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Involvement of KLF11 in hepatic glucose metabolism in mice via suppressing of PEPCK-C expression.

Zhang H, Chen Q, Jiao T, Cui A, Sun X, Fang W, Xie L, Liu Y, Fang F, Chang Y - PLoS ONE (2014)

Bottom Line: Abnormal hepatic gluconeogenesis is related to hyperglycemia in mammals with insulin resistance.Despite the strong evidences linking Krüppel-like factor 11 (KLF11) gene mutations to development of Type 2 diabetes, the precise physiological functions of KLF11 in vivo remain largely unknown.Our data strongly indicated the involvement of KLF11 in hepatic glucose homeostasis via modulating the expression of PEPCK-C.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, School of Basic Medicine, Anhui Medical University, Hefei, China ; National Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China.

ABSTRACT

Background: Abnormal hepatic gluconeogenesis is related to hyperglycemia in mammals with insulin resistance. Despite the strong evidences linking Krüppel-like factor 11 (KLF11) gene mutations to development of Type 2 diabetes, the precise physiological functions of KLF11 in vivo remain largely unknown.

Results: In current investigation, we showed that KLF11 is involved in modulating hepatic glucose metabolism in mice. Overexpression of KLF11 in primary mouse hepatocytes could inhibit the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase (cytosolic isoform, PEPCK-C) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), subsequently decreasing the cellular glucose output. Diabetic mice with overexpression of KLF11 gene in livers significantly ameliorated hyperglycemia and glucose intolerance; in contrast, the knockdown of KLF11 expression in db/m and C57BL/6J mice livers impaired glucose tolerance.

Conclusions: Our data strongly indicated the involvement of KLF11 in hepatic glucose homeostasis via modulating the expression of PEPCK-C.

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Related in: MedlinePlus

Overexpression of KLF11 in db/db mice alleviates hyperglycemia. (A) Blood glucose level in control Ad-GFP or Ad-KLF11-injected db/db mice 7 days after injection under fasting conditions (n = 6/group, 6-hr fasting). (B) qRT-PCR showing the mRNA levels of gluconeogenic genes in the livers of the same mice as in (A) (n = 6/group). (C) Western blot analysis showing the protein levels of PEPCK-C in in the livers of the same mice as in (A) (n = 6/group). GADPH was used to show the similar amount of protein loaded in different lanes (Left panel). The relative intensities of PEPCK-C bands on the Western blot were determined using NIH Image 1.62 software and normalized using GADPH band intensity (Right panel). (D–F) Glucose tolerance tests (GTTs) (D), pyruvate-tolerance tests (PTT) (E), Insulin tolerance tests (ITTs) (F) in control Ad-GFP or Ad-KLF11 -injected db/db mice 5 days after injection (n = 6/group). All data are presented as mean ± SEM, with statistical analysis performed by repeated-measures two-way ANOVA (*p<0.05, **P<0.01, ***P<0.001). (G) Db/db mice were injected with Ad-KLF11 or Ad-GFP. After 5 days, the mice were fasted overnight and anesthetized with tribromoethanol followed by IP injection of 5 U of insulin or saline (as a control). Ten minutes later, the animals were sacrificed, and their liver protein lysates were subjected to Western blot analysis (Left panel). The relative intensities of Phospho-Akt, total Akt, phosphor-GSK3β and total GSK3β were quantitated by densitometry analysis of their bands on film. The results are expressed as the ratios of phospho-Akt/total Akt and phosphor-GSK3β/total GSK3β (statistical analysis of Western blot data from 4 mice under each condition; Right panel). The data shown are the means ± SEM. Statistical significance was determined using a two-tailed Student’s t-test (*P<0.05, **P<0.01).
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pone-0089552-g004: Overexpression of KLF11 in db/db mice alleviates hyperglycemia. (A) Blood glucose level in control Ad-GFP or Ad-KLF11-injected db/db mice 7 days after injection under fasting conditions (n = 6/group, 6-hr fasting). (B) qRT-PCR showing the mRNA levels of gluconeogenic genes in the livers of the same mice as in (A) (n = 6/group). (C) Western blot analysis showing the protein levels of PEPCK-C in in the livers of the same mice as in (A) (n = 6/group). GADPH was used to show the similar amount of protein loaded in different lanes (Left panel). The relative intensities of PEPCK-C bands on the Western blot were determined using NIH Image 1.62 software and normalized using GADPH band intensity (Right panel). (D–F) Glucose tolerance tests (GTTs) (D), pyruvate-tolerance tests (PTT) (E), Insulin tolerance tests (ITTs) (F) in control Ad-GFP or Ad-KLF11 -injected db/db mice 5 days after injection (n = 6/group). All data are presented as mean ± SEM, with statistical analysis performed by repeated-measures two-way ANOVA (*p<0.05, **P<0.01, ***P<0.001). (G) Db/db mice were injected with Ad-KLF11 or Ad-GFP. After 5 days, the mice were fasted overnight and anesthetized with tribromoethanol followed by IP injection of 5 U of insulin or saline (as a control). Ten minutes later, the animals were sacrificed, and their liver protein lysates were subjected to Western blot analysis (Left panel). The relative intensities of Phospho-Akt, total Akt, phosphor-GSK3β and total GSK3β were quantitated by densitometry analysis of their bands on film. The results are expressed as the ratios of phospho-Akt/total Akt and phosphor-GSK3β/total GSK3β (statistical analysis of Western blot data from 4 mice under each condition; Right panel). The data shown are the means ± SEM. Statistical significance was determined using a two-tailed Student’s t-test (*P<0.05, **P<0.01).

Mentions: To determine whether KLF11 regulated hepatic gluconeogenesis in vivo, we performed genetic constitution experiments in db/db mice in which the endogenous expression of KLF11 was low. Our previous results indicated that exogenous myc-tagged KLF11 was effectively expressed in the liver mediated by adenovirus Ad-KLF11, but not in the other tissues, including muscle and adipose tissue [16]. We found that Ad-KLF11 injection of db/db mice had significantly lower fasting plasma glucose levels compared with control adenovirus (Ad-GFP) (Fig. 4A). Consistent with the decreased blood glucose levels, the expression of gluconeogenic genes, including PGC-1α and its downstream target genes PEPCK-C and G6pase, were decreased in the livers of Ad-KLF11-injected mice (Fig. 4B), and western blot analysis indicated that PEPCK-C protein levels were also decreased with KLF11 overexpression (Fig. 4C), indicating that the decrease in hepatic gluconeogenesis contributed to the lower glucose levels. Glucose tolerance tests (GTTs) showed that a modest induction of KLF11 in db/db mouse livers markedly improved glucose intolerance after an intraperitoneal glucose injection compared with control Ad-GFP-injected db/db mice (Fig. 4D). A pyruvate-tolerance test (PTT) demonstrated that de novo hepatic glucose production was reduced in Ad-KLF11-treated db/db mice (Fig. 4E). However, Insulin tolerance tests (ITTs) showed modestly increased insulin sensitivity in Ad-KLF11-infected db/db mice (Fig. 4F). AUC was reduced for GTT and PTT and ITT modestly decreased but did not reach statistical difference for ITT in mice with hepatic overexpression of KLF11 (Figure S1 A–C). These effects through KLF11 overexpression were accompanied by increased phosphorylation of Akt and its downstream target GSK3β in response to acute intraperitoneal insulin injections, whereas the total Akt and GSK3β protein levels remained unchanged (Figure 4G). These results suggest that the gain of function of KLF11 in the db/db mouse livers reduced hepatic glucose production, and improved glucose intolerance, but lesser extent insulin sensitivity.


Involvement of KLF11 in hepatic glucose metabolism in mice via suppressing of PEPCK-C expression.

Zhang H, Chen Q, Jiao T, Cui A, Sun X, Fang W, Xie L, Liu Y, Fang F, Chang Y - PLoS ONE (2014)

Overexpression of KLF11 in db/db mice alleviates hyperglycemia. (A) Blood glucose level in control Ad-GFP or Ad-KLF11-injected db/db mice 7 days after injection under fasting conditions (n = 6/group, 6-hr fasting). (B) qRT-PCR showing the mRNA levels of gluconeogenic genes in the livers of the same mice as in (A) (n = 6/group). (C) Western blot analysis showing the protein levels of PEPCK-C in in the livers of the same mice as in (A) (n = 6/group). GADPH was used to show the similar amount of protein loaded in different lanes (Left panel). The relative intensities of PEPCK-C bands on the Western blot were determined using NIH Image 1.62 software and normalized using GADPH band intensity (Right panel). (D–F) Glucose tolerance tests (GTTs) (D), pyruvate-tolerance tests (PTT) (E), Insulin tolerance tests (ITTs) (F) in control Ad-GFP or Ad-KLF11 -injected db/db mice 5 days after injection (n = 6/group). All data are presented as mean ± SEM, with statistical analysis performed by repeated-measures two-way ANOVA (*p<0.05, **P<0.01, ***P<0.001). (G) Db/db mice were injected with Ad-KLF11 or Ad-GFP. After 5 days, the mice were fasted overnight and anesthetized with tribromoethanol followed by IP injection of 5 U of insulin or saline (as a control). Ten minutes later, the animals were sacrificed, and their liver protein lysates were subjected to Western blot analysis (Left panel). The relative intensities of Phospho-Akt, total Akt, phosphor-GSK3β and total GSK3β were quantitated by densitometry analysis of their bands on film. The results are expressed as the ratios of phospho-Akt/total Akt and phosphor-GSK3β/total GSK3β (statistical analysis of Western blot data from 4 mice under each condition; Right panel). The data shown are the means ± SEM. Statistical significance was determined using a two-tailed Student’s t-test (*P<0.05, **P<0.01).
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pone-0089552-g004: Overexpression of KLF11 in db/db mice alleviates hyperglycemia. (A) Blood glucose level in control Ad-GFP or Ad-KLF11-injected db/db mice 7 days after injection under fasting conditions (n = 6/group, 6-hr fasting). (B) qRT-PCR showing the mRNA levels of gluconeogenic genes in the livers of the same mice as in (A) (n = 6/group). (C) Western blot analysis showing the protein levels of PEPCK-C in in the livers of the same mice as in (A) (n = 6/group). GADPH was used to show the similar amount of protein loaded in different lanes (Left panel). The relative intensities of PEPCK-C bands on the Western blot were determined using NIH Image 1.62 software and normalized using GADPH band intensity (Right panel). (D–F) Glucose tolerance tests (GTTs) (D), pyruvate-tolerance tests (PTT) (E), Insulin tolerance tests (ITTs) (F) in control Ad-GFP or Ad-KLF11 -injected db/db mice 5 days after injection (n = 6/group). All data are presented as mean ± SEM, with statistical analysis performed by repeated-measures two-way ANOVA (*p<0.05, **P<0.01, ***P<0.001). (G) Db/db mice were injected with Ad-KLF11 or Ad-GFP. After 5 days, the mice were fasted overnight and anesthetized with tribromoethanol followed by IP injection of 5 U of insulin or saline (as a control). Ten minutes later, the animals were sacrificed, and their liver protein lysates were subjected to Western blot analysis (Left panel). The relative intensities of Phospho-Akt, total Akt, phosphor-GSK3β and total GSK3β were quantitated by densitometry analysis of their bands on film. The results are expressed as the ratios of phospho-Akt/total Akt and phosphor-GSK3β/total GSK3β (statistical analysis of Western blot data from 4 mice under each condition; Right panel). The data shown are the means ± SEM. Statistical significance was determined using a two-tailed Student’s t-test (*P<0.05, **P<0.01).
Mentions: To determine whether KLF11 regulated hepatic gluconeogenesis in vivo, we performed genetic constitution experiments in db/db mice in which the endogenous expression of KLF11 was low. Our previous results indicated that exogenous myc-tagged KLF11 was effectively expressed in the liver mediated by adenovirus Ad-KLF11, but not in the other tissues, including muscle and adipose tissue [16]. We found that Ad-KLF11 injection of db/db mice had significantly lower fasting plasma glucose levels compared with control adenovirus (Ad-GFP) (Fig. 4A). Consistent with the decreased blood glucose levels, the expression of gluconeogenic genes, including PGC-1α and its downstream target genes PEPCK-C and G6pase, were decreased in the livers of Ad-KLF11-injected mice (Fig. 4B), and western blot analysis indicated that PEPCK-C protein levels were also decreased with KLF11 overexpression (Fig. 4C), indicating that the decrease in hepatic gluconeogenesis contributed to the lower glucose levels. Glucose tolerance tests (GTTs) showed that a modest induction of KLF11 in db/db mouse livers markedly improved glucose intolerance after an intraperitoneal glucose injection compared with control Ad-GFP-injected db/db mice (Fig. 4D). A pyruvate-tolerance test (PTT) demonstrated that de novo hepatic glucose production was reduced in Ad-KLF11-treated db/db mice (Fig. 4E). However, Insulin tolerance tests (ITTs) showed modestly increased insulin sensitivity in Ad-KLF11-infected db/db mice (Fig. 4F). AUC was reduced for GTT and PTT and ITT modestly decreased but did not reach statistical difference for ITT in mice with hepatic overexpression of KLF11 (Figure S1 A–C). These effects through KLF11 overexpression were accompanied by increased phosphorylation of Akt and its downstream target GSK3β in response to acute intraperitoneal insulin injections, whereas the total Akt and GSK3β protein levels remained unchanged (Figure 4G). These results suggest that the gain of function of KLF11 in the db/db mouse livers reduced hepatic glucose production, and improved glucose intolerance, but lesser extent insulin sensitivity.

Bottom Line: Abnormal hepatic gluconeogenesis is related to hyperglycemia in mammals with insulin resistance.Despite the strong evidences linking Krüppel-like factor 11 (KLF11) gene mutations to development of Type 2 diabetes, the precise physiological functions of KLF11 in vivo remain largely unknown.Our data strongly indicated the involvement of KLF11 in hepatic glucose homeostasis via modulating the expression of PEPCK-C.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, School of Basic Medicine, Anhui Medical University, Hefei, China ; National Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China.

ABSTRACT

Background: Abnormal hepatic gluconeogenesis is related to hyperglycemia in mammals with insulin resistance. Despite the strong evidences linking Krüppel-like factor 11 (KLF11) gene mutations to development of Type 2 diabetes, the precise physiological functions of KLF11 in vivo remain largely unknown.

Results: In current investigation, we showed that KLF11 is involved in modulating hepatic glucose metabolism in mice. Overexpression of KLF11 in primary mouse hepatocytes could inhibit the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase (cytosolic isoform, PEPCK-C) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), subsequently decreasing the cellular glucose output. Diabetic mice with overexpression of KLF11 gene in livers significantly ameliorated hyperglycemia and glucose intolerance; in contrast, the knockdown of KLF11 expression in db/m and C57BL/6J mice livers impaired glucose tolerance.

Conclusions: Our data strongly indicated the involvement of KLF11 in hepatic glucose homeostasis via modulating the expression of PEPCK-C.

Show MeSH
Related in: MedlinePlus