Highlights

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Gly-Low reduces food intake and weight gain while preserving muscle mass

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Gly-Low works independently of traditional caloric restriction to extend lifespan in mice

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Gly-Low treatment improves insulin sensitivity and glucose homeostasis in mice

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Gly-Low acts through hypothalamic signaling to elicit appetite and weight control

Summary

Non-enzymatic reactions in glycolysis produce methylglyoxal (MGO), a reactive precursor to advanced glycation end-products (AGEs), which has been hypothesized to drive obesity, diabetes, and aging-associated pathologies. A combination of nicotinamide, α-lipoic acid, thiamine, pyridoxamine, and piperine (Gly-Low) lowers the deleterious effects of glycation by reducing MGO and the MGO-derived AGE, MG-H1, in mice. Gly-Low supplementation in the diet reduces food consumption, decreases body weight while preserving muscle mass, improves insulin sensitivity, and increases survival in leptin receptor-deficient (Lepr**db) and wild-type C57B6/J mice. Transcriptional, protein, and functional analyses demonstrate that Gly-Low inhibits appetite stimulation through ghrelin and AMP-activated protein kinase (AMPK) signaling pathways in the hypothalamus, leading to reduced hunger responses. Consistent with these molecular findings, Gly-Low inhibits ghrelin-mediated hunger responses. As a late-life intervention, Gly-Low slows hypothalamic aging signatures, improves glucose homeostasis and motor coordination, and increases lifespan, suggesting its potential benefits in ameliorating age-associated decline.

Graphical abstract

Keywords

1. aging
2. methylglyoxal
3. obesity
4. diabetes
5. ghrelin
6. leptin
7. hypothalamus
8. glycation
9. lifespan
10. appetite

Research topic(s)

1. CP: Metabolism

Introduction

Despite the substantial efforts of public health, the incidence of obesity is growing worldwide.1 Obesity reduces life expectancy by increasing the risk of several diseases.2 Lifestyle changes have gained popularity to combat increasingly sedentary lifestyles and excess caloric intake, but dietary improvements remain challenging for most individuals.3,4 Part of this challenge is due to homeostatic mechanisms governing food intake and energy expenditure to resist body weight loss.5 Ghrelin and leptin are hormones that regulate food intake by promoting appetite and satiety, respectively.6,7,8 Chronic dietary excess can disrupt leptin and ghrelin signaling impairing homeostatic regulation of food intake and thus promoting obesity.9

Food overconsumption and obesity are contributing factors to chronic hyperglycemia, which can alter glycolytic flux and thus increase the production of reactive α-dicarbonyls, such as methylglyoxal (MGO).10,11 MGO is an unavoidable byproduct of anaerobic glycolysis and is generated through the non-enzymatic degradation of glycolytic intermediates, dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P).12 Once formed, MGO reacts non-enzymatically with biomolecules such as proteins, lipids, and DNA to form advanced glycation end-products (AGEs).13,14 These covalent adducts have many deleterious consequences, such as impairing protein function, disrupting tissue architecture by forming extracellular crosslinks, activating inflammatory signaling cascades, altering cell-cell communication, and damaging nucleic acids.11 Accumulation of these adducts, which occurs slowly with age or rapidly in hyperglycemic and obese individuals, drives many age-, obesity-, and hyperglycemia-associated pathologies. Cellular protection against AGEs occurs by endogenous glyoxalase enzymes, which detoxify MGO and thereby prevent downstream AGE formation.10,15 We recently demonstrated using Caenorhabditis elegans that accumulation of AGEs also increases food intake.16 We therefore hypothesize that therapeutically enhancing detoxification of AGEs may protect against obesity and obesity-associated pathologies.

Results

A natural compound screen identifies compounds that reduce glycation stress

Hyperglycemia, as often occurs in diabetes, accelerates AGE formation and its accumulation in various tissues, contributing to pathologies such as nephropathy, retinopathy, cardiomyopathy, neuropathy, and vascular injury.17 In C. elegans, the glod-4 mutant, which lacks the endogenous MGO detoxification pathway, develops diabetic-like manifestations, including peripheral neuropathy and reduced lifespan.18 To identify interventions protective against AGE-associated pathologies, we previously conducted a high-throughput screen of 640 natural compounds (TimTex, NPL640) using the glod-4 model. We further tested 11 hits from our screen, as well as compounds from relevant glyoxalase-associated literature (thiamine19 and pyridoxamine20) in a cell culture model of glycation stress, as measured by rescuing neurite length retraction of rat dopaminergic (N27) neurons following exposure to MGO (Figure S1A). We found that a combination of five compounds: α-lipoic acid, nicotinamide, piperine, pyridoxamine, and thiamine, termed Gly-Low, conferred better protection against MGO relative to the single compounds (Figure S1B).

To test Gly-Low’s therapeutic potential, we tested it in the diabetic leptin receptor-deficient mouse model, Lepr**db, which rapidly develops obesity, hyperglycemia, and glucose intolerance, alongside increased glycation precursors and their adducts in diabetes-relevant tissues such as the kidneys, heart, and liver.21 We started 8-week-old male Lepr**db mice on a regular, low-fat diet supplemented with Gly-Low for 16 weeks (Figure 1). LC/MS analysis of plasma demonstrated that Gly-Low significantly lowered absolute levels of both MGO (61% decrease) and its protein-bound arginine adduct, MG-H1 (41% decrease), compared to controls (Figure 1A), confirming its glycation reducing effects in vivo.

Figure 1 Glycation-lowering compounds (Gly-Low) lower glycolytic byproducts as well as rescue hyperphagia and obesity-associated pathologies in a diabetic, leptin receptor-deficient (Lepr**db) mouse model

(A) Absolute levels of glycolytic byproduct, MGO (left), and its arginine adduct, MG-H1 (right), were reduced in the plasma of male mice treated with Gly-Low. n = 5 per treatment group, measured by LC/MS.

(B and C) Weekly food consumption (B) and body weights (C) of male Lepr**db mice fed diets containing varying concentrations of Gly-Low were altered in a dose-dependent manner. Food measurements of n = 3 cages of group-housed animals per treatment (n = 5 in the control group). Body weights of n = 12 mice per treatment group (n = 17 in the control group). Bar graphs (right) show food consumption differences (B) and percent change in body weight from starting diets (C) at their respective timepoint relative to control.

(D) Percent change from baseline in fat (left) and lean mass (right) of Lepr**db mice on their respective diet.

(E) Wet weights of inguinal fat deposits and livers of Lepr**db mice.

(F) Representative images of H&E-stained liver sections from control and 1× Gly-Low-treated mice showing the presence of large lipid vacuoles (arrowhead). Quantification of liver lipid vacuoles, n = 3–4 livers per treatment group, 4 fields# of view per animal.

(G) Random (non-fasted) and fasted (16-h) blood glucose levels up to a max of 600 mg/dL. n = 12 per treatment group (n = 17 in the control, with deaths throughout the study [1 death before 8 weeks, 8 deaths before 16 weeks]).

(H) Survival curves of control mice and Gly-Low-treated mice prior to their experimental endpoint. p = 0.007 (log rank Mantel-Cox), p = 0.007 (Gehan-Breslow-Wilcoxon). Significance: ns (not significant), ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005. Statistical analyses performed by unpaired t test. a, b, and c designate statistical significance (p < 0.05) between the control group and 1×, 0.5×, and 0.25× Gly-Low treatments, respectively. Data are represented as mean ± SEM.

Glycation-lowering compounds reduce food intake and diabetic pathologies in Lepr**db mice

All concentrations of Gly-Low (0.25×, 0.5×, and 1×) reduced food consumption during the first 7 weeks on the diet, with a dose-dependent effect observed for the first 6 weeks (Figure 1B). The loss of statistical significance for lower doses (0.5× and 0.25×) after 6 weeks may be attributed to the increased variability in food consumption among control fed mice later in the study (Figure S2A). Gly-Low had an early dose-dependent effect on body weight gain that remained significant only for the highest dose (1×) over time (Figure 1C). By 8 weeks of age, which is the age at which treatment began, Lepr**db mice were already overweight. During the treatment period, control-fed Lepr**db mice continued gaining weight, reaching an average of 45 grams by 8 weeks of treatment, roughly 50% heavier than wild-type (C57B6/J) mice, reflecting severe obesity. In contrast, 1× Gly-Low-treated mice lost an average of 22% of their starting weight, stabilizing at 30 grams, which is comparable to wild-type male mice of the same age.22 Mice fed the 0.5× Gly-Low diet maintained their weight, while those on 0.25× Gly-Low continued gaining weight, failing to prevent pathological obesity.

To determine whether weight loss was due to fat or lean mass reduction, we performed dual X-ray absorptiometry (DXA), which directly measures bone and fat mass and calculates lean mass. After 8 weeks of treatment, control-fed mice gained an average of 10% fat mass while 1× Gly-Low-treated mice lost an average of 13% (Figure 1D, left). Only the highest dose (1×) of Gly-Low preserved lean mass, while lower doses failed to prevent lean mass loss (Figure 1D, right). To assess whether increased activity or metabolic rate contributed to weight loss of 1× Gly-Low-treated mice, we performed metabolic cage testing, which revealed no significant differences in activity or metabolic rate between groups (Figures S2B and S2C).

Given the pronounced reduction in food intake with 1× Gly-Low treatment, we performed several tests to assess potential food aversion. A formal conditioned taste aversion (CTA) test was not feasible due to incompatible solubility of Gly-Low compounds for IP injection. Instead, we used food restriction and re-feeding experiments and tested Gly-Low in palatable diets. In wild-type (C57B/6J) mice, re-feeding rates after an 18-h fast were statistically similar whether mice were re-fed a control diet or a Gly-Low-supplemented standard or high-fat (HFD) diet (Figures S3A and S3B). Additionally, 0.5× Gly-Low reduced food consumption in both wild-type and Lepr**db mice (Figure S3C). However, when 0.5× Gly-Low was incorporated into a palatable HFD, it had no effect on food consumption in wild-type mice (Figure S3C). These findings, combined with long-term treatment data (∼10 months) showing converging food consumption rates over time (Figure 2B), suggest that Gly-Low does not induce taste aversion. However, we acknowledge that formal CTA tests remain the gold standard for taste aversion testing and therefore cannot rule out the possibility that taste aversion contributes to Gly-Low’s effect on food intake. Although Gly-Low affected feeding behavior and body weight across multiple concentrations, we focused subsequent in vivo analyses on the highest dose due to its effectiveness and robust reductions in circulating AGE levels.

Figure 2 Gly-Low and its constituents reduce body weights and food consumption in male and female C57B6/J mice, independent of major pituitary hormones and hypothalamic leptin signaling

(A and B) Body weights (A) and food consumption (B) of male wild-type mice chronically (24 weeks) treated with Gly-Low were reduced compared to control mice.

(C and D) Body weights (C) and food consumption (D) of male wild-type mice acutely (1 week) treated with Gly-Low were reduced compared to control mice.

(E and F) Body weights (E) and food consumption (F) of female wild-type mice acutely (1 week) treated with Gly-Low were reduced compared to control mice.

(G and H) Body weights (G) and food consumption (H) of male wild-type mice acutely (1 week) treated with individual compounds of Gly-Low compared to control mice. Boxplots to the right show changes from starting to ending weights within the respective treatment group.

(I and J) Plasma levels of growth hormone (GH), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), and prolactin (PRL) from male mice acutely treated with Gly-Low constituents (I) and from mice acutely treated with Gly-Low (J).

(K) Food consumption rates (kcal) of wild-type mice fed a control diet or a Gly-Low diet were reduced following an injection of leptin compared to those given a saline vehicle control. Bar graphs (right) show differences in food consumption between treatment groups at 1 and 32 h after an injection of leptin compared to their saline food consumption. Significance: ns p > 0.05, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005. Statistical analyses: paired t test for (G); unpaired t test for all other panels. Data are represented as mean ± SEM.

Consistent with the reduced adiposity observed by DXA analysis, Gly-Low-treated mice had significantly smaller inguinal fat pads (Figure 1E). Liver weights were also significantly lower in Gly-Low-treated mice, and histological analysis showed 89.8% reduction in large lipid vacuoles, indicating improved lipid homeostasis (Figures 1E and 1F).

In agreement with reduced glycation burden, Gly-Low-treated mice displayed significantly improved glycemic control, with 9.9% lower fasted glucose and 28.4% lower random blood glucose after 8 weeks, which persisted throughout treatment (Figure 1G). This was accompanied by reduced polyuria, measured by weighing home cages to determine cage soiling (Figure S2D). Polyuria in diabetes results from excessive glucose in the urine due to excessive blood sugar levels.23 In mouse models of diabetes, excessive urine output can contribute to differences in cage weight due to excessive soiling of bedding and nesting material and thus serve as a proxy for changes in polyuria.24 Notably, water intake was unchanged between Gly-Low- and control-fed mice, confirming that reduced soiling was not due to lower fluid consumption (Figure S2E). Furthermore, Gly-Low-treated mice exhibited reduced proteinuria, a marker of diabetic kidney damage.25 Control-fed mice displayed increased urinary protein levels over time, indicative of worsening kidney function, whereas, Gly-Low-treated mice had reduced urinary protein, suggesting a protective role within the kidney (Figure S2F).

The benefits of Gly-Low in reducing hyperphagia and diabetes-associated pathology translated to a complete rescue of early mortality (Figure 1H). Previous studies report a median lifespan of 349 days for male Lepr**db mice, with continuous mortality after 16 weeks of age.26 In our study, 18% of control-fed mice died by 23 weeks of age (15 weeks on diet), and chance of survival dropped to 52.9% by 26 weeks. In contrast, Gly-Low-treated mice had no recorded deaths during this period. Collectively, these findings in leptin receptor-deficient mice highlight the therapeutic potential of Gly-Low in treating various obesity- and diabetes-associated conditions by reducing overconsumption, associated glycation burden, and glycation-associated pathologies.

Gly-Low reduces food intake in a leptin-independent manner

Following experimentation in a leptin receptor-deficient mouse model (Lepr**db), we tested the highest dose of Gly-Low in 4-month-old wild-type (C57BL/6J) mice. Long-term treatment of male wild-type mice resulted in similar reductions and subsequent maintenance of body weights (Figure 2A) and food consumption (Figure 2B). After 6-month treatment, Gly-Low-treated mice lost an average of 13.2% of their body weight, while control-fed mice gained an average of 16.9%. As observed in Lepr**db mice, metabolic cage testing confirmed that weight loss was primarily due to reduced food intake rather than changes in activity or metabolic rate (Figures S3D and S3E). To test for sex specificity, we conducted a 1-week acute treatment in age-matched, wild-type males and females, both of which showed similar reductions in body weight and food consumption (Figures 2C–2F). After treatment, young (3-month-old) males lost an average of 11.7% of their body weights, while females lost an average of 11.1%, indicating that Gly-Low’s effects on food consumption and body weight are not sexually dimorphic.

Next, we determined which Gly-Low constituents drive its effects on food consumption and/or body weight. Each compound comprising Gly-Low was incorporated into a standard low-fat diet at the same dose as in the 1× Gly-Low diet, and young (6-month-old) wild-type male mice were acutely treated for 1 week. At the end of treatment, α-lipoic acid and nicotinamide treatment caused significant weight loss, while thiamine caused significant weight gain (Figure 2G). Notably, α-lipoic acid had the strongest effect on food consumption, with mice consuming roughly 32% less food than control-fed mice and driving the most significant weight loss (Figure 2H). Interestingly, this effect was less pronounced in Gly-Low-treated mice, suggesting that other components temper α-lipoic acid’s appetite-suppressing effects. These findings indicate that α-lipoic acid is the primary driver of appetite suppression, while nicotinamide and thiamine contribute to weight change through additional mechanisms.

To determine how Gly-Low and its individual components influence feeding behavior and body weight, we measured circulating hormone levels of the hypothalamic-pituitary axis, which directly regulate body weight maintenance or feeding behavior. Using a multiplex hormone panel, we analyzed growth hormone (GH), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), and prolactin (PRL). GH secretion decreases during feeding.27 GH levels were significantly elevated in piperine-treated mice and trended lower in nicotinamide-treated mice (Figure 2I, left). However, GH levels were unchanged in Gly-Low-treated mice (Figure 2J, left), suggesting that piperine’s effects are tempered by co-administration with other components of Gly-Low. Hypothyroidism is often observed during reduced TSH production and release, leading to decreased basal energy expenditure and increased body weights.28 Surprisingly, TSH levels trended lower in nicotinamide- and α-lipoic acid-treated mice (Figure 2I, middle left) but were unaffected in Gly-Low-treated mice (Figure 2J, middle left). ACTH regulates cortisol and androgen production.29 Following feeding, plasma levels of ACTH are significantly reduced.30 All Gly-Low components trended toward lower ACTH levels, and the full Gly-Low cocktail caused a significant reduction in circulating levels of ACTH (Figures 2I and 2J, middle right panels). These findings suggest additive effects of the individual components. PRL levels were significantly lower in nicotinamide-treated mice and trended lower in α-lipoic acid-, pyridoxamine-, and thiamine-treated mice. However, PRL levels were unchanged in Gly-Low-treated mice. These findings indicate that Gly-Low’s individual components influence feeding-related hormones differently, with additive or negating effects when combined. α-lipoic acid appears to be the primary driver of appetite suppression, while nicotinamide may regulate body-weight independently of food consumption. This aligns with observations in Lepr**db mice, where nicotinamide alone reduced body weight without affecting food intake (Figure S2G). These results suggest that similar mechanisms underlie Gly-Low’s effects across different models.

To determine whether Gly-Low acts independently of leptin signaling in wild-type mice, we injected exogenous leptin into 6-month-old wild-type males and measured food consumption compared to saline-injected controls. Exogenous leptin stimulates pSTAT3 signaling within hypothalamic neurons, and a readout of this signaling can be measured by a reduction in feeding behavior. Both Gly-Low- and control-fed mice showed similar reductions in food consumption following leptin injections, indicating intact leptin signaling (Figure 2K). Gly-Low-treated mice showed a greater initial increase in food consumption (1 h post-injection), but differences between groups disappeared by 32 h post-injection. pSTAT3 immunostaining in hypothalamic neurons within the arcuate nucleus confirmed that Gly-Low-treated mice exhibited a comparable leptin response to controls (Figure S3F). These findings demonstrate that Gly-Low reduces food intake through a leptin-independent mechanism.

Gly-Low reduces glycolysis and enhances cellular detoxification pathways in the hypothalamus

The hypothalamus surrounds the third ventricle, allowing it to sense circulating nutrients and hormones from the periphery to regulate body-weight homeostasis.31,32 Thus, we interrogated the hypothalamus for molecular changes induced by Gly-Low. We assessed changes in hypothalamic transcripts using bulk RNA-sequencing and performed protein analysis on hypothalamic lysates from 3-month-old wild-type mice untreated or acutely treated (1 week) with Gly-Low.

We investigated how Gly-Low influenced the expression of pathways relevant to glycation production and clearance. We found that Gly-Low-treated mice showed a general reduction in the expression of glycolytic genes, except for Tpi and Pgk1, whose increased activity is predicted to lower MGO production33,34 (Figures 3A and S4A). In contrast, genes directly responsible for MGO detoxification (glyoxalase genes) and a larger set of general cellular detoxification genes showed increased expression in Gly-Low-treated mice (Figures 3A and S4A). Consistent with our transcriptomic findings, targeted plasma metabolomics data revealed decreased glycolytic metabolites and elevated pentose phosphate pathway (PPP) metabolites. (Figure S4B). The PPP regenerates NADPH, which is necessary for replenishing glutathione.35,36 This suggests that Gly-Low increases the potential for MGO-specific and generalized cellular detoxification. We hypothesize that Gly-Low may reduce glycation burden by reducing its production and enhancing its clearance. Furthermore, these findings in the hypothalamus of wild-type mice complement our findings that Gly-Low reduces systemic burden of the glycation precursor, MGO, and its predominant AGE, MGH1, in the blood of Lepr**db mice.

To investigate how Gly-Low may impact feeding behavior, we analyzed the 1,407 genes differentially expressed in the hypothalamus of Gly-Low-treated vs. control mice for changes to canonical feeding behavior genes. Interestingly, two well-characterized regulators of hunger and satiety, Agrp and Pomc, were consistently changed in the direction typically observed in hungry mice37 (Figure 3B). Analysis of this set of positive and negative regulator feeding genes in Gly-Low-treated mice indicates that no one particular pathway was expressed at higher or lower levels than expected (Figure S4C).

Figure 3 Gly-Low treatment alters genes responsible for appetite stimulation and blunts hypothalamic ghrelin and downstream AMPK signaling

(A) Heatmaps showing transcript fold changes of genes involved in MGO production (glycolysis) and clearance (glyoxalase) in the hypothalamus of wild-type male mice acutely (1 week) treated with Gly-Low.

(B) Heatmap showing transcript fold changes of negative and positive appetite regulators in the hypothalamus of mice acutely (1 week) treated with Gly-Low.

(C) Plasma levels of acylated ghrelin were unchanged with Gly-Low treatment.

(D and E) Plasma levels of ghrelin-receptor antagonist, LEAP-2 (D), and ghrelin-stimulated growth factor, IGF-1 (E), were reduced with Gly-Low treatment.

(F and G) Exogenous ghrelin increased food consumption compared to saline injected controls in control (F) but not Gly-Low-treated (G) mice.

(H) Schematic depicting activation or inhibition of mTOR signaling and ribosomal translation by hormone/growth factor or ghrelin-dependent GHSR signaling, respectively.

(I) Food consumption rates (kcal/g BW) following IP injection of AMPK activator, AICAR, compared to saline vehicle controls were increased in control-fed mice but significantly decreased in Gly-Low-fed mice.

(J) Western blots of hypothalamic lysates from mice acutely (1 week) treated with Gly-Low. Protein expression of phosphorylated AMPK (top), phosphorylated AKT (middle), and phosphorylated S6 kinase (bottom) relative to their un-phosphorylated protein levels with quantification (right). Uncropped western blot images with ladder are included in supplemental information.

(K) Transcripts from ribosomal genes were significantly upregulated in hypothalamic lysates of mice acutely (1 week) treated with Gly-Low compared to control mice. Significance: ns p > 0.05, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005. Statistical analyses performed by unpaired t test. a and b designate statistical significance (p < 0.05) between AICAR-injected and saline-injected groups fed either a control diet or Gly-Low diet, respectively. Data are represented as mean ± SEM.

Gly-Low inhibits ghrelin signaling and alters activation of hypothalamic AMPK

The stomach-derived hormone ghrelin is a common signaling molecule to both the Agrp and Pomc pathways. Ghrelin is a peptide produced in the stomach during fasting and times of hunger.38,39 Following production, the circulating hormone binds its receptor, the GH secretagogue receptor (Ghsr),40 within the hypothalamus to engage signaling pathways that stimulate food intake.41,42 To determine whether Gly-Low affected ghrelin production, we measured levels of acylated ghrelin in the plasma of acutely treated 4-month-old male wild-type mice. We found no significant difference in ghrelin levels between the treatment groups (Figure 3C). Ghrelin signaling is inhibited by competitive binding of the ghrelin receptor by the liver-derived hormone LEAP243 or when ghrelin production is reduced during feeding.44,45 However, LEAP-2 levels were significantly lower in Gly-Low-treated mice (40% lower; Figure 3D), suggesting the absence of competitive Ghsr binding. Ghrelin signaling stimulates the release of GH,39 which in turn stimulates the release of IGF-1 in a well-studied GH/IGF-1 endocrine axis within the hypothalamus and pituitary gland.46 To test if this ghrelin-related pathway is disrupted in Gly-Low-treated mice, we measured insulin-like growth factor (IGF-1) in the plasma of control and Gly-Low-treated mice. Consistent with disrupted ghrelin signaling, IGF-1 levels were significantly lower in Gly-Low-treated mice relative to control mice (10.5% lower; Figure 3E). To test if ghrelin signaling is indeed disrupted in Gly-Low-treated mice, we subjected control and Gly-Low-treated mice to exogenous acylated ghrelin, known to activate ghrelin signaling, increase appetite, and induce feeding behavior.47 In control mice, those injected with ghrelin consumed significantly more food (average of 75% increase) relative to PBS-injected controls post-IP injection (Figure 3F). In contrast, Gly-Low-treated mice failed to respond to ghrelin injections with increased food consumption (an average of 23% less food consumed relative to PBS-injected controls; Figure 3G). These findings suggest a role for impaired ghrelin signaling in the reduced food intake observed upon Gly-Low treatment.

Upon ghrelin signaling, AMPK is phosphorylated and activated48 (Figure 3H). To determine whether Gly-Low suppresses hypothalamic AMPK activation, we challenged 5-month-old male mice with the AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), after an 18-h fast. IP-injections of AICAR increased food consumption in mice adapted to a control diet but decreased food consumption in mice adapted to a Gly-Low diet (Figure 3I). To assess hypothalamic AMPK activity, we measured ACC phosphorylation (pACC), a direct AMPK substrate in hypothalamic lysates. In mice adapted to a control diet, pACC levels significantly increases with fasting and trended higher 1 h post-AICAR injection (Figure S4D). However, this response was reversed in mice adapted to a Gly-Low diet. Together, these findings indicate that Gly-Low inhibits AMPK activation in response to both energy depletion (high AMP:ATP ratio) associated with fasting and in response to AMP analogs like AICAR.

Gly-Low increases ribosomal protein S6 phosphorylation in the hypothalamus

In parallel with Ghsr-dependent AMPK signaling, growth factors, such as GH and IGF-1, counteract ghrelin by binding their respective hypothalamic receptors, promoting AKT phosphorylation (pAKT; Figure 3H). pAKT stimulates mechanistic target of rapamycin (mTOR), leading to S6 kinase phosphorylation, enhanced protein synthesis, and appetite suppression.49 Importantly, activated (phosphorylated) AMPK directly and indirectly inhibits mTOR activity, highlighting the antagonistic relationship between these two pathways.50,51,52 Consistent with impaired ghrelin signaling and reduced AMPK activity, pAMPK levels were significantly lower in hypothalamic lysates from 4-month-old Gly-Low-treated mice, while phosphorylated S6 (pS6) levels were increased (Figure 3J). To further assess mTOR/S6 pathway activation, we analyzed hypothalamic RNA-seq data, which revealed a widespread upregulation of ribosomal transcripts (Figure 3K). This suggests enhanced ribosomal biogenesis and protein translation, hallmarks of mTOR/S6 signaling. Supporting this, Fcf1 and Nip7, key regulators of ribosomal rRNA processing, were also significantly upregulated in Gly-Low-treated mice (Figure S4E). Interestingly, despite increased pS6 levels, pAKT was significantly reduced in the hypothalami of Gly-Low-treated mice (Figure 3J), consistent with lower IGF-1 levels. This suggests that mTOR activation in Gly-Low-treated mice likely results from reduced inhibition by AMPK rather than AKT stimulation. These findings suggest that Gly-Low reduces food consumption by impairing appetite-stimulating ghrelin signaling and modulating AMPK-associated appetite-stimulating signaling.53,54

Gly-Low rescues aging-associated dysregulations in glucose homeostasis, motor coordination, and extends lifespan as a late-life treatment

Glycation stress increases with age, accelerating aging-associated conditions.55,56,57 To test whether Gly-Low (1× dosage) mitigates age-related decline, we assessed glucose metabolism and motor function in middle-aged (12 months old; 8 months of treatment) and aged (24-5 months old; 4–5 months of treatment) wild-type male mice. Like young wild-type mice, aged mice treated with Gy-Low exhibited a rapid reduction in food intake and body weight relative to control-fed counterparts. While food intake was significantly reduced during the first 12 weeks of treatment, and body weight declined initially, then weight stabilized and remained lower than controls for the remainder of the experiment (Figures S4F and S4G). Similar to improved glucose homeostasis observed in Lepr**db mice, Gly-Low-treated middle-aged mice showed improved glucose metabolism during a glucose tolerance test (Figure 4A). Following an injection of exogenous glucose, Gly-Low-treated mice displayed a 47.0% lower maximum blood glucose level and a 45.4% lower area under the curve (AUC) compared to controls. Additionally, insulin response testing revealed that Gly-Low-treated mice had a 62.7% greater reduction in blood glucose from baseline, compared to 33.5% in controls (Figure 4B). With age, wild-type C57B6/J mice develop dysregulated glucose homeostasis, reflected in increased fasted and non-fasted blood glucose levels.58 Gly-Low significantly reduced fasting blood glucose levels in both middle-aged and aged mice (Figures 4C and 4E). To assess age-related motor function decline, we performed rotarod testing, commonly used to evaluate sensorimotor function and neuromuscular function, which declines with age.59,60,61 Gly-Low-treated mice outperformed controls in both middle-aged and aged cohorts (Figures 4D and 4F), indicating preserved motor coordination with treatment.

Figure 4 Gly-Low rescues aging-associated dysregulation in glucose homeostasis, motor coordination, and extends lifespan as a late-life intervention

(A) Gly-Low-treated mice had significantly reduced surges in glucose levels during a glucose tolerance test (GTT) compared to control mice. AUC analysis (right).

(B) Glucose levels were decreased to a greater degree in mice treated with Gly-Low than control mice when administered a bolus of insulin during insulin tolerance testing (ITT). AUC analysis (right).

(C and E) Fasted blood glucose levels were reduced in middle-aged (12 months) (C) and aged (25 months) (E) wild-type male mice treated with Gly-Low compared to control mice.

(D and F) Rotarod performance was improved in middle-aged (12 months) (D) and aged (25 months) (F) Gly-Low-treated mice compared to control mice.

(G) Kaplan-Meier curve showing lifespan extension as a late-life intervention (beginning at 24 months of age) in wild-type male Gly-Low-treated compared to control mice.

(H) Scatterplot showing the log2 fold changes of genes significantly (p < 0.05) altered in the hypothalamus of aged (25 months) Gly-Low-treated vs. aged (25 months) control and aged (25 months) control vs. young (3 months) control mice. The regression line is shown in red. The Pearson correlation coefficient, r, is shown in the top right quadrant. Significance: ∗p < 0.05, ∗∗p < 0.005, ∗∗∗∗p < 0.00005. Statistical analyses performed by unpaired t test. Data are represented as mean ± SEM.

Given its effects on functional aging, we next examined whether Gly-Low extends lifespan as a late-life intervention. A cohort of 24-month-old male C57BL/6J mice was placed on a Gly-Low diet, with natural lifespan and deaths recorded, in which mice were only removed from the study based on stringent health parameters. Survival analysis (Kaplan-Meier curve) showed that control mice had a median lifespan of 825 days (105 days post-intervention), whereas Gly-Low-treated mice lived a median of 888 days (173 days post-intervention; Figure 4G). This represents an 8.25% increase in median lifespan (p = 0.0199, Mantel-Cox) and a 5.23% increase in maximum lifespan when treatment started at 24 months of age. This also translates to a 60.7% increase in survival time post-intervention. Unlike caloric restriction, which does not extend lifespan when initiated late in life, Gly-Low’s effects are likely not solely driven by reduced food intake.62,63

Since hypothalamic aging drives whole-body aging,64,65,66,67,68 we assessed transcriptional differences in the hypothalamus of aged (25 months) Gly-Low-treated and control mice. From the differentially expressed genes found in this dataset, we performed linear regression against the genes identified when we compared aged control mice to young (3-month) control mice (Figure 4H). This regression shows a negative correlation (r = 0.8, p < 0.0001) between the two comparisons, suggesting that some changes in hypothalamic transcription seen in normal aging are reversed with a late-life intervention of Gly-Low.

Discussion

Here, we highlight Gly-Low as a potential therapeutic for obesity- and diabetes-associated pathologies using various mouse models. We selected the compounds that make Gly-Low based on their ability to protect against glycation stress. For that reason, we chose to test Gly-Low in the well-studied, glycation-burdened Lepr**db mouse model of hyperphagia and obesity.21 As hypothesized, Gly-Low significantly reduced systemic levels of glycation stress, reducing levels of the glycation precursor, MGO, and its arginine-adduct AGE, MG-H1. Our findings, along with previous work in C. elegans, support the potential of glycation lowering compounds as a therapeutic strategy against diabetic pathologies.18,69 MGO is a well-known target for aging and age-related diseases,55,56,57 but it has been difficult to target pharmacologically.69 Here, we show that Gly-Low likely reduces MGO and MG-H1 through multiple mechanisms, including lowering glycolytic production and enhancing cellular detoxification pathways.

Beyond reducing glycation stress, we found that Gly-Low suppresses appetite, leading to reduced caloric intake without loss in relative muscle mass. Given that Lepr**db mice are both glycation burdened and hyperphagic, Gly-Low expectedly improved multiple pathological phenotypes. However, Gly-Low also had health-promoting effects in young, middle-aged, and aged wild-type mice, suggesting broader therapeutic potential. Our data suggests that Gly-Low’s health benefits likely stem from both its glycation-lowering and calorie-reducing effects, which are closely linked. We previously found that MG-H1 increases feeding in C. elegans,16 while others have shown that MGO-modified bovine serum albumin induces insulin resistance, weight gain, and shortens lifespan in mice.70 In a clinical trial, glyoxalase-activating compounds that detoxify MGO reduced body weight and improved insulin sensitivity.27 We propose that Gly-Low influences feeding behavior through multiple mechanisms, including suppression of the appetite-stimulating ghrelin pathway and activating the appetite-suppressing mTOR pathway in the hypothalamus. While our data supports mTOR pathway activation, further studies measuring translation flux and ribosomal biogenesis are needed to fully validate the extent of mTOR activation.

Caloric restriction (CR) is one of the most potent and widely conserved interventions for increasing healthspan and lifespan across species.71 While some of Gly-Low’s benefits may come from calorie reduction, our findings suggest that it acts through distinct mechanisms. One key difference is Gly-Low’s ability to extend lifespan even when initiated late in life. CR’s lifespan benefits depend on the age at which CR is initiated, with late-life CR showing little or no effect on longevity.62,63 Lipman et al. reported no median lifespan extension in rats subjected to 33% CR starting at 18 months62 and demonstrated that CR rats had increased mortality rates compared to ad libitum (AL) fed rats (61% and 47%, respectively; p = 0.6), concluding that there may be a stage in the aging process after which CR no longer increases longevity.62 Additionally, Hahn et al. compared large cohorts of mice (800 animals) chronically (beginning at 12 weeks of age) fed an AL diet to those chronically fed an AL diet prior to switching to a dietary restriction (DR) diet at 24 months of age.63 They reported that late on-set DR caused no measurable increase in survival in a large fraction of old animals, with observed increases being largely driven by a single breeding cohort.63 In contrast, we found that Gly-Low treatment at 24 months increased both median and maximum lifespan, with mice voluntarily eating 13.4%–29.6% less than controls. While our study differs from traditional CR paradigms, these findings warrant further investigation. We hypothesize that Gly-Low promotes metabolic health and anti-aging effects by altering hypothalamic signaling, which, in turn, regulates both feeding behavior and systemic aging phenotypes.64,65,66,67 Our transcriptional analysis of the hypothalamus in aged Gly-Low-treated mice indicates that many age-related gene expression changes are reversed by treatment (Figure 4H).

While CR is known to improve metabolic health and slow aging,72 long-term CR is difficult to maintain in humans.73 We propose that lowering glycation stress with therapies such as Gly-Low may offer a sustainable alternative to enforced CR by providing voluntary calorie reduction, direct tissue protection, and slowing of aging-related outcomes linked to glycation stress due to methylglyoxal and related precursors.

Limitations of the study

Although the current study indicates that Gly-Low inhibits hypothalamic ghrelin and AMPK signaling to mechanistically contribute to the observed reduction in feeding behavior, we cannot fully exclude the potential contribution of taste aversion. In support of our hypothesis, we provide data that Gly-Low did not affect immediate feeding behavior in mice that were food restricted for 18 h and then re-introduced to either a control diet or a diet containing Gly-Low, with alterations in food intake only emerging after several hours (Figures S3A and S3B). However, standard CTA protocols were not conducted in our cohorts. Future studies employing rigorous CTA assessments will be necessary to distinguish between Gly-Low’s effect on feeding behavior and potential aversive responses. Additionally, while our data highlight Gly-Low’s impact on mitigating age-associated decline in male mice, we did not collect data from aged female mice. Given the significant influence of hormonal changes during aging in females, future studies should evaluate Gly-Low’s efficacy in female aging models. Finally, the individual contributions of Gly-Low’s components to glucose metabolism, ghrelin signaling, and aging remain to be fully defined. While we assessed the effect of individual compounds on food consumption rates, body weights, and abbreviated hormonal analysis, future studies testing individual components in varied combinations will be essential to determine whether certain combinations produce synergistic effects for specific outcomes.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Dr. Pankaj Kapahi ([email protected]).

Materials availability

This study did not generate new unique reagents or mouse lines.

Data and code availability

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FASTQ files for raw bulk RNA-seq were deposited at the Gene Expression Omnibus (GEO) and are publicly available as of the date of publication. Accession numbers are listed in the key resources table.

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Raw metabolomics files were deposited at the Mass Spectrometry Interactive Virtual Environment (MassIVE) and are publicly available as of the date of publication. Accession numbers are listed in the key resources table.

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This paper does not report original code.

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Any additional information required to reanalyze the data reported in this paper is available from the lead contact and or corresponding author upon request.

Acknowledgments

We thank A. Bartke and D. Medina for discussion and experimental procedure development, B. Schilling and S. Melov for useful discussion, A. Lopez-Ramirez for brain dissections and contribution to the work, and C. Patterson for contribution to cohort husbandry and experimentation. We thank the Kapahi Lab at Buck Institute for helpful discussion and critique and the employees of the Phenotyping Core and Morphology Core at the Buck Institute. We acknowledge support from the National Institute of Health: T32AG000266-23 (to K.R.K.), R01AG038688 (to P.K.), R01AG068288 (to P.K.), R01AG061165 (to P.K.), the Hevolution Foundation (to P.K.), the Donner Foundation (to P.K.), and the Larry L. Hillblom Foundation (to P.K.).

Author contributions

P.K., S.K., J.J.G., J.C.N., and L.M.E. were involved in conceptualization and supervision. L.A.W., K.R.K., P.K., L.M.E., and J.C.N. drafted the manuscript. L.W. and K.R.K. were involved in analysis and visualization. L.A.W., K.R.K., J.R., N.B., M.V., M.M.S., D.O.F., P.S., J.B., D.S., and L.E.N. were involved in experimental procedures and live animal experimentation. S.-J.C. was involved in blinded pathological assessment. J.J.G. and D.O.F. were involved in experimental quantification and analysis of α-dicarbonyls. All authors have read and agreed to the final version of the manuscript.

Declaration of interests

L.A.W., N.B., and P.K. are patent holders of GLYLO, a supplement licensed to Juvify Bio by the Buck Institute. P.K. is the founder of Juvify Bio.

STAR★Methods

Key resources table

REAGENT or RESOURCESOURCEIDENTIFIER Antibodies Mouse Akt (pan) (40D4)Cell SignalingCat# 2920; RRID: AB_1147620 Rabbit monoclonal pAKT Ser473 (D9E)Cell SignalingCat# 4060; RRID: AB_2315049 Rabbit monoclonal S6 Ribosomal protein (5G10)Cell SignalingCat# 2217; RRID: AB_331355 Rabbit monoclonal pS6 Ribosomal protein (Ser 240/244) (D68F8)Cell SignalingCat# 5364; RRID: AB_10694233 Rabbit monoclonal AMPKαCell SignalingCat# 2532; RRID: AB_330331 Rabbit monoclonal pAMPKα (Thr172) (D4D6D)Cell SignalingCat# 50081; RRID: AB_2799368 Rabbit monoclonal mTOR (7C10)Cell SignalingCat# 2983; RRID: AB_2105622 Rabbit monoclonal β-Actin (13E5)Cell SignalingCat# 4970; RRID: AB_2223172 Rabbit monoclonal Acetyl-CoA Carboxylase (ACC) (3662)Cell SignalingCat# 3662; RRID: AB_2219400 Rabbit polyclonal Phospho-Acetyl-CoA Carboxylase (pACC) (Ser79) (D7D11)Cell SignalingCat#11818; RRID: AB_2687505 Chemicals, peptides, and recombinant proteins Alpha-lipoic acidSigmaCat# 62320-25G-F NicotinamideSigmaCat# 72345-50G Thiamine hydrochlorideSigmaCat# T4625-10G Pyridoxamine dihydrochlorideChem-IPEX INT’LCat# 1461 PiperineSigmaCat# 1003442421 InsulinSigma-AldrichCat# I2643-50MG D-GlucoseSigma-AldrichCat# G8270-100G Ghrelin peptidesPhoenix PharmaceuticalsCat #031-30 Methylglyoxal (MGO)Sigma AldrichCat #M0252 AICARToronto Research ChemicalsCat# A611700 Critical commercial assays LEAP-2 (38–77) (Human)/LEAP-2 (37/76) (Mouse) ELISA KitPhoenix PharmaceuticalsCat #075-40 Rat/Mouse Ghrelin active ELISA KitSigma AldrichCat #EZRGRA-90K Mouse IGF-1 ELISA KiitCrystal ChemCat #80574 MILLIPLEX MAP Mouse Pituitary Magnetic Bead Panel - Endocrine Multiplex AssayMillipore SigmaCat# MPTMAG-4