Loss of Slc12a2 specifically in pancreatic β-cells drives metabolic syndrome in mice

Abstract

The risk of type-2 diabetes and cardiovascular disease is higher in subjects with metabolic syndrome, a cluster of clinical conditions characterized by obesity, impaired glucose metabolism, hyperinsulinemia, hyperlipidemia and hypertension. Diuretics are frequently used to treat hypertension in these patients, however, their use has long been associated with poor metabolic outcomes which cannot be fully explained by their diuretic effects. Here, we show that mice lacking the diuretic-sensitive Na+K+2Cl-cotransporter-1 Nkcc1 (Slc12a2) in insulin-secreting β-cells of the pancreatic islet (Nkcc1βKO) have reduced in vitro insulin responses to glucose. This is associated with islet hypoplasia at the expense of fewer and smaller β-cells. Remarkably, Nkcc1βKO mice excessively gain weight and progressive metabolic syndrome when fed a standard chow diet ad libitum. This is characterized by impaired hepatic insulin receptor activation and altered lipid metabolism. Indeed, overweight Nkcc1βKO but not lean mice had fasting and fed hyperglycemia, hypertriglyceridemia and non-alcoholic steatohepatitis. Notably, fasting hyperinsulinemia was detected earlier than hyperglycemia, insulin resistance, glucose intolerance and increased hepatic de novo gluconeogenesis. Therefore, our data provide evidence supporting the novel hypothesis that primary β-cell defects related to Nkcc1-regulated intracellular Cl-homeostasis and β-cell growth can result in the development of metabolic syndrome shedding light into additional potential mechanisms whereby chronic diuretic use may have adverse effects on metabolic homeostasis in susceptible individuals.

Fig 1. The Ins1^Cre line deletes target alleles exclusively in β-cells of the pancreatic islet.

A-H. Representative pancreas sections of Ins1^Cre:Nkcc1^lox/lox:Tomato mice. Islets were coimmunolabeled against RFP (A and E), insulin (Ins, C and G), glucagon (Gcg, B) and somatostatin (Sst, F) to identify β-, α- or δ-cells, respectively. Overlay images of A-C and E-G are shown in D and H, respectively. I-L. Representative pancreas sections of Nkcc1^βKO mice coimmunolabeled against Nkcc1 (I), Gcg (J) and Ins (K) showing β-cell-specific Nkcc1 deletion in the overlay image (L). Arrowheads in L indicate Nkcc1 immunoreactivity in Ins- or Gcg-negative cells. M. Shown are exons 6–11 (filled boxes) of the mouse Slc12a2 gene and Lox sites (empty arrowheads). Filled arrowheads indicate PCR primers 106w/220f and 206w/320f designed to amplify 5’ Lox sites as 220bp and 320bp bands, respectively. The primer triplet 106w/220f/450r co-amplifies 220bp and 450bp fragments corresponding to the Nkcc1^lox/lox genotype of non-β cells and Cre/Lox-recombined alleles of β-cells, respectively. N. PCR of islet genomic DNA from Nkcc1^βKO mice showing amplicons of expected sizes by using the primers indicated in M. O. Represented are exons 1–5 (filled arrows) of Nkcc1 mRNAs and the RT-PCR primer pair 400s/400a used to produce Nkcc1 amplicons of 400bp. P. Representative RT-PCR experiments using total islet RNA from Nkcc1^lox/lox and Nkcc1^βKO mice. Nkcc1 mRNA expression detected as amplicons of expected sizes mainly in Nkcc1^lox/lox samples.

Fig 2. Loss of Nkcc1 in β-cells reduces islet insulin secretion and β-cell mass.

A. Insulin secretory responses to low (5.5mM) and high (12.5mM) glucose of islets from 22w old Nkcc1^βKO and control mice (Nkcc1^lox/lox) in the presence of vehicle (DMSO) or 10μM bumetanide (BTD), as indicated. Results are expressed as the mean ± SEM of insulin secreted relative to total islet insulin content (n = 7–8, *p<0.05). B-F. Morphometry analysis performed on pancreas sections from Nkcc1^βKO and control mice (Nkcc1^lox/lox) at the indicated ages and immunolabeled against insulin. Shown are the number of β-cells per islet (B), mean β-cell volume (C, pL), β-cell mass (D, mg), islet area (E, % pancreas section) and the number of β-cell clusters (representing ≤5 β-cells/cluster) per mm² of pancreas tissue section (F). The data in B-C represents the mean ± SEM corresponding to >700 individual islets identified in 19–21 tissue sections obtained from male mice (n = 3) of the indicated genotypes and ages. Each point in D-F represents the mean values per single tissue section (*p<0.05).

Fig 3. Hepatic insulin receptor expression, signaling and de novo gluconeogenesis in Nkcc1^βKO mice.

A, B. Expression pattern of insulin receptors (Insr, 95kDa), Akt (60kDa) and G6Pc (40kDa) and phospho-activation of Akt (pAkt) in liver extracts of 10w, 20w and 30w Nkcc1^βKO and control mice (Ins1^Cre) fed (A) or fasted 16h (B). Shown are representative immunoblots loaded to represent 2 mice (n = 3–4 per genotype, age and condition). As loading control, we used β-actin (45kDa). C. Semi-quantitative densitometry analysis of hepatic Insr expression levels relative to β-actin expressed in arbitrary units (au). Shown are the mean ± SEM of 3 independent blots corresponding to 3 male mice of the indicated genotypes, ages and condition (*p<0.05). D. Blood glucose excursions (mg/dl) during alanine tolerance tests (ATT) performed in 16h fasted Nkcc1^βKO and control mice (Ins1^Cre) at the indicated ages (mean ± SEM, n = 9–10, *p<0.05). The areas under each curve (mg/ml/min) are indicated as insets in D.

Fig 4. Absolute BW, gain, composition and adipose tissue morphometry of Nkcc1^βKO mice.

A. Growth of Nkcc1^βKO and control (Nkcc1^lox/lox) mice fed ad libitum a chow diet. Data recorded as net weekly BW mass (g) starting at weaning until mice reached 30w of age. Plotted are the mean ± SEM (n = 9–16, *p<0.01). B. Weekly BW gain (g/week) of Nkcc1^βKO and control (Nkcc1^lox/lox) mice computed by subtracting BW at a given week age to that of the previous week. Each point represents data from a single mouse (n = 9–16, *p<0.01). C, D. Indicated are the mean ± SEM values corresponding to net fat mass (C, g) and lean mass (D, g) of Nkcc1^βKO and control (Nkcc1^lox/lox) mice at the indicated ages (n = 9–16, *p<0.01). E. Mean cross sectional area (μm²) of adipocytes morphometrically determined by analyzing retroperitoneal white fat tissue sections from 10w and 30w old Nkcc1^βKO and control (Nkcc1^lox/lox) mice (n = 3). Each point represents the mean adipocyte area found in a single non-overlapping digital image randomly taken from tissue sections (n = 6–9) of the indicated genotypes and ages (*p<0.001). F. Relative mean adipocyte size distribution computed from the data in E.

Fig 5. Plasma lipids, hepatic index, liver fat content and liver histopathology of Nkcc1^βKO mice.

A, B. Plasma glycerol (A, mg/L) and triglycerides (B, TG mg/dl) of 10w and 30w old Nkcc1^βKO and control (Ins1^Cre) mice fasted 16h. Results represent the mean ± SEM (n = 4–5, *p<0.01). C, D. Plotted are the hepatic index (C) calculated as wet liver mass (g) relative to total BW (g), and the net fat content (mg) per gram of liver tissue (D) of Nkcc1^βKO and control (Nkcc1^lox/lox) mice at the indicated ages. Results are expressed as the mean ± SEM (n = 5–6, *p<0.001). E-H. Shown are representative H&E-stained liver sections of 10w (E-F) or 30w old (G-H) control (Nkcc1^lox/lox, E and G) and Nkcc1^βKO (F and H) mice. The squares in E-H are shown magnified in the images below each one of them to depict histopathology changes including mild steatosis around a central vein (cv) in 10w old Nkcc1^βKO mice and hypertrophic hepatocytes (dashed-lined cells), micro- and macro-vesicular fat deposits in 30w Nkcc1^βKO mice, consistent with a more severe steatosis phenotype (see S4 Fig). Bars indicate 20μm.

Fig 6. Plasma insulin, blood glucose, glucose tolerance and insulin sensitivity of Nkcc1^βKO mice.

A, B. Plasma insulin (A, pmol/L) and whole blood glucose (B, mg/dl) of 10w, 20w and 30w old Nkcc1^βKO and control (Ins1^Cre) mice fed or fasted 16h. Results represent the mean ± SEM (n = 17–28, *p<0.05). C, D. Blood glucose excursions (mg/dl) during glucose tolerance tests (GTT, C) performed in 6h fasted Nkcc1^βKO and control (Ins1^Cre) mice of the indicated ages (mean ± SEM, n = 9–14, *p<0.05) and the areas under the curve (D, mg/ml/min) of those responses. E. Blood glucose responses to exogenous insulin during insulin tolerance tests (ITT) performed in 6h fasted Nkcc1^βKO and control (Ins1^Cre) mice at 10w, 20w and 30w of age. Each point represents the mean ± SEM (n = 9–16, *p<0.05).