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. 2022 Oct;21(10):e13711.
doi: 10.1111/acel.13711. Epub 2022 Sep 19.

Hexosamine pathway activation improves memory but does not extend lifespan in mice

Kira Allmeroth  1 , Matías D Hartman  1 , Martin Purrio  1 , Andrea Mesaros  1 , Martin S Denzel  1   2   3   4
Affiliations

Hexosamine pathway activation improves memory but does not extend lifespan in mice

Kira Allmeroth et al. Aging Cell. 2022 Oct.

Abstract

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long-term effects of chronic HBP activation in mammals. Thus, we asked if HBP activation affects metabolism, coordination, memory, and survival in mice. N-acetyl-D-glucosamine (GlcNAc) supplementation in the drinking water had no adverse effect on weight in males but increased weight in young females. Glucose or insulin tolerance was not affected up to 20 months of age. Of note, we observed improved memory in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc treatment. To assess the effects of genetic HBP activation, we overexpressed the pathway's key enzyme GFAT1 and a constitutively activated mutant form in all mouse tissues. We detected elevated levels of the HBP product UDP-GlcNAc in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.

Keywords: GFAT1; hexosamine biosynthetic pathway; memory; metabolism; mouse survival.

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Conflict of interest statement

The authors declare no potential conflict of interests.

Figures

FIGURE 1
FIGURE 1
Hexosamine biosynthetic pathway activation by GlcNAc feeding does not have adverse effects in mice. (a) Schematic representation of the hexosamine biosynthetic pathway. The rate‐limiting enzyme glutamine fructose‐6‐phosphate amidotransferase (GFAT) is depicted in purple, GlcNAc is marked in blue. GNPDA: Glucosamine‐6‐phosphate isomerase 1; GNA1: Glucosamine‐6‐phosphate N‐acetyltransferase; PGM3: Phosphoacetylglucosamine mutase; UAP1: UDP‐N‐acetylhexosamine pyrophosphorylase; GALE: UDP‐glucose 4‐epimerase; HK: Hexokinase; NAGK: N‐acetyl‐D‐glucosamine kinase. (b) Drinking volume of control (white) and GlcNAc‐treated mice (blue) of both sexes at 3 and 9 months of age. Data are presented as mean ± SD (n ≥ 7). Two‐way ANOVA, Tukey's post‐test; *p < 0.05; ns: Not significant. (c) GlcNAc consumption in mg/kg body weight per day of mice of both sexes at 3 and 9 months of age. Data are presented as mean ± SD (n ≥ 7). Unpaired t‐test; ns: Not significant. (d) Relative UDP‐HexNAc levels in hemibrains of control (white) and GlcNAc‐treated mice (blue) of both sexes. Data are presented as mean ± SEM (n = 6). Unpaired t‐test; ns: Not significant. (e) Blood glucose concentration at 0 (fasting), 15, 30, 60, and 120 min after intraperitoneal injection of glucose solution (2 g/kg body weight) of control (black) and GlcNAc‐treated mice (blue) of both sexes at 10 months of age. Data are presented as mean ± SD (n ≥ 7). Multiple unpaired t‐tests; ns: Not significant (f) area under the curve (AUC) calculated using data shown in (e). Data are presented as mean ± SD (n ≥ 7). Unpaired t‐test; ns: Not significant (g) Body weight of control (black) and GlcNAc‐treated mice (blue) of both sexes from 3 to 30 months of age. The data point at 19 months (females) is missing since the body weight measurements were not performed for this time point. Data are presented as mean ± SD (n ≥ 13). Multiple unpaired t‐tests; **p < 0.01; *p < 0.05; only significant changes are indicated
FIGURE 2
FIGURE 2
GlcNAc supplementation does not influence the fitness of mice. (a) Mean force measured in a grip strength test with two paws of control (white) and GlcNAc‐treated mice (blue) of both sexes at 6 months of age. (b) Mean force measured in a grip strength test with four paws of control (white) and GlcNAc‐treated mice (blue) of both sexes at 6 months of age. (c) Maximal time on a rotarod of control (white) and GlcNAc‐treated mice (blue) of both sexes at 6 months of age. (a‐c) Data are presented as mean ± SEM (n ≥ 13). (d) Maximal distance on a treadmill of control (white) and GlcNAc‐treated mice (blue) of both sexes from at 6 months of age. Data are presented as mean ± SD (n ≥ 13). (a‐d) Unpaired t‐test; ns: Not significant (e) Lifespan analysis of control (black) and GlcNAc‐treated mice (blue) of both sexes (females: N = 69; males: N = 82). (f) Cumulative incidence calculated by Gray's test based on the lifespan analysis shown in (e). Df: Degrees of freedom
FIGURE 3
FIGURE 3
GlcNAc feeding improves memory of young male mice. (a) XY activity measured in the open field test of control (white) and GlcNAc‐treated mice (blue) of both sexes at 6 months of age. (b) Percent of distance spent in the center of the open field of control (white) and GlcNAc‐treated mice (blue) of both sexes at 6 months of age. (a‐b) Data are presented as mean ± SD (n ≥ 12). (c) Percent of time spend in the target quadrant, (d) Latency of the first platform visit, and (e) Number of platform visits upon removal of the hidden platform in the Morris water maze test of control (white) and GlcNAc‐treated mice (blue) of both sexes at 4 months of age. (c‐e) Data are presented as mean ± SD (n = 12). (a‐e) Unpaired t‐test; **p < 0.01; *p < 0.05; ns: Not significant
FIGURE 4
FIGURE 4
HBP activation by huGFAT1 wt OE does not influence body weight in mice. (a) Schematic representation of the transgene. The expression cassette was inserted in the Rosa26 locus (conditional knock‐in allele). Upon cre‐mediated deletion of the transcription termination cassette, FLAG‐HA tagged human GFAT1 is expressed under the control of the chicken β‐actin promoter (constitutive knock‐in allele). (b) Western blot analysis of GFAT1 and HA expression in primary fibroblasts isolated from newborn mice (n = 1). β‐actin was used as loading control. (c) Relative UDP‐HexNAc levels in primary fibroblasts. (d) Relative UDP‐GlcNAc levels in hemibrain isolated from 3 months old control and huGFAT1 wt OE mice of both sexes. (c‐d) Data are presented as mean ± SEM (n ≥ 5). One‐way ANOVA, Dunnett's post‐test; ***p < 0.001; ns: Not significant. (e) Body weight of control and huGFAT1 wt OE mice of both sexes from 12 to 27 months of age. Data are presented as mean ± SD (females: n ≥ 4; Males: n ≥ 7; Details about the number of mice used at each time point is provided in Table S1). Body weight of female WT and CMV‐cre+/− mice is also shown in figure S3d. Two‐way ANOVA, Dunnett's post‐test. Statistical significance was calculated compared with CMV‐cre+/− mice at each time point; only significant changes are indicated. *p < 0.05
FIGURE 5
FIGURE 5
HBP activation by huGFAT1 wt OE does not affect fitness of mice. (a) Mean force measured in a grip strength test with two paws of control and huGFAT1 wt OE mice of both sexes at 3, 15, and 22 months of age. (b) Maximal time on a rotarod of control and huGFAT1 wt OE mice of both sexes at 3, 15, and 21 months of age. (a‐b) Data are presented as mean ± SEM (n ≥ 2). (c) Maximal distance on a treadmill of control and huGFAT1 wt OE mice of both sexes at 4, 16, and 22 months of age. Data are presented as mean ± SD (n ≥ 4). (a‐c) Two‐way ANOVA, Dunnett's post‐test. Statistical significance was calculated compared to CMV‐cre+/− mice at each time point; only significant changes are indicated. **p < 0.01; *p < 0.05 (d) Lifespan analysis of control and huGFAT1 wt OE mice of both sexes (females: n ≥ 68; males: n ≥ 80). Survival of WT and CMV‐cre+/− mice is also shown in figure S4b. (e) Cumulative incidence calculated by Gray's test based on the lifespan analysis shown in (d). Df: Degrees of freedom
FIGURE 6
FIGURE 6
HBP activation by huGFAT1 wt OE does not influence spontaneous locomotor and exploratory behavior or memory of mice. (a) XY activity measured in the open field test of control and huGFAT1 wt OE mice of both sexes at 3, 15, and 21 months of age. (b) Percent of distance spent in the center of the open field of control and huGFAT1 wt OE mice of both sexes at 3, 15, and 21 months of age. (c) Percent of alternations measured in the Y maze test of control and huGFAT1 wt OE mice of both sexes at 3, 15, and 21 months of age. (a‐c) Data are presented as mean ± SD (n ≥ 4). Two‐way ANOVA, Dunnett's post‐test. Statistical significance was calculated compared to CMV‐cre+/− mice at each time point; only significant changes are indicated. ***p < 0.001; **p < 0.01; *p < 0.05 (d) Percent of time spent in the target quadrant, (e) Latency of the first platform visit, and (f) Number of platform visits upon removal of the hidden platform in the Morris water maze test of control and huGFAT1 wt OE mice of both sexes at 4 months of age. (d‐f) Data are presented as mean ± SD (n ≥ 6). One‐way ANOVA, Dunnett's post‐test; ns: Not significant
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