Deletion of the mammalian INDY homolog mimics aspects of dietary restriction and protects against adiposity and insulin resistance in mice

Abstract

Reduced expression of the Indy (I'm Not Dead, Yet) gene in D. melanogaster and its homolog in C. elegans prolongs life span and in D. melanogaster augments mitochondrial biogenesis in a manner akin to caloric restriction. However, the cellular mechanism by which Indy does this is unknown. Here, we report on the knockout mouse model of the mammalian Indy (mIndy) homolog, SLC13A5. Deletion of mIndy in mice (mINDY(-/-) mice) reduces hepatocellular ATP/ADP ratio, activates hepatic AMPK, induces PGC-1α, inhibits ACC-2, and reduces SREBP-1c levels. This signaling network promotes hepatic mitochondrial biogenesis, lipid oxidation, and energy expenditure and attenuates hepatic de novo lipogenesis. Together, these traits protect mINDY(-/-) mice from the adiposity and insulin resistance that evolve with high-fat feeding and aging. Our studies demonstrate a profound effect of mIndy on mammalian energy metabolism and suggest that mINDY might be a therapeutic target for the treatment of obesity and type 2 diabetes.

Figure 1

mIndy tissue distribution and functional characteristics. A and B) mINDY transport kinetics for citrate (A) and succinate (B). HEK293 cells were transfected with the plasmids pIndy-mouse.3.1 or empty expression vector pcDNA3.1(+). Uptake assays were performed in HEK-mIndy cells and HEK-Co/418 cells transfected with the empty expression vector pcDNA3.1(+) serving as control cells. Net-uptake is expressed as the difference between the uptake of substrates into HEK-mIndy cells and HEK-Co/418 cells (n=3-6 for each concentration). K_m values were determined by fitting the data to a non-linear regression curve fit. C) mIndy mRNA tissue expression in mINDY^+/+ and mINDY^−/− mice (n=3-4). D) [¹⁴C]-citrate clearance in mINDY^+/+ and mINDY^−/− mice in vivo (n=6). E) Relative mINDY-substrate plasma concentrations. Plasma citrate concentration is increased in the mINDY^−/− mice, in which the cellular uptake of citrate is reduced (n=7-8),***P<0.01, all error bars represent SEM, see also Figures S1 and S2

Figure 2

mINDY mice characteristics. A) Time course of body weight over a nine months period (n=9-15). Differences in body weight increase with time. B) Representative photographs of mINDY^+/+ and mINDY^−/− mice. Body lengths of mINDY^+/+ and mINDY^−/− mice are given in Supplemental Figure S3. *P<0.05, #P<0.01, all error bars represent SEM, see also Figure S3.

Figure 3

mINDY mice are protected from HFD induced obesity. A) Body weight of mINDY^+/+, mINDY^+/−and mINDY^−/− mice on a 6 week HFD (n=8). B) Whole body fat content and lean body mass after a 6 week HFD in the indicated phenotype, as assessed by ¹H magnetic resonance spectroscopy (n=8). Body fat is reduced in HFD fed mINDY^−/− mice. C) Plasma β-hydroxybutyrate, a marker of hepatic lipid oxidation, is increased in HFD fed mINDY^−/− mice (n=7) D) Lipid oxidation from [1-¹⁴C]oleate is increased in primary hepatocytes mINDY^−/− mice (n=5). E) State III oxygen consumption in liver homogenate from mINDY^−/− mice is increased (n=3). F) Lipid synthesis from [¹⁴C]citrate is reduced by ~ 90% in mINDY^−/− primary hepatocytes (n=6). *P<0.05, #P<0.01, all error bars represent SEM, see also Figure S4

Figure 4

Protection from HFD induced hepatic steatosis. A) Representative H&E stains (top), Oil-Red-O stains (middle) and electron microscopic magnifications of liver samples from 6 week HFD fed mice of the indicated genotype, L=lipid droplets, M=mitochondria, N=nucleus. B) Liver triglyceride concentrations are reduced in the mINDY^−/− mice after a 6 week HFD (n=8). C) Hepatic membrane protein kinase ε content is reduced in mINDY^−/− mice (n=4) D) Hepatic diacylglycerol (DAG), and ceramide concentrations as assessed by GC/MS/MS (n=7-8), all error bars represent SEM.

Figure 5

In vivo glucose metabolism in mINDY mice after a 6 week HFD. A) Venous glucose concentrations during an intraperitoneal glucose tolerance test (IPGTT, 1mg/kg BW glucose) in overnight fasted mice of the indicated genotype (n=7-8). B) Venous insulin concentrations during the IPGTT (n=7-8). C) Plasma glucose concentrations during hyperinsulinemic-euglycemic clamp studies (n=7-8). D) Glucose infusion rate during hyperinsulinemic-euglycemic clamp studies is increased with deletion of mIndy (n=6-9). E) Endogenous glucose production in the basal and the clamped state (n=6-9). F) Peripheral glucose uptake during the hyperinsulinemic-euglycemic clamp studies (n=6-9). G) ¹⁴C-2-deoxyglucose uptake into gastrocnemius muscle during the hyperinsulinemic-euglycemic clamp is increased with deletion of mIndy (n=5) H) Gastrocnemius muscle diacylglycerol (DAG) content assessed by LC/MS/MS is reduced in mINDY^−/− mice (n=5). *P<0.05, #P<0.01 by two way ANOVA, all error bars represent SEM, also see Table S3.

Figure 6

Loss of mIndy enhances mitochondrial metabolism. A) Hepatic gene set enrichment analysis in young mINDY^−/− mice compared to young mINDY^+/+ mice on a regular chow (n=4-5). B) Representative hepatic mitochondria (M) with similar EM-magnification. C) Mitochondrial density (mitochondrial number/counted cell volume) as assessed by the point counting method by a sample-blinded specialist using electron microscope magnification in liver slices of the indicated phenotype (n=3) D) Hepatic ATP content (left) and ATP/ADP ratio assessed with ³¹P-MRS after a 24hr fast are reduced in mINDY^−/− mice (n=5) E) Representative immunoblots of hepatic AMP activated protein kinase (AMPK) alpha Thr172 phosphorylation/ total AMPK content (n=6). F) Representative immunoblots of hepatic PGC-1α expression (n=6), all error bars represent SEM, see also Figure S5

Figure 7

Older mINDY mice are protected from adiposity and insulin resistance. A) Body weight in two-month old (young) and eight-month old mice (old) of the indicated phenotype (n=5-8). B) Proportion of whole body fat to body weight as assessed by ¹H magnetic resonance spectroscopy in two-month old (young) and eight-month old mice (old) of the indicated phenotype (n=5-8). The percentage of whole body fat did not change significantly in young compared to old mINDY^−/− mice. C) Proportion of total lean body mass as assessed by ¹H magnetic resonance spectroscopy in two-month old (young) and eight-month old mice (old) of the indicated genotype (n=5-8). D) Glucose infusion rate during the hyperinsulinemic euglycemic clamp study in three-month old (young) and eight-month old (old) mice of the indicated genotype (n=5-8). Older mINDY^−/− were protected from aging related insulin resistance. E) Basal endogenous glucose production in three-month old (young) and eight-month old (old) mice of the indicated genotype (n=6-7). F) Clamp endogenous glucose production in three-month old (young) and eight-month old (old) mice of the indicated genotype (n=6-7). G) Peripheral glucose uptake during the hyperinsulinemic-euglycemic clamp study in three-month old (young) and eight-month old (old) mice of the indicated genotype (n=6-7). H) ¹⁴C-2-deoxyglucose uptake into gastrocnemius muscle during the hyperinsulinemic-euglycemic clamp studies in eight-month old mINDY^+/+ and mINDY^−/− mice (n=6-7), all error bars represent SEM, ns=not significant.