly enriched for “mitogen-activated protein kinase (MAPK)
   cascade” and “FGF receptor 1 signaling” pathways and included the gene
   Itgav encoding an integrin subunit shown to modulate FGF
   signaling^[124]24. Furthermore, module sn-glia-M2, which included Gfap
   and Vim (markers of reactive astrocytes^[125]25), was upregulated
   across all three cell populations (Fig. [126]1h), suggesting a
   pan-reactive glial response. Module sn-glia-M3 was strongly induced in
   tanycytes and contains the gene Sprr1a (Fig. [127]1i), which marks a
   unique subset of tanycytes situated at the junction of the arcuate
   nucleus (ARC) and the median eminence (ME)^[128]16.

   Whereas these three modules were strongly induced Day 1 after icv FGF1
   injection, each of these responses was nearly normalized by Day 5, with
   a different set of modules showing increased expression relative to
   vehicle at this time point. Among the modules upregulated Day 5 were
   sn-glia-M4 and sn-glia-M5, which were activated specifically in
   astrocytes and ependymal cells, respectively (Fig. [129]1h). These data
   suggest that the transcriptomic response to FGF1 evolves over time in a
   manner that differs fundamentally between astrocyte, tanycyte, and
   ependymal cell populations.

Tanycyte activation is sustained through Day 5 following icv FGF1 injection

   As tanycyte and ependymal cell responses diverged with the passage of
   time following icv FGF1 injection, we generated a larger
   single-cell RNA-sequencing (scRNA-seq) Day 5 dataset, which comprised
   63,904 hypothalamic cells from a separate cohort of icv FGF1-injected
   Lep^ob/ob mice and PF, icv vehicle-injected controls (n = 6/group).
   After quality control and doublet removal, cells were clustered into 14
   cell populations (Supplementary Fig. [130]1). On average, we recovered
   1802 UMIs and 966 unique genes/cell across all cell populations. Within
   this larger dataset, we first inspected cell types lining the third
   ventricle (3V) (Supplementary Fig. 2). Subclustering identified the
   four major tanycyte subpopulations: α1-, α2-, β1-, and β2-tanycytes
   (Fig. [131]2a). Because tanycyte cell identity and function vary with
   dorsal–ventral distribution along the ventricular wall, we anticipated
   finding a continuum of gene expression moving dorsally up the 3V. We
   therefore plotted all tanycyte cells in principal component (PC) space,
   which revealed a one-dimensional curve, reminiscent of what is seen in
   terminally differentiated cells with spatial zonation^[132]26. We next
   fit a principal curve to our data and assigned each cell a value on
   that curve, henceforth referred to as “pseudoventricle score”
   (Fig. [133]2b). Application of WGCNA across this tanycyte population
   identified 35 modules (Supplementary Data [134]5), of which 7 showed a
   significant difference following icv FGF1 treatment compared to vehicle
   across at least 5 consecutive points along the pseudoventricle. Module
   sc-tany-M1 showed a strong significant (linear mixed-effects model;
   FDR < 0.05) increase in expression excluding locations in which
   β2/β1-tanycyte marker expression was high. This module contained the
   gene Vim and enriched for pathways such as “glycolysis” and
   “gluconeogenesis” (Fig. [135]2c, d), suggesting a shift in metabolic
   processes in these cells. Immunostaining for vimentin confirmed a
   robust increase in both ependymal and tanycyte vimentin content 1 week
   after icv FGF1 injection at the predicted ventricle heights. Combined
   with a marked increase in the number of tanycyte processes projecting
   into adjacent hypothalamic parenchyma, these findings are highly
   suggestive of a phenotype switch in these cells (Fig. [136]2e).
   Furthermore, we found that sc-tany-M2, which contained genes with stem
   cell-like properties^[137]27,[138]28, was increased specifically in the
   ventral part of the pseudoventricle, and sc-tany-M3 exhibited a
   significant FGF1-induced increase in expression across nearly the
   entire pseudoventricle and comprised genes involved in protein
   synthesis thus mirroring the response of tanycytes on Day 1. Modules
   sc-tany-M5 and sc-tany-M6 were expressed dorsally along the
   pseudoventricle, included Cd24a and Igfbp5, respectively, confirmed by
   in situ hybridization in cells lining the 3V (Supplementary
   Fig. [139]3)^[140]29, and identified in progenitor cells in the
   ventricular–subventricular zone^[141]30. Genes of the peroxiredoxin
   family characteristic of sc-tany-M23 exhibited a distinct expression
   pattern characteristic of α2-tanycytes, suggesting a role for this
   tanycyte subset in the hypothalamic response to oxidative stress that
   is upregulated by FGF1 (Fig. [142]2f). Additional studies are warranted
   to determine the contribution made by various tanycyte subsets to
   FGF1-induced diabetes remission (for more detailed results and a full
   list of tanycyte and ependymal modules, please refer to
   [143]Supplementary Notes, Supplementary Fig. [144]4, and Supplementary
   Data [145]4 and [146]5).

Fig. 2. Tanycyte module expression maps to the spatial distribution lining
the third ventricle.

   [147]Fig. 2
   [148]Open in a new tab

   a UMAP clustering of tanycytes (β2-, β1-, α1-, α2-Tanycytes) from the
   Day 5 single-cell RNA-sequencing (scRNA-seq) dataset. Cell type
   annotations were inferred from a published dataset^[149]16. b Tanycyte
   lineage cells plotted on principal components 1 and 2 revealed a
   trajectory (left). The colors represent the subtype of each cell from
   a. A principal curve fit across the trajectory was used to assign a
   pseudoventricle score to each cell corresponding to the spatial
   location of that cell type along third ventricular (3V) wall. Density
   plot of each tanycyte subpopulation plotted along the pseudoventricle
   revealed a continuum of gene expression and non-distinct shifts in the
   expression of marker genes along the pseudoventricle. c Scaled module
   expression of sc-tany-M1 in FGF1-treated (black) and vehicle (gray)
   tanycytes (linear mixed-effects model; bin size of 1; mean ± SEM;
   *p < 0.05) (Bonferroni adjusted) from the Day 5 scRNA-seq dataset
   (n = 6 mice/group). Exact adjusted p values are provided in
   Supplementary Data [150]5. ME module eigengene. d Gene–gene network
   plot of the correlation of top 15 genes in module sc-tany-M1 (left) and
   associated pathways (right). Genes mentioned in the text are labeled in
   red. e Representative images of immunolabeling of vimentin in diabetic
   Lep^ob/ob mice 1 week after vehicle or icv FGF1 injection. Pixel
   dimensions: 0.27 × 0.27 × 2.00 microns. Quantification: dots represent
   volume of vimentin-positive voxels assessed by iMARIS for individual
   animals (mean from 2 sections per animal, n = 4/group), mean ± SEM.
   Unpaired t test, two tailed, *p = 0.022 (not adjusted). Source data are
   provided as a Source data file. f Scaled module expression of
   sc-tany-M2, sc-tany-M3, sc-tany-M5, sc-tany-M6, and sc-tany-M23 with 5
   consecutive points of difference in expression along the
   pseudoventricle between FGF1-treated (black) and vehicle (gray)
   tanycytes (linear mixed-effects models; bin size of 1; mean ± SEM;
   *p < 0.05) (Bonferroni adjusted) from the Day 5 scRNA-seq dataset
   (n = 6 mice/group). Exact adjusted p values are provided in
   Supplementary Data [151]5.

Evolution of the astrocyte response to icv FGF1

   In response to various stimuli, the astrocyte phenotype can change
   rapidly and dramatically^[152]13. To date, neurotoxic, neuroprotective,
   pan-reactive, neuron-interactive, and neuronal activity-induced
   astrocyte phenotypes have been reported^[153]25,[154]31. To test
   whether astrocyte responses on Day 1 or Day 5 after icv FGF1 treatment
   correspond to any of these phenotypes, we first performed WGCNA on
   astrocytes in the snRNA-seq dataset. Of the 7 identified modules, 3
   were significantly increased on Day 1 (sn-astro-M1–sn-astro-M3) and 1
   module was significantly increased on Day 5 (sn-astro-M4; Fig. [155]3a;
   linear mixed-effects models; FDR < 0.05; Supplementary Data [156]6).
   The 3 upregulated astrocyte modules on Day 1 enriched for markers of
   pan-reactive astrocytes, while sn-astro-M2 and sn-astro-M3
   also enriched for markers of neuroprotective astrocytes (Fig. [157]3a).
   Although sn-astro-M1 did not enrich for markers of either neurotoxic or
   neuroprotective astrocytes, its hub gene Npas3 (Fig. [158]3b) is a
   transcription factor belonging to the bHLH-PAS superfamily known to be
   involved in FGF signaling, hypoxia, circadian rhythm, and metabolic
   pathways and has been proposed as a link between mental illness and
   metabolic disorders^[159]32.

Fig. 3. Evolution of the astrocyte response to icv FGF1.

   [160]Fig. 3
   [161]Open in a new tab

   a Identification of sn-astro-M1–sn-astro-M3 upregulated at Day 1 and
   sn-astro-M4 upregulated at Day 5 in FGF1-treated mice (linear
   mixed-effects model, FDR < 0.05) using WGCNA on astrocytes from the
   snRNA-seq datasets. Enrichment of DEGs in astrocytes in response to
   lipopolysaccharide (neurotoxic, A1), middle cerebral artery occlusion
   (neuroprotective, A2), or overlapping genes (PAN)^[162]25 or enrichment
   of genes induced by neuron interaction (NeurI) or neuron activity
   (NeurA)^[163]31. Point size is scaled by −log[10](p value) (Bonferroni
   adjusted). b Correlation of top 15 genes of sn-astro-M1–sn-astro-M4.
   Node size; correlation of the given gene with the module eigengene
   (kME). c Number of DEGs for non-neuronal subclusters identified in the
   Day 5 scRNA-seq dataset with repeated downsampling (10 cells/group/cell
   type/iteration, 100 iterations; n = 6 mice/group), median (IQR).
   Summary statistics provided as a Source data file. Macro macrophages,
   Peri pericytes, ABC arachnoid barrier cells, SMC smooth muscle cells,
   VLMC vascular leptomeningeal cells. d Volcano plot of pseudobulk DEGs
   in astrocytes Day 5, scRNA-seq dataset between FGF1 and vehicle. Blue,
   genes with FDR < 0.05; red, genes with FDR < 0.05 and |log[2] fold
   change| > 0.25; green, genes with |log[2] fold change| > 0.25; gray,
   genes below both thresholds. e Astrocytes WGCNA (Day 5; scRNA-seq)
   identified three significant upregulated (sc-astro-M1–sc-astro-M3;
   linear mixed-effects model; FDR < 0.05) and three downregulated
   (sc-astro-M5, sc-astro-M6, sc-astro-M9) modules in FGF1-treated mice,
   median (IQR). Lower panel: hypergeometric enrichment analysis of
   single-nucleus RNA-sequencing (snRNA-seq) and scRNA-seq astrocyte
   modules. Point size is scaled by −log[10](p value) (Bonferroni
   adjusted; n = 6 mice/group). f Correlation of top 15 genes in modules
   sc-astro-M1 and sc-astro-M3. g Representative images of
   immunoreactivity of aquaporin 4 (Aqp4; red) and glial fibrillary acid
   protein (GFAP; green) from coronal sections of hypothalamus from
   diabetic Lep^ob/ob mice 1 week after either vehicle or FGF1 icv
   injection. Lower panels: higher magnification of white inset. Scale bar
   100 μm (top), 50 μm (lower). Pixel dimensions:
   0.27 × 0.27 × 2.00 microns. Quantification: dots represent volume of
   Aqp4- or GFAP-positive voxels for individual animals (mean from 2
   sections per animal, n = 4/group), mean ± SEM. Unpaired t test, two
   tailed, *p = 0.037, **p = 0.003 (not adjusted). Source data are
   provided as a Source data file. Genes mentioned in the text are labeled
   in red.

   In contrast, sn-astro-M4, which was upregulated on Day 5 post-FGF1
   treatment, significantly enriched for neuron-interacting (NeurI) genes,
   suggestive of increased astrocyte–neuronal interaction. Among hub genes
   for sn-astro-M4 was Sparcl1, a matricellular protein implicated in
   synaptic plasticity^[164]33, as well as genes encoding proteins
   involved in synaptic glutamate (Ndrg2) and potassium (Kcnj10)
   homeostasis (Fig. [165]3b). This module enriched for the terms
   “ammonium ion metabolic process,” “PPAR signaling pathway,” and “lipid
   metabolic process” (Supplementary Data [166]6), consistent with a model
   in which FGF1 helps to reduce neuronal stress^[167]34 through increased
   neuron–astrocyte metabolic coupling.

   To confirm the snRNA-seq findings on Day 5 post-treatment and to gain
   more insight in the glial response to icv FGF1, we again utilized the
   larger scRNA-seq dataset. The relatively high number of resampled DEGs
   in astrocytes at Day 5 compared to the other cell glial cell types
   confirmed that, in these cells, the transcriptional response at Day 5
   was more robust than at Day 1 (Fig. [168]3c). Pseudobulk analysis of
   astrocytes identified 186 DEGs on Day 5 (|log[2] fold change (FC)| > 1;
   FDR < 0.05), of which 94 were upregulated and 92 downregulated, with
   Gfap, Aqp4, and Vim among the top upregulated genes (Fig. [169]3d;
   Supplementary Data [170]7). Using WGCNA, we identified 6 distinct
   astrocyte modules associated with FGF1 treatment on Day 5 (linear
   mixed-effects models; beta > 0.005; FDR < 0.05)
   (sc-astro-M1–sc-astro-M6; Fig. [171]3e; Supplementary Data [172]8), 3
   of which were upregulated by icv FGF1. Hypergeometric enrichment
   revealed a significant overlap between sn-astro-M4 and sc-astro-M1 (10
   genes in common) as well as with sc-astro-M3 (7 genes in common;
   Fig. [173]3e). Module sc-astro-M3 included genes involved in regulating
   homeostasis of both synaptic glutamate (Slc1a2, Slc1a3) and GABA
   (Slc6a11), as well as the major astrocyte water channel Aqp4
   (Fig. [174]3f). Module sc-astro-M1 included genes regulating synapse
   formation (Sparcl1, Sparc), dendritic growth (Psap), and other
   neuroprotective functions (Cpe, Clu, Plat). These findings confirm the
   astrocyte response on Day 5 and are suggestive of multiple
   neuroprotective responses that range from changes in interstitial fluid
   homeostasis to direct regulation of synapse/dendrite formation
   (Supplementary Data [175]8).

   To validate and extend these transcriptomic findings, we used
   immunohistochemistry (IHC) to determine whether expression of Aqp4 (a
   shared upregulated gene at Day 5 in all astrocyte analyses and
   datasets) was increased at the protein level in astrocytes. We found
   that, in a separate cohort of diabetic Lep^ob/ob mice, Aqp4 protein
   content was increased 1 week after icv FGF1 injection, particularly in
   the mediobasal hypothalamus (MBH; Fig. [176]3g). We also detected a
   robust increase of glial fibrillary acidic protein (GFAP; encoded by
   Gfap) immunostaining in astrocytes 1 week following icv FGF1 injection,
   confirming the FGF1-induced astrocyte modules sc-astro-M3 and
   sn-astro-M2 containing Gfap. Combined with the robust, star-shaped
   astrocyte morphological change induced by FGF1 (Fig. [177]3g), these
   findings are suggestive of an astrocyte phenotype switch. Clarification
   of contribution made by these astrocyte responses to FGF1-induced
   glucose lowering is warranted.

Unique temporal signatures in oligodendrocytes induced by icv FGF1

   At a transcriptional level, oligodendrocytes displayed a distinct
   temporal response to FGF1. While evidence of minor immune activation
   was seen in mature oligodendrocytes on Day 1 (Supplementary
   Data [178]3), icv FGF1 had minimal effects on oligodendrocyte precursor
   cells (OPCs) at this early time point. By Day 5, however, our
   resampling test indicated a strong response in both oligodendrocytes
   and committed to differentiate oligodendrocyte precursors (COPs)
   (Fig. [179]3c). To better understand these responses, we extracted all
   oligodendrocyte lineage cells from our scRNA-seq dataset and mapped
   them to an existing dataset with a more detailed annotation of
   transcriptional oligodendrocyte heterogeneity^[180]35 (Fig. [181]4a).
   Retaining only cells that mapped with high confidence between datasets,
   we detected a significant increase in populations of both COPs and
   newly formed oligodendrocytes (NFOLs) in FGF1-treated mice
   (Fig. [182]4b) but not in mature oligodendrocytes.

Fig. 4. Icv FGF1 induces a population of differentiating oligodendrocytes.

   [183]Fig. 4
   [184]Open in a new tab

   a UMAP clustering of cells in the oligodendrocyte lineage from the Day
   5 scRNA-seq dataset. Cell type annotations were inferred from a
   published dataset^[185]35. b Dots represent mean number of cells per
   animal and lines represent mean ± SEM. Unpaired t test, two tailed,
   *p < 0.022, **p < 0.0074 (Bonferroni adjusted; n = 6 mice/group); ns
   not significant. Source data for each subcluster are provided as a
   Source data file. c UMAP of cells in the oligodendrocyte lineage
   labeled according to maturation in Monocle-derived pseudotime. d UMAP
   of cells expressing Gpr17, a maturing oligodendrocyte marker. e
   RNAScope detection of Gpr17 expression in the hypothalamus of Lep^ob/ob
   mice 5 days after icv FGF1 or vehicle. f Quantification of Gpr17+ cells
   from the ARC area (2 sections per animal, icv vehicle (n = 4) and icv
   FGF1 (n = 5)), mean ± SEM. Unpaired t test, two tailed, *p = 0.005 (not
   adjusted). Source data are provided as a Source data file. NFOL newly
   formed oligodendrocyte, MFOL myelin-forming oligodendrocyte, MOL1
   myelinating oligodendrocyte.

   We next used Monocle2^[186]36 to sort these cells along their likely
   developmental trajectories. The pseudotime trajectory confirmed the
   effect of icv FGF1 to increase the pool of cells at an intermediate
   pseudotime (Fig. [187]4c), corresponding to an accumulation of cells
   differentiating from OPCs toward mature oligodendrocytes. Using
   RNAScope to detect Gpr17 expression as a proxy for COPs and NFOLs
   (Fig. [188]4d) in a separate cohort of Lep^ob/ob mice, we confirmed an
   increase in the number of these oligodendrocyte lineage cells in the
   ARC of icv FGF1-injected mice compared to vehicle-treated, PF controls
   (Fig. [189]4e), a response that appeared to extend beyond the MBH.
   Together, these results suggest that oligodendrocyte differentiation
   was induced by icv FGF1 in the hypothalamus of diabetic Lep^ob/ob mice.
   Although oligodendrocytes are well known to ensheath axons and provide
   metabolic support, and although glucose has been shown to be rapidly
   metabolized by oligodendrocytes^[190]37, we are unaware of evidence
   implicating oligodendrocyte lineage cells in the control of glucose
   homeostasis. Whether and how these cells might contribute to sustained
   FGF1-induced diabetes remission warrants additional study.

Effect of FGF1 on neurons

   To confidently identify established hypothalamic neuronal subtypes in
   diabetic Lep^ob/ob mice, we propagated labels from published
   hypothalamic single-cell datasets^[191]16–[192]18 (see “Methods”) and
   subclustered all confidently mapped cells into 15 hypothalamic
   neuron-specific cell populations (Fig. [193]5a). These neurons (11,986)
   were subclustered and manually annotated based on highly enriched genes
   and revealed a small cluster of Pomc-expressing cells (Supplementary
   Fig. [194]5a). Surprisingly, no neurons in our dataset were assigned
   with high confidence to previously characterized Pomc populations.

Fig. 5. Effects of icv FGF1 on Agrp neurons.

   [195]Fig. 5
   [196]Open in a new tab

   a UMAP clustering of neurons from the snRNA-seq dataset that
   confidently mapped to published datasets^[197]16–[198]18. Original
   cell type annotations from the published datasets are indicated in
   brackets. b Numbers of DEGs for neuron subclusters identified using
   repeated downsampling (10 cells/group/cell type/iteration, 100
   iterations; n = 3 mice/group), median (IQR). c Volcano plot of
   pseudobulk DEGs in Agrp neurons from the Day 1 snRNA-seq dataset
   between FGF1 and vehicle. Red, genes with FDR < 0.05 and |log[2] fold
   change| > 0.25; green, genes with an |log[2] fold change| > 0.25; gray,
   genes below both thresholds. d Gene Ontology (biological process; red)
   and REACTOME (blue) terms enriched for DEGs in Agrp neurons at Day 1.
   Circle size denotes gene overlap with the identified geneset. e
   Principal component analysis of Agrp neurons sampled at Days 1 and 5
   (vehicle and FGF1) revealed strong separation from other groups at PC4
   and PC5, respectively (n = 3 mice/group Day 1, n = 2 mice/group Day 5),
   median (IQR). f Gene Ontology term (biological processes) of highly
   loading genes on PC5. g Normalized expression of Agrp and Npy in Agrp
   neurons across the 4 groups (FGF1, Days 1 and 5; vehicle, Days 1 and 5;
   n = 3 mice/group Day 1, n = 2 mice/group Day 5). h Top panel:
   representative electron microscopic images of coverage of synapses
   between axonal boutons (B) and dendritic spines (S) by peri-synaptic
   astrocytic in the ARC area of Lep^ob/ob mice. Bottom panel:
   immunostaining for Npy. Scale bar 500 nm. Quantification: dots
   represent percentage of astrocytic coverage for individual animals
   (mean of 30 synapses per animal, n = 4/group), mean ± SEM. Unpaired t
   test, two tailed, ***p < 0.0001 (not adjusted). i Contacts between
   GFAP(+) astrocytes (green) and Agrp(+) neurons in the ARC area
   (equivalent to outline in g) 1 week following icv FGF1 or vehicle
   injection of Lep^ob/ob. Yellow represents co-labeled GFAP-positive and
   Agrp-positive voxels. Scale bar 8 μm. Pixel dimensions:
   0.057 × 0.057 × 0.500 microns. Quantification: dots represent mean
   contact volume over high power field (hpf: 116.5 × 116.5 microns) for
   individual animals (2 sections per animal, n = 4/group), mean ± SEM.
   Unpaired t test, two tailed, **p = 0.0093 (not adjusted). Source data
   and summary statistics are provided as a Source data file.

Agouti-related peptide (Agrp) neurons are the most responsive neuronal
subtype

   To estimate which neuronal populations were most affected 1 day after
   icv FGF1 injection, we quantified the amount of DEGs for each neuronal
   subtype using a repeated downsampling approach^[199]19. This analysis
   revealed Agrp neurons to be the most FGF1-responsive neuronal
   population, with no other subsets (including Pomc neurons) having a
   median number of DEGs > 1 by Day 1 (Fig. [200]5b; Supplementary
   Fig. [201]5b). Utilizing the pseudobulk approach including all Agrp
   neurons from Day 1 (n = 313), we identified 47 DEGs (|log[2] FC| > 1;
   FDR < 0.05), of which 37 were upregulated (Fig. [202]5c; Supplementary
   Data [203]9). To identify putative biological pathways activated by
   FGF1 in Agrp neurons, we subjected upregulated and downregulated DEGs
   identified in Agrp neurons to geneset enrichment analysis (GSEA) and
   found that, in Agrp neurons, FGF1 increases the expression of genes
   involved in both synapse assembly and regulation of membrane potential
   (Fig. [204]5d).

   To investigate whether the response in Agrp neurons evolved over time,
   we compared Agrp neuron expression profiles between Day 1 and Day 5
   after icv injection. To identify genes that drive variance in Agrp
   neurons across these two time points, we performed PC analysis (PCA) to
   identify those components which best separate the four groups (vehicle
   vs. FGF1, Day 1 vs. Day 5). In these neurons, the strongest differences
   in loading values were in PC4 and PC5, which, respectively, explained
   1.9% and 1.7% of the variation in Agrp neurons and separated FGF1-Day 1
   and FGF1-Day 5 Agrp neurons from the other Agrp neurons (Fig. [205]5e).
   The top loading genes on PC4 overlapped with genes identified as being
   significantly upregulated on Day 1, including Pcdh15 and Kcnq3,
   suggesting that the effect of FGF1 to acutely induce expression of
   these genes in Agrp neurons had waned by Day 5 (Supplementary
   Data [206]10). Pathway enrichment analysis for negatively loading genes
   on PC5 (genes with increased expression in Agrp neurons in the FGF1 Day
   5 group) was suggestive of changes in postsynaptic regulation and
   function (Fig. [207]5f). Furthermore, Agrp and Npy were the two most
   strongly positively loading genes on PC5, and expression levels of both
   genes were strongly reduced on Day 5 compared to all other groups
   (Fig. [208]5g). By comparison, neither Agrp nor Npy expression were
   reduced on Day 1 relative to PF, vehicle-treated controls, suggesting
   that the initial response of Agrp neurons to icv FGF1 injection (Day 1)
   involves alteration of genes implicated in the control of membrane
   potential, whereas the later response (Day 5) is predominated by
   decreased Agrp and Npy gene expression. Whether these findings are
   indicative of sustained, FGF1-induced Agrp neuron inhibition warrants
   additional study.

FGF1 increases astrocytic contact with neurons

   In light of both the important role played by astrocytes to modulate
   synaptic function^[209]13–[210]15 and our evidence of a robust
   transcriptomic and histochemical response of astrocytes to FGF1, we
   next sought to test whether synaptic ensheathment by astrocyte
   processes is affected by FGF1. Electron microscopic analysis of the ARC
   area on samples obtained following icv injection of either vehicle or
   FGF1 in a separate cohort of Lep^ob/ob mice (n = 4/group) revealed an
   increase in coverage of synaptic boutons on dendritic spines by
   peri-synaptic astrocytic processes that included, but was not limited
   to, neurons positive for Neuropeptide Y (NPY; which is co-expressed
   with Agrp in ARC neurons; Fig. [211]5h).

   Because these findings were associated with reduced Agrp and Npy
   expression levels, and because astrocytes are reported to modulate the
   tone of neurons in the melanocortin system^[212]15, we next asked
   whether physical interactions between astrocytes and Agrp neurons are
   induced by icv FGF1 injection. Consistent with this hypothesis,
   co-staining for GFAP and Argp performed 1 week following icv FGF1 (or
   vehicle) injection in a separate cohort of Lep^ob/ob mice reveals a
   marked increase of cellular contacts between GFAP(+) astrocytes and
   Agrp neurons induced by FGF1 in the ARC area (Fig. [213]5i). These
   findings point to a model in which the astrocyte response to FGF1
   involves changes in synaptic function within hypothalamic neurocircuits
   relevant to glucose homeostasis. Such an effect need not be limited to
   Argp neurons, and further studies are warranted to determine whether
   and how enhanced glia–neuron interactions in the hypothalamus might
   contribute to the sustained antidiabetic action of FGF1.

Dependence of FGF1-induced diabetes remission on melanocortin receptor
signaling

   Reduced melanocortin signaling is linked to diabetes pathogenesis in
   both rodents and humans^[214]38–[215]41. Because Agrp neurons are the
   most responsive neurons after FGF1 injection, and since these neurons
   function as endogenous inhibitors of melanocortin signaling, we next
   asked whether sustained antidiabetic action of FGF1 depends on intact
   melanocortin signaling. To this end, we tested whether either mice
   lacking the melanocortin 4 receptor (Mc4r; Mc4r^−/−) or agouti (KK-A^y)
   mice (in which obesity and T2D (depending on the background strain)
   result from ectopic overexpression of Agouti, an Agrp homolog^[216]42)
   are resistant to the sustained antidiabetic response to icv FGF1
   injection. We found that, whereas both Mc4r knockout and KK-A^y mice
   exhibited the rapid reduction of food intake and BG levels typical of
   Lep^ob/ob mice following icv injection of FGF1 (3 µg)^[217]4
   (Figs. [218]1a and [219]6a, b), BG levels returned to baseline within
   10–14 days in both mouse strains, unlike the prolonged normoglycemia
   induced by icv FGF1 injection in Lep^ob/ob mice^[220]4. Whereas intact
   melanocortin signaling is not required for initial anorexia and glucose
   lowering, these findings suggest that it is required for the sustained
   antidiabetic action induced by icv FGF1 injection.

Fig. 6. FGF1-induced sustained diabetes remission is melanocortin dependent.

   [221]Fig. 6
   [222]Open in a new tab

   a Blood glucose levels and food intake in diabetic Mc4r^−/− (Mc4r
   knockout) mice injected with icv FGF1 (3 µg) or vehicle (n = 8/group).
   b Blood glucose levels and food intake in agouti (KK-A^y) mice icv
   injected with FGF1 (3 µg) (n = 12) or vehicle (n = 9). Vehicle-injected
   mice were not pair-fed to the intake of mice receiving FGF1. c Blood
   glucose levels and food intake of diabetic Lep^ob/ob mice treated with
   either a single icv injection of FGF1 (3 µg) or vehicle, followed
   immediately by continuous icv infusion of either vehicle or the Mc4r
   antagonist SHU9119 (5 nmol/day) for 28 days. Veh+Veh (n = 8), FGF1+Veh
   (n = 9), Veh+SHU (n = 6), FGF1+SHU (n = 8). Data are represented as
   mean ± SEM, source data are provided as a Source data file.

   To test this hypothesis further, we generated four groups of Lep^ob/ob
   mice, each of which received two separate icv treatments: a single icv
   injection of either FGF1 (3 µg) or vehicle, followed immediately by
   continuous icv infusion of either vehicle or the Mc4r antagonist,
   SHU9119 (5 nmol/day) for 28 days. Whereas food intake, body weight
   (BW), and BG levels did not change in icv vehicle-injected mice,
   regardless of whether they also received continuous icv infusion of
   vehicle or SHU9119 (Fig. [223]6c; Supplementary Fig. [224]6), icv FGF1
   injection elicited both the expected initial reduction of food intake
   and the long-lived normalization of glycemia when it was coupled to
   continuous icv infusion of vehicle. When icv FGF1 injection was
   combined with continuous SHU9119 infusion, however, remission of
   hyperglycemia was not sustained even though the transient effects of
   icv FGF1 to reduce food intake, BW, and BG levels remained intact
   (Fig. [225]6c; Supplementary Fig. [226]6). Statistical analysis
   ([227]Supplementary Notes) confirmed that, whereas icv SHU9119 infusion
   had no effect on transient reductions of food intake and glycemia
   induced by FGF1, it fully blocked sustained diabetes remission (Chi
   square (3) = 10.3, p = 0.016 (not adjusted)). These findings
   collectively demonstrate that, although increased melanocortin
   signaling is not required for the acute, transient effects of icv FGF1
   injection on energy balance and glycemia, it is required for sustained
   diabetes remission.

Long-term hypothalamic changes induced by icv FGF1

   To investigate the durability of transcriptional responses elicited by
   icv FGF1, we generated bRNA-seq datasets from hypothalami of
   independent cohorts of Lep^ob/ob mice on Days 1, 5, and 42 post-icv
   FGF1 injection and compared the results to PF, vehicle-treated
   controls. DEG analysis revealed profound transcriptional changes at Day
   1 that dissipated by Day 42 (|log[2] FC| > 0.5; FDR < 0.05)
   Fig. [228]7a–c; Supplementary Data [229]11). Using rank–rank
   hypergeometric overlap analysis (RRHO)^[230]43 to identify
   transcriptome-wide patterns across the three time points, we identified
   a robust overlap in genes that were upregulated both at Days 1 and 5 as
   well as genes that were upregulated at Day 1 and downregulated at Day
   5. As expected, among the upregulated genes at both time points were
   Gfap, Vim, and S100a10 (another glial marker gene) consistent with the
   snRNA-seq analyses of a shared reactive glia response, while Aqp4 and
   Gpr17 and Bmp4 (a COP marker gene) were upregulated only at Day 5
   likewise confirming the single-cell analyses (Fig. [231]7c). Pathway
   enrichment analysis of co-upregulated genes indicated extracellular
   matrix remodeling and integrin signaling. Likewise, genes upregulated
   at Day 1 and not coherently regulated at Day 5 enriched for ribosome
   biogenesis, mirroring the dissipating response seen in tanycytes and
   ependymal cells. The Day 5 vs. Day 42 comparison revealed a strong
   overlap between genes upregulated at Day 5 and downregulated at Day 42.
   These genes enriched for pathways indicative of glial activity (e.g.,
   “cilium assembly” and “gliogenesis”), suggesting that most of the
   robust glial responses observed on Days 1 and 5 had waned by Day 42
   (Fig. [232]7b).

Fig. 7. FGF1-induced long-term changes in the hypothalamus.

   [233]Fig. 7
   [234]Open in a new tab

   a Number of DEGs (|log[2] fold change| > 0.5; FDR < 0.05) from bulk
   RNA-sequencing (bRNA-seq) of hypothalami from Lep^ob/ob mice injected
   with icv FGF1 or vehicle and harvested Days 1 (FGF1 n = 10, vehicle
   n = 4), 5 (FGF1 n = 9, vehicle n = 7), and 42 (n = 5 mice/group) after
   injection. Control vehicle mice were pair-fed to FGF1-injected mice. b
   Rank–rank hypergeometric overlap maps of the degree of overlapping
   genes between Day 1 and Day 5 (top) and between Day 5 and Day 42
   (bottom). Venn diagrams and top overlapping genes (middle panel).
   Enrichment of Gene Ontology biological process terms of overlapping
   genes (right panel). c Normalized counts (mean ± SEM, n = 5 mice/group)
   of the selected glial genes from the bRNA-seq datasets Days 1, 5, and
   42 after icv FGF1 or vehicle injection. d Normalized counts
   (mean ± SEM, n = 5 mice/group) of Agrp, Npy, Mef2c, and Pomc expression
   Days 1, 5, and 42 after icv FGF1 or vehicle injection. e Targeted
   relative gene expression of Agrp, Npy, and Pomc. Unpaired t test, two
   tailed, *p = 0.04 (not adjusted, n = 5 mice/group); ns not significant.
   Source data are provided as a Source data file. f Threshold-free
   geneset enrichment analysis (Gene Ontology biological processes terms)
   of DEGs identified in bRNA-seq data at Days 1, 5, and 42. Point size is
   scaled by −log[10](p value) (Bonferroni adjusted) and is colored based
   on the normalized enrichment score (NES). g Representative electron
   microscopic images of synapses of Lep^ob/ob mice 5 days after icv
   injection of either vehicle or FGF1. syn synaptic active zone, DCV
   dense-core vesicle, SV small light-core vesicle. Scale bar 500 nm.
   Quantification: dots represent mean %DCV/synapse per mouse (25 synapses
   per animal, n = 4/group). Unpaired t test, two tailed, *p = 0.01 (not
   adjusted); ns not significant. Source data are provided as a Source
   data file. Data are presented as mean ± SEM.

   However, Agrp expression remained significantly downregulated in
   FGF1-treated mice compared to controls on Day 42 (log[2]
   FC = −1.33 ± 0.39; p = 6.0 × 10^−4; Bonferroni significance threshold,
   p < 0.05/30). This analysis also detected Pomc expression at low levels
   at each of the 3 time points, with a significant increase detected 1
   day after FGF1 treatment (log[2] FC = −1.66 ± 0.46; p = 2.9*10^−4;
   Bonferroni significance threshold, p < 0.05/30). While this finding
   raises the possibility of Pomc neuron activation as an early component
   of the neuronal response to FGF1, by Day 42 Pomc levels had returned to
   those of vehicle-treated controls. To validate these observations, we
   performed a targeted analysis using reverse transcription polymerase
   chain reaction (RT-PCR), which confirmed that, 42 days after a single
   icv injection of FGF1 in Lep^ob/ob mice, Agrp expression was indeed
   reduced compared to vehicle controls, albeit not significantly when
   adjusting for multiple testing (p = 0.042; Bonferroni significance
   threshold, p < 0.05/3). Expression of Npy mRNA showed a similar trend
   at this late time point, but the effect did not achieve statistical
   significance, while Pomc expression was unchanged (Fig. [235]7d, e).

   Among the most upregulated genes on Day 42 in Lep^ob/ob mice receiving
   FGF1 was Mef2c (Fig. [236]7d), which encodes a transcription factor
   that influences synaptic plasticity^[237]44. Furthermore,
   threshold-free GSEA on the Day 42 data indicated increased activity in
   neuronal pathways including “long-term synaptic potentiation”,
   “glutamate receptor signaling” and “regulation of synaptic plasticity”,
   while genes involved in “regulation of transmission of nerve impulse”
   were decreased (Fig. [238]7f; Supplementary Data [239]12). Because
   long-term presynaptic silencing can cause synaptic accumulation of
   dense-core vesicles (DCVs; neuronal organelles that contain
   neuropeptides)^[240]45,[241]46, we investigated whether synaptic
   content of DCVs is affected by icv FGF1 injection. Thus electron
   microscopy was employed to evaluate 110 synapses from the MBH of
   Lep^ob/ob mice (n = 4/group) that received icv injection of either FGF1
   or vehicle and were sacrificed 28 days later. The average number of
   DCVs/synapse/mouse was increased in mice receiving FGF1 by 67%
   (p = 0.010; Fig. [242]7g). Taken together, these findings offer
   indirect support for a model in which FGF1 induces sustained changes in
   presynaptic plasticity (afferent and/or local inputs) in the ARC area
   that contribute to long-term changes in circuit computations involved
   in central control of glucose homeostasis. Based on this model, we
   speculate that Mef2c induction by FGF1 contributes to a sustained
   reduction in the excitability of hypothalamic neurocircuits that raise
   the BG level when activated, leading to synaptic DCV accumulation and
   potentially contributing to sustained amelioration of hyperglycemia.
   Future studies are warranted to investigate this model.

Discussion

   Until recently, the brain’s ability to ameliorate hyperglycemia in a
   sustained manner in diabetic animals was unrecognized. Based on the
   untapped therapeutic potential of such an effect, the current work was
   undertaken to identify and characterize the response of diverse
   hypothalamic cell types to FGF1, with the ultimate goal of linking
   these responses to the sustained antidiabetic action of this peptide.
   To this end, we combined an unbiased, discovery-based transcriptomics
   approach with histochemistry, electron microscopy, and in vivo
   pharmacological and physiological studies. We identified dramatic
   transcriptional and morphological changes in both tanycytes and
   astrocytes in the days following icv FGF1 injection. At the
   ultrastructural level, increased astrocyte coverage of synaptic boutons
   was detected on dendritic spines of ARC neurons, including Agrp
   neurons. The transcriptome of Agrp neurons was also strongly altered
   following icv FGF1 administration, including a sustained inhibition of
   both Agrp and Npy expression. These observations raise the possibility
   that Agrp neuron function is altered as a consequence of FGF1 action on
   glial cells, and additional work is warranted to asses this
   possibility. If so, we would infer that the sustained glucose-lowering
   effects of central FGF1 involve actions on one or more hypothalamic
   glial cell types that in turn alter glucoregulatory neurocircuit
   function in a highly durable manner. To our knowledge, such an effect
   has not previously been reported.

   Of particular interest was the response of tanycytes, which line the 3V
   and project long filamentous processes into adjacent hypothalamic
   nuclei involved in glucose homeostasis. These cells have
   glucose-sensing properties and are implicated as participants in
   hypothalamic control of metabolism^[243]9,[244]10,[245]28,[246]47.
   Among the canonical signal transduction pathways activated downstream
   of FGF receptor activation is the MAPK-ERK pathway, and we recently
   reported that icv FGF1 injection rapidly induces MAPK-ERK signaling in
   tanycytes and ependymal cells lining the 3V^[247]5. It is therefore
   unsurprising that, within 24 h of icv FGF1 injection, tanycytes and
   ependymal cells were enriched for co-regulated genes known to be
   involved in the ERK cascade (including Spry2 and Spred1^[248]12).
   Moreover, since tanycytes are implicated in ATP-dependent brain glucose
   sensing^[249]47, it is of interest that genes involved in glycolysis
   (which generates cytosolic ATP) were also upregulated in tanycytes at
   Day 5. Additional study is warranted to ascertain the contribution of
   these robust tanycyte responses to FGF1-induced normalization of
   glucose homeostasis.

   In astrocytes, the most consistently upregulated genes are involved in
   neuroprotection. These include Aqp4, Sparcl1, and the glutamate
   transporters Slc1a2 and Slc1a3, as well as Clu, Cpe, and Ntrk2. The
   latter is of interest because it encodes TrkB, the receptor for
   brain-derived neurotrophic factor (BDNF). Like FGF1, BDNF
   administration can ameliorate hyperglycemia in mouse models of T2D
   whether administered peripherally^[250]48 or when given icv at lower
   doses^[251]49, and BDNF is also implicated as an axon guidance cue for
   ARC neurons innervating adjacent hypothalamic areas during postnatal
   development^[252]50. Upregulation of TrkB in astrocytes is therefore of
   interest as a potential way to influence glucoregulatory neurocircuit
   organization.

   Astrocytes also provide essential homeostatic support to neurons via
   multiple mechanisms. Among these are peri-synaptic astrocyte processes
   (PAPs) which, together with the presynaptic and postsynaptic terminals,
   comprise the “tri-partite synapse”^[253]13. As PAPs are essential both
   to normal synaptic function and to neuroprotection following brain
   injury^[254]25, our finding that PAP coverage of dendritic spines on
   ARC neurons was increased 5 days after icv FGF1 injection identifies a
   potential mechanism whereby FGF1 influences synaptic function of
   hypothalamic neurons involved in glucose homeostasis. This possibility
   is further strengthened by our finding that genes involved in
   astrocyte–neuron crosstalk^[255]31 were also upregulated in astrocytes
   and that FGF1 markedly increased cellular contacts between astrocytes
   and Agrp neurons.

   With the observations that (1) morphological changes of hypothalamic
   astrocytes (astrogliosis) induced by high-fat feeding^[256]15,[257]51
   are associated with altered activity of both Pomc and Agrp
   neurons^[258]15 and (2) impaired melanocortin signaling is implicated
   in the pathogenesis of T2D in both humans and rodent
   models^[259]38–[260]41, we hypothesized that the melanocortin system is
   a target for sustained diabetes remission induced by FGF1. Consistent
   with this notion, we report that sustained diabetes remission is not
   observed when icv FGF1 is given to diabetic mice with genetic
   impairment of melanocortin signaling (agouti mice, Mc4r^−/− mice), nor
   is it observed when melanocortin signaling is blocked pharmacologically
   in Lep^ob/ob mice (by icv infusion of an Mc4r antagonist). Combined
   with evidence that this effect of melanocortin blockade was observed
   despite the fact that FGF1-induced anorexia was unaffected, these
   findings imply that, whereas the sustained antidiabetic action of FGF1
   is melanocortin dependent, the transient anorexic response is not.
   Based on these collective findings, we hypothesize that the
   antidiabetic action of FGF1 involves robust effects on hypothalamic
   glial cell types that in turn trigger sustained changes in the
   melanocortin system and perhaps other, as yet unidentified,
   glucoregulatory neurocircuits.

   One limitation of the current work is that we identified only a small
   subset of Pomc neurons, possibly owing to the marked reduction of Pomc
   gene expression characteristic of Lep^ob/ob mice^[261]52,[262]53.
   Nevertheless, we did observe an increase of Pomc expression in the
   bRNA-seq data on Day 1. If this response was accompanied by increased
   α-melanocyte-stimulating hormone release and binding to Mc4r, the
   resultant increase of melanocortin signaling might have played a role
   in the response to FGF1, potentially augmented by concomitant Agrp
   neuron inhibition. Additional investigation into the roles played by
   Pomc and Agrp neurons in the sustained antidiabetic action of FGF1 will
   be of considerable interest.

   Another limitation inherent in transcriptomics research is that induced
   genes may or may not be translated into protein. We addressed this
   issue by employing IHC, in situ hybridization, and electron microscopy,
   but additional work is needed to clarify the physiological relevance of
   many of the observed transcriptional responses. Outstanding questions
   include: Which transcriptomic responses and cell types are direct
   targets for the effect of FGF1 on the melanocortin system? Which are
   affected secondarily? How do these changes evolve over time? What other
   glucoregulatory neurocircuits might be similarly affected?; And, which
   of these cellular responses are relevant to how the brain senses the
   circulating glucose level? Studies to address these questions are an
   important priority.

   That a single icv injection of FGF1 is sufficient to re-set the
   defended level of glycemia to normal in rodent models of
   T2D^[263]4,[264]5,[265]11 is a finding of substantial therapeutic
   relevance. Ongoing efforts to bridge the gap between diverse cellular
   responses to FGF1 and the associated normalization of glycemia may
   ultimately uncover novel strategies for achieving sustained diabetes
   remission.

Methods

Mice

   All procedures were either performed in accordance with the NIH Guide
   for the Care and Use of Laboratory Animals and were approved by the
   Institutional Animal Care and Use Committee at the University of
   Washington, Novo Nordisk Research Center Seattle, and St. Joseph’s
   Hospital and Medical Center or performed with approved protocols from
   The Danish Animal Experiments Inspectorate permit number
   2014–15–0201–00181 and the University of Copenhagen project number
   P16-122. Male, 6–8-week-old Lep^ob/ob (B6.Cg-Lep^ob/J), 6-week-old
   KK-A^y (KK.Cg-A^y/J), and Mc4r^−/− (B6; 129S4-Mc4r^tm1lowl/J) mice were
   purchased from Jackson Laboratories (Bar Harbor, ME, USA) and
   8-week-old Lep^ob/ob (B6.V-Lep^ob/JRj) mice were purchased from Janvier
   Labs, France.

   Surgerized animals were individually housed under specific
   pathogen-free conditions in a temperature-controlled room, with 75–80%
   humidity and with a 12:12 h light:dark cycle and provided with ad
   libitum access to water and Purina 5008 chow (Animal Specialties, Inc.,
   CA, USA) or Altromin 1310 chow (Brogaarden, Denmark) unless otherwise
   stated. Mice were allowed to acclimatize at least 7 days before
   surgery. After surgery, Mc4r^−/− mice were allowed to acclimatize for 2
   weeks and subsequently put on a high-fat, high-sucrose diet (F1850,
   Cholesterol (0.15%), Paste; Bio-Serv, NJ, USA) to induce hyperglycemia.

Surgeries and icv injections

   Cannulation of the lateral ventricle (LV; 26-ga, Plastics One, Roanoke,
   VA) were performed under isoflurane anesthesia in either Lep^ob/ob or
   KK-A^y mice, using the following stereotaxic coordinates: −0.7 mm
   posterior to bregma; 1.3 mm lateral, and 1.3 mm below the skull
   surface^[266]4. Animals received buprenorphine hydrochloride (Reckitt
   Benckiser Pharmaceuticals Inc., Richmond, VA) or Rimadyl (5 mg/kg,
   Pfizer, NY, USA) postoperatively and were allowed to recover for at
   least 7 days before study, while food intake and BW were recorded. Mice
   whose BW had not recovered 7 days after surgery were excluded from the
   study.

   Mice were closely monitored to ensure matched BW and BG levels between
   study groups. Lep^ob/ob, Mc4r^−/−, and KK-A^y mice that did not exceed
   BG of 250 mg/dl for 3 consecutive days were not considered diabetic and
   were excluded from the study. Recombinant human FGF1 (hFGF1 from Novo
   Nordisk A/S, Denmark) was dissolved in phosphate-buffered saline (PBS)
   at a concentration of 1.5 μg/μl. Mice were injected with either hFGF1
   or vehicle (PBS) over 60 s in a final volume of 2 μl using a 33-gauge
   needle extending 0.8 mm beyond the tip of the icv cannula. Food intake,
   BW, and BG were recorded daily for mice receiving hFGF1 for 5 days and
   vehicle control mice were pair-fed to the mean amount eaten by
   hFGF1-injected mice for 5 days.

   To determine whether the effect of icv FGF1 to induce sustained
   remission of hyperglycemia in Lep^ob/ob mice is dependent on
   melanocortin signaling, we commenced infusion of either saline vehicle
   or the melanocortin receptor antagonist SHU9119 (SHU, Bachem Americas,
   Inc., CA) immediately after administering a single icv injection of
   either saline vehicle or recombinant mouse FGF1 (3 µg; Prospec, NJ,
   USA) in a volume of 2.5 µl to male Lep^ob/ob mice in a volume of 2.5 μl
   as follows. Thus four groups of animals were studied: Group 1: vehicle
   (saline) + vehicle, Group 2: vehicle + FGF1 (3 µg), Group 3: SHU
   (5 nmol) + FGF1, and Group 4: SHU + vehicle. Vehicle or SHU
   (5 nmol/day) was continuously administered to Groups 1–2 or Groups 3–4,
   respectively, for 4 weeks using an osmotic minipump (ALZET, DURECT
   Corporation, CA) that was connected to an icv injector (PlasticOne)
   with polyethylene tubing (Tygon) and subsequently implanted
   subcutaneously to enable direct infusion of either vehicle or SHU. The
   injector was inserted into the LV cannula and fixed with both instant
   and orthodontic adhesives immediately following a single icv injection
   of either vehicle or FGF1.

Generation of single-nucleus suspensions

   Hypothalamic punches (2 mm) (Day 1, n = 3/group; Day 5, n = 2/group)
   were rapidly extracted between 9 and 12 a.m., dissected, and then
   placed ventral surface up into a chilled stainless steel brain matrix.
   A single coronal section (1.5-mm thick) block was obtained between the
   rostral and the caudal ends of the Circle of Willis, followed by a
   hypothalamic punch (2 mm), which was immediately frozen on dry ice and
   stored at −80 °C until further processing. For the single-nuclei
   dissociation, frozen hypothalamic punches were recovered in 1 ml
   ice-cold EZ PREP buffer (Sigma) and homogenized using 2 ml glass dounce
   tissue grinders; 10 times with pastel A and 10 times with pastel B,
   followed by 5-min incubation on ice. Homogenate was transferred to
   clean tubes and centrifuged at 500 × g for 5 min at 4 °C. Supernatant
   was removed followed by an additional 5 min incubation on ice in 1 ml
   EZ PREP buffer and centrifuged at 500 × g for 5 min at 4 °C. The pellet
   was washed first in 1 ml PBS (Gibco) with 1.0% bovine serum albumin
   (BSA) and centrifuged at 500 × g for 5 min at 4 °C followed by a 500 ml
   PBS + 0.1% wash containing LIVE/DEAD stain (Life Technologies) to ease
   nuclei visualization. Debris and myelin were removed from the
   resuspended nuclei by a 1.80 M sucrose gradient centrifugation step
   according to the manufacturer’s protocol (Nuclei PURE Prep Isolation
   Kit, Sigma). Nuclei were subsequently washed in 1 ml PBS + 1.0% BSA,
   filtered through a 40-mm mesh (Falcon), centrifuged at 500 × g for
   5 min at 4 °C, and resuspended in 50–150 ml PBS + 1.0% BSA. Nuclei
   concentration was estimated by counting on a hemocytometer (iChip) and
   kept on ice until single-cell encapsulation.

Generation of single-cell suspensions

   Mice (n = 6/per group) were euthanized and brains were rapidly
   extracted, cooled in ice-cold Dulbecco’s modified Eagle’s medium/F12
   media (Gibco, Thermo Fisher Scientific) for 5 min. Dissection of the
   brain was performed as above followed by a triangular section of the
   MBH using a scalpel. Dissected tissue was digested using a Neural
   Tissue Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany)
   with manual dissociation with the following modifications: sections
   were immediately placed in preheated (37 °C) enzyme mix 1 (1425 ml
   Buffer X + 37.5 ml Enzyme E) and incubated in closed tubes for 15 min
   at 37 °C under slow, continuous rotation. Then enzyme mix 2 (15 ml
   Buffer Y + 7.5 ml Enzyme A) was added followed by mechanically
   dissociated using a wide-tipped, fire-polished Pasteur pipette by
   pipetting up and down 10 times slowly. After a 10-min incubation under
   slow, continuous rotation, using a series of 2 fire-polished Pasteur
   pipettes with incrementally smaller openings the tissue was gently
   dissociated until no visible pieces were observed. Cell suspensions
   were centrifuged at 300 × g for 10 min at room temperature (RT) and
   resuspended in 500 ml Hanks’ Balanced Salt Solution (HBSS) (catalog #
   14025092, Gibco, Thermo Fisher Scientific) and 0.04% (w/v) BSA (catalog
   # A9418, Sigma-Aldrich). Following an additional wash (centrifugation
   at 300 × g for 5 min and re-suspension in 100 µl HBSS + 0.04% BSA),
   cells were filtered through a 40-mm mesh, and 50 ml HBSS + 0.04% BSA
   was added through the filter and transferred to clean tubes. The cell
   suspension was centrifuged at 300 × g for 5 min and resuspended in
   50–150 ml HBSS + 0.04% BSA. Cell concentrations were estimated on a
   NucleoCounter NC-3000 (Chemometec, Denmark) and kept on ice until
   single-cell encapsulation.

Generation of bulk RNA-seq data

   Following euthanasia, brains (Day 1, n = 4 vehicle; n = 10 FGF1; Day 5,
   n = 7 vehicle; n = 9 FGF1; Day 42, n = 42/group) were dissected as for
   snRNA-seq and immediately frozen on dry ice and stored at −80 °C until
   further processing. RNA was isolated using the Qiagen RNeasy Mini Kit
   (catalog # 74104), followed by high capacity cDNA reverse transcription
   (from Applied Biosystems, catalog # 4368813), and finally analysis was
   carried the using Agilent RNA 6000 Nano Kit. The RNA sample
   concentration was between 200 and 350 ng/μl, with 10–15 μg purified RNA
   from each individual sample. All RNA samples showed a good purity
   (A260/280 ratios are between 2.0 and 2.1), and Bioanalyzer results
   demonstrated a good quality of RNA for all samples (RIN numbers > 9.2).
   Sample mRNA libraries were prepared using the Illumina mRNA True Strand
   Kit (Covance Genomics Lab, Redmond, WA; Seattle Genomics Core, Seattle,
   WA) and sequenced on either an Illumina HiSeq 2500 system to generate
   100 bp paired end libraries with 20–30 million reads (Covance Genomics
   Lab, Redmond, WA) or Illumina NovaSeq 6000 system on an S1 flow cell
   yielding approximately 35 million (2 × 50 bp) paired end reads.

Single-cell RNA sequencing

   Single-cell cDNA libraries were generated using the Chromium
   single-cell platform and the 3′ v2 Reagent Kit according to the
   manufacturer’s protocol (10× Genomics, USA). Single-cell libraries were
   sequenced on a NextSeq 500 platform (3 samples (whole single cells) and
   2 samples (single nuclei) on one flow cell) to obtain 100- and 32-bp
   paired end reads using the following read length: read 1, 26 cycles,
   read 2, 98 cycles and i7 index, 8 cycles.

Single-cell RNA-seq and single-nucleus RNA-seq data processing

   Cell Ranger version 1.2 (10× Genomics, USA) was used to de-multiplex
   and quantify UMIs. A count matrix was generated for each sample with
   default parameters and genes were mapped to the mouse reference genome
   GRCm38 (mm10, part of the Cell Ranger software package; for the
   scRNA-seq dataset) or an adapted reference from mouse reference genome
   including introns (for the snRNA-seq dataset), following the steps
   outlined on the 10× Genomics website
   ([267]https://support.10xgenomics.com/single-cell-gene-expression/softw
   are/pipelines/latest/advanced/references#premrna). Barcodes were