Profound hypoglycemia in starved, ghrelin-deficient mice is caused by decreased gluconeogenesis and reversed by lactate or fatty acids

Abstract

When mice are subjected to 7-day calorie restriction (40% of normal food intake), body fat disappears, but blood glucose is maintained as long as the animals produce ghrelin, an octanoylated peptide that stimulates growth hormone secretion. Mice can be rendered ghrelin-deficient by knock-out of the gene encoding either ghrelin O-acyltransferase, which attaches the required octanoate, or ghrelin itself. Calorie-restricted, fat-depleted ghrelin O-acyltransferase or ghrelin knock-out mice fail to show the normal increase in growth hormone and become profoundly hypoglycemic when fasted for 18-23 h. Glucose production in Goat(-/-) mice was reduced by 60% when compared with similarly treated WT mice. Plasma lactate and pyruvate were also low. Injection of lactate, pyruvate, alanine, or a fatty acid restored blood glucose in Goat(-/-) mice. Thus, when body fat is reduced by calorie restriction, ghrelin stimulates growth hormone secretion, which allows maintenance of glucose production, even when food intake is eliminated. In humans with anorexia nervosa or kwashiorkor, ghrelin and growth hormone are known to be elevated, just as they are in fat-depleted mice. We suggest that these two hormones prolong survival in starved humans as they do in mice.

FIGURE 1.

Comparison of WT and Ghrl^−/− mice fed a chow diet or subjected to calorie restriction. Male littermates (8 weeks old) were housed in individual cages and fed ad libitum with a chow diet or subjected to 60% calorie restriction as described under “Experimental Procedures.” Body weight (A) and blood glucose (B) were measured daily at 5:30 p.m. (30 min before feeding). C–E, mice were euthanized at 5:30 p.m. (before feeding) on day 9 of calorie restriction, after which plasma levels of ghrelin (C), des-acyl ghrelin (D), and growth hormone (E) were determined. Each value represents the means ± S.E. of data from six mice. The asterisks denote the levels of statistical significance (Student's t test) between WT and Ghrl^−/− mice. *, p < 0.05; ***, p < 0.001.

FIGURE 2.

Response to starvation in WT and Goat^−/− mice after 7 days of calorie restriction. Male WT and Goat^−/− littermates (8 weeks old) were subjected to 60% calorie restriction for 7 days as described under “Experimental Procedures,” having received their last food at 6 p.m. on day 7. On day 8, blood was obtained at the indicated time for measurement of glucose (A), ghrelin (B), des-acyl ghrelin (C), and growth hormone (D). Food was given on day 8 at 6 p.m. (arrow). Each value represents the mean ± S.E. of data from four mice. The asterisks denote the levels of statistical significance (Student's t test) between WT and Goat^−/− mice. *, p < 0.05; ***, p < 0.001.

FIGURE 3.

Changes in plasma parameters in WT and Goat^−/− mice after 5 days of calorie restriction. Male WT and Goat^−/− littermates (8 weeks old) were subjected to 60% calorie restriction for 5 days as described under “Experimental Procedures,” having received their last food at 6 p.m. on day 5. On day 6, blood was obtained at the indicated time for measurement of glucose (A), insulin (B), glucagon (C), free fatty acids (D), β-hydroxybutyrate (E), lactate (F), and pyruvate (G). Food was given on day 6 at 6 p.m. (arrow). Each value represents the mean ± S.E. of data from six mice except for glucagon, which was measured using pooled samples from six mice. The asterisks denote the levels of statistical significance (Student's t test) between WT and Goat^−/− mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Response of calorie-restricted WT and Goat^−/− mice to injections of gluconeogenic precursors. Male WT and Goat^−/− littermates (8 weeks old) were subjected to 60% calorie restriction for 7–9 days as described under “Experimental Procedures.” On day 7, 8, or 9 as indicated, WT (A, C, and E) and Goat^−/− (B, D, and F) mice were injected intraperitoneally with either NaCl or one of the following gluconeogenic precursors: sodium lactate (A and B), sodium pyruvate (C and D), or alanine (E and F). Each mouse received four injections of either NaCl or the indicated gluconeogenic precursor (18 μmol/g of body weight/injection). Each intraperitoneal injection was given at hourly intervals beginning at 2 p.m. in a volume of 150–180 μl (gluconeogenic precursors were dissolved in water and adjusted to pH 7.2). The arrows denote the times of injections. A–F, blood glucose was measured immediately before each injection and at 6 p.m., 1 h after the last injection. Each value represents the mean ± S.E. of data from five mice. The asterisks denote the levels of statistical significance (Student's t test) between WT and Goat^−/− mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Response of calorie-restricted WT and Goat^−/− mice to injections of octanoate. Male WT and Goat^−/− littermates (8 weeks old) were subjected to 60% calorie restriction for 7 days as described under “Experimental Procedures.” On day 7, WT (left panel) and Goat^−/− (right panel) mice were injected intraperitoneally with each mouse receiving two injections (2 and 4 p.m.) of equimolar amounts of either NaCl or sodium octanoate (3 μmol/g of body weight/injection) in a volume of 140–170 μl (octanoate was dissolved in water to a final concentration of 0.3 m and adjusted to pH 7.6). Blood glucose was measured at the indicated time (just prior to injections). Each value represents the mean ± S.E. of data from five mice. The asterisks denote the levels of statistical significance (Student's t test) between WT and Goat^−/− mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The arrows denote the times of injections.

FIGURE 6.

Comparison of WT and Goat^−/− mice fed a high fat diet and then subjected to calorie restriction. Four-week-old male littermates were fed ad libitum either a chow diet or a high fat diet for 4 weeks and then subjected to calorie restriction by feeding each mouse 1.4 g of chow diet each day as described under “Experimental Procedures.” Body composition (A and B), plasma free fatty acids (C and D), plasma β-hydroxybutyrate (E and F), and blood glucose (G and H) were measured every 1–2 days at 5:30 p.m. (30 min prior to feeding). Each value represents the mean ± S.E. of data from six mice.

FIGURE 7.

Whole body glucose production in calorie-restricted WT and Goat^−/− mice in the absence or presence of octanoate. Eight-week-old male littermates were implanted with jugular vein catheters 4 days before 60% calorie restriction as described under “Experimental Procedures.” On day 6 of calorie restriction, the mice were injected intraperitoneally with either NaCl or sodium octanoate (3 μmol/g of body weight/injection). Each mouse received two injections (2 and 4 p.m.) as described in Fig. 6. After the last injection (4 p.m.), each mouse received a continuous infusion of [3-³H]glucose (31 dpm/fmol). The infusion rate was 0.3 μCi/min from 0–20 min, followed by 0.1 μCi/min from 20–90 min, after which blood was obtained at the indicated time for measurement of its content of glucose (A and B) and ³H radioactivity (C and D). Specific activity of blood glucose (E and F) and whole body glucose production (G and H) were then calculated as described under “Experimental Procedures.” Each value represents the mean ± S.E. of data from six mice. The asterisks denote the levels of statistical significance (Student's t test) between mice injected with octanoate and mice injected with NaCl. **, p < 0.01; ***, p < 0.001.