The broad physiological impact of the 5:2 diet, a popular weight-loss strategy, is poorly understood.  We have shown that this diet fails to induce adult hippocampal neurogenesis or neurogenesis-dependent behaviour in mice¹ and induces weight loss in males^, while elevating lipid storage in both sexes².  Here we examine the impact of this dietary strategy on skeletal morphology and biomechanical performance and indices of reproductive axis activity.

 

Adolescent (7 week-old) male and female wild-type (WT) and loxTB-GHSR (growth hormone secretagogue receptor (GHSR)-null) littermate mice, were fed standard rodent chow in either ad libitum (AL) or 5:2 (5 days AL:2 non-consecutive 24hr fasting periods) pattern for 3 or 6 weeks (n=6-9 per group) (Animal procedures performed in accordance with the Animals (Scientific Procedures) Act, 1986 (UK), the Cardiff University Animal Welfare Ethical Review Body (AWERB) and reported as per the ARRIVE guidelines).  Femoral length was quantified with a hand-held micrometer, with biomechanical strength and proximal trabecular architecture assessed by 3-point bending and micro-CT^3,4.  Testes, seminal vesicles and ovaries were weighed post mortem, with spermatozoa and follicular development quantified in histological sections.  Circulating LH, FSH, testosterone and oestrogen (E2) were quantified by ELISA in terminal plasma samples.  All data reported are mean ± SEM, with statistical comparisons performed by Students unpaired t-test or 2-way ANOVA with Sidak’s multiple comparison post hoc test.

 

Three weeks of the 5:2 diet had no effect on femoral length, mid-diaphysial diameter or cross-sectional area.  In contrast, lateral and medial wall thicknesses were elevated and reduced by 13% respectively in 5:2-fed males (P=0.044; P=0.002), with anterior and posterior wall thicknesses unchanged.  Despite the significant influence of sex (P=0.0036), neither overall biomechanical strength, tissue strength (ultimate tensile stress) nor the geometric contribution to strength (2^nd moment of area) were significantly changed by the 5:2 diet.  However, the 5:2 diet induced a 12% elevation in trabecular bone density (BV/TV; P=0.009), accompanied by a 10% reduction in lattice fragmentation (Tb.Pf; P=0.0495).  Trabecular surface (BS/BV), number (Tb.N), thickness (Tb.Th), separation (Tb.Sp), and cross-sectional shape (Structure-modal index (SMI)) were unaffected.

Three weeks of the 5:2 diet reduced seminal vesicle weight by 16% (P=0.016), without affecting testicular weight or spermatozoa counts.  Although mean circulating LH was halved (P=0.095) and mean circulating FSH was 92% of that in AL-fed male mice (P=0.077), circulating testosterone was unaffected.  However, by 6 weeks, circulating testosterone was reduced by 88% (P=0.023).  This was reversed in GHSR-null males.  In females, 3 weeks of the 5:2 diet had no significant effect on ovarian weight or the number or morphology of primary follicles, graafian follicles or corpora lutea.  Mean circulating oestrogen were 75% of that in AL-fed females (P=0.075), with circulating LH halved (P=0.262) and FSH unaffected.  After 6 weeks, the 5:2 diet had no impact on circulating oestrogen in either WT or GHSR-null mice.

 

Thus, infradian stimulation of the ghrelin axis by the 5:2 diet induces a collapse in circulating testosterone in male mice.  When combined with previous evidence of enhanced lipid storage, this weight loss strategy elicits some undesirable physiological consequences.