Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

The transcriptional coactivator PGC-1α has been implicated in the regulation of multiple metabolic processes. However, the previously reported metabolic phenotypes of mice deficient in PGC-1α have been inconsistent. PGC-1α exists as multiple isoforms, including variants transcribed from an alternative first exon. We show here that alternative PGC-1α variants are the main entity that increases PGC-1α during exercise. These variants, unlike the canonical isoform of PGC-1α, are robustly upregulated in human skeletal muscle after exercise. Furthermore, the extent of this upregulation correlates with oxygen consumption. Mice lacking these variants manifest impaired energy expenditure during exercise, leading to the development of obesity and hyperinsulinemia. The alternative variants are also upregulated in brown adipose tissue in response to cold exposure, and mice lacking these variants are intolerant of a cold environment. Our findings thus indicate that an increase in PGC-1α expression, attributable mostly to upregulation of alternative variants, is pivotal for adaptive enhancement of energy expenditure and heat production and thereby essential for the regulation of whole-body energy metabolism.

The PDK1-FoxO1 signaling in adipocytes controls systemic insulin sensitivity through the 5-lipoxygenase-leukotriene B4 axis.

Although adipocytes are major targets of insulin, the influence of impaired insulin action in adipocytes on metabolic homeostasis remains unclear. We here show that adipocyte-specific PDK1 (3'-phosphoinositide-dependent kinase 1)-deficient (A-PDK1KO) mice manifest impaired metabolic actions of insulin in adipose tissue and reduction of adipose tissue mass. A-PDK1KO mice developed insulin resistance, glucose intolerance, and hepatic steatosis, and this phenotype was suppressed by additional ablation of FoxO1 specifically in adipocytes (A-PDK1/FoxO1KO mice) without an effect on adipose tissue mass. Neither circulating levels of adiponectin and leptin nor inflammatory markers in adipose tissue differed between A-PDK1KO and A-PDK1/FoxO1KO mice. Lipidomics and microarray analyses revealed that leukotriene B4 (LTB4) levels in plasma and in adipose tissue as well as the expression of 5-lipoxygenase (5-LO) in adipose tissue were increased and restored in A-PDK1KO mice and A-PDK1/FoxO1KO mice, respectively. Genetic deletion of the LTB4 receptor BLT1 as well as pharmacological intervention to 5-LO or BLT1 ameliorated insulin resistance in A-PDK1KO mice. Furthermore, insulin was found to inhibit LTB4 production through down-regulation of 5-LO expression via the PDK1-FoxO1 pathway in isolated adipocytes. Our results indicate that insulin signaling in adipocytes negatively regulates the production of LTB4 via the PDK1-FoxO1 pathway and thereby maintains systemic insulin sensitivity.

Hyperglycemia induces skeletal muscle atrophy via a WWP1/KLF15 axis.

Diabetes mellitus is associated with various disorders of the locomotor system including the decline in mass and function of skeletal muscle. The mechanism underlying this association has remained ambiguous, however. We now show that the abundance of the transcription factor KLF15 as well as the expression of genes related to muscle atrophy are increased in skeletal muscle of diabetic model mice, and that mice with muscle-specific KLF15 deficiency are protected from the diabetes-induced decline of skeletal muscle mass. Hyperglycemia was found to upregulate the KLF15 protein in skeletal muscle of diabetic animals, which is achieved via downregulation of the E3 ubiquitin ligase WWP1 and consequent suppression of the ubiquitin-dependent degradation of KLF15. Our results revealed that hyperglycemia, a central disorder in diabetes, promotes muscle atrophy via a WWP1/KLF15 pathway. This pathway may serve as a therapeutic target for decline in skeletal muscle mass accompanied by diabetes mellitus.

Role of S6K1 in regulation of SREBP1c expression in the liver.

The transcription factor sterol regulatory element-binding protein 1c (SREBP1c) plays an important role in the control of fatty acid metabolism in the liver. Evidence suggests that mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contributes to the regulation of SREBP1c expression, but signaling downstream of mTORC1 remains unclear. We have now shown that medium rich in branched-chain amino acids stimulates expression of the SREBP1c gene in cultured hepatocytes in a manner sensitive both to rapamycin, a pharmacological inhibitor of mTORC1, and to a short hairpin RNA (shRNA) specific for S6 kinase 1 (S6K1), a downstream effector of mTORC1. The phosphorylation of S6K1 was increased in the liver of obese db/db mice. Furthermore, depletion of hepatic S6K1 in db/db mice with the use of an adenovirus vector encoding S6K1 shRNA resulted in down-regulation of SREBP1c gene expression in the liver as well as a reduced hepatic triglyceride content and serum triglyceride concentration. These results thus suggest that S6K1 regulates SREBP1c expression both in cultured hepatocytes and in mouse liver, and that increased hepatic activity of S6K1 contributes at least in part to the pathogenesis of obesity-induced hepatic steatosis and hypertriglyceridemia.

Role of KLF15 in regulation of hepatic gluconeogenesis and metformin action.

OBJECTIVE: An increase in the rate of gluconeogenesis is largely responsible for the hyperglycemia in individuals with type 2 diabetes, with the antidiabetes action of metformin being thought to be achieved at least in part through suppression of gluconeogenesis. RESEARCH DESIGN AND METHODS: We investigated whether the transcription factor KLF15 has a role in the regulation of gluconeogenesis and whether KLF15 participates in the antidiabetes effect of metformin. RESULTS: Here we show that KLF15 regulates the expression of genes for gluconeogenic or amino acid-degrading enzymes in coordination with the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha. Liver-specific ablation of KLF15 in diabetic mice resulted in downregulation of the expression of genes for gluconeogenic or amino acid catabolic enzymes and in amelioration of hyperglycemia. Exposure of cultured hepatocytes to metformin reduced the abundance of KLF15 through acceleration of its degradation and downregulation of its mRNA. Metformin suppressed the expression of genes for gluconeogenic or amino acid-degrading enzymes in cultured hepatocytes, and these effects of metformin were attenuated by restoration of KLF15 expression. Administration of metformin to mice inhibited both the expression of KLF15 and glucose production in the liver, the latter effect also being attenuated by restoration of hepatic KLF15 expression. CONCLUSIONS: KLF15 plays an important role in regulation of the expression of genes for gluconeogenic and amino acid-degrading enzymes and that the inhibitory effect of metformin on gluconeogenesis is mediated at least in part by downregulation of KLF15 and consequent attenuation of the expression of such genes.

Role of the E3 ubiquitin ligase gene related to anergy in lymphocytes in glucose and lipid metabolism in the liver.

Gene related to anergy in lymphocytes (GRAIL) is an E3 ubiquitin ligase that regulates energy in T-lymphocytes. Whereas, the relevance of GRAIL to T lymphocyte function is well established, the role of this protein in other cell types remains unknown. Given that GRAIL is abundant in the liver, we investigated the potential function of GRAIL in nutrient metabolism by generating mice in which the expression of GRAIL is reduced specifically in the liver. Adenovirus-mediated transfer of a short hairpin RNA specific for GRAIL mRNA markedly reduced the amounts of GRAIL mRNA and protein in the liver. Blood glucose levels of the mice with hepatic GRAIL deficiency did not differ from those of control animals in the fasted or fed states. However, these mice manifested glucose intolerance in association with a normal increase in plasma insulin levels during glucose challenge. The mice also manifested an increase in the serum concentration of free fatty acids, whereas the serum levels of cholesterol and triglyceride were unchanged. The hepatic abundance of mRNAs for glucose-6-phosphatase, catalytic (a key enzyme in hepatic glucose production) and for sterol regulatory element-binding transcription factor 1 (an important transcriptional regulator of lipogenesis) was increased in the mice with hepatic GRAIL deficiency, possibly contributing to the metabolic abnormalities of these animals. Our results thus demonstrate that GRAIL in the liver is essential for maintenance of normal glucose and lipid metabolism in living animals.

Diversity of mouse proton-translocating ATPase: presence of multiple isoforms of the C, d and G subunits.

Vacuolar-type proton-translocating ATPases (V-ATPases), multimeric proton pumps, are involved in a wide variety of physiological processes. For their diverse functions, V-ATPases utilize a specific subunit isoform(s). Here, we reported the molecular cloning and characterization of three novel subunit isoforms, C2, d2 and G3, of mouse V-ATPase. These isoforms were expressed in a tissue-specific manner, in contrast to the ubiquitously expressed C1, d1 and G1 isoforms. C2 was expressed predominantly in lung and kidney, and d2 and G3 specifically in kidney. We introduced these isoforms into yeasts lacking the corresponding genes. Although the G3 and d2 did not rescue the vmaDelta phenotype, d1 and the two C isoforms functionally complemented the Deltavma6 and Deltavma5, respectively, indicating that they are bona fide subunits of V-ATPase.

A human gene, ATP6E1, encoding a testis-specific isoform of H(+)-ATPase subunit E.

We have identified a novel human gene, ATP6E, encoding an E subunit isoform of vacuolar-type proton-translocating ATPase (V-ATPase). ATP6E1 was mapped to approximately 2p16-p12 on chromosome 2, and has a simple genomic organization: a noncoding exon and a coding one for an E1 isoform separated by a 6.1 kb intron, with boundaries following the GT-AG rule. Transcription initiation sites were found at -375 and -158 bases upstream of the translation initiation codon. Northern blotting analysis demonstrated that ATP6E1 is specifically transcribed in testis as 1.1 kb and 2.2 kb mRNAs, whereas the previously reported ATP6E2 (E2) is expressed in all tissues tested. E1 exhibited 76.9% identity with ubiquitously expressed E2, and both isoforms functionally complemented null mutations of the yeast counterpart VMA4, indicating that they are bona fide subunits of the V-ATPase complex.

A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification.

The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase. Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.