Evidence of a role for insulin-like growth factor binding protein (IGFBP)-3 in metabolic regulation - PubMed (original) (raw)

Evidence of a role for insulin-like growth factor binding protein (IGFBP)-3 in metabolic regulation

P M Yamada et al. Endocrinology. 2010 Dec.

Abstract

IGF-binding protein (IGFBP)-3 is a metabolic regulator that has been shown to inhibit insulin-stimulated glucose uptake in murine models. This finding contrasts with epidemiological evidence of decreased serum IGFBP-3 in patients with type 2 diabetes. The purpose of this study was to clarify the role of IGFBP-3 in metabolism. Four-week-old male IGFBP-3(-/-) and control mice were subjected to a high-fat diet (HFD) for 12 wk. IGFBP-3(-/-) mice were heavier before the initiation of HFD and at the end of the study period. Resting metabolic rate was significantly decreased in knockout mice; however, respiratory exchange ratio was not significantly different. Fasting blood glucose and insulin levels were significantly elevated in IGFBP-3(-/-) mice. However, IGFBP-3(-/-) mice had relatively normal glucose tolerance because the relative glucose excursion over time was not different between the groups. During hyperinsulinemic clamps, IGFBP-3(-/-) mice had increased basal hepatic glucose production, but after insulin stimulation, no differences in hepatic glucose production were observed. A second cohort of older IGFBP-3(-/-) mice on HFD displayed unexpected evidence of hepatic steatosis. In summary, glucose tolerance and clamp testing indicate that IGFBP-3(-/-) mice preserve insulin sensitivity despite evidence of increased basal glucose turnover and hepatic steatosis. We provide evidence that genetic deletion of IGFBP-3 modulates hepatic carbohydrate and lipid metabolism.

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Figures

Figure 1

Figure 1

Targeted disruption of the Igfbp3 gene locus. A, Targeting strategy used to disrupt the Igfbp3 locus. Homologous recombination (represented by ×) between the targeting vector and the Igfbp3 gene results in the replacement of the first coding exon with the selection cassette. The _Acc_65I and _Nde_I restriction enzyme cut sites used in the Southern blot analysis are indicated. B, Southern hybridization indicating proper gene targeting in the embryonic stem cell clones. Using a 5′ external probe on _Acc_65I-digested genomic DNA produces a 20.2-kb WT band and a 14.6-kb targeted band. Using a 3′ external probe on _Nde_I-digested genomic DNA produces a 10.2-kb WT band and a 7.6-kb targeted band. Clone 2G6 was selected for blastocyst injections. Lex1 represents untransfected embryonic stem cell DNA.

Figure 2

Figure 2

Circulating IGFBP-3 and related analytes. The 30-wk-old male mice were analyzed after HFD. A, Serum IGFBP-3 was measured using an in-house ELISA and immunoblotting (inset); B, ALS was measured using ELISA; C, reduced IGF-I levels in IGFBP-3−/− mice was expected response because IGFBP-3 dramatically extends the half-life of IGF-I; D, GH levels are not different; E, IGFBP-2 is significantly elevated in IGFBP-3−/− mice; F, IGFBP-1 was measured after a 5-h fast. *, P < 0.05; **, P < 0.01. Means ±

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are presented; n = 5–7 per group.

Figure 3

Figure 3

Body weight and food intake. A, Male and female IGFBP-3−/− and WT mice were weighed at weaning (3 wk old) (n = 90–130 per group); B, male IGFBP-3−/− are heavier than male WT mice at all time points using Holm’s sequential Bonferroni adjustment (n = 30–40 per group; HFD was initiated at 4 wk of age); C, male IGFBP-3−/− mice consumed less food at wk 7 and 9 when the significance level was adjusted with Holm’s sequential Bonferroni method. Using unadjusted significance levels (P < 0.05), IGFBP-3−/− mice had significantly lower food consumption from wk 7–10 and wk 12; n = 30–40. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Means ±

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are presented.

Figure 4

Figure 4

Basal glucose levels, GTT, and ITT. A, Fasting blood glucose was measured in male mice at 4 wk of age before HFD, after a 5-h fast; B, after 12 wk of HFD, fasting blood glucose levels doubled in IGFBP-3−/− mice; C, absolute blood glucose levels were significantly higher in IGFBP-3−/− mice; D, blood glucose was not different during the GTT when glucose levels were expressed relative to baseline levels (n = 10 per group); E, basal insulin concentrations were higher in IGFBP-3−/− mice, but no differences were observed 30 min after glucose injection (n = 10 per group); F, absolute blood glucose levels were not different during the ITT; G, blood glucose levels were reduced in IGFBP-3−/− mice at 150, 180, and 210 min after insulin injection when glucose levels were expressed relative to baseline during ITT, adjusted with Holm-Bonferroni adjustment (n = 5–6 per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Means ±

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are presented.

Figure 5

Figure 5

Hepatic insulin sensitivity. A, In 16-wk-old male mice after HFD, basal HGP is significantly elevated in IGFBP-3−/− mice, but no differences were observed during insulin stimulation (n = 7–8 per group); B, during insulin stimulation, PEPCK was suppressed in the KO compared with WT as detected with immunoblotting (inset) and densitometric analyses; C, glucose infusion rate was not significantly different (n = 7–8 per group); D, no differences in total and insulin-stimulated glucose disposal rate (n = 7–8 per group); E, 16-wk-old male IGFBP-3−/− mice have greater quadriceps (Quad) and tibialis anterior (Tib Ant) muscle mass in the fasted state (n = 8–11 per group). *, P < 0.05. Means ±

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are presented.

Figure 6

Figure 6

Hepatic lipid metabolism. A, The 16-wk-old IGFBP-3−/− male mice have heavier liver mass in the fasted state (n = 15–20 per group); B, FAS was up-regulated in IGFBP-3−/− mice under insulin stimulation as shown with immunoblotting (inset) and densitometric analyses, and p-JNK was up-regulated in IGFBP-3−/− mice as shown with immunoblotting and densitometric analyses in the fed state; C, greater hepatic oil red O staining in 30-wk-old male IGFBP-3−/− mice after HFD. Magnification, ×100. *, P < 0.05; **, P < 0.01. Means ±

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are presented.

Figure 7

Figure 7

Adipose tissue, triglycerides, and adiponectin. A, In 16-wk-old IGFBP-3−/− male mice, plasma triglycerides (TG) were reduced in the fed state but not different in the fasted state (n = 5–7 per group); B, reduced WAT mass in the IGFBP-3−/− group in a fed state (n = 15–20 per group); C, IGFBP-3−/− mice have reduced adiponectin (ADN) levels as measured with ELISA in both fed and fasted states (n = 5 per group) and as measured with immunoblotting in fed plasma (inset). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Means ±

sem

are presented.

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