Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta - PubMed (original) (raw)
. 2003 Jul;112(2):197-208.
doi: 10.1172/JCI16885. Epub 2003 Jul 3.
Stephen J Orena, Kristina Rafidi, Anthony J Torchia, Jeffrey L Stock, Audrey L Hildebrandt, Timothy Coskran, Shawn C Black, Dominique J Brees, Joan R Wicks, John D McNeish, Kevin G Coleman
Affiliations
- PMID: 12843127
- PMCID: PMC164287
- DOI: 10.1172/JCI16885
Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta
Robert S Garofalo et al. J Clin Invest. 2003 Jul.
Abstract
The serine/threonine kinase Akt/PKB plays key roles in the regulation of cell growth, survival, and metabolism. It remains unclear, however, whether the functions of individual Akt/PKB isoforms are distinct. To investigate the function of Akt2/PKBbeta, mice lacking this isoform were generated. Both male and female Akt2/PKBbeta-null mice exhibit mild growth deficiency and an age-dependent loss of adipose tissue or lipoatrophy, with all observed adipose depots dramatically reduced by 22 weeks of age. Akt2/PKBbeta-deficient mice are insulin resistant with elevated plasma triglycerides. In addition, Akt2/PKBbeta-deficient mice exhibit fed and fasting hyperglycemia, hyperinsulinemia, glucose intolerance, and impaired muscle glucose uptake. In males, insulin resistance progresses to a severe form of diabetes accompanied by pancreatic beta cell failure. In contrast, female Akt2/PKBbeta-deficient mice remain mildly hyperglycemic and hyperinsulinemic until at least one year of age. Thus, Akt2/PKBbeta-deficient mice exhibit growth deficiency similar to that reported previously for mice lacking Akt1/PKBalpha, indicating that both Akt2/PKBbeta and Akt1/PKBalpha participate in the regulation of growth. The marked hyperglycemia and loss of pancreatic beta cells and adipose tissue in Akt2/PKBbeta-deficient mice suggest that Akt2/PKBbeta plays critical roles in glucose metabolism and the development or maintenance of proper adipose tissue and islet mass for which other Akt/PKB isoforms are unable to fully compensate.
Figures
Figure 1
Generation of Akt2-deficient mice. (a) Illustration depicting strategy for homologous recombination in DBA/1_lac_J ES cells. The locations of the 3′ probe and XbaI recognition sites used for Southern blot screening of the ES cells are indicated. The PGK-neo cassette was inserted in the same orientation as the Akt2 gene. (b) Southern blot analysis of DNA isolated from ES cells transfected with the Akt2 targeting vector. Genomic DNA from six ES cell clones was isolated and digested with XbaI restriction endonuclease and hybridized with the external 1.0-kb BamHI/XbaI 3′ probe. This probe will recognize a 7.0-kb endogenous, 3.0-kb targeted, and 2.7-kb Akt2 pseudogene XbaI fragment. The probe recognizes the pseudogene due to the presence of a 3′ exon, also contained in the Akt2 pseudogene sequence. Note that only clone 2 contains the targeted allele. (c) PCR genotyping analysis of F2-generation mice. Two PCR reactions are performed on each sample; one is specific for a targeted allele (top panel) and the other is specific for the Akt2 locus within the knockout region (bottom panel). Taken together, all three genotypes, wild-type, heterozygous, and homozygous (+/+, +/–, and –/–), can be determined. (d) Western blot analysis of protein extracts from brains isolated from Akt20+/+ and Akt20–/– mice. The blot to the left was hybridized with an anti-Akt1 Ab and the one to the right with an anti-Akt2 Ab.
Figure 2
Adipose tissue mass is decreased in Akt2-null mice. Representative cross-sectional images of wild-type (WT) and Akt2-null (KO) male (a and b) and female (c and d) mice subjected to micro-CT analysis of in situ adipose tissue mass. (a and c) The inguinal subcutaneous (red) and epididymal/gonadal (green) depots and (b and d) the retroperitoneal (red) and mesenteric (green) depots are demarcated for illustration of the gross effect of the Akt2 deficiency on adipose tissue mass.
Figure 3
Adipose tissue mass is decreased in Akt2-null mice. Adipose tissue mass of wild-type and Akt2-null mice was determined by micro-CT scanning in four regional depots, the inguinal subcutaneous (Ing), epididymal/gonadal (Epi/Gon), retroperitoneal (RP), and mesenteric (Mes) regions. Adipose tissue mass was significantly (P < 0.05) reduced in both female (a) and male (b) Akt2-null mice. In female Akt2-null mice adipose depot mass was reduced 80–90% in all depots measured. In male Akt2-null mice adipose depot mass was reduced 65–75% in the inguinal and epididymal depots, and more than 95% in the retroperitoneal and mesenteric depots.
Figure 4
Seven-week-old Akt2-null mice exhibit fasting hyperglycemia and glucose intolerance in an oral glucose-tolerance test. Blood samples were taken from overnight-fasted Akt2-null (open symbols) and wild-type (filled symbols) mice at time zero. Mice were immediately given an oral dose of glucose (1 g/kg), and blood was sampled at the indicated times. Plasma glucose levels were significantly elevated in both male and female Akt2-null mice (open circles and diamonds, respectively) relative to wild-type male and female mice (filled circles and diamonds, respectively) at time zero and 30 minutes following the glucose load.
Figure 5
Hyperglycemia and hyperinsulinemia in male and female Akt2-null mice. Plasma glucose (a) and insulin (b) levels were determined every 14 days in male wild-type (filled circles, n = 11), male Akt2-null (open circles, n = 12), female wild-type (filled diamonds, n = 9), and female Akt2-null (open diamonds, n = 13) mice.
Figure 6
Diabetic phenotype of male Akt2-null mice. (a) Plasma glucose (filled symbols) and insulin (open symbols) levels in three male Akt2-null mice from Figure 5 exhibiting β cell failure (see Figure 9). (b) The remaining nine male Akt2-null mice were mildly hyperglycemic (filled diamonds) while becoming increasingly hyperinsulinemic (open squares), or insulin resistant, with age.
Figure 7
Male Akt2-null mice become severely hypoinsulinemic and hyperglycemic by 8 months of age. A group (n = 6) of 8-month-old male Akt2-null (KO) mice were hypoinsulinemic (a) and extremely hyperglycemic (b) relative to age-matched wild-type males.
Figure 8
Muscle glucose uptake is impaired in Akt2-null mice. The 2-deoxyglucose (2–DG) uptake into isolated soleus muscles from male control and Akt2-null mice was determined in the absence of insulin (basal, white bars) or in the presence of a submaximal (1 nM, gray bars) or maximal (100 nM, black bars) concentration of insulin. *P < 0.05 versus corresponding basal; **P < 0.01 versus corresponding basal; 0#P < 0.05 versus corresponding insulin-treated control sample.
Figure 9
Insulin immunohistochemical analysis of β cells in male Akt2-null mice. Representative pancreatic islets from (a) a 7-week-old wild-type DBA/1lacJ mouse, (b) a 7-week-old Akt2-null mouse, and (c) a 24-week-old Akt2-null mouse displaying the hypoinsulinemic/hyperglycemic phenotype (Figure 6a). The remaining β cells of the latter mouse were interspersed between the exocrine pancreatic cells and exhibited little to no cytoplasmic staining.
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