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. 2013 Jul;62(7):2183-94.
doi: 10.2337/db12-1311. Epub 2013 Feb 19.

_targeting pyruvate carboxylase reduces gluconeogenesis and adiposity and improves insulin resistance

Naoki Kumashiro  1 , Sara A Beddow, Daniel F Vatner, Sachin K Majumdar, Jennifer L Cantley, Fitsum Guebre-Egziabher, Ioana Fat, Blas Guigni, Michael J Jurczak, Andreas L Birkenfeld, Mario Kahn, Bryce K Perler, Michelle A Puchowicz, Vara Prasad Manchem, Sanjay Bhanot, Christopher D Still, Glenn S Gerhard, Kitt Falk Petersen, Gary W Cline, Gerald I Shulman, Varman T Samuel
Affiliations

_targeting pyruvate carboxylase reduces gluconeogenesis and adiposity and improves insulin resistance

Naoki Kumashiro et al. Diabetes. 2013 Jul.

Abstract

We measured the mRNA and protein expression of the key gluconeogenic enzymes in human liver biopsy specimens and found that only hepatic pyruvate carboxylase protein levels related strongly with glycemia. We assessed the role of pyruvate carboxylase in regulating glucose and lipid metabolism in rats through a loss-of-function approach using a specific antisense oligonucleotide (ASO) to decrease expression predominantly in liver and adipose tissue. Pyruvate carboxylase ASO reduced plasma glucose concentrations and the rate of endogenous glucose production in vivo. Interestingly, pyruvate carboxylase ASO also reduced adiposity, plasma lipid concentrations, and hepatic steatosis in high fat-fed rats and improved hepatic insulin sensitivity. Pyruvate carboxylase ASO had similar effects in Zucker Diabetic Fatty rats. Pyruvate carboxylase ASO did not alter de novo fatty acid synthesis, lipolysis, or hepatocyte fatty acid oxidation. In contrast, the lipid phenotype was attributed to a decrease in hepatic and adipose glycerol synthesis, which is important for fatty acid esterification when dietary fat is in excess. Tissue-specific inhibition of pyruvate carboxylase is a potential therapeutic approach for nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes.

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Figures

FIG. 1.
FIG. 1.
Hepatic pyruvate carboxylase (PC) protein expression levels relate to glycemic levels in humans. Hepatic PC mRNA expression in human livers compared with fasting plasma glucose concentration (A) and HbA1c (B). Hepatic PC protein expression in human livers compared with fasting plasma glucose concentration (C) and HbA1c, along with representative bands (D). PC mRNA and protein are expressed as a relative increase to the lowest expression in the data set (n = 20). VDAC, voltage-dependent anion channel.
FIG. 2.
FIG. 2.
Pyruvate carboxylase (PC) ASO decreased PC expression in liver and epididymal adipose tissue. PC mRNA in liver (A) and epididymal adipose tissue (B). PC protein, with representative bands, is shown in liver (C) and epididymal adipose tissue (D). **P < 0.01 and ***P < 0.001 compared with control ASO group in the same diet condition. #P < 0.05 and ###P < 0.001 compared with control ASO group in regular chow-fed condition (n = 3–4 per group in regular chow-fed condition; n = 9–10 per group in HFF condition). All rats were killed and tissues were taken at 4 weeks of treatment. VDAC, voltage-dependent anion channel.
FIG. 3.
FIG. 3.
Pyruvate carboxylase (PC) ASO decreased plasma glucose concentration and did not decrease insulin secretion. Fasting plasma glucose (A) and insulin concentration (B) in the regular chow-fed rats (n = 7–10 per group). C: Ad lib–fed plasma glucose concentration in the regular chow-fed rats (n = 5 per group). Results of mixed-meal tolerance test in the regular chow-fed rats for plasma glucose (D), plasma insulin (E), and plasma C-peptide (F) (n = 7–10). G: Pyruvate tolerance test in the regular chow-fed and HFF rats. ○ are control ASO and ● are PC ASO in regular chow-fed rats (both n = 9). △ are control ASO and ▲ are PC ASO in HFF rats (both n = 8). *P < 0.05, **P < 0.01, and ***P < 0.001 between control and PC ASO in regular chow-fed rats; ##P < 0.01 and ###P < 0.001 between control and PC ASO in HFF rats. Experiments were done at 4–5 weeks of treatment.
FIG. 4.
FIG. 4.
Pyruvate carboxylase (PC) ASO reduced adiposity and hepatic steatosis in HFF rats. A: Body weight (BW) time course in regular chow-fed (n = 10–11 per group) and HFF rats (n = 12 per group). Epididymal adipose tissue weight (B), hepatic triglyceride content (C), and muscular triglyceride content (D) at 4 weeks of treatment in HFF rats (n = 9–10 per group). *P < 0.05 and **P < 0.01 compared with control ASO group in HFF condition. RC, regular chow.
FIG. 5.
FIG. 5.
Pyruvate carboxylase (PC) ASO improves hepatic insulin sensitivity in HFF rats. Fasting plasma glucose (A) and insulin concentration (B) (n = 9 per group). C: Basal endogenous glucose production (n = 9 per group). Plasma glucose concentration (D) and glucose infusion rate time course (E) during hyperinsulinemic-euglycemic (4 mU/kg per min) clamp, respectively (n = 7–8 per group). Insulin-stimulated peripheral glucose metabolism (F), endogenous glucose production (G), and percentage suppression of endogenous glucose production (H) during clamp (n = 7–8 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control ASO group. Experiments were done at 4–5 weeks of treatment.
FIG. 6.
FIG. 6.
Pyruvate carboxylase (PC) ASO decreased hepatic DAG content and PKCε activation and increased hepatic Akt phosphorylation in HFF rats. A: Hepatic DAG content (n = 9–10 per group). *P < 0.05 compared with control ASO group. B: PKCε activation. The average of control ASO group was set as 1 (n = 5 per group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. $$P < 0.001 compared with control ASO group. C: Akt phosphorylation (Ser473). The average expression of control ASO group in the basal condition was set as 1 (n = 5 per group). #P < 0.05 and ###P < 0.001 compared with control ASO group in basal condition. **P < 0.01 compared with control ASO group in clamp condition. All tissues were taken at 4–5 weeks of treatment.
FIG. 7.
FIG. 7.
Pyruvate carboxylase (PC) ASO reduced hepatic and adipose glycerol synthesis. A: Whole-body lipolysis as assessed by glycerol turnover in HFF rats (n = 8–9 per group). Palmitate oxidation (B) and oleate oxidation (C) assay with primary hepatocytes isolated from HFF-treated and ASO-treated rats (n = 5 per group). D: In vivo hepatic de novo fatty acid synthesis in HFF rats (n = 9–10 per group). Hepatic (E) and adipose glycerol synthesis (F) in HFF rats (n = 7–8 per group). G: Summary of this study. *P < 0.05 compared with control ASO group. All experiments were done at 4–5 weeks of treatment.
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