Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific Gs alpha deficiency

Abstract

The G protein G(s)alpha is essential for hormone-stimulated cAMP generation and is an important metabolic regulator. We investigated the role of liver G(s)-signaling pathways by developing mice with liver-specific G(s)alpha deficiency (LGsKO mice). LGsKO mice had increased liver weight and glycogen content and reduced adiposity, whereas survival, body weight, food intake, and metabolic rates at ambient temperature were unaffected. LGsKO mice had increased glucose tolerance with both increased glucose-stimulated insulin secretion and increased insulin sensitivity in liver and muscle. Fed LGsKO mice were hypoglycemic and hypoinsulinemic, with low expression of hepatic gluconeogenic enzymes and PPARgamma coactivator-1. However, LGsKO mice maintained normal fasting glucose and insulin levels, probably due to prolonged breakdown of glycogen stores and possibly increased extrahepatic gluconeogenesis. Lipid metabolism was unaffected in fed LGsKO mice, but fasted LGsKO mice had increased lipogenic and reduced lipid oxidation gene expression in liver and increased serum triglyceride and FFA levels. LGsKO mice had very high serum glucagon and glucagon-like peptide-1 levels and pancreatic alpha cell hyperplasia, probably secondary to hepatic glucagon resistance and/or chronic hypoglycemia. Our results define novel roles for hepatic G(s)-signaling pathways in glucose and lipid regulation, which may prove useful in designing new therapeutic targets for diabetes and obesity.

Figure 1

Generation of LGsKO mice. (A) The upstream portion of the wild-type Gnas allele (E1⁺) including alternative first exon 1A and G_sα exons 1, 2, and 3 is shown at the top, with the positions of the 5′ and 3′ probes used for Southern blot analysis shown above. The scale is in kilobases, with position 0 being the G_sα translational start site. The E1^neo-fl allele is shown below E1⁺, with loxP sites represented as triangles. E1^neo-fl mice were mated with EIIa-cre mice to generate mice with the E1^fl allele. Repeated mating of E1^fl and Alb-cre–transgenic mice produced LGsKO (E1^fl/flAlb-cre⁺) mice with liver-specific deletion of G_sα exon 1 (E1^–) in both alleles. S, SacI; Bg, BglII; Neo, neomycin resistance gene. (B) Southern blot analysis of founder mice (2 left lanes) or offspring of E1^neo-fl mice crossed with EIIa-cre mice (2 right lanes) after SacI digestion and hybridization with the 5′ probe. Genotypes are indicated above each lane. (C) Immunoblot analysis of protein extracts (60 μg/lane) of various tissues from E1^+/+, E1^fl/fl, and LGsKO mice, using a G_sα-specific antibody. The doublet represents the long and short forms of G_sα produced by alternative splicing of exon 3. (D) Immunoblot of liver (left) and kidney (right) extracts from control (C) and LGsKO mice (L) with anti–phospho-CREB (CREB-P; top row) and anti-CREB Abs (bottom row). Pairs are indicated by the lines above.

Figure 2

Body weight, organ weights, and body composition. (A) Body weights of male LGsKO mice (black bars) and controls (white bars) at indicated ages (n = 16/group). (B) Body composition determined by NMR analysis (n = 5–6/group). (C) Organ weights expressed as percent of total body weights (n = 4–6/group). WAT, epididymal WAT. *P < 0.05; **P < 0.01.

Figure 3

Glucose metabolism in fed and fasted LGsKO mice. (A) Blood glucose was measured at indicated times before and after glucagon administration in LGsKO (filled triangles) and control (filled squares) mice (n = 5/group). Serum glucose (B), insulin (C), and corticosterone (Cortico; D) levels in fed (left) and fasted (right) LGsKO and control mice (n = 5–7/group). (E) Urine norepinephrine (NE) and epinephrine (Epi) levels corrected for urine creatinine in overnight-fasted mice (n = 9/group). (F) Liver glycogen content in fed and 16-hour- and 24-hour-fasted mice (n = 6–7/group). *P < 0.05; **P < 0.01.

Figure 4

Glucose tolerance, insulin sensitivity, and secretion. (A) Blood glucose (top) and serum insulin (bottom) was measured at indicated time points before and after i.p. glucose administration (2 mg/g body weight) in overnight-fasted LGsKO (filled triangles) and control (filled squares) mice (n = 13–15/group). (B) Short-term glucose tolerance test using 3 mg/g glucose (n = 6/group). (C) Blood glucose was measured before and after i.p. insulin administration (n = 3/group). (D) Whole-body glucose fluxes in LGsKO and control mice during euglycemic-hyperinsulinemic clamp studies (n = 3–7/group). EGP, endogenous glucose production. Basal and clamp glucose and insulin levels are shown on the right. (E) Tissue glucose uptake rates during clamp studies. (F) Glycogen synthesis rates in gastrocnemius (Gastroc) and quadriceps (Quad) muscles and liver during clamp studies. (G) Immunoblot of muscle (left) and liver (right) extracts from control and LGsKO mouse with anti–phospho-Akt1 (top) and anti-Akt Abs (bottom). *P < 0.05; **P < 0.01.

Figure 5

Lipid metabolism in LGsKO mice. Serum (A) FFA and (B) triglyceride (TG) levels in LGsKO (black bars) and control (white bars) mice (n = 5–7/group). (C) Liver triglyceride content (n = 7/group). (D) Triglyceride secretion rates (n = 4–5/group). *P < 0.05.

Figure 6

Food intake, energy expenditure, and activity studies. (A) Food intake measured over 14 days in LGsKO (black bars) and control (white bars) mice (n = 6/group). (B) Resting and total O₂ consumption, (C) total and ambulatory (Amb) activity levels, and (D) resting and total RER measured over 24 hours at 22°C (left) and 30°C (right) (n = 6/group). Time course of total O₂ consumption rate (E), total RER (F), and total activity(G) over a 45-hour period (fed, hours 1–21; fasting, hours 22–45) at 22°C in LGsKO (filled triangles) and control (filled squares) mice (n = 6/group). To the right of each graph are panels showing the mean ± SEM of each parameter during the fed and fasting periods.*P < 0.05; **P < 0.01. vO₂, volume of O₂ consumed.

Figure 7

Gene expression studies. Gene expression determined by real-time quantitative PCR and normalized to β-actin expression is shown for each gene as percent of expression in LGsKO mice relative to paired control littermates. (A and B) Liver gene expression. (C and D) Kidney gene expression. (E and F) WAT gene expression. A, C, and E show results in randomly fed mice; B, D, and F show results after 24 hours of fasting. MCAD, medium-chain fatty acid dehydrogenase. n = 5–8 per group. *P < 0.05 or **P < 0.01 versus control.

Figure 8

Serum glucagon and GLP-1 levels and pancreatic islet histology. Serum glucagon (A) and GLP-1 (B) levels (in log scale) in fed LGsKO and control mice (n = 4–5 per group; **P < 0.01). (C) H&E-stained sections of pancreatic islets (arrows) from LGsKO mice (right) and controls (left) at ×50 and ×100 original magnification. For LGsKO at ×100, most of the field consists of a single islet. (D) Immunohistochemistry of pancreatic sections from control (top row) and LGsKO mice (bottom 2 rows) with insulin Ab (green, left column), glucagon Ab (red, middle column), and merged images (right column). A positively staining pancreatic duct is indicated with an asterisk.