Suppression of hyperinsulinaemia in growing female mice provides long-term protection against obesity - PubMed (original) (raw)

Suppression of hyperinsulinaemia in growing female mice provides long-term protection against obesity

Nicole M Templeman et al. Diabetologia. 2015 Oct.

Abstract

Aims/hypothesis: Hyperinsulinaemia is associated with obesity but its causal role in the onset of obesity remains controversial. In this study, we tested the hypothesis that transient attenuation of diet-induced insulin hypersecretion in young mice can provide sustained protection against obesity throughout adult life.

Methods: Using 'genetically humanised' mice lacking both alleles of rodent-specific Ins1, we compared mice heterozygous for the ancestral insulin gene Ins2 with Ins2(+/+) controls. Female Ins1(-/-):Ins2(+/-) and Ins1(-/-):Ins2(+/+) littermates were fed chow or high-fat diet (HFD). Insulin secretion, metabolic health variables and body mass/composition were tracked for over 1 year. We examined islet function and adipose transcript levels of adipogenic, lipogenic and lipolytic genes at two time points.

Results: In control Ins1(-/-):Ins2(+/+) mice, HFD resulted in elevated fasting and glucose-stimulated insulin secretion between 8 weeks and 27 weeks of age. Hyperinsulinaemia was reduced by nearly 50% in Ins1(-/-):Ins2(+/-) mice during this period, without lasting adverse effects on glucose homeostasis. This corresponded with attenuated weight gain and adiposity. White adipose tissue from Ins1(-/-):Ins2(+/-) mice had fewer large lipid droplets, although transcriptional changes were not detected. Importantly, Ins1(-/-):Ins2(+/-) mice remained lighter than Ins1(-/-):Ins2(+/+) littermates despite reaching an equivalent degree of hyperinsulinaemia on HFD by 52 weeks.

Conclusions/interpretation: These data demonstrate that attenuation of hyperinsulinaemia in young, growing female mice provides a long-lasting protection against obesity. This protection persists despite a late-onset emergence of hyperinsulinaemia in HFD-fed Ins1(-/-):Ins2(+/-) mice. Given the evolutionary conserved roles of insulin, it is possible that suppressing hyperinsulinaemia early in life may have far-reaching consequences on obesity in full-grown adult humans.

Keywords: Adolescence; Diet-induced obesity; Glucose homeostasis; Insulin; Knockout mice; Type 2 diabetes.

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Figures

Fig. 1

Fig. 1

Transiently attenuated HFD-induced hyperinsulinaemia in Ins1 −/−:Ins2 +/− mice. (a) Experimental design of Ins1 −/−:Ins2 +/+ and Ins1 −/−:Ins2 +/− littermates fed CD or HFD. (b, c) Islet Ins2 mRNA is corrected against β-actin mRNA and normalised to CD-fed Ins1 −/−:Ins2 +/+ mice at 25 weeks (n = 3–5) (b), or HFD-fed Ins1 −/−:Ins2 +/+ mice at 50 weeks (n = 3) (c). (d, e) Insulin content in islets from mice at 25 (d) and 50 weeks (e). (f–h) At 25 (f, g) and 50 weeks (h), insulin secretion by 150 islets perifused with basal 3 mmol/l glucose followed by stimulatory 15 mmol/l glucose (Glu) or 30 mmol/l KCl, with AUC (insets; _y_-axis units, pmol/l × min) depicted, including phases I/II of glucose stimulation (n = 3). (i, j) Fasted insulin (n = 17–21) (i) and C-peptide (n = 5–6) (j) at 27 weeks is from in vivo sampling. Dark blue, CD-fed Ins1 −/−:Ins2 +/+ mice; dark purple, HFD-fed Ins1 −/−:Ins2 +/+ mice; light blue, CD-fed Ins1 −/−:Ins2 +/− mice; light purple, HFD-fed Ins1 −/−:Ins2 +/− mice. Data are means ± SEM. *p ≤ 0.05, CD vs HFD; † p ≤ 0.05, Ins1 −/−:Ins2 +/+ vs Ins1 −/−:Ins2 +/−

Fig. 2

Fig. 2

Longitudinal glucose homeostasis. Periodic measurements of glucose-stimulated insulin secretion (n = 17–21) (a), blood glucose responses to intraperitoneal glucose (n = 29–34) (b) and insulin analogue (n = 29–34) (c) are shown, together with AUC or area over curve (AOC) (insets, _y_-axis units, pmol/l × min [a], mmol/l × min [b] and % × min [c]) and fasted blood glucose (n = 15–18, most time points) (d). Dark blue, CD-fed Ins1 −/−:Ins2 +/+ mice; dark purple, HFD-fed Ins1 −/−:Ins2 +/+ mice; light blue, CD-fed Ins1 −/−:Ins2 +/− mice; light purple, HFD-fed Ins1 −/−:Ins2 +/− mice. Data are means ± SEM. *p ≤ 0.05, CD vs HFD; † p ≤ 0.05, Ins1 −/−:Ins2 +/+ vs Ins1 −/−:Ins2 +/−

Fig. 3

Fig. 3

Attenuated obesity in HFD-fed Ins1 −/−:Ins2 +/− mice. (a) Body mass was tracked in pups (n = 19–29, inset) and weaned mice (n = 29–33, most time points). (b) DEXA-measured fat mass is shown (n = 8–11). (c, d) Staining (magnification ×50) for perilipin (yellow) and DAPI (blue) in gonadal WAT of 25-week-old mice (c) is quantified (n = 3) in (d) as frequency per size category. (e, f) Gene expression in gonadal (e) and inguinal (f) WAT mRNA is corrected against Hprt and normalised to CD-fed Ins1 −/−:Ins2 +/+ mice. Dark blue, CD-fed Ins1 −/−:Ins2 +/+ mice; dark purple, HFD-fed Ins1 −/−:Ins2 +/+ mice; light blue, CD-fed Ins1 −/−:Ins2 +/− mice; light purple, HFD-fed Ins1 −/−:Ins2 +/− mice. Data are means ± SEM. *p ≤ 0.05, (*) p = 0.051, CD vs HFD; † p ≤ 0.05, (†) p = 0.054, Ins1 −/−:Ins2 +/+ vs Ins1 −/−:Ins2 +/−; *+/+ p ≤ 0.05, CD-fed vs HFD-fed Ins1 −/−:Ins2 +/+ mice; *+/− p ≤ 0.05, CD-fed vs HFD-fed Ins1 −/−:Ins2 +/− mice; †HFD p ≤ 0.05, HFD-fed Ins1 −/−:Ins2 +/+ vs Ins1 −/−:Ins2 +/− mice

Fig. 4

Fig. 4

Plasma lipids and metabolic factors. Fasted levels of cholesterol (a), triacylglycerols (b), NEFA (c), leptin (d), resistin (e), interleukin 6 (f), GIP (g), peptide YY (h) and glucagon (i) in 40-week-old mice (n = 8–12). Dark blue, CD-fed Ins1 −/−:Ins2 +/+ mice; dark purple, HFD-fed Ins1 −/−:Ins2 +/+ mice; light blue, CD-fed Ins1 −/−:Ins2 +/− mice; light purple, HFD-fed Ins1 −/−:Ins2 +/− mice. Data are means ± SEM. *p ≤ 0.05, CD vs HFD; *+/+ p ≤ 0.05, CD-fed vs HFD-fed Ins1 −/−:Ins2 +/+ mice; †HFD p ≤ 0.05, HFD-fed Ins1 −/−:Ins2 +/+ vs Ins1 −/−:Ins2 +/− mice

Fig. 5

Fig. 5

Energy homeostasis and brown adipose tissue. (a–d) In HFD-fed 17-week-old mice (n = 6–8), 24 h activity (a), food intake (b), respiratory exchange ratio (c) and energy expenditure (d) were averaged across 48–84 h; the dark period is shown in grey. (e, f) In 25-week-old mice (n = 5–7), BAT depot mass is shown as absolute values (e) and proportional to body mass (f). (g) mRNA levels of genes expressed in BAT are corrected against Tbp and normalised to levels in CD-fed Ins1 −/−:Ins2 +/+ mice. Dark blue, CD-fed Ins1 −/−:Ins2 +/+ mice; dark purple, HFD-fed Ins1 −/−:Ins2 +/+ mice; light blue, CD-fed Ins1 −/−:Ins2 +/− mice; light purple, HFD-fed Ins1 −/−:Ins2 +/− mice. Energy expenditure is shown as estimated marginal means ± SEM, adjusted for covariates of lean and fat mass; other data are simple means ± SEM. *p ≤ 0.05, CD vs HFD; † p ≤ 0.05, Ins1 −/−:Ins2 +/+ vs Ins1 −/−:Ins2 +/−

Fig. 6

Fig. 6

Long-term effects of HFD and INSULIN2. Mice were implanted with pumps releasing INSULIN2 or vehicle for 4 weeks (indicated by grey hatching). (a) Fasted insulin levels, n = 3. (b) Body mass, n = 3. (c–e) At 50 weeks, characterisation included WAT depot weight proportional to body mass (c) and gonadal WAT staining (magnification ×50) for perilipin (yellow) and DAPI (blue) (d) (n = 3; quantification is shown in e as frequency per size category). (f–h) mRNA levels of genes expressed in gonadal WAT (f), inguinal WAT (g) and BAT (h) are corrected against Tbp or Hprt, and normalised to levels in Ins1 −/−:Ins2 +/+ mice. Dark purple, vehicle-treated Ins1 −/−:Ins2 +/+ mice; light purple, vehicle-treated Ins1 −/−:Ins2 +/− mice; hatched bars and triangles, INSULIN2-treated Ins1 −/−:Ins2 +/− mice. Data are means ± SEM. ‡ p ≤ 0.05, for the indicated comparisons; (‡) p = 0.058 in (f) for group effect; and (‡) p ≤ 0.057 in (h) for the indicated comparisons

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