Ketogenic diet sex-dependent effects on rat bone marrow cells during development and β-HB protection in hypoglycemia

TL;DR

A ketogenic diet affects rat bone marrow cells differently in males and females, and ketone bodies like β-HB can protect BMCs in low glucose conditions.

Contribution

This study reveals sex-dependent effects of a low-protein ketogenic diet on BMCs and the protective role of β-HB in hypoglycemia.

Findings

Low-protein KD supports osteogenic differentiation in female rat BMCs but reduces bone regenerative potential in males.

β-HB supplementation in low glucose conditions promotes BMC mineralization and reverses negative effects on BMC viability and inflammation.

Sex differences in BMC responses suggest caution in using KD or fasting for bone-related therapies.

Abstract

The ketogenic diet (KD) is a high-fat, moderate-protein, and low-carbohydrate diet. Initially prescribed for drug-resistant epilepsy, KD has become popular for weight reduction in patients with diabetes and obesity, who are often affected by reduced bone mass. However, KD’s impact on bone marrow cells remains largely unexplored. Here, we show the effects of low protein KD on bone marrow cells (BMCs) during pregnancy, lactation, and postnatal development in 30-day-old Wistar rat offspring. KD consumption in female juvenile rat offspring supported BMC osteogenic differentiation and inhibited osteoclast activity, while in male rat BMCs it reduced bone regenerative potential. This was observed despite a strongly reduced body weight in both sexes. The addition of the primary ketone body β-hydroxybutyrate (β-HB) to juvenile and adult rat BMC cultures in a low glucose culture medium effectively promoted extracellular matrix mineralization and proliferation of rat BMCs and reversed the negative impact of low glucose on BMC viability, inflammation, and osteoclast activity. Given the above, we recommend considering the potential sex differences when implementing restrictive diets and their consequences during pregnancy. Our results also highlight the distinct effects of low glucose and β-HB on the osteogenesis of juvenile and adult rat BMCs, which suggests caution in considering short-term KD or fasting for bone-related therapies.

The online version contains supplementary material available at 10.1038/s41598-026-40169-3.

Figures

Ketogenic diet (KD) affects the regenerative potential of bone marrow cells (BMCs) harvested from 30-day-old female and male rat offspring. (a) Body weight of 30-day-old rat offspring maintained on a KD or normal diet (ND). (b) mRNA levels of selected markers for stemness (*Sox2*, *Oct4*), osteoblast (*Col1a1*, *Opg*, *Bsp*, *Opn*) and osteoclast (*Trap*, *Ctsk*, *Mmp-9*) activity, and inflammatory cytokines (*Il-1β*,* Tnf-α*,* Il-6*,* Il-10*) in isolated BMCs. Adherent BMCs were analyzed using RT-PCR 4 days after bone marrow harvesting. The results are presented as relative mRNA expression co

Ketogenic diet (KD) affects the osteogenic potential of rat bone marrow cells (BMCs) harvested from 30-day-old female and male offspring. BMCs were cultured for 7 days in standard osteogenic medium (OM) consisting of ascorbic acid, dexamethasone, and β-glycerophosphate. The mRNA levels of selected osteoblastic markers were analyzed using RT-PCR. The results are presented as relative quantification (RQ) of mRNA levels vs. mRNA levels in control ND male and female BMCs. Two-way ANOVA with a post hoc Tukey test, * *p* < 0,05, *p* < 0,001, * *p* < 0,0001 compared between samples marked in brackets

The addition of β-hydroxybutyrate (β-HB) to juvenile (a,c,e) and adult (b,d,f) rat osteogenic BMC cultures under low glucose conditions supports osteogenesis in vitro and reverses the negative impact of low glucose. (a-f) BMCs were cultured in osteogenic media (OM) containing glucose at 5.5 mM (control), 2 mM (low glucose), or 2 mM glucose + 5 mM β-HB (ketosis-mimicking conditions). (a-b) 7-day BMC cultures were analyzed for mRNA levels of selected osteoblastic, osteoclastic and pro-inflammatory markers. Real-time RT-PCR results are presented as relative mRNA expression vs. control BMCs. (c–d)

Full text

Introduction

The ketogenic diet (KD) is a restrictive diet based on high-fat, adequate-protein, and very-low-carbohydrate content that results in rapid weight loss. Acetyl-CoA (acetyl coenzyme A) is a central metabolite produced in the mitochondria from a glucose conversion product - pyruvate, β-oxidation of fatty acids, or amino acid catabolism. Acetyl-CoA is mainly oxidized in the citric acid cycle (TCA) to generate energy, but it is also used as a substrate for ketogenesis in hepatocytes, fatty acid synthesis in adipocytes, and histone and transcription factor acetylation^1^. As a result of ketogenesis in the liver, ketone bodies are produced by the conversion of acetyl-CoA to acetoacetate (AcAc), which is decarboxylated to acetone or dehydrogenated to β-hydroxybutyrate (β-HB)^2^. Ketogenesis is upregulated by glucagon^3,4^ and suppressed by insulin^5,6^. In the absence of glucose, ketone bodies serve as an alternative energy source since cells are capable of converting ketone bodies (i.e., β-HB and AcAc) back to acetyl-CoA, which then enters the TCA cycle^2^. Ketone bodies reach 0.5-3 mM levels during 2-day starvation, a restrictive diet or prolonged exercise, but they can also increase to 5–7 mM after 2–3 weeks of fasting or a ketogenic diet and up to 20 mM in untreated diabetic ketoacidosis^7,8^. Diets that restrict caloric supply or mimic calorie restriction (i.e., KD) sometimes serve as an alternative to drug treatment therapies.

KD has been used to treat intractable epilepsy since 1920^9^. In recent decades, KD has become increasingly popular for obesity, diabetes, and neurological and metabolic disorders^10^. However, the effects of KD on bone regenerative potential are not well recognized. Studies using rat models have been limited so far to adult male rats subjected to the KD for up to 12 weeks^11–14^. KD has been shown to result in bone loss, biomechanical reduction in appendicular bones^11^, and delayed posterolateral lumbar spine fusion^12^. The KD may also increase serum levels of tartrate-resistant acid phosphatase (TRAP) and decrease alkaline phosphatase (ALP), along with decreased bone density^13,14^. Whereas in an osteoarthritis knee joint model, KD reduced subchondral bone damage and inflammation^15^. Nevertheless, the impact of KD has been studied in relatively small patient groups and without dissecting sex-related effects. Carter et al. reported that a 3-month Atkins-like diet did not increase bone turnover, as assessed by urinary N-telopeptide and serum bone-specific ALP levels^16^. Similarly, no effect on bone mass was reported in children with epilepsy who were on a modified Atkins diet for 24 months^17^. Interestingly, a 3-week KD in race walkers (majority of males) had a negative impact on serum markers for bone remodeling (CTX, P1NP, OC)^18^, whereas an 8-week KD in resistance-trained women had some positive effects on bone mass density^19^.

Somatic stem cells are important for maintaining tissue functions in postnatal life, undergoing both self-renewal and differentiation. Restrictive diets such as KD may modulate the latter processes. KD has been shown to enhance intestinal stem cell self-renewal^20 ^and increase the proliferation of muscle satellite cells in Duchenne disease^21^. Yet, the only reported effect of KD administered in vivo on bone marrow stem cell (BMSC) isolates is reduced ALP activity and extracellular matrix (ECM) mineralization after 7 days of BMSC osteogenic culture^13,22^. Given that β-HB is the predominant ketone body produced during ketosis, the supplementation of culture medium with β-HB can mimic ketosis in vitro. Zhao et al. reported that β-HB treatment of murine osteoblast MC3T3-E1 cultures resulted in increased osteocalcin expression and ECM mineralization and prevented bone mass density reduction in ovariectomized rats^23^. In contrast, Saito et al. observed a positive effect of acetate on the osteogenic progression of MC3T3-E1 cultures, whereas β-HB diminished ALP activity and ECM mineralization^24^. Notably, β-HB has been reported to attenuate osteolysis in mice exposed to simulated microgravity^25^, decrease osteoclast differentiation^25,26^, suppress M1^27^, and promote M2 macrophage polarization^28,29^. In view of the above, KD and β-HB may impact bone marrow stem cells and their regenerative potential.

In this study, we present the effects of a KD implemented during pregnancy, lactation, and postnatal development on bone marrow cells (BMCs) in juvenile 30-day-old Wistar rat offspring. We show in vivo and in vitro sex-dependent differences after KD implementation in the osteogenic and regenerative potential of BMCs. We also explored the osteogenic role of primary ketone body β-hydroxybutyrate supplementation in juvenile and adult rat BMC cultures in decreased glucose culture conditions. Since KD has become a popular weight-reduction remedy in patients with diabetes and obesity who often display reduced bone mass, the observed sex-dependent differences after KD and negative effects of low glucose concentration should be considered in clinical practice.

Results

Ketogenic diet affects bone marrow cell regenerative potential in 30-day-old rat offspring

The ketogenic diet was implemented in female Wistar rats and their offspring during pregnancy, lactation, and up to 30 days after birth. The KD negatively affected the offspring’s body weight in both sexes (∼50 g) compared to offspring raised on a normal diet (∼75 g) (Fig. 1a). Bone marrow was harvested from femurs of male and female offspring subjected to a KD or normal diet (ND). Adherent BMC cells 4 days after isolation were analyzed for gene expression (Fig. 1b) or directly cultured under experimental conditions (Figs. 1c-d and 2). We detected that KD increased the mRNA levels of the stemness markers Sox2 and Oct4 in male BMCs but decreased the latter in female BMCs. Notably, KD female BMCs displayed the highest mRNA levels of the osteoblastic markers bone sialoprotein (Bsp) and osteopontin (Opn), whereas KD male BMCs showed decreased mRNA levels of collagen type I (Col1a1), osteoprotegerin (Opg), and Bsp. The mRNA expression of osteoclast-related tartrate-resistant acid phosphatase (Trap) was the lowest in KD female BMCs and the highest in KD male BMCs, but KD BMCs of both sexes displayed decreased mRNA levels of other osteoclastic markers, i.e., cathepsin K (Ctsk) and matrix metalloproteinase 9 (Mmp-9). Regardless of sex, KD BMCs also exhibited lower mRNA levels of pro-inflammatory (interleukin 1β, Il-1β, tumor necrosis factor α, Tnf–α) and anti-inflammatory (interleukin 6, Il-6, interleukin 10, Il-10) cytokines (Fig. 1b, see Supplementary Fig. 1 for bar graph version). To assess the cell proliferation rate, BMCs were cultured for up to 9 days. We observed that KD male BMCs proliferated faster than ND male BMCs and both KD and ND female BMCs. KD female BMCs displayed an increased proliferation rate at culture day 6 compared to ND female BMCs (Fig. 1d). To verify stem cell differentiation in BMCs from the ND and KD groups, we cultured BMCs in osteogenic, adipogenic, and chondrogenic differentiation media for 21 days (Fig. 1c). The investigated BMC groups differentiated into all three lineages. We observed that KD in male BMCs lowered osteogenic and chondrogenic potential. Enlarged osteocalcin-positive osteoblasts or abundant Alcian-stained proteoglycans were observed in KD female BMCs cultured in either osteogenic or chondrogenic conditions, respectively (Fig. 1c). Thus, our results suggest that KD affects BMCs of 30-day-old female offspring by supporting osteogenic progression and reducing stemness, whereas in male offspring, KD decreases BMC osteogenic potential and increases stemness markers and osteoclast-related enzyme mRNA expression levels.

Fig. 1. Ketogenic diet (KD) affects the regenerative potential of bone marrow cells (BMCs) harvested from 30-day-old female and male rat offspring. (a) Body weight of 30-day-old rat offspring maintained on a KD or normal diet (ND). (b) mRNA levels of selected markers for stemness (Sox2, Oct4), osteoblast (Col1a1, Opg, Bsp, Opn) and osteoclast (Trap, Ctsk, Mmp-9) activity, and inflammatory cytokines (Il-1β,* Tnf-α*,* Il-6*,* Il-10*) in isolated BMCs. Adherent BMCs were analyzed using RT-PCR 4 days after bone marrow harvesting. The results are presented as relative mRNA expression compared to ND male BMCs. For all markers, ND vs. KD is significant (p < 0,05) in both females and males (see Supplementary Fig. 1). (c) Osteogenic (OC, osteocalcin, magenta colored), adipogenic (FABP4, yellow colored), and chondrogenic (Alcian blue staining) potential of BMCs cultured for 21 days in the respective differentiation media. Nuclei are stained with DAPI (cyan colored). Scale bar represents 100 μm. The quantification of fluorescent-positive cells and stained area was performed using ImageJ (lower panel). Two-way ANOVA with a post hoc Tukey test, * p < 0,05, *** p < 0,0001 compared between samples marked in brackets. (d) Proliferation of BMC isolates cultured for 9 days in standard culture medium. Three-way ANOVA with a post hoc Tukey test, * p < 0,05 compared to ND groups.

Ketogenic diet enhances the osteogenic potential of female but not male BMCs of rat offspring

We determined that KD implemented in vivo increased osteogenic and decreased osteoclastic markers in BMCs harvested from 30-day-old female rat offspring (Fig. 1). We further explored the impact of KD on the in vitro osteogenic potential of BMCs from rat offspring and possible sex-related differences. BMCs were cultured for 7 days in osteogenic medium (OM) consisting of ascorbic acid, dexamethasone, and β-glycerophosphate, followed by mRNA expression analyses of osteogenic markers (Fig. 2). However, it should be noted that BMCs were cultured in a cell culture medium that did not sustain ketosis in vitro (Figs. 1c-d and 2), which could attenuate some of the effects of the KD in vivo. Under standard culture conditions (control), KD male BMCs expressed significantly lower mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), Opn, and Bsp than ND male BMCs. In osteogenic cultures, KD male BMCs decreased Col1a1 and Opg mRNA expression further, whereas ND male BMCs decreased the mRNA levels for all the studied osteogenic markers versus the respective control culture conditions. However, osteogenic stimulation in male BMCs was comparable between the KD and ND groups, despite the reduced Bsp expression in the KD group. In contrast, control KD female BMC cultures displayed significantly higher mRNA levels of Runx2, Osx, Bsp, and Opn than ND female BMCs. Osteogenic stimulation of KD female BMCs further increased the expression of Runx2, Col1a1, and Opg (Fig. 2). Thus, we confirmed that implementing KD in pregnant and lactating rat mothers increases the osteogenic potential of BMCs in female offspring only.

Fig. 2. Ketogenic diet (KD) affects the osteogenic potential of rat bone marrow cells (BMCs) harvested from 30-day-old female and male offspring. BMCs were cultured for 7 days in standard osteogenic medium (OM) consisting of ascorbic acid, dexamethasone, and β-glycerophosphate. The mRNA levels of selected osteoblastic markers were analyzed using RT-PCR. The results are presented as relative quantification (RQ) of mRNA levels vs. mRNA levels in control ND male and female BMCs. Two-way ANOVA with a post hoc Tukey test, * p < 0,05, ** p < 0,001, *** p < 0,0001 compared between samples marked in brackets.

β-Hydroxybutyrate (β-HB) supports in vitro osteogenesis and reverses the negative impacts of low glucose in juvenile and adult rat bone marrow cells

In addition to the effects of KD during pre- and postnatal development on rat BMCs from 30-day offspring (Figs. 1 and 2), we examined selected in vitro effects of β-HB, the main ketone body, both on 30-day-old rat offspring BMCs and male adult rat BMCs, which had been all raised on ND (Fig. 3). BMCs were cultured continuously (starting from day 1) in osteogenic medium containing either control (5.5 mM, 1 g/L glucose), low glucose (2 mM), or low glucose (2 mM) + 5 mM β-HB – the latter representing ketosis-mimicking conditions.

After 7 days of juvenile rat BMC osteogenic cultures in low glucose conditions, the expression of osteoblastic (Bsp,* Opn*,* Opg*) and osteoclastic (Mmp-9,* Trap*) markers remained comparable to control conditions in male juvenile rat BMCs (Fig. 3a). Whereas in female juvenile rat BMCs, the expression of osteoblastic (Opn,* Opg*) and osteoclastic (Mmp-9,* Trap*) markers decreased. In both sexes, increased expression of the inflammatory cytokine Il-1β was detected. β-HB supplementation in low glucose medium increased Bsp, Opn, and Opg expression in both male and female juvenile rat BMCs compared to low glucose and control conditions. However, the response was stronger in female BMCs than in male BMCs. Interestingly, in male BMCs we observed increased Mmp-9 levels under ketosis-mimicking conditions (2 mM glucose + 5 mM β-HB) but not in female BMCs. In both sexes, β-HB addition reduced Il-1β expression to undetectable levels. Additionally, compared to male BMCs, the overall mRNA levels of osteoblastic markers in female BMCs were higher, including those in the control group (Fig. 3a). In 10-day osteogenic cultures, we observed reduced ECM mineralization under low glucose conditions, with a significant decrease compared to the control in male BMCs (Fig. 3c). The addition of β-HB to the low glucose medium increased mineralization in female BMCs, whereas a weaker response was observed in male BMCs. There was no significant change in the viability of juvenile BMCs under either β-HB or low glucose conditions (data not shown). After 7 days of adult rat BMC osteogenic culture, β-HB addition to low glucose medium significantly increased the mRNA levels of Bsp compared to the control and low glucose culture conditions without β-HB (Fig. 3b). β-HB in low glucose medium also enhanced the expression of Opn, Col1a1, and Opg compared to the control. In low glucose medium, the mRNA levels of Opn, Col1a1, and Opg were significantly higher than in low glucose medium supplemented with β-HB. However, in low glucose conditions, we detected increased mRNA levels of both osteoclastic (Trap, Ctsk) and pro-inflammatory markers (Tnf–α, Il-1β) that were significantly reduced after β-HB addition (Fig. 3b). Furthermore, low glucose conditions not only decreased BMC viability but also reduced ECM mineralization in 10-day adult rat BMC cultures compared to the control (Fig. 3d). The addition of β-HB to low glucose BMC cultures restored cell viability and increased mineral deposition twice as much as in control cultures (Fig. 3d). Since our results suggested that the implementation of KD in vivo enhances proliferation of offspring BMCs (Fig. 1d), we analyzed the colony forming units in 7-day standard and osteogenic BMC cultures from female and male juvenile rats, as well as adult males, under control or low glucose, or low glucose with β-HB (Fig. 3e-f). We determined that low glucose conditions enhanced BMC proliferation in all investigated osteogenic BMC cultures, whereas ketosis-mimicking conditions further increased colony area only in adult male rat BMCs. The latter effects were then enhanced by osteogenic medium with the largest colonies formed in osteogenic BMC cultures treated with β-HB.

Compared to juvenile BMCs, adult BMCs displayed a stronger response to low glucose conditions. In adult cells, low glucose induced higher expression levels of osteogenic markers such as Opn, Col1a1, and Opg and more strongly increased osteoclast markers, including Trap and Ctsk expression (Fig. 3a-b). Moreover, the reduction in extracellular matrix mineralization in low glucose conditions was more notable in adult BMCs than in juvenile BMCs (Fig. 3c-d). In contrast, the response to β-HB was stronger in adult BMCs than in juvenile BMCs, effectively compensating for the effects of low glucose conditions. Thus, our findings imply that β-HB promotes osteogenesis and restores the negative impact of low glucose on BMCs in an age-dependent context.

Fig. 3. The addition of β-hydroxybutyrate (β-HB) to juvenile (a,c,e) and adult (b,d,f) rat osteogenic BMC cultures under low glucose conditions supports osteogenesis in vitro and reverses the negative impact of low glucose. (a-f) BMCs were cultured in osteogenic media (OM) containing glucose at 5.5 mM (control), 2 mM (low glucose), or 2 mM glucose + 5 mM β-HB (ketosis-mimicking conditions). (a-b) 7-day BMC cultures were analyzed for mRNA levels of selected osteoblastic, osteoclastic and pro-inflammatory markers. Real-time RT-PCR results are presented as relative mRNA expression vs. control BMCs. (c–d) The mineral deposition (Alizarin Red S staining) in 10-day BMC cultures (the scale bar represents 100 μm), followed by colorimetric quantification of extracted dye, normalized to BMC viability (right panel). (e-f) Colony forming unit (CFU) assay (crystal violet staining) in 7-day BMC cultures (upper panel), followed by densitometric quantification (ImageJ) of colony area and number (lower panel). (a–f) One- and two-way ANOVAs with a post hoc Tukey test, * p < 0,05, ** p < 0,001, *** p < 0,0001 compared to control (5.5 mM glucose) or between samples marked by brackets or lines.

Discussion

In this work, we demonstrate for the first time the effects of a continuous ketogenic diet during pregnancy, lactation, and postnatal development in 30-day-old Wistar rat offspring. Although the offspring of KD-fed rats had reduced body mass (Fig. 1a), our studies indicate sex-dependent differences in the regenerative potential of bone marrow cells (BMCs) harvested from femurs. We also demonstrate that β-HB, the main ketone body, effectively induces osteogenesis and restores the negative effects of low glucose conditions in BMC cultures of juvenile and male adult rats.

After in vivo KD implementation, female rat BMC isolates exhibited increased expression of osteogenic markers (Bsp,* Opn*) and decreased expression of osteoclastic markers (Trap,* Ctsk*,* Mmp-9*) and stemness markers (Sox2,* Oct4*). The opposite trend was observed in KD male BMC isolates with increased expression of stemness markers (Sox2,* Oct4*) and osteoclastic markers (Trap) but decreased osteogenic markers (Opg,* Bsp*,* Col1a1*) (Fig. 1b). We also confirmed the ability of isolated BMCs to differentiate into all three lineages (osteogenic, adipogenic, and chondrogenic). Male KD BMCs displayed a lower number of cells that had undergone osteogenic and chondrogenic differentiation than male ND BMCs (Fig. 1c). Notably, the elevated expression of osteogenic markers in KD female BMCs in standard culture conditions (Runx2,* Osx*,* Bsp*,* Opn*) was further enhanced by osteogenic culture conditions, as evidenced by Opg and Col1a1 mRNA levels (Fig. 2). Our findings regarding the negative KD impact on BMCs in juvenile male rats are consistent with studies conducted only on adult male rats, showing that KD implementation for 8–12 weeks lowered bone mass density^11–15 ^and impaired in vitro BMSC osteogenic differentiation^13^. However, some indications in other studies support our results indicating that KD in female rat offspring supports bone formation and inhibits osteoclast activity in BMCs. Clinical trials have shown that KD treatment in resistance-trained women has some beneficial effect on their bone mass^19^, but in most male athletes an increase in serum osteolysis-related markers has been reported^18^. Additionally, the implementation of KD compromised trabecular and cortical bone density more severely in adult ovariectomized mice relative to control female mice^30^, suggesting a role of female hormones in inhibiting osteoclast activity. Furthermore, a short-term Atkins diet and low-protein KD treatments have been reported to reduce trabecular bone volume and serum IGF-1 levels in male adult rats but not in female adult rats^31^. However, our study demonstrates long-term (pre- and postnatal) sex-dependent effects of KD on BMC regenerative potential in juvenile 30-day-old rats (Fig. 1). In juvenile Wistar rats, males exhibit higher testosterone concentrations than females, while estradiol concentrations remain equivalent in both sexes^32^. Therefore, it is plausible that the observed sex-dependent differences in juvenile rat BMC osteogenesis are associated with testosterone rather than estradiol. Testosterone is known to reduce adipogenesis^33 ^and increase lipolysis^34^. Given that KD, despite reducing weight, elevates adiposity in female rats but not in males^31^, it suggests that the potential involvement of testosterone may be linked to reported higher ketone body levels in females^35 ^and sex-dependent differences in energy distribution. The latter requires further verification in rat offspring, as our previous studies are limited to the measurement of ketone bodies in pregnant rats^36^. We believe that dissecting the sex-dependent differences in KD implementation in juvenile as well as in adult animals is of key importance for future therapeutic KD applications.

It should be noted that the KD used in this study was low in protein (8%), and the micronutrient composition differed between ND and KD. Rats require higher protein intake (20%) during gestation and lactation^37^. Maternal protein deficiency during gestation may result in delayed development and a smaller body weight of offspring^38^. We observed that a low-protein KD during pre- and postnatal growth reduced body weight in both sexes of offspring (Fig. 1a) and delayed overall bone development (data not shown). In a previous study, KD was also found to delay neurological development^36^. These observed changes may be due not only to high fat and low carbohydrate content in the diet but also to low protein intake, since adequate protein levels are important for maintaining bone health^39^. However, some studies reported lowered proliferation and alkaline phosphatase levels in BMCs from the rat offspring of dams maintained on a low (9%) protein diet compared to a normal (18%) protein diet^40^. We show contradictory results, demonstrating that low-protein KD increases the proliferation of BMCs and enhances the osteogenic potential of BMCs from female offspring. Therefore, we believe that the positive effects we observed on BMC osteogenic potential are due to the high-fat diet.

Since the ketone body β-HB has been shown to stimulate osteogenesis of a mouse osteoblast cell line in standard culture conditions^23^, we also addressed the effects of β-HB in conjunction with low glucose BMC culture conditions. It was verified that at the end of pregnancy, rats have glucose levels of 40 mg/dL and overall ketone body levels of 5–7 mM^36^. Therefore, we cultured juvenile (male and female) and adult male rat BMCs in low glucose (2 mM) medium supplemented with 5 mM β-HB (Fig. 3). Our results showed that low glucose conditions in juvenile osteogenic BMC culture decreased the expression of osteogenic markers (Opn, Opg) in females but not in males (Fig. 3a). Supplementation with β-HB effectively increased the expression of osteogenic markers (Bsp,* Opn*,* Opg)* in both sexes, with a stronger response observed in females. Other sex-dependent differences included increased mRNA marker expression of an osteoclastic marker (Mmp-9) in males and significantly increased ECM mineralization only in females (Fig. 3a, c). Notably, we observed similar dependencies in BMCs isolated from juvenile rats kept on KD in vivo (Fig. 1) and BMCs from rats on ND cultured in vitro with ketosis-mimicking conditions (Fig. 3).

In adult male BMCs, our results showed that reduced glucose culture conditions disrupted ECM mineralization in 10-day BMC cultures (Fig. 3d) vs. control BMC cultures, despite increased mRNA expression of the osteogenic markers Opn,* Col1a1 and Opg* (Fig. 3b). We also detected increased mRNA expression of osteoclastic enzymes (Trap,* Ctsk*) in adult male BMCs cultured in reduced glucose conditions that has not been reported before. Notably, the addition of β-HB to low glucose culture medium in adult male BMC cultures decreased the expression of osteoclastic markers (Trap,* Ctsk*), enhanced the expression of osteoblastic markers Bsp,* Opn*,* Col1a1 and Opg* and led to higher ECM mineralization compared to control osteogenic cultures (Fig. 3). The observed effect may be linked to β-HB increasing glucose uptake^41^. β-HB can also modulate epigenetic modifications such as histone acetylation, which can alter gene expression^42^, but the molecular mechanisms of β-HB in osteogenesis and osteoclastogenesis need further research. The observed sex-dependent responses to low glucose conditions and β-HB in juvenile rat BMC cultures (Fig. 3) align with other findings suggesting that glucose homeostasis is regulated differently in males and females^43^. Our results also show that with age, low glucose conditions had a more negative impact on the osteogenic potential of male rat BMCs, while β-HB supplementation more effectively enhanced the osteogenic response (Fig. 3). Collectively, our results indicate that β-HB attenuates the negative effects of reduced glucose levels on bone formation, suggesting that the negative effects of KD on bone homeostasis may depend on the balance between glucose and β-HB.

Furthermore, KD increased the proliferation of BMCs isolated from rat offspring and cultured for up to 9 days (Fig. 1d). Using a colony-forming unit assay, we determined that adult male BMCs proliferated faster and formed larger colonies when subjected to ketosis-mimicking conditions (i.e., 2 mM glucose + 5 mM β-HB) in osteogenic cultures (Fig. 3f) vs. reduced glucose or control cultures. β-HB also restored adult male BMC viability decreased by reduced glucose conditions in 10-day osteogenic cultures (Fig. 3d), which may be beneficial for bone regeneration. Moreover, reduced glucose conditions also increased the proliferation of BMCs from juvenile male and female rats in osteogenic cultures (Fig. 3e). The observed increased colony formation in osteogenic BMC cultures may be due to the ascorbic acid^44 ^that was added to the osteogenic medium. Some reports have also indicated KD-induced cellular senescence, often associated with cell growth arrest^45^, whereas other reports have shown that KD potentiates proliferation of e.g., muscle satellite cells^21 ^and intestinal stem cells^20^.

Finally, we determined that 7-day low glucose BMC cultures led to increased mRNA expression of pro-inflammatory cytokines (Tnf–α,* Il-1**β*) vs. control cultures in medium containing 5.5 mM glucose (Fig. 3a-b). Similar pro-inflammatory responses to hypoglycemia were observed in monocytes^46^. Our study showed that β-HB supplementation attenuated pro-inflammatory cytokines to the levels detected in control cultures (Fig. 3a-b). In BMCs analyzed directly after isolation from rat offspring maintained on KD, the mRNA expression of both pro- (Tnf–α, Il–1β) and anti-inflammatory (Il-10,* Il-6*) cytokines was reduced in both sexes (Fig. 1b). Some reports suggest KD involvement in ameliorating inflammation in osteoarthritis^15^. This supports our findings indicating that β-HB contributes to the protective and immunomodulatory role of KD in a low-carbohydrate diet (Fig. 3).

Collectively, our study reveals that KD, despite reducing body weight in juvenile rat offspring, supports osteogenesis and inhibits osteoclastogenesis in female rat BMCs, whereas in male rat BMCs it reduces their bone regenerative potential. We suggest that the observed sex-dependent differences in juvenile rat BMCs in response to KD are probably not associated with estradiol levels, but this and other metabolic factors need further research. Furthermore, our study indicates the necessity to consider sex-dependent differences when applying a ketogenic diet for any potential future treatments. Beyond the above, we demonstrate that β-HB promotes proliferation and bone matrix mineralization of juvenile and adult rat BMCs and reverses the negative impact of reduced glucose conditions on in vitro BMC viability, inflammation, and osteoclast activity. Thus, we believe our study should prompt further research on the distinct effects of low glucose conditions and β-HB during bone regeneration, especially when applying short-term KD or fasting to support bone-related therapies.

Methods

Animals on a ketogenic diet and bone marrow harvesting

All methods are reported in the paper in accordance with ARRIVE guidelines (https://arriveguidelines.org). The animals used in this study were Wistar rats originating from the husbandry of the Department of Experimental Neuropathology of the Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow. All animal studies were conducted in accordance with international standards and approved by the 2nd Local Institutional Animal Care and Use Committee in Krakow (approval no. 255/2020). Prior to the experiments, all animals were fed a normal diet for rodents (ND, Morawski Labofeed H Standard). Thirty pregnant 2-month-old female white Wistar rats (280 g) on gestation day 0 were randomly divided into two groups. Female rats were maintained on a normal diet (ND) or a ketogenic diet (KD, snif^®^ EF R/M with 80% fat, Table 1) during pregnancy (21 days), lactation, and up to 30 days post-partum. 30-day-old offspring were subjected to either ND or KD throughout pre- and postnatal development. No differences in litter size or structure were observed in pregnant animals kept on KD compared to those on ND. Single offspring on KD were euthanized due to reduced body mass. Bone marrow was harvested from 30-day-old rat offspring (5 males and 5 females) and five 2-month-old adult male rats in accordance with the 3Rs (replacement, reduction and refinement) rule following decapitation, which was performed for other neurodevelopmental research studies. Detailed developmental body mass changes were also verified in our previous study^36^. Bone marrow was washed from the marrow cavities of both femurs with culture medium (MEM Alpha, 10% FBS, 1% antibiotics) using a syringe. Collected bone marrow cells (BMCs) were cultured in vitro for 4 days to obtain adherent cells for further analysis.

Table 1. Diet composition (% of the dry mass).Normal dietKetogenic dietFat4.2%79.2%Protein22%8%Carbohydrates67.1%2.2%Fiber3.5%5%Calcium0.95%0.79%Phosphorus0.75%0.57%Magnesium0.29%0.17%Potassium0.95%0.79%Vitamin D1000 IU/kg1500 IU/kg

Cell culture

Bone marrow cells (BMCs) were expanded in growth medium consisting of 89% MEM Alpha (Thermo Fisher Scientific), 10% FBS (Thermo Fisher Scientific) and 1% ZellShield antibiotics (Minerva Biolabs). Cell cultures were maintained in a culture incubator at 37 °C in a 5% CO_2_ humidified atmosphere; culture media were exchanged every 3 days, and cells were passaged using 0.25% trypsin/EDTA (Thermo Fisher Scientific) before they reached full confluence. For experiments, BMCs were seeded at a density of 20,000/cm^2^.

Experimental cell culture treatments

The multipotency of BMCs was verified using a Rat Mesenchymal Stem Cell Functional Identification Kit (#SC020, R&D Systems) according to the manufacturer’s instructions.

For osteogenic differentiation, complete growth medium was supplemented with 100 µg/ml ascorbic acid, 10^–7^ M dexamethasone and 10 mM β-glycerophosphate (all from Sigma Aldrich).

For ketosis-mimicking conditions BMCs were continuously cultured in DMEM (no glucose, Thermo Fisher Scientific), 10% FBS, and 1% antibiotics, and supplemented with 2 mM glucose (Gibco) and/or 5 mM β-hydroxybutyrate (Sigma Aldrich). Control cells were cultured with growth medium consisting of DMEM (1 g/L glucose, Thermo Fisher Scientific), 10% FBS, and 1% antibiotics.

In all experimental treatments, BMCs were treated starting from day 1 culture, and the culture media with supplements were exchanged every 3 days.

RT-PCR

Total RNA was extracted using TRI Reagent (Zymo Research). Equal amounts of RNA were reverse transcribed using a high-capacity cDNA Reverse Transcription kit (Applied Biosystems). The PCR amplifications were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). Each reaction mixture contained 50 ng of cDNA and TaqMan probes with TaqMan Universal Master Mix (Thermo Fisher Scientific). The following probes were used: Sox2 Rn01286286_g1, Oct4 Rn06413993_s1, Bsp Rn00561414_m1, Col1a1 Rn01463848_m1, Opn Rn00681031_m1, Opg Rn00563499_m1, Runx2 Rn01512298_m1, Osx Rn02769744_s1, Mmp-9 Rn00579162_m1, Trap Rn00569608_m1, Ctsk Rn00580723_m1, Il-6 Rn01410330_m1, TNF-alpha Rn99999017_m1, Il-1beta Rn00580432_m1, Il-10 Rn00563409_m1, and Gapdh Rn01775763_g1. Relative expression levels were obtained with the 2^−ΔΔCT^ method.

Cell viability and proliferation

Cell viability was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, MTS Assay) according to the manufacturer’s instructions (Figs. 1d and 3c-d). For the cell proliferation assay, we repeatedly measured cell viability for 15 min at particular time points, and after washing the cells with phosphate-buffered saline, the culture was continued. The absorbance was measured at 492 nm using a SpectraMax iD3 Molecular Devices reader.

Mineralization of extracellular matrix

After the cell viability assay, the cells were washed with PBS, fixed with 100% methanol and then stained for 30 min with 1% (water solution) Alizarin Red S (ARS). For quantitative analysis, cells were washed with distilled water and the ARS dye was extracted with 5% perchloric acid. The absorbance of the extracted dye was measured at 490 nm using a SpectraMax iD3 Molecular Devices reader. The results were normalized to cell viability.

CFU assay

BMCs were seeded at a density of 1,000 cells/well in 12-well plates. After 7 days of culture, the cells were washed with PBS and fixed with 4% formalin solution. For colony staining, 0.5% crystal violet solution in 25% methanol was used. The number and size of colony forming units were measured in ImageJ.

Imaging

After experimental treatments, the cells were fixed with 4% paraformaldehyde and probed with anti-osteocalcin or anti-FABP4 antibodies from the Rat Mesenchymal Stem Cell Functional Identification Kit (#SC020, R&D Systems) according to the manufacturer’s instructions. Cell nuclei were stained with DAPI (Sigma Aldrich). For chondrogenic differentiation assessment, after cryostat sectioning (15 μm), spheroids were stained with 1% Alcian Blue in 3% acetic acid. Images were acquired with a ZEISS Axiovert 5 Microscope at 100x magnification. The quantification of fluorescent-positive cells and stained area was performed using ImageJ.

Statistical analysis

All experiments were performed in triplicate, data were collected as the mean +/- SD, and data were analyzed for statistical significance using Mann-Whitney tests or one, two or three-way analysis of variance (ANOVA) followed by Tukey’s tests for multiple comparisons.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1